Assay Methods

SERUM ASSAYS

8-hydroxy-2'-deoxyguanosine (8-OHdG) more info +

The Northwest Life Sciences (NWLSS^TM) 8-OHdG ELISA assay uses a competitive format wherein a murine monoclonal antibody to 8-OHdG (Primary Antibody) and sample or standard are added to a microtiter plate which has been precoated with 8-OHdG. Sample or calibrator 8-OHdG competes with plate-bound 8-OHdG for binding with the antibody. Accordingly, higher concentrations of sample or calibrator lead to reduced binding of the antibody to the 8OHdG coated on the plate. A subsequent wash step removes any free 8-OHdG/antibody adduct leaving stationary plate bound 8-OHdG complexed to antibody for later detection. Anti-murine antibody conjugated to horse radish peroxidase (HRP-Conjugate) is then added to the plate. HRP-conjugate binds to remaining murine anti-8-OHdG and unbound HRP-conjugate is removed in another wash step. Addition of 3,3',5,5'tetramethylbenzidine (TMB Substrate) results in blue color development proportional to the amount of anti 8-OHdG antibody bound to the plate and inversely proportional to theconcentration 8-OHdG in original samples or calibrators applied to the plate. The reaction is terminated by addition of phosphoric acid (Stop Solution) producing yellow color with measurable absorbance at 450 nm.

ACTH more info +

The determination of ACTH should be performed on EDTA plasma. We need 200 uL per determination. To assay the specimen in duplicate, 400 μL of EDTA plasma is required.

This ACTH immunoassay (ALPCO Diagnostics, Salem, HN) is a two-site ELISA [Enzyme Linked ImmunoSorbent Assay] for the measurement of the biologically active 39 amino acid chain of ACTH. A goat polyclonal antibody to human ACTH, purified by affinity chromatography, and a mouse monoclonal antibody to human ACTH are specific for well-defined regions on the ACTH molecule. One antibody is prepared to bind only the C-terminal ACTH 34-39 and this antibody is biotinylated. The other antibody is prepared to bind only the mid-region and N-terminal ACTH 1-24 and this antibody is labeled with horseradish peroxidase [HRP] for detection. In this assay, calibrators, controls, or patient samples are simultaneously incubated with the enzyme labeled antibody and a biotin coupled antibody in a streptavidin-coated microplate well. At the end of the assay incubation, the microwell is washed to remove unbound components and the enzyme bound to the solid phase is incubated with the substrate, tetramethylbenzidine (TMB). An acidic stop solution is then added to stop the reaction and convert the color to yellow. The intensity of the yellow color is directly proportional to the concentration of ACTH in the sample. A dose response curve of absorbance unit vs. concentration is generated using results obtained from the calibrators. Concentrations of ACTH present in the controls and patient samples are determined directly from this curve.

ACTH levels were measured in one hundred and thirty-four (134) apparently normal individuals in the U.S. with the ACTH ELISA. The values obtained ranged from 7.0 to 63 pg/mL. Based on statistical tests on skewness and kurtosis, the population, when transformed logarithmically, follows the normal or Gaussian distribution. The geometric mean + 2 standard deviations of the mean were calculated to be 6.17 to 58.2 pg/mL. The assay range (Standard curve) is 0.0- 165 pg/mL. The assay measures analyte concentrations to 165 pg/mL with a minimum reportable concentration (lowest standard) of 5 pg/mL, and a LLD (sensitivity) of 0.22 pg/mL. The inter assay coefficient of variation reported by the manufacturer is 7.1% at 42.3 pg/mL and 6.9% at 287.8 pg/mL. The intra assay coefficient of variation reported by the manufacturer is 6.7% at 42.2 pg/mL and 2.3% at 269.9 pg/mL.

Adiopnectin more info +

Chemicon (Millipore ) Adiponectin ELISA Kit, human (non boiling) kit is enzyme-linked immunosorbent assay (ELISA) for quantitative determination of Adiponectin in human serum or plasma. Monoclonal antibody specific for human Adiponectin has been precoated onto 96-well microplate. Standards and samples are pipetted into the wells and any adiponectin present is bound by immobilized antibody. Bound adiponectin is captured by biotinylated anti-human adiponectin polyclonal antibody. HRP conjugated streptavidin is added. After washing, a substrate solution is added. The colors develop in proportion to the bound adiponectin quantity. The color development is stopped and the intensity of color is measured. The assay is linear from 2-25 mg/dL. We report the following interassay coefficient of variation: 8.4% at 17.73 ng/mL, 2.4% at 29.13 ng/mL and 6.2% at 39.1 ng/mL. We report the following intraassay coefficient of variation: 7.4 % at 17.73 ng/mL, 0.9 % at 29.13 ng/mL and 1.8 % at 39.1 ng/mL

Androstenedione more info +

We will be using a commercially available enzyme-linked immunosorbent assay (ELISA) from Diagnostic Systems Laboratories (DSL) for the in vitro quantitative measurement of Androstenedione in human serum. The DSL Androstenedione Coated Well ELISA kit Features a direct competitive immunoassay with no required sample extraction or hydrolysis. There is no detectable cross-reactivity with closely related compounds (androsterone, DHEA, DHEA-S or testosterone). The expected values are 0.6 to 2.5 ng/mL in females. The assay measures analyte concentrations from 0.1 to 10 ng/mL with a minimum detectable concentration of 0.1 ng/mL, and a Sensitivity of 0.03 ng/mL. The inter assay coefficient of variation is 3.9% at 0.98 ng/mL, 3.0% at 6.1 ng/mL. The intra assay coefficient of variation is 2.1 % at 0.98 ng/mL, 1.3% at 6.1 ng/mL.

AntiThrombin III (ATIII) more info +

Affinity-purified antibody to ATIII is coated onto the wells of a microtiter plate. The plates are washed. Plasma or other fluids containing ATIII are applied. The coated antibody will capture the ATIII in the sample. After washing the plate to remove unbound material, a second anti-ATIII antibody labeled with peroxidase is added to the plate to bind to the captured ATIII. After washing the plate to remove unbound labeled antibody, the peroxidase activity is expressed by incubation with o-phenylenediamine (OPD). After a fixed development time, the reaction is quenched with the addition of sulfuric acid. The color produced is quantified using a microplate reader. The color generated is proportional to the concentration of ATIII present in the sample.

Beta-Endorphin (β-EP) more info +

The human Beta-Endorphin (β-EP) assay (Elabscience, E-EL-H0572) assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for β-EP has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and (β-EP present is bound by the immobilized antibody. An enzyme-linked monoclonal antibody specific for β-EP is added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of β-EP bound in the initial step. The color development is stopped and the intensity of the color is measured, Absorbance at 450nm is produced in proportion to the amount of β-EP bound in the initial step. The assay has a range (Standard curve) of 15.63 - 1000 pg/mL. We report the following coefficients of variation. Inter-assay CV: 12.8% at 85 pg/mL, 10.2% at 252 pg/mL. Intra-assay CV: 5.1% at 85 pg/mL, 6,2% at 252 pg/mL.

Brain-Derived Neurotropic Factor (BDNF) more info +

The human Brain-Derived Neurotropic Factor (BDNF) assay (R&D Systems, DBD00) assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for BDNF has been pre-coated onto a microplate. Standards and pre-diluted samples are pipetted into the wells and BDNF present is bound by the immobilized antibody. An enzyme-linked monoclonal antibody specific for BDNF is added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of BDNF bound in the initial step. The color development is stopped and the intensity of the color is measured, Absorbance at 450nm is produced in proportion to the amount of BDNF bound in the initial step. The assay has a range (Standard curve) of 62.5 - 4000 pg/mL. We report the following coefficients of variation. Inter-assay CV: 5.8% at 1081 pg/mL, 11.3% at 2669 pg/mL. Intra-assay CV: 1.8% at 1081 pg/mL, 4.4% at 2669 pg/mL.

C-Reactive protein (CRP) more info +

The CRP Ultra Wide Range Reagent Kit is a latex-enhanced turbidimetric in vitro immunoassay. CRP in the sample binds to the specific anti-CRP antibody, which has been adsorbed to latex particles, and aggLutinates. The agglutination is detected as an absorbance change when read on an automated clinical chemistry analyzer (at 570 nm). The magnitude of the change is proportional to the quantity of CRP in the sample. The actual concentration is then determined by interpolation from a calibration curve prepared from calibrators of known concentrations.

C-Reactive protein (hsCRP) more info +

C-Reactive protein is performed on the Alfa-Wasserman ACE analyzer. CRP in the sample reacts with the anti-CRP sensitized latex particles in the hs-CRP antibody reagent to form antigen-antibody complexes that agglutinate the latex particles. The increase in absorbance, measured monochromatically at 592 nm, during a fixed time interval, is directly proportional to the amount of CRP in the sample. The ACE hs-CRP assay is linear from 0.25 to 10.0 mg/L. The MDL is 0.1 mg/dL (2 SD greater than the 0 Standard). We report the following Intra-assay precision expressed as CV: 5.7 % (0.5 mg/L), 2.0% (2.3 mg/L) and 1.2% (9.8 mg/L). We report the following Inter-assay precision expressed as CV: 7.0 % (0.5 mg/L), 3.3% (2.2 mg/L) and 1.2% (9.8 mg/L).

Cholesterol more info +

After de-esterification by cholesterol esterase, the concentration in serum is directly proportional to the amount of quinoneimine produced from cholesterol oxidase action on cholesterol resulting in the production of hydrogen peroxide. The hydrogen peroxide then acts to oxidatively couple p-hydroxybenzioc acid (catalyzed by peroxidase) and 4-aminoantipyrine producing the red quinoneimine chromophore which absorbes light strongly at 505 nm. The ACE cholesterol assay is linear from 2 to 600 mg/dL. The MDL is 2 mg/dL (2 SD greater than the 0 Standard). The manufacturer provides the following Intra-assay precision expressed as CV: 2.2 % (139 mg/dL), 1.1% (212 mg/dL) and 2.0% (290 mg/dL). The manufacturer provides the following Inter-assay precision expressed as CV: 2.3 % (139 mg/dL), 1.6% (212 mg/dL) and 2.1% (290 mg/dL).

CMV (Cytomegalovirus) IgG ELISA more info +

The Wampole Laboratories Cytomegalovirus (CMV) IgG ELISA Kit is a solid phase enzyme-linked immunosorbent assay (ELISA). Microtiter wells as a solid phase are coated with CMV antigen. Diluted patient specimens and controls are pipetted into these wells. During incubation CMV-specific antibodies of positive specimens and controls are bound to the immobilized antigens. After a washing step to remove unbound sample and control material horseradish peroxidase conjugated goat anti-human IgG antibodies are dispensed into the wells. During a second incubation this anti-IgG conjugate binds specifically to IgG antibodies resulting in the formation of enzyme-linked immune complexes. After a second washing step to remove unbound conjugate the immune complexes formed (in case of positive results) are detected by incubation with TMB substrate and development of a blue color. The blue color turns into yellow by stopping the enzymatic indicator reaction with sulfuric acid. The intensity of this color is directly proportional to the amount of CMV-specific IgG antibody in the patient specimen. Absorbance at 450 nm is read using an ELISA microtiter plate reader.

Complement C3 more info +

Complement C3 is performed on the Alfa-Wasserman ACE analyzer using the K-ASSAY (KAMIYA BIOMEDICAL COMPANY) complement C3 reagents. Complement C3 in the sample reacts with the anti- complement C3 sensitized latex particles in the complement C3 antibody reagent to form antigen-antibody complexes that agglutinate the latex particles. The increase in absorbance, measured monochromatically at 592 nm, during a fixed time interval, is directly proportional to the amount of complement C3 in the sample. The ACE complement C3 assay is linear from 30 to 350 mg/dL. The MDL is 30 mg/dL (lowest standard). The manufacturer reports the following Intra-assay precision expressed as CV: 3.33 % (33.7 mg/dL), 2.22% (75.6 mg/dL) and 1.94% (112.4 mg/dL). The manufacturer reports the following Inter-assay precision expressed as CV: 3.63 % (35.4 mg/dL), 2.31% (84.3 mg/dL) and 1.48% (114.4 mg/dL).

 

Complement C4 more info +

Complement C4 is performed on the Alfa-Wasserman ACE analyzer using the K-ASSAY (KAMIYA BIOMEDICAL COMPANY) complement C4 reagents. Complement C4 in the sample reacts with the anti- complement C4 sensitized latex particles in the complement C4 antibody reagent to form antigen-antibody complexes that agglutinate the latex particles. The increase in absorbance, measured monochromatically at 592 nm, during a fixed time interval, is directly proportional to the amount of complement C4 in the sample. The ACE complement C4 assay is linear from 8 to 80 mg/dL. The MDL is 8 mg/dL (lowest standard). The manufacturer reports the following Intra-assay precision expressed as CV: 5.17 % (5.7 mg/dL), 2.10% (36.3 mg/dL) and 3.38% (56.8 mg/dL). The manufacturer reports the following Inter-assay precision expressed as CV: 3.72 % (6.51 mg/dL), 3.87% (30.6 mg/dL) and 2.10% (53.2 mg/dL).

 

Cortisol more info +

The human Cortisol assay (ALPCo, 11-CORHU-E01) assay employs the quantitative competitive enzyme immunoassay technique. An antibody specific for Cortisol has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and cortisol present is bound by the immobilized antibody. This competes with enzyme-labeled cortisol for available binding sites. Following a wash to remove any unbound enzyme reagent, a substrate solution is added to the wells and color develops. The color development is stopped and the intensity of the color is measured. Absorbance at 450nm is inversely proportional to the amount of cortisol bound in the initial step. The assay has a range (Standard curve) of 0.5 - 60 ug/dL. We report the following coefficients of variation. Inter-assay CV: 2.9% at 39.6 ug/dL, 6.1% at 16.2 ug/dL. Intra-assay CV: 2.6% at 39.6 ug/dL, 4.4% at 16.2 ug/dL.

Cortisol more info +

The Cortisol assay is a competitive immunoassay run on Siemen's ACS-180 automated analyzer using chemiluminescent technology. Cortisol in the patient sample competes with acridinium ester-labeled cortisol in the Lite Reagent for binding to polyclonal rabbit anti-cortisol antibody in the Solid Phase Reagent. The polyclonal rabbit anti-cortisol antibody is bound to monoclonal mouse anti-rabbit antibody, which is covalently coupled to paramagnetic particles in the Solid Phase. 20 uL of serum is required for the assay in addition to sufficient dead volume for aspiration and repeat analysis. The assay range is 1.45 - 78.4 ug/dL and the assay is standardized analytically and confirmed by gas chromatography- mass spectroscopy. We report the following coefficients of variation. Inter-assay CV: 9.4% at 4.3 ug/dL, 8.5% at 12.6 ug/dL, 7.5% at 27.1 ug/dL. Intra-assay CV: 5.4% at 4.3 ug/dL, 4.9% at 12.6 ug/dL, 4.1% at 27.1 ug/dL.

Cotinine more info +

The Cotinine Immunoassay (CalBiotech) is a competitive ELISA [Enzyme-Linked ImmunoSorbent Assay] for the measurement of Cotinine. Samples are added to a cotinine coated microwell and allowed to compete for HRP-labeled anti-cotinine antibody binding sites. At the end of the assay incubation, the microwell is washed to remove unbound components and the enzyme bound to the solid phase is incubated with the substrate, tetramethylbenzidine (TMB). An acidic stopping solution is then added to stop the reaction and converts the color to yellow. The intensity of the yellow color is indirectly proportional to the concentration of Cotinine in the sample. A dose response curve of absorbance unit vs. concentration is generated using results obtained from the calibrators. Concentrations of Cotinine present in the controls and patient samples are determined directly from this curve. The assay requires 10 uL of serum or urine for duplicate analysis. The reportable undiluted assay range (standard curve) is 5-100 ng/mL. The intra-assay coefficient of variation is 7% (0.72 ng/mL) and 8% (285 ng/mL). The inter-assay coefficient of variation is 14.1% (0.72 ng/mL) and 14.6% (263 ng/mL).

Creatinine Assay more info +

Creatinine is a catabolic end-product produced by an irreversible loss of water from the creatine molecule and is an intermediate in energy generation for muscle contraction. The ability of creatinine to form a red-orange complex with alkali picrate (the Jaffee Reaction) has, since its introduction, formed the basis for most serum creatinine assays. A number of modifications and manipulations to increase the specificity of the reaction have been introduced including adsorption onto Lloyd ’s Reagent, sample pretreatment and protein precipitation.

In the Alfa-Wasermann ACE creatinine Assay the amount of red-orange complex formed is measured during a fixed time interval. This kinetic approach provides better specificity then those methods previously mentioned because of the difference in the rate of color frormation between creatinine and interfering substances. 20 μL of serum is required for the assay in addition to sufficient dead volume for aspiration and repeat. The assay is linear from 2-25 mg/dL.

CRH/CRF more info +

This competitive Enzyme Immunoassay kit (manufactured by Accurate Chemican & Scientific Corp.) is designed to detect CRH in human serum. Peptide antibody and non-biotinylated peptide (either standard or unknown) are place in a well and mixed. The peptide antibodies bind to the specially treated walls of the well, and the non-biotinylated peptide bind to the peptide antibodies. After the pre-incubation of peptide antibody and non-biotinylated peptide (either standard or unknown), the biotinylated peptide is added. The biotinylated peptide competes for the antibody binding sites with the standard peptide or the unknown sample peptide. After incubation, unbound biotinylated peptide is removed by washing and streptavidin-conjugated horseradish peroxidase (SA-HRP) is added and allowed to bind to the immobilized primary antibody-biotinylated peptide complex. After washing away excess SA-HRP, TMB (3,3 ’,5,5 ’-tetramethyl benzidine dihydrochloride) is allowed to react with the bound HRP. The color intensity that develops depends on the quantity of biotinylated peptide bound to the immobilized antibody. When more non-biotinylated peptide competes for the limited amount of antibody, less biotinylated peptide/SA-HRP can be immobilized and less color is produced by the substrate.

Cystatin C more info +

Cystatin C is a member of family 2 of the cystatin superfamily. It inhibits many cysteine proteases such as papain and Cathepsins B, H, K, L and S. It is ubiquitous in human tissues and body fluids. A point mutation in the gene coding for the 120 amino acid mature Cystatin C causes a hereditary form of amyloid angiopathy in which the protein variant (Leu 68 to Gln) is deposited in the cerebral arteries, leading to fatal cerebral hemorrhage.

The Quantikine Human Cystatin C Immunoassay (R&D Systems) is a 4.5 hour solid-phase ELISA designed to measure human Cystatin C in cell culture supernates, serum, plasma, saliva, urine, and human milk. This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for Cystatin C has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any Cystatin C present is bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked monoclonal antibody specific for Cystatin C is added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of Cystatin C bound in the initial step. The color development is stopped and the intensity of the color is measured. Assay range 3.12-100 ng/mL. Fifty assays were evaluated and the minimum detectable dose (MDD) of Cystatin C ranged from 0.030 - 0.227 ng/mL. The mean MDD was 0.102 ng/mL. The MDD was determined by adding two standard deviations to the mean optical density value of twenty zero standard replicates and calculating the corresponding concentration.

Dehydroepiandrosterone (DHEA) more info +

The DHEA EIA assay from Diagnostic Systems Laboratories, Inc., (DSL-10-9000) coated Well EIA assay kit features a direct competitive immunoassay with no required sample extraction or hydrolysis. There is negligible cross-reactivity with closely related compounds (DHEA-S, androstenedione, androsterone, testosterone or androstanediol). The assay employs the basic principle of enzyme immunoassay involving competition between unlabeled DHEA and enzyme-labeled DHEA for a fixed number of rabbit polyclonal antibody binding sites. Standards, samples, enzyme-labeled DHEA, and the DHEA-specific antibody are pipetted into the wells of a microtiterplate precoated with goat-anti-rabbit antibody. After an incubation period allowing the antigen-antibody complexes to be immobilized by the secondary antibody on the plate, the plate is washed removing any unbound substances. A chromogen solution is added to the wells, and after color has developed due to the action of the enzyme, the reaction is stopped and the color is quantified by reading the light absorbance with a plate reader. The amount of enzyme-labeled antigen bound to the antibody, and thus the color intensity, is inversely proportional to the concentration of unlabeled DHEA present. The assay range is 0.2 - 30 ng/mL with a lower limit of detection of 0.1 ng/mL. The expected values are 1.3 to 9.8 ng/mL in females. The inter assay coefficient of variations are 17.5% at 2.2 ng/mL and 20.7% at 6.8 ng/mL. The intra assay coefficient of variations are 8.9% at 2.2 ng/mL, and 10.7% at 6.8 ng/mL.

Dehydroepiandrosterone sulphate (DHEAS) more info +

Dehydroepiandrosterone sulphate (DHEAS) is a steroid produced by the adrenal cortex, the concentration of which changes significantly throughout the human lifespan. We have developed an automated, ACS:180-based chemiluminescent assay an automated chemiluminescent assay using the Bayer Diagnostics ACS:180 to determine the levels of DHEAS in human serum(1). This chemiluminescent immunoassay involves the competitive binding of a Dimethylacridinuim ester (DMAE) labeled DHEAS derivative to a rabbit anti-DHEAS antibody. The solid phase is Goat anti-rabbit IgG conjugated to superparamagnetic particles (PMP). 10 uL of serum is required for the assay in addition to sufficient dead volume for aspiration and repeat analysis. The assay range is 1.52 to 1020 ug/dL and the assay is standardized against DHEA-s obtained from Steraloids. The detection level of this assay is approximately 1.9 ug/dL. Closely related steroids ((danazol, cholesterol, pregnenolone, 17a-hydroxyprogesterone, corticosterone, 17a-estradiol, 17b-estradiol, testosterone, estriol, 11-deoxycortisol, norethindrone, estrone, aldosterone, estradiol glucuronide and cortisol) show little cross reactivity in the assay indicating high specificity. The assay demonstrates acceptable parallelism of diluted serum samples and recovery of added exogenous hormone. DHEA cross reacts 192%, but the low levels of serum DHEA render this inconsequential. The expected values are 150-500 ug/dL for normally cycling females and 10-350 ug/dL for postmenopausal females. The intra assay coefficient of variation is 8.02% (n=261) and inter assay coefficient of variation is 11.34% (53.32 ug/dL, n=37) and 9.74% (250.21 ug/dL, n=37).

EBV (Epstein-Barr Virus) IgG more info +

The DRG Epstein-Barr Virus (EBNA-1) IgG ELISA Kit is a solid phase enzyme-linked immunosorbent assay (ELISA). Microtiter wells as a solid phase are coated with inactivated EBV nuclear antigen. Diluted patient specimens and ready-for-use controls are pipetted into these wells. During incubation EBNA-1-specific antibodies of positive specimens and controls are bound to the immobilized antigens. After a washing step to remove unbound sample and control material horseradish peroxidase conjugated anti-human IgG antibodies are dispensed into the wells. During a second incubation this anti-IgG conjugate binds specifically to IgG antibodies resulting in the formation of enzyme-linked immune complexes. After a second washing step to remove unbound conjugate the immune complexes formed (in case of positive results) are detected by incubation with TMB substrate and development of a blue color. The blue color turns into yellow by stopping the enzymatic indicator reaction with sulfuric acid. The intensity of this color is directly proportional to the amount of EBNA-1-specific IgG antibody in the patient specimen. Absorbance at 450 nm is read using an ELISA microtiter plate reader.

Endothelin-1 (ET-1), more info +

Endothelin-1 (ET-1), a peptide of 21 amino acid (aa) residues, is a pleiotropic molecule best known for its action as a potent vasoconstrictor. Originally isolated from porcine aortic endothelial cells, ET-1 is one of a family of three proteins encoded by distinct genes that also includes Endothelin-2 (ET-2) and Endothelin-3 (ET-3) (2, 3). ET-2 and ET-3 differ from ET-1 by 2 and 6 amino acids, respectively. All members of the Endothelin family contain two essential disulfide bridges and six conserved aa residues at the C-terminus. Human ET-1 is initially synthesized as a pre-pro-polypeptide of 212 amino acids. It is proteolytically cleaved by a signal peptidase to produce pro-ET-1 and further processed by a Furin-like protease to yield a 38 aa peptide termed Big ET-1. Big ET-1 is then cleaved by the membrane-bound metalloprotease Endothelin-converting enzyme (ECE-1), producing the potent 21 aa mature form ET-1 (aa 1-21). Alternatively, ET-1 may exist in an active 31 aa form (ET-1 (aa 1-31)) following cleavage of Big ET-1 by chymase. The vascular endothelium is an abundant source of ET-1. It may also be expressed by leukocytes, smooth muscle cells, mesangial cells, cardiac myocytes, and astrocytes. ET-1 can be induced in endothelial cells by many factors including mechanical stimulation, various hormones, and pro-inflammatory cytokines (16). Production is inhibited by nitric oxide (NO), Prostacyclin, and atrial natriuretic peptide (ANP).

Two receptors for the Endothelin family have been cloned and designated ETA and ETB. ETA and ETB belong to the large family of heptahelical G protein-coupled receptors. The ETA receptor shows a higher affinity for ET-1 than for ET-2 and lowest affinity for ET-3, while the ETB receptor shows approximately equal affinity for each of the three Endothelins. ETA is primarily responsible for the vasoconstrictor effects of ET-1 and is expressed by blood vessel smooth muscle cells. The ETB receptor is also present in smooth muscle and the endothelia of blood vessels, kidney, lung, and brain. ET-1 has the ability to activate an array of signaling cascades including classical phosphatidylinositol turnover pathways leading to downstream PKC activation and Ca²⁺ mobilization. Other potential signaling mediators activated or produced by ET-1 include PI 3-kinase/Akt, NO, FAK, and Rho GTPases. ET-1 signaling may also be mediated indirectly via transactivation of the EGF receptor leading to downstream signaling by Ras and MAP kinases. Injection of a single dose of ET-1 produces an initial decrease in systemic blood pressure followed by a prolonged increase in blood pressure. Blockade of Endothelin receptors with a systemic injection of an ETA/ETB antagonist causes progressive vasodilation, and elevated levels of ET-1 are found in some forms of human hypertension. ET-1 also stimulates cardiac contraction and the growth of cardiac myocytes, regulates the release of vasoactive substances, and stimulates smooth muscle cell mitogenesis. It also acts as a pro-survival factor for endothelial cells and regulates secretion by hypothalamic and pituitary cells. ET-1 may control inflammatory responses by promoting the adhesion and migration of neutrophils and stimulating the production of pro-inflammatory cytokines. It has also been implicated in cancer progression at several levels including regulating the proliferation and migration of tumor cells and acting as a pro-angiogenic factor. In addition, ET-1 has putative roles in other pathologies including septic shock, atherosclerosis, heart failure, renal insufficiency, pulmonary hypertension, and cerebrovascular conditions associated with subarachnoid hemorrhage.

The Quantikine Endothelin-1 (ET-1) immunoassay is a solid phase ELISA designed to measure ET-1 in cell culture supernates, serum, plasma, and urine. It contains synthetic ET-1 and antibodies raised against synthetic ET-1. This immunoassay has been shown to accurately quantitate synthetic and naturally occurring ET-1. This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for ET-1 has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and the immobilized antibody binds any ET-1 present. After washing away any unbound substances, an enzyme-linked monoclonal antibody specific for ET-1 is added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of ET-1 bound in the initial step. The color development is stopped and the intensity of the color is measured. This assay recognizes natural and synthetic Endothelin-1. There is no detectable cross-reactivity with most closely related compounds. Human Endothelin-2 shows 23.4% cross-reactivity in this assay. Normal human serum values are expected to be approximately 1.2 pg/mL. The assay measures analyte concentrations from 0.39 to 25 pg/mL with a minimum detectable concentration of 0.087 pg/mL. and a sensitivity of 0.08 ng/mL. The inter assay coefficient of variation provided by the manufacturer is 7.6% at 3.0 pg/mL, 5.3% at 14.4 pg/mL. The intra assay coefficient of variation provided by the manufacturer is 4.0% at 3.0 pg/mL, 1.9% at 14.7 pg/mL.

Estradiol (E2) ACS OLE more info +

Serum Estradiol (E2) concentrations will be measured with a modified, off-line ACS: 180 (E2-6) immunoassay(3) (Bayer Diagnostics Corp, Tarrytown , NY). This assay is a competitive chemiluminescence immunoassay; estradiol in the sample is allowed to competitively bind to a monoclonal mouse antibody labeled with acridinium ester. Unbound antibody then is allowed to bind to an estradiol derivative coupled to paramagnetic particles. An inverse relationship exists between the amount of estradiol present in the sample and the amount of relative light units detected by the luminometer. This assay is standardized analytically and confirmed by gas chromatography-mass spectrometry. The assay requires 400 uL of sample for duplicate analysus. Expected values for menstruating women in the follicular phase are 28 to 172 pg/mL, luteal phase are 55 to 246 pg/mL, postmenopausal are 13 to 93 pg/mL, and males are 21 to 76 pg/mL; the assay measures estradiol concentrations up to 250 pg/mL with a minimum detectable concentration of 1 pg/mL. Closely related compounds (Estriol, Estrone, 17 Beta-Estradiol, 3 Beta-D-Glucuronide, Ethinyl Estradiol, Bromocriptine, Clomiphene and Danazol) show insignificant crossreactivity. Inter- and intra-assay coefficients of variation are 13.8% and 8.5%, respectively, over the assay range.

The following steroids were tested for cross-reactivity in the assay system: aldosterone, cholesterol, cortisone, cortisol, danazol, dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), estradiol, estradiol-glucuronate, estriol, estrone, progesterone, corticosterone, testosterone, norethindrone, pregnenolone sulfate, 11-deoxycortisol, 17-hydroxypregnenolone and 17-hydroxy-progesterone. Cross-reactivity of less than 1% was observed for estriol (0.28%), estrone (0.75%), 17-estradiol (0.04%), estradiol glucuronide (0.09%), and aldosterone (0.05%). All others showed <0.009% cross-reaction in the assay.

Estradiol (E2) Centaur more info +

Serum Estradiol (E2) concentrations are measured using the Estradiol-6 III immunoassay performed on the ADVIA Centaur instrument (Siemens HealthCare Diagnostics). This assay is a competitive chemiluminescence immunoassay whereby Estradiol in the patient sample competes with acridinium ester-labeled estradiol in the Lite Reagent for a limited amount of rabbit anti-estradiol antibody in the Antibody Reagent. Rabbit anti-estradiol is captured by mouse anti-rabbit IgG, which is coupled to paramagnetic particles in the Solid Phase. An inverse relationship exists between the amount of estradiol present in the sample and the amount of relative light units detected by the luminometer. This assay is standardized using internal standards manufactured analytically which are traceable to gas chromatography-mass spectroscopy. The assay requires 150 uL of sample for duplicate analysis not including requisite dead volume. Expected values for menstruating women in the follicular phase are 18.9 to 246.7 pg/mL, luteal phase are 22.4 to 256 pg/mL, postmenopausal are not detectable to 44.5 pg/mL, and males are 11.6 to 41.2 pg/mL; the assay measures estradiol concentrations up to 1000 pg/mL with a minimum detectable concentration of 7.0 pg/mL. Inter-assay coefficients of variation are 11.0% (102.9 pg/mL), 7.0% (225.9 pg/mL) and 3.8% (615.9 pg/mL). Intra-assay coefficients of variation are 3.9% (102.9 pg/mL), 5.0% (225.9 pg/mL) and 1.4% (615.9 pg/mL).

Estriol (E3) more info +

The DSL-10-3700 ACTIVE^® Ultra-Sensitive Unconjugated Estriol EIA Kit uses the competitive binding enzyme immunoassay format. In the assay, Standards, Controls and unknowns containing unconjugated estriol are incubated with biotin-labeled estriol and rabbit anti-estriol antiserum in microtitration wells coated with goat anti-rabbit gamma globulin where the unlabeled and biotin-labeled antigens compete for a limited number of anti-estriol binding sites. After incubation and washing, the wells are incubated with streptavidin-horse radish peroxidase, which binds to the biotinylated estriol. The unbound streptavidin-HRPO is washed, followed by incubation with the substrate tetramethylbenzidine (TMB). An acidic stopping solution is then added and the degree of enzymatic turnover of the substrate is determined by dual wavelength absorbance measurement at 450 and 620 nm. The expected values in pregnant females are ND-0.48 ng/mL (1^st trimester), 0.20-4.43 ng/mL (2^nd trimester) and 1.47-13.92 ng/mL (3^rd trimester). This method features a standard range of 0.1 to 30 ng/mL, and a minimum detectable concentration of 0.1 ng/mL. The manufacturer reports the following: Inter-assay CV: 6.7% at 0.66 ng/mL, 5.3% at 3.27 ng/mL and 5.7% at 7.55 ng/mL.

Estrone (E1) more info +

We will be using a commercially available enzyme-linked immunosorbent assay (ELISA) from Diagnostic Systems Laboratories (DSL) for the in vitro quantitative measurement of Estrone (E1) in human serum. The DSL Estrone assay is a direct assay which uses a highly specific antiserum formulated to eliminate the need for laborious sample preparation schemes. The expected values in females range from 0,1 to 7 ng/mL in normally cycling women and from non-detectable to 0.6 in post menopausal women (without HRT). This method features a wide dynamic standard range of 0.05 to 90 ng/mL, and a minimum detectable concentration of 0.01 ng/mL. We report the following: Inter-assay CV: 12.7% at 1.2 ng/mL, 10.8% at 9.2 ng.mL, Intra-assay CV: 6.7% at 1.2 ng/mL, 2.9% at 9.2 ng.mL.

Estrone Sulfate (E1S) more info +

We will be using a commercially available enzyme-linked immunosorbent assay (ELISA) from Diagnostic Systems Laboratories (DSL) for the in vitro quantitative measurement of Estrone Sulfate (E1S) in human serum. The DSL Estrone Sulfate assay is a direct assay which uses a highly specific antiserum formulated to eliminate the need for laborious sample preparation schemes. The expected values in females range from 0,1 to 7 ng/mL in normally cycling women and from non-detectable to 0.6 in post menopausal women (without HRT). This method features a wide dynamic standard range of 0.05 to 90 ng/mL, and a minimum detectable concentration of 0.01 ng/mL. We report the following: Inter-assay CV: 12.7% at 1.2 ng/mL, 10.8% at 9.2 ng.mL, Intra-assay CV: 6.7% at 1.2 ng/mL, 2.9% at 9.2 ng.mL

F2 Isoprostane more info +

[iPF_2α-III EIA Kit; 8-epi Prostaglandin F_2α EIA Kit; 8-Isoprostane ELISA Kit; 8-iso Prostaglandin F_2α EIA]

This Cayman assay is based on the competition between 8-isoprostane and an 8-isoprosrane- Acetlycolinesterase (AChE) conjugate (8-Isoprostane Tracer) for a limited number of 8-iosprostane-specific rabbit antiserum binding sites. Because the concentration of the 8-Isoprostane Tracer is held constant while the concentration of 8-isoprostane varies, the amount of 8-Isoprostane Tracer that is able to bind to the rabbit antiserum will be inversely proportional to the concentration of 8-isoprosrane in the well. This rabbit anriserum-8-isoprostane (either free or tracer) complex binds to the rabbit IgG mouse monoclonal antibody that has been previously attached to the well. The plate is washed to remove any unbound reagents and then Ellman's Reagent (which contains the substrate to AChE) is added to the well. The product of this enzymatic reaction has a distinct yellow color and absorbs strongly at 412 nm. The intensity of this color, determined spectrophorometrically, is proportional to the amount of 8-Isoprostane Tracer bound to the well. which is inversely proportional to the amount of free 8-isoprostane present in the well during the incubation.

Plasma. serum, urine, whole blood, as well as other heterogeneous mixtures can interfere In the assay. It is best to check for interference before embarking on a large number of sample measurements. To test for interference, dilute one or two test samples to obtain at least two different dilutions of each sample between -5 and 200 pg/ml (i.e., 20-80% B/Bo). The two different dilutions of the sample show good correlation (differ by 20% or less) in the final calculated 8-isoprostane concentration, purification is not required. If you do not see good correlation of the different dilutions. purification is advised. Cayman offers an 8-Isoprosrane Affinity Column and Affinity Sorbent are recommended as the easiest and most convenient purification format for 8-isoprosrane. The affinity column purification procedures have been validated with plasma and urine samples.

Fibrinogen more info +

The AssayMax Human Fibrinogen ELISA kit is designed for detection of human FBG in plasma. This assay employs a quantitative competitive sandwich enzyme immunoassay technique that measures FBG in less than 3 hours. A murine antibody specific for FBG has been pre-coated onto a 96-well microplate with removable strips. FBG in standards and samples is competed by a biotinylated FBG sandwiched by the immobilized antibody and streptavidin-peroxidase conjugate. All unbound material is then washed away and a peroxidase enzyme substrate is added. The color development is stopped and the intensity of the color is measured.

Follicle Stimulating Hormone (FSH) more info +

Serum Follicle Stimulating Hormone (FSH) concentrations will be measured with a two-site chemiluminescence (sandwich) immunoassay which uses constant amounts of two antibodies that have a specificity for the intact FSH molecule(1); a polyclonal sheep anti-FSH antibody labeled with acridinium ester and a monoclonal mouse anti-FSH antibody covalently coupled to paramagnetic particles. Separation, aspiration, and deionized water wash steps separate bound from free. A direct relationship exists between the amount of FSH in the sample and the relative light units detected by the luminometer (photomultiplier tube). The test results are determined from a calibration curve using standards obtained from Bayer Diagnostics which are referenced to the WHO 2nd IRP 78/549 standard. The expected values for the follicular phase are 1.1 to 9.6 mIU/mL, midcycle are 2.3 to 20.9 mIU/mL, luteal phase are 0.8 to 7.5 mIU/mL, and postmenopausal are 34.4 to 95.8 mIU/mL; the assay requires 100 uL of sample and measures FSH concentrations up to 200 mIU/mL with a minimum detectable concentration of 0.3 mIU/mL. For serum, inter- and intra-assay coefficients of variation are 8.1% and 3.5 %, respectively and, for urine, they are 10.9% and 3.9%, respectively.

Galectin-3 more info +

Galectin is widely distributed in nematodes, insects, and porifers, as well as vertebrates, and it has also been found to be present in true fungi. Galectin does not just occur in the cytoplasm, it is also present in the nucleus, on the cell surface, in the extracellular matrix, etc., and it is thought to be involved in many biological phenomena, including development, differentiation, morphogenesis, tumor metastasis, cell death, and RNA splicing. Galectin-3 is a β-galactoside-binding protein that has been named IgE-binding protein, CBP35, CBP30, Mac-2, L-29, L-31, L-34, etc., and structurally it is a chimera-type lectin composed of a sugar-chain-binding domain (galectin domain) and a non-lectin domain. The Human Galectin-3 Assay Kit from Immuno-Biological Laboratories Co (Takasaki-Shi, Gunma) is a solid phase sandwich ELISA using 2 kinds of high specific antibodies. Standards and samples are pipetted into the wells and any Galectin-3 present is bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked polyclonal antibody specific for Galectin-3 is added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of Galectin-3 bound in the initial step. Tetra Methyl Benzidine (TMB) is used as coloring agent (Chromogen). The color development is stopped and the intensity of the color is measured. The assay range is 117.19 - 7,500 pg/mL and the LLD is 44 pg/mL. The manufacturer reports the following coefficients of variation. Inter-assay CV: 4.7% (5,403.15 pg/mL) , 7.4% (1,247.61 pg/mL), 8.8% (275.73 pg/mL). Intra-assay: 4.0% (5,363.51 pg/mL), 4.0% (1,229.51 pg/mL), 5.5% (267.41 pg/mL). We report the following Inter-assay CV: 1.2% (4395 pg/mL) , 0.8% (5267 pg/mL).

GGT (γGT, Glutamyltransferase) more info +

ACE y-GT Reagent is intended for the quantitative determination of gamma-glutamyltransferase activity in serum using the ACE clinical chemistry systems. The enzyme y-glutamyltransferase (GGT; EC 2.3.2.2) is a C-terminal peptidase. It is specific for free terminal glutamic acid carboxyl groups in proteins or peptides. The glutamic acid cleaved from the protein (or peptide) may be transferred to another protein (or peptide), liberated as the free glutamic acid, or transferred to the donor peptide moiety. It is believed that all of the above transfers can occur in vivo. Early in vitro assays used substrate as the acceptor for glutamyl, but these could not be performed kinetically. Later methods used γ-glutaml-p-nitroanilide as substrate and glycylglycine as the preferred acceptor. Although kinetic, these methods were limited by the low solubility of γ-glutamyl-p-nitroanilide. The ACE -GT Assay is a modification of Theodorsen, et al.', in which the more soluble -glutamyl-3-carboxy-4-nitroanilide is the donor substrate. The reaction of γ-glutamyltransferase with this substrate results in the formation of 5-amino-2-nitrobenzoate, which can be measured spectrophotometrically at 408 nm.

In this method, γ-GT in serum catalyzes the transfer of the γ-glutamyl group from L-γ glutamyl-3-carboxy-4-nitroanilide to the glycylglycine in the reagent:

L-γ-glutamyl-3-carboxy-4-nitroanilide and glycylglycine is converted to 5-amino-2 nitrobenzoate and γ-glutamyl-glycylglycine. The product, 5-amino-2-nitrobenzoate, absorbs strongly at 408 nm. The rate of increase in absorbance, monitored bichromatically at 408 nm/ 486 nm, is directly proportional to the γ-GT activity in the sample.

Ghrelin (total) more info +

The Milipore (St. Charles, Missouri) ghrelin assay is a Sandwich ELISA based on: 1) capture of human ghrelin molecules (both active and des-octanoyl forms) in the sample by anti-human ghrelin IgG and immobilization of the resulting complex to the wells of a microtiter plate coated by a pre-titered amount of anchor antibodies, 2) and the simultaneous binding of a second biotinylated antibody to ghrelin, 3) wash away of unbound materials, followed by conjugation of horseradish peroxidase to the immobilized biotinylated antibodies, 4) wash away of free enzyme, and 5) quantification of immobilized antibody-enzyme conjugates by monitoring horseradish peroxidase activities in the presence of the substrate 3,3 ’,5,5 ’-tetramethylbenzidine. The enzyme activity is measured spectrophotometrically by the increased absorbency at 450 nm, corrected from the absorbency at 590nm, after acidification of formed products. Since the increase in absorbency is directly proportional to the amount of captured total human ghrelin in the unknown sample, the concentration of total ghrelin can be derived by interpolation from a reference curve generated in the same assay with reference standards of known concentrations of human ghrelin. The manufacturer reports an LoD of 100 pg/mL. The standard curve includes points at 0.0, 100, 200, 500, 1000, 2000 and 5000 pg/mL. We report the following interassay coefficient of variation: 9.0% at 1765 pg/mL. We report the following intraassay coefficient of variation: 3.4% at 1765 pg/mL.

Glucose more info +

Glucose in serum reacts with adenosine triphosphate (ATP) and magnesium with the formation of glucose-6-phosphate (G-6-P) and adenosine diphosphate (ADP). Glucose-6-phosphate dehydrogenase catalyses the oxidation of G-6-P with NAD⁺ to form 6-phosphogluconate and NADH. NADH strongly absorbs at 340 nm. The amount of NADH produced and measured is directly proportional to the amount of glucose concentration in the serum sample. The ACE glucose assay is linear from 1 to 750 mg/dL. The MDL is 1 mg/dL. The manufacturer provides the following Intra-assay precision expressed as CV: 1.1 % (58 mg/dL), 0.9% (158 mg/dL) and 0.9% (273 mg/dL). The manufacturer provides the following Inter-assay precision expressed as CV: 1.4 % (58 mg/dL), 1.7% (158 mg/dL) and 1.2% (273 mg/dL). We report the following Intra-assay precision expressed as CV: 1.3 % (94 mg/dL) and 1.2% (305 mg/dL). We report the following Inter-assay precision expressed as CV: 2.1 % (94 mg/dL) and 2.5% (305 mg/dL).

High Density Lipoprotein (HDL) more info +

The HDL assay is performed on the Alfa-Wasserman ACE analyzer and utilizes a detergent to solubilize only the HDL particles, releasing HDL to react with cholesterol oxidase in the presence of chromogen (N, N-bis (4-sulfobutyl)-m-touidine) to produce a chromophor that absorbes at 592/692. HDL concentration in serum is directly proportional to the amount of chromophor produced. The ACE HDL assay is linear from 2 to 125 mg/dL. The MDL is 2 mg/dL (2 SD greater than the 0 Standard). The manufacturer provides the following Intra-assay precision expressed as CV: 2.6% (37 mg/dL), 2.3% (53 mg/dL) and 1.9% (69 mg/dL). The manufacturer provides the following Inter-assay precision expressed as CV: 3.2 % (36 mg/dL), 4.5% (53 mg/dL) and 3.9% (69 mg/dL).

Human Chorionic Gonadotropin (hCG) more info +

Serum human chorionic gonadotropin (hCG) concentrations are measured with a two-site chemiluminescent immunoassayon the ADVIA Centaur (Bayer Diagnostics, Inc.) using constant amounts of two antibodies. One antibody is a polyclonal goat anti-hCG which has been affinity purified and labeled with acridinium ester (DMAE). The second antibody is a purified mouse monoclonal covalently coupled to paramagnetic particles. The two antibodies are specific for different epitopes that are present on both the free  and the  subunit of intact hCG. The assay measures analyte concentrations to 1000 mIU/mL with a minimum detectable concentration of 2.0 mIU/mL. The Inter-assay coefficient of variation provided by the manufacturer is 2.0% (6.9 mIU/mL), 4.3% (14.6 mIU/mL), 2.7% (20.6 mIU/mL), 2.7% (161.2 mIU/mL), 2.4% (493.0 mIU/mL) and 1.7% (638.5 mIU/mL); intra-assay coefficient of variation is 3.9% (6.9 mIU/mL), 2.7% (14.6 mIU/mL), 2.3% (20.6 mIU/mL), 2.8% (161.2 mIU/mL), 2.1% (493.0 mIU/mL) and 2.6% (638.5 mIU/mL).

ICAM more info +

This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for sICAM-1 has been pre-coated onto a microplate. Standards, samples, Controls and Conjugate are pipetted into the wells and any sICAM-1 present is sandwiched by the immobilized antibody and the enzyme-linked monoclonal antibody specific for sICAM-1. Following a wash to remove any unbound substances and/or antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of sICAM-1 bound. The color development is stopped and the intensity of the color is measured.

IL-6 more info +

The Human IL-6 Chemiluminescent Immunoassay (R&D Systems, Q60000B) is a plate assay which employs the quantitative sandwich enzyme immunoassay technique using a monoclonal antibody specific for IL-6 which has been pre-coated onto a microplate. Standards, QC ’s and samples are pipetted into the wells and any IL-6 present is bound by the immobilized antibody during a 2 hr incubation on an orbital shaker at RT. After washing away any unbound substances, an enzyme-linked polyclonal antibody specific for IL-6 is added to the wells and incubated for 3 hrs on an orbital shaker at RT. Following a wash to remove any unbound antibody-enzyme reagent, an enhanced luminol/peroxide substrate solution is added to the wells, incubated for 5-20 min and then read in a microplate luminometer (ICN Titertek Luminoscan set to read for 2.0 seconds) which measures the intensity of the light emitted. Light is produced in proportion to the amount of IL-6 bound in the initial step. The counts are transferred to a computer program (StatLia) for analysis with a range of 0.5 - 200 pg/mL. We report the following coefficients of variation. Inter-assay CV: 15.8% at 24.2 pg/mL, 19.3% at 67.4 pg/mL, 14.8 at 6.1 pg/mL, 13.3 at 144.2 pg/mL. Intra-assay 2.6% at 24.2 pg/mL, 3.0% at 67.4 pg/mL, 3.5 at 6.1 pg/mL, 2.8 at 144.2 pg/mL.

IL-6R more info +

The Human IL-6 sR immunoassay from R&D Systems (DR600) uses the quantitative sandwich enzyme immunoassay method. A monoclonal antibody specific for IL-6 sR has been pre-coated onto a microtiterplate. Standards and samples are pipetted into the wells and any IL-6 sR present is bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked polyclonal antibody specific for IL-6 sR is added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of IL-6 sR bound in the initial step. The color development is stopped and the intensity of the color is measured. The assay range is 31.2 - 2000 pg/mL. We report the following coefficients of varation. Inter-assay CV: 15.1% at 64.6 pg/mL, 5.8% at 406 pg/mL, 6.3% at 838 pg/mL. Intra-assay 2.6% at 64.6 pg/mL, 1.2% at 406 pg/mL, 4.8% at 838 pg/mL.

Inhibin-A more info +

We will be using a commercially available enzyme-linked immunosorbent assay (ELISA) from Diagnostic Systems Laboratories (DSL) for the in vitro quantitative measurement of Inhibin-A in human serum. The DSL Inhibin-A. Coated Well ELISA kit Features a direct competitive immunoassay with no required sample extraction or hydrolysis. Sample wells which have been coated with anti-Inhibin β_A subunit antibody. After incubation and washing, the wells are treated with another anti-Inhibin alpha subunit detection antibody labeled with the enzyme horseradish peroxidase (HRP). There is no detectable cross-reactivity with closely related compounds and the assay does not detect inhibin free alpha subunits. The assay measures analyte concentrations to 1000 pg/mL with a minimum detectable concentration of 10 pg/mL. The assay range (standard curve) is 10-1000 pg/mL. The inter-assay coefficient of variation provided by the manufacturer is 7.4 % at 123 pg/mL, 3.0 % at 322 pg/mL and 4.0 % at 601 pg/mL. The intra-assay coefficient of variation is 4.8% at 132 pg/mL, 1.4 % at 243 pg/mL and 3.0% at 539 pg/mL. CLASS Laboratory inter-assay coefficient of variation is 6.8% at 101 pg/mL, 3.6% at 412 pg/mL, 18.8% at 17 pg/mL and 24.2% at 14 pg/mL. CLASS Laboratory intra-assay coefficient of variation is 3.4% at 101 pg/mL, 3.1% at 412 pg/mL, 18.8% at 17 pg/mL and 19.4% at 14 pg/mL.

Inhibin-B more info +

We will be using a commercially available enzyme-linked immunosorbent assay (ELISA) from Diagnostic Systems Laboratories (DSL) for the in vitro quantitative measurement of Inhibin-B in human serum. The DSL Inhibin-B Coated Well ELISA kit Features a direct competitive immunoassay with no required sample extraction or hydrolysis. Sample wells are coated with anti-Inhibin _B antibody. The detection system consists of a biotinylated anti-Inhibin  and strepavidin-labeled horseradish peroxidase. There is no detectable cross-reactivity with closely related compounds and the assay does not detect inhibin free subunits. The assay requires 100 uL sample (duplicates) and measures analyte concentrations to 1000 pg/mL with a minimum detectable concentration of 7 pg/mL. The assay range (standard curve) is 10-1000 pg/mL. The Inter-assay coefficient of variation provided by the manufacturer is 7.6% at 50.1 pg/mL, 6.3% at 188.4 pg/mL and 6.2% at 355 pg/mL; intra-assay coefficient of variation is 3.5% at 69 pg/mL, 4.6% at 274 pg/mL and 5.6% at 472 pg/mL.

Inhibin-B Gen II ELISA more info +

We will be using a commercially available enzyme-linked immunosorbent assay (ELISA) from Beckman Coulter (A81301) for the in vitro quantitative measurement of Inhibin-B in human serum. Inhibin B Gen II ELISA uses a pair of antibodies that specifically recognize only the functional dimeric Inhibin B molecule. The Beckman Coulter Inhibin-B Coated Well ELISA kit is direct competitive immunoassay with no required sample extraction or hydrolysis. Sample wells are coated with coated with anti-Activin B antibody. After incubation and washing, the wells are incubated with biotinylated anti-Inhibin α-subunit detection antibody. The detection system consists of strepavidin-labeled horseradish peroxidase. The degree of enzymatic turnover of the substrate is determined by dual wavelength absorbance measurement at 450nm as primary test filter and 630 nm as primary reference filter. The absorbance measured is directly proportional to the concentration of Inhibin B in the samples. There is no detectable cross-reactivity with closely related compounds and the assay does not detect inhibin free subunits. The assay requires 50 uL sample (in duplicate) and measures analyte concentrations to 1000 pg/mL with a minimum detectable concentration of 10 pg/mL. The assay range (standard curve) is 10-1000 pg/mL. The lowest amount of Inhibin B in a sample that can be detected with a 95% probability is 2.6 pg/mL. The estimated minimum dose achieved at 20% total imprecision is 4.8 pg/mL. The Inter-assay coefficient of variation provided by the manufacturer is 5.6% at 19.3 pg/mL, 3.7% at 76.0 pg/mL and 3.7% at 275.3 pg/mL; intra-assay coefficient of variation is 3.8% at 19.3 pg/mL, 2.4% at 76.0 pg/mL and 2.2% at 275.3 pg/mL

Insulin more info +

The ADVIA Centaur Insulin assay is a two-site sandwich immunoassay using direct chemiluminescent technology which uses constant amounts of two antibodies. The first antibody, in the Lite Reagent, is a monoclonal mouse anti-insulin antibody labeled with acridinium ester. The second antibody, in the Solid Phase, is a monoclonal mouse anti-insulin antibody, which is covalently coupled to paramagnetic particles. A direct relationship exists between the amount of insulin present in the patient sample and the amount of relative light units (RLUs) detected by the system. The manufacturer reports an LoD of 0.5 mU/L. We report the following interassay coefficient of variation: 4.2% at 12 ng/mL, 3.5% at 78 and 4.7% at 167 mU/L. We report the following intraassay coefficient of variation: 2.1% at 12 ng/mL, 1.5% at 78 and 2.7% at 167 mU/L.

Insulin-Like Growth Factor Binding Protein-3 (IGFBP3) more info +

We will be using a commercially available enzyme-linked immunosorbent assay (ELISA) from Diagnostic Systems Laboratories (DSL) for the in vitro quantitative measurement of Insulin-Like Growth Factor Binding Protein-3 (IGFBP3) in human serum. The DSL IGFBP3 Coated Well ELISA kit Features a direct immunoassay with no required sample extraction. This assay is a highly specific two-site "sandwich" assays with no cross-reactivity with other human IGFBPs and no interference from IGF-I or IGF-II. The expected values are 27 to 56 ng/dL in females. The assay measures analyte concentrations up to 100 ng/mL with a minimum detectable concentration of 2.0 ng/mL. The inter assay coefficient of variation provided by the manufacturer is 11.4% at 5.6 ng/mL and 8.2% at 66 ng/mL.

Insulin-Like Growth Factor-1 (IGF-1) more info +

We will be using a commercially available enzyme-linked immunosorbent assay (ELISA) from Diagnostic Systems Laboratories (DSL,Webster City, TX) for the in vitro quantitative measurement of Insulin-Like Growth Factor-1 (IGF-1) in human serum. This direct measurement of IGF-I shows excellent correlation with acid-column chromatography and acid-ethanol extraction IGF-1 methods. It is configured as a highly specific two-site assay utilizing a coated-microplate format. There is no detectable interference by the IGF-binding proteins, insulin, proinsulin, or IGF-II. The expected values increase with age and are expected to range from 130 to 200 ng/mL in this population group (age 40-80). The inter-assay coefficient is 6.8%( 90 ng/mL) and 6% (426 ng/mL). The intra-assay precision is excellent 104 (8.6%), 268 (4.5%) and 524 (6.3%). The range of the assay is 7.8-580 ng/mL.

LDL more info +

The LDL assay utilizes a detergent to solubilize only the non-LDL particles, which are consumed by cholesterol oxidase in a non-color forming reaction. The remaining LDL is solubilized with a second detergent and released to react with cholesterol oxidase in the presence of chromogen (N, N-bis (4-sulfobutyl)-m-touidine) to produce a chromophor that absorbes at 544/692. LDL concentration in serum is directly proportional to the amount of chromophor produced . The ACE LDL assay is linear from 3 to 800 mg/dL. The MDL is 3 mg/dL (2 SD greater than the 0 Standard). The manufacturer provides the following Intra-assay precision expressed as CV: 2.2 % (95 mg/dL), 2.6% (146 mg/dL) and 1.4% (196 mg/dL). The manufacturer provides the following Inter-assay precision expressed as CV: 2.6 % (95 mg/dL), 3.2% (146 mg/dL) and 3.2% (196 mg/dL).

Leptin more info +

We will be using a commercially available enzyme-linked immunosorbent assay (ELISA) from R&D Systems (Minneapolis, MN) for the in vitro quantitative measurement of Leptin in human serum. The RD Coated Well ELISA kit Features a direct competitive immunoassay with no required sample extraction or hydrolysis. This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for Leptin has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any Leptin present is bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked monoclonal antibody specific for Leptin is added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of Leptin bound in the initial step. The color development is stopped and the intensity of the color is measured. The manufacturer reports an LoD of 0.078 pg/mL. The standard curve includes points at 0.0, 0.125, 0.25, 0.5, 1.0, 2.0, 5.0, 10.0, and 20.0 ng/mL. We report the following interassay coefficient of variation: 15.0% at 1.8 ng/mL and 7.6% at 7.9 ng/mL. We report the following intraassay coefficient of variation: 12.8% at 1.8 ng/mL and 2.4% at 7.9 ng/mL.

Leukotriene B₄ (LTB₄) more info +

Leukotriene B₄ (LTB₄) is a major product of arachidonic acid metabolism formed via the 5-lipoxygenase pathway. Leukotriene B₄ stimulates leukocyte functions including lysosomal enzyme release, adhesion and aggregation of polymorphonuclear leukocytes. A potent mediator of inflammatory diseases and immunoregulation, Leukotriene B₄ also increases the cytotoxic activity of human natural killer cells. Elevated levels of Leukotriene B₄ are associated with bronchial vasoconstriction and increased intracellular calcium levels.

The Leukotriene B₄ (LTB₄) is a plate assay which employs a competitive enzyme immunoassay technique using a monoclonal antibody which has been pre-coated onto a microplate. After an extraction and pre-purification step, standards, QC ’s and samples, an antibody specific to LTB4 and a tracer of Acteylcholinesterase linked to LTB4 are pipetted into the wells and LTB4 present is bound by the immobilized antibody during incubation. After washing, the concentration of analyte is determined by measuring the enzymatic activity of the AChE by adding Ellmen ’s reagent. The product is measures at 412nm. The intensity of the color produced is inversely directly proportional to the amount of bound conjugate and the concentration of LTB4. The manufacturer reports the following coefficients of variation. Inter-assay CV: xxx% at xxx pg/mL, and xxx% at xxx pg/mL.

Luteinizing Hormone (LH) more info +

Serum Luteinizing Hormone (LH) concentrations will also be measured with a two-site chemiluminescent immunoassay by Bayer diagnostics(1;2), using the same technique as the FSH assay; using two monoclonal antibodies, One antibody is directed at the alpha subunit and the other at the beta subunit (one coupled to paramagnetic particles and the other labeled with DMAE) with specificity for intact LH. The expected values are for normally menstruating females is 0.5-75 mIU/mL, for postmenopausal females 16-54 mIU/mL is expected. The assay measures analyte concentrations to 200 mIU/mL with a minimum detectable concentration of 0.07 mIU/mL. For serum, inter- and intra-assay coefficients of variation for serum are projected to be 6.8% and 2.8 %, respectively and for urine, they are projected to be 10.7% and 4.8%, respectively.

Matrix metalloproteinase-2 (MMP-2) more info +

Matrix metalloproteinases (MMPs), also called matrixins, constitute a family of zinc and calcium-dependent endopeptidases that function in the breakdown of the extracellular matrix and in the processing of a variety of biological molecules. The Human MMP-2 immunoassay (DMP2F0) from R&D Systems (Minneapolis, MN) uses the quantitative sandwich enzyme immunoassay technique. A polyclonal antibody specific for MMP-2 has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any MMP-2 present is bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked polyclonal antibody specific for MMP-2 is added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of MMP-2 bound in the initial step. The color development is stopped and the intensity of the color is measured.

The assay range is 0.78 - 50 ng/mL and the LLD is 0.047 ng/mL. The manufacturer reports the following coefficients of variation. Inter-assay CV: 9.8% (3.96 ng/mL) , 6.9% (12.4 ng/mL), 5.6% (19.6 ng/mL). Intra-assay: 5.5% (3.30 ng/mL), 5.4% (12.1 ng/mL), 5.8% (18.9 ng/mL). We report the following Inter-assay CV: 13.0% (3.9 ng/mL) , 4.2% (11.8 ng/mL), 0.3% (17.9 ng/mL).

MIS/AMH more info +

Mullerian Inhibiting Substance (MIS), also known as Anti-Mullerian Inhibiting Substance (AMH), is a 145-kd homodimeric glycoprotein composed of two 72 kDa monomers. It acts by means of a plasma membrane receptor. We used a commercially available enzyme-linked immunosorbent assay (ELISA) from Beckman Coulter (Marseille, France) for the in vitro quantitative measurement of Mullerian Inhibiting Substance/Anti-Mullerian Hormone (MIS/AMH) in human serum. The Beckman Coulter MIS/AMH Coated Well ELISA kit features a two site sandwich type immunoassay with no sample extraction. Sample wells are coated with a primary monoclonal antibody. The detection system consists of a biotinylated secondary monoclonal antibody and strepavidin-peroxidase. The biotinlyated antibody binds to the solid phase antibody-antigen complex and, in turn binds the conjugate. After incubation, the wells are washed and the antigen complex bound to the well detected by addition of a chromatogenic substrate. Color intensity is directly proportional to AMH concentration. There is no detectable cross-reactivity with closely related compounds. The assay requires 50 uL of serum or plasma for duplicate analysis and measures analyte concentrations from a minimum detectable concentration of 1 pM (1 ng/mL corresponds to 7.14 pM) to 150 pM with an assay range (standard curve) of 3-150 pM. The inter-assay coefficient of variation reported by the manufacturer is <14.2%; the intra-assay coefficient of variation reported by the manufacturer is <12.3%. In our hands the inter-assay CV was 15.3% and the intra-assay CV 5.6%. All analytical characteristics are stated in ng/mL. To convert to:

1 ng/mL = 7.14 pM

MIS/AMH more info +

Mullerian Inhibiting Substance (MIS), also known as Anti-Mullerian Inhibiting Substance (AMH), is a 145-kd homodimeric glycoprotein composed of two 72 kDa monomers. It acts by means of a plasma membrane receptor. We will be using a commercially available enzyme-linked immunosorbent assay (ELISA) from Diagnostic Systems Laboratories (DSL, Webster TX) for the in vitro quantitative measurement of Mullerian Inhibiting Substance/Anti-Mullerian Hormone (MIS/AMH) in human serum. The DSL MIS/AMH Coated Well ELISA kit Features a direct competitive immunoassay with no required sample extraction or hydrolysis. Sample wells are coated with a primary antibody. The detection system consists of a biotinylated secondary antibody and strepavidin-labeled horseradish peroxidase. There is no detectable cross-reactivity with closely related compounds. The assay requires 200uL for duplicate analysis and measures analyte concentrations from a minimum detectable concentration of 0.017 to 10 ng/mL with an assay range (standard curve) of 0.05-10 ng/mL. The manufacturer recommends that samples be assayed in triplicate. Inter-assay coefficient of variation provided by the manufacturer is 8.0% at 0.15 ng/mL, 4.8% at 0.85 ng/mL and 6.7% at 4.28 ng/mL (mean = 6.5%); intra-assay coefficient of variation is 4.6% at 0.14 ng/mL, 2.4% at 0.84 ng/mL and 3.3% at 4.41 ng/mL (mean= 4.0%).

Pre-pubertal males(4) are elevated >5 ng/mL and fall to 2-5 ng/mL post-pubery. Pre-pubertal females are undetectable, rise to 2-5 ng/mL post-puberty, then fall again to undetectable post-menopause

MIS/AMH Gen II more info +

Anti-Mullerian Inhibiting Substance (AMH), also known as Mullerian Inhibiting Substance (MIS), is a 145-kd homodimeric glycoprotein composed of two 72 kDa monomers. It acts by means of a plasma membrane receptor. We used a commercially available enzyme-linked immunosorbent assay (ELISA) from Beckman Coulter (A73818 Marseille, France) for the in vitro quantitative measurement of Anti-Mullerian Hormone/ Mullerian Inhibiting Substance (AMH/MIS) in human serum. The Beckman Coulter Gen II AMH/MIS Coated Well ELISA kit features a two site sandwich type immunoassay with no sample extraction. Sample wells are coated with a primary monoclonal antibody. The detection system consists of a biotinylated secondary monoclonal antibody and strepavidin-horseradish peroxidase. The biotinlyated antibody binds to the solid phase antibody-antigen complex and, in turn binds the conjugate. After incubation, the wells are washed and the antigen complex bound to the well detected by addition of the chromatogenic substrate tetramethylbenzidine (TMB). Color intensity is directly proportional to AMH concentration. There is no detectable cross-reactivity with closely related compounds. The assay requires 40 uL of serum or plasma for duplicate analysis and measures analyte concentrations at a minimum detectable concentration of 0.08 ng/mL (0.57 pM)(1 ng/mL corresponds to 7.14 pM). The reportable assay range (standard curve) is 0.16-22.5 ng/mL. The intra-assay coefficient of variation reported by the manufacturer is 3.7% (3.82 ng/mL), 5.4% (4.42 ng/mL), 3.6% (14.03 ng/mL) and 3.4% (16.45 ng/mL); the intra-assay coefficient of variation reported by the manufacturer is 4.4% (3.82 ng/mL), 5.6% (4.42 ng/mL), 4.5% (14.03 ng/mL) and 4.0% (16.45 ng/mL). In our hands the intra-assay CV was 5.1% (2.99 ng/mL) and 1.7% (9.59 ng/mL); the inter-assay CV was 6.1% (2.99 ng/mL) and 5.4% (9.59 ng/mL)

Monocyte chemotactic protein more info +

The Quantikine Monocyte chemotactic protein (MCP-1) immunoassay is a solid phase ELISA designed to measure MCP-1 in cell culture supernates, serum, plasma, and urine. It contains recombinant human MCP-1 and antibodies raised against recombinant human MCP-1. This immunoassay has been shown to accurately quantitate synthetic and naturally occurring MCP-1. This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for MCP-1has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and the immobilized antibody binds any MCP-1present. After washing away any unbound substances, an enzyme-linked monoclonal antibody specific for MCP-1 is added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of MCP-1 bound in the initial step. The color development is stopped and the intensity of the color is measured. This assay recognizes natural and recombinant human MCP-1. The assay measures analyte concentrations from 31.2 to 2000 pg/mL with a minimum detectable concentration of 1.7 pg/mL. The inter assay coefficient of variation provided by the manufacturer is

7.8% at 77 pg/mL, 4.7% at 364 pg/mL and 4.9% at 1121pg/mL. The intra assay coefficient of variation provided by the manufacturer is 6.7% at 74 pg/mL, 5.8% at 352 pg/mL and 4.4% at 1076 pg/mL.

Osteocalcin (OST) more info +

Osteocalcin or bone Gla protein (B.G.P) is the major non-collagen protein of the bone matrix. It has a molecular weight of 5800 Da and contains 49 amino-acids, including 3 residues of gamma carboxyl glutamic acid. Osteocalcin is synthesized in the bone by the osteoblasts. After production, it is partly incorporated in the bone matrix and the rest is found in the blood circulation. The exact physiological function of osteocalcin is still unclear. A large number of studies show that the circulating levels of osteocalcin reflect the rate of bone formation.

The Osteocalcin (Intact) ELISA is a solid phase Enzyme Amplified Sensitivity Immunoassay performed on breakable microtiterplates. The assay uses monoclonal antibodies (MAbs) directed against distinct epitopes of human osteocalcin. Calibrators and samples react with the capture monoclonal antibody (MAb 1) coated on microtiter well and with a monoclonal antibody (MAb 2) labelled with horseradish peroxidase (HRP). After an incubation period allowing the formation of a sandwich: coated MAb 1 - human osteocalcin - MAb 2 - HRP, the microtiterplate is washed to remove unbound enzyme labelled antibody. Bound enzyme-labelled antibody is measured through a chromogenic reaction. Chromogenic solution (TMB ready for use) is added and incubated. The reaction is stopped with the addition of Stop Solution and the microtiterplate is then read at the appropriate wavelength. The amount of substrate turnover is determined colourimetrically by measuring the absorbance, which is proportional to the osteocalcin concentration. A calibration curve is plotted and OST concentration in samples is determined by interpolation from the calibration curve.

We will be using a commercially available enzyme-linked immunosorbent assay (ELISA) from ALPCO Diagnostics for the in vitro quantitative measurement of Osteocalcin in human serum. The ALPCO Osteocalcin Coated Well ELISA kit Features a direct competitive immunoassay with no required sample extraction or hydrolysis. There is no detectable cross-reactivity with closely related compounds. This method detects intact human osteocalcin. N-terminal and C-terminal fragments have been tested at their maximum levels found in normal and pathological samples, were added to a low and a high value calibrator. No cross reactivity was observed at these concentrations. Normal values are expected between 5 to 25 ng/ml. The assay measures analyte concentrations from 1.56 to 75 ng/mL with a minimum detectable concentration of 1.56 ng/mL, and a Sensitivity of 0.08 ng/mL. The inter assay coefficient of variation provided by the manufacturer is 3.5% at 11.8 ng/mL, 5.6% at 27.7 ng/mL. The intra assay coefficient of variation provided by the manufacturer is 4.7% at 11.4 ng/mL, 3.1% at 28.2 ng/mL.

Osteoprotegerin (OPG) more info +

The Osteoprotegerin Immunoassay (R&D Systems) is a two-site ELISA [Enzyme-Linked ImmunoSorbent Assay] for the measurement of Osteoprotegerin. A mouse monoclonal antibody to human OPG, purified by affinity chromatography, and a biotin-labeled goat polyclonal antibody to human OPG are specific for well defined regions on the OPG molecule. At the end of the assay incubation, the microwell is washed to remove unbound components and the enzyme bound to the solid phase is incubated with the substrate, tetramethylbenzidine (TMB). An acidic stopping solution is then added to stop the reaction and converts the color to yellow. The intensity of the yellow color is directly proportional to the concentration of OPG in the sample. A dose response curve of absorbance unit vs. concentration is generated using results obtained from the calibrators. Concentrations of OPG present in the controls and patient samples are determined directly from this curve. The assay requires 100 uL of serum for duplicate analysis. The reportable assay range (standard curve) is 62.5 - 4000 pg/mL. The intra-assay coefficient of variation is 1.1% (1582 pg/mL) and 0.9% (1426 pg/mL). The inter-assay coefficient of variation 6.3% (1582 pg/mL) and 8.9% (1426 pg/mL).

Oxytocin Assay more info +

Oxytocin was measured by competitive enzyme immunoassay using the Correlate-EIA Oxytocin kit (Assay Designs, Ann Arbor, MI). Sample or standard is incubated overnight at 4degC with covalently-linked alkaline phosphatase-oxytocin label and a rabbit anti-oxytocin polyclonal antibody in a microtiter plate coated with goat anti-rabbit IgG. On the second day, excess reagents are washed away and p-nitrophenylphosphate substrate is added. After a one-hour room temperature incubation, the reaction is stopped and the plate read at 405nm. The concentration of oxytocin in the sample is inversely proportional to the measured optical density and is quantified by comparison with a standard curve.

PAI-1 more info +

The IMUBIND® Tissue PAI-1 ELISA kit bu American Diagnostica, Inc., is an enzyme-linked immunoassay for the determination of human PAI-1 in tissue extracts and cell culture supernatants. The assay detects latent (inactive) and active forms of PAI-1 and PAI-1 complexes and is insensitive to PAI-2. This assay is for research use only. It is not intended for diagnostic or therapeutic procedures. EXPLANATION OF THE TEST Plasminogen activator inhibitor type 1 (PAI-1), a primary regulator in fibrinolysis,1,2 has been found in a number of different cell types, i.e. macrophages/monocytes, platelets, vascular endothelial and adipose tissue of the heart and lungs.3 PAI-1 has been implicated along with urokinase-type plasminogen activator (uPA) as a prognostic factor for relapse-free and overall survival in breast cancer and gastric cancer patients. In patients diagnosed with primary breast cancer, PAI-1 ranks second to only lymph node status as a prognostic factor for Disease-free Survival (DFS) and Overall Survival (OS), with the established prognostic factors of node status, grading, tumor size and hormone receptor status.7 In ancillary node-negative patients, PAI-1 ranks highest with the established factors of tumor size, grading and hormone receptor status and experimental factors such as uPA (Urokinase Plasminogen Activator), Cathepsin D and S-Phase Fraction.8 It has been shown that a combination of PAI-1 and uPA further advances the delineation of patients having a low or high risk for relapse independent of the classical risk factors. Therefore by combining PAI-1 and uPA levels in tissue extracts a more individualized estimation of prognosis becomes possible in breast cancer patients.4-8 PAI-1 positivity may also serve as a prognostic factor for completely resected gastric cancer patients.9

PRINCIPLE OF THE METHOD

The IMUBIND Tissue PAI-1 ELISA employs a murine anti-human PAI-1 antibody as the capture antibody. Samples incubate in microwells coated with the monoclonal anti-

human PAI-1 and are detected with a biotinylated anti-human PAI-1 antibody that recognizes the bound PAI-1 molecules. Adding streptavidin conjugated horseradish peroxidase (HRP) completes the formation of the antibody-enzyme detection complex. The addition of a perborate/3, 3', 3, 5' - tetramethylbenzidine (TMB) substrate, and its subsequent reaction with HRP creates a blue colored solution. Sensitivity is increased by addition of a sulfuric acid stop solution, yielding a yellow color. PAI-1 levels are quantified by measuring solution absorbances at 450 nm and comparing the values with those of a standard curve. This method requires 40 ul of sample for duplicate analysis and features a standard range of 12.5 to 50 ng/mL, and a minimum detectable concentration of 2.2 ng/mL. Inter-assay coefficient of variation provided by the manufacturer is 9.0% at 19 ng/mL and 6.9% at 40 ng/mL; intra-assay coefficient of variation is 6.6% at 19 ng/mL and 5.4% at 40 ng/mL.

PIIINP more info +

Type III collagen is derived from a larger protein, type III procollagen, which has propeptide extensions at both ends of the molecule. Some of the aminoterminal propeptides are set free during the synthesis and deposition of type III collagen, and some are retained in the molecules which remain on the surface of the collagen fibrils. This antigen, when found in serum, can thus be derived from the synthesis (including late processing) of new type III collagen or from the degradation of existing type III collagen fibrils. The Orion Diagnostica (Espoo, Finland) UniQ PIIINP RIA kit is based on the competitive radioimmunoassay technique. A known amount of labelled PIIINP and an unknown amount of unlabelled PIIINP in the sample compete for the limited number of high affinity binding sites of the antibody. After separating the free antigen, the amount of labelled PIIINP in the sample tube is inversely proportional to the amount of PIIINP in the sample. The concentrations in unknown samples are obtained from a calibration curve. The assay range is1 - 50 ug/L and the LLD is 0.3 ug/L. The manufacturer reports the following coefficients of variation. Inter-assay CV: 6.5% (2.7 ug/L), 4.5% (6.8 ug/L), 7.2% (12.2 ug/L). Intra-assay: 3.0% (2.8 ug/L), 7.0% (6.6 ug/L), 4.1% (11.9 ug/L). We report the following Inter-assay CV: 3.1% (4.6 ug/L).

Progesterone more info +

The serum Progesterone assay is a competitive chemiluminescent immunoassay. This serum assay uses two antibodies, a rabbit anti-progesterone labeled with Dimethlyacridiniumester (DMAE) and the other, an anti-rabbit IgG. covalently coupled to Superparamagnetic Particles (PMP). A serum sample of 20μL of serum is required for the assay that is withdrawn by the instrument, aspirated to the sample cuvette, and the appropriate reagents are added. Free label is separated from bound by means of a magnetic separation step and the residual trace measured. Relative light units (RLU) are inversely proportional the concentration of progesterone in the sample. The reporting range for the Progesterone assay is 0.1-40 ng/mL (standard curve: 0.1-68.5 ng/mL). Progesterone standards have been confirmed by GC-MS. Our results have shown the following coefficients of variation: Inter-assay CV: 7.30 %, Intra-assay CV: 3.0%.

Prolactin more info +

The serum Prolactin assay is a two-site chemiluminometric (sandwich) immunoassay, which uses constant amounts of two antibodies that have specificity for the intact Prolactin molecule. In the first step of this assay, 25μL of serum sample and 100 μL of first antibody, a monoclonal mouse anti-Prolactin antibody covalently coupled to dimethylacridinium ester, are incubated for 5 minutes at 37 ˚C. This is followed by the addition of 450 μL of the second antibody, a monoclonal mouse anti-Prolactin antibody covalently coupled to paramagnetic particles and incubated for 2.5 minutes at 37 ˚C. Separation, aspiration, and deionized water wash steps separate bound from free. A direct relationship exists between the amount of Prolactin in the sample and the relative light units detected by the luminometer (photomultiplier tube). The test results are determined from a calibration curve derived from the standard, WHO 3rd IRP 84/500. The expected values for nonpregnant females are 3.3 to 26.7 ng/mL, pregnant are 5.3 to 215.0 ng/mL, postmenopausal are 2.4 to 24.0 ng/mL, and men are 2.8 to 29.9 ng/mL; the assay measures Prolactin concentrations up to 200 ng/mL with a minimum detectable concentration of 0.3 ng/mL. The inter assay coefficient of variations are 3.6% at 2.7 ng/mL and 4.0% at 34 ng/mL. The intra assay coefficient of variations are 2.5% at 2.7 ng/mL, 2.8% at 34 ng/mL and 3.8% at 121 ng/mL.

RANKL more info +

The RANKL Immunoassay (R&D Systems) is a two-site ELISA [Enzyme-Linked ImmunoSorbent Assay] for the measurement of RANKL. An antibody to human RANKL and a biotin-labeled antibody to human RANKL are specific for well defined regions on the RANKL molecule. At the end of the assay incubation, the microwell is washed to remove unbound components and the enzyme bound to the solid phase is incubated with the substrate, tetramethylbenzidine (TMB). An acidic stopping solution is then added to stop the reaction and converts the color to yellow. The intensity of the yellow color is directly proportional to the concentration of RANKL in the sample. A dose response curve of absorbance unit vs. concentration is generated using results obtained from the calibrators. Concentrations of RANKL present in the controls and patient samples are determined directly from this curve. The assay requires 100 uL of serum for duplicate analysis. The reportable assay range is 10 - 5000 pg/mL. The intra-assay coefficient of variation is 1.6% (851 pg/mL) and 2.3% (1168 pg/mL). The inter-assay coefficient of variation is 9.5% (851 pg/mL) and 7.6% (1168 pg/mL).

RANKL more info +

RANKL (receptor activator of nuclear factor (NF)-kB ligand; also: osteoprotegerin ligand, OPGL), its cellular receptor, receptor activator of NF-kB (RANK), and the decoy receptor, osteoprotegerin (OPG) have been identified as the key molecular regulation system for bone remodelling. RANKL, a member of the tumor necrosis factor (TNF) family, is the main stimulatory factor for the formation of mature osteoclasts and is essential for their survival. RANKL is produced by osteoblastic lineage cells and activated T lymphocytes. It activates its specific receptor RANK which is located on osteoclasts and dendritic cells. The effects of RANKL are counteracted by OPG which is secreted by various tissues and acts as an endogenous soluble receptor antagonist. sRANKL is a part of the TNF superfamily. Two isoforms are produced by alternate splicing, a type II membrane protein (MW 35.5 kD), and a secreted molecule (MW 27.7kD). The RANKL Immunoassay (ALPCo Diagnostics) is a two-site ELISA [Enzyme-Linked ImmunoSorbent Assay] for the measurement of RANKL. An antibody to human RANKL and a biotin-labeled antibody to human RANKL are specific for well defined regions on the RANKL molecule. At the end of the assay incubation, the microwell is washed to remove unbound components and the enzyme bound to the solid phase is incubated with the substrate, tetramethylbenzidine (TMB). An acidic stopping solution is then added to stop the reaction and converts the color to yellow. The intensity of the yellow color is directly proportional to the concentration of RANKL in the sample. A dose response curve of absorbance unit vs. concentration is generated using results obtained from the calibrators. Concentrations of RANKL present in the controls and patient samples are determined directly from this curve. The assay requires 100 uL of serum for duplicate analysis. The reportable assay range (standard curve) is 1.56 - 60 pg/mL. The intra-assay coefficient of variation reported by the manufacturer is 0.9% (618 pg/mL) and 3.5% (2346 pg/mL). The intra-assay coefficient of variation reported by the manufacturer is 9.3% (618 pg/mL) and 7.1% (2306 pg/mL). Conversion factor: 1 pg/mL= 0.016 pmol/L (MW 60 kDa trimer).

Serotonin more info +

The Serotonin Immunoassay (ALPCo) is a competitive ELISA [Enzyme-Linked ImmunoSorbent Assay] for the measurement of Serotonin. Samples are acylared and then added to a serotonin coated microwell and allowed to compete for HRP-labeled anti-serotonin antibody binding sites. At the end of the assay incubation, the microwell is washed to remove unbound components and the enzyme bound to the solid phase is incubated with the substrate, tetramethylbenzidine (TMB). An acidic stopping solution is then added to stop the reaction and converts the color to yellow. The intensity of the yellow color is indirectly proportional to the concentration of Serotonin in the sample. A dose response curve of absorbance unit vs. concentration is generated using results obtained from the calibrators. Concentrations of Serotonin present in the controls and patient samples are determined directly from this curve. The assay requires 25 uL of serum for duplicate analysis. The reportable assay range (standard curve) is 15-2500 ng/mL. The intra-assay coefficient of variation is 4.7% (106 ng/mL) and 3.1% (301 ng/mL). The inter-assay coefficient of variation is 9.0% (106 ng/mL) and 13.2% (301 ng/mL).

Sex Hormone Binding Globulin (SHBG) more info +

The Sex Hormone Binding Globulin (SHBG) assay is a competitive immunoassay run on the Bayer Diagnostic ACS:180 automated analyzer (Bayer Diagnostics Corp, Tarrytown , NY) using chemiluminescent technology(1). The assay was developed de novo and uses a rabbit polyclonal anti-SHBG antibody, SHBG labeled with dimethylacridinium ester (DMAE), and goat anti-rabbit Immunoglobulin G (IgG) coupled with paramagnetic particles (PMP). 40 μL of serum is required for the assay in addition to sufficient dead volume for aspiration and repeat. The expected values are from 20 to 130 nM. The reporting range for the SHBG assay is 10-150 nM (actual assay range: 1.95 to 250 nM). The assay is standardized against SHBG obtained from Wein Industries (Succasunna, NJ). We report the following: Inter-assay CV: 17.9%, Intra-assay CV: 9.3%.

Sex Hormone Binding Globulin (SHBG) CENTAUR more info +

The Sex Hormone Binding Globulin (SHBG) assay is a competitive immunoassay run on the Bayer Diagnostic ACS:180 automated analyzer (Bayer Diagnostics Corp, Tarrytown , NY) using chemiluminescent technology(1). The assay was developed de novo and uses a rabbit polyclonal anti-SHBG antibody, SHBG labeled with dimethylacridinium ester (DMAE), and goat anti-rabbit Immunoglobulin G (IgG) coupled with paramagnetic particles (PMP). 40 μL of serum is required for the assay in addition to sufficient dead volume for aspiration and repeat. The expected values are from 20 to 130 nM. The reporting range for the SHBG assay is 10-150 nM (actual assay range: 1.95 to 250 nM). The assay is standardized against SHBG obtained from Wein Industries (Succasunna, NJ). We report the following: Inter-assay CV: 17.9%, Intra-assay CV: 9.3%.

Serum Oxidized LDL more info +

Both the Mercodia Oxidized LDL ELISA and the new Oxidized LDL Competitive ELISA use the specific antibody 4E6. The 4E6 antibody is directed against a conformational epitope in the apo B-100 moiety of LDL that is generated as a consequence of aldehyde substitution of the lysine residues of apoB-100. The Oxidized LDL ELISA is a solid phase two-site enzyme immunoassay. It is based on the direct sandwich technique in which two antibodies are directed against separate epitopes of the oxidized LDL apo B molecule. The specific antibody 4E6 binds the sample oxidized LDL to the microtiter well. After washing the plate the bound antigen is detected by an anti-apo B enzyme conjugate. The manufacturer reports an LoD of <1mU/L (3SD above the 0.0 calibrator). The standard curve included points at 0.0, 1.8, 3.6, 6.7, 14, and 28 mU/L. We report the following interassay coefficient of variation: 16.7% at 5.9 mU/L and 6.9% at 12.6 mU/L. We report the following intraassay coefficient of variation: 6.9% at 5.9 mU/L and 4.4% at 12.6 mU/L. 149 randomly selected individuals in Stockholm, Sweden provided a mean oxLDL of 61 mU/L, range 26-117.

Mercodia Oxidized LDL ELISA is a solid phase two-site enzyme immunoassay. It is based on the direct sandwich technique in which two monoclonal antibodies are directed against separate antigenic determinants on the oxidized apolipoprotein B molecule. During incubation oxidized LDL in the sample reacts with anti-oxidized LDL antibodies bound to microtitration well. After washing, which removes non-reactive plasma components, a peroxidase conjugated anti-human apolipoprotein B antibody recognizes the oxidized LDL bound to the solid phase. After a second incubation and a simple washing step that removes unbound enzyme labeled antibody, the bound conjugate is detected by reaction with 3,3 ’, 5,5 ’-tetramethylbenzidine (TMB). The reaction is stopped by adding acid to give a colorimetric endpoint, then read spectrophotometrically.

sPLA2 Activity more info +

Secretory phospholipase A₂ (sPLA2) activity is measured by a radiometric C-14

assay. In principle, the DOC/PC assay measures the hydrolysis of fatty acids from the 2-acyl bond of phospholipids catalyzed by phospholipase A2 enzyme. Substrate lipid micelles are prepared as follows: for each tube 1.8 uL (0.045 uCi) of C-14-PC is required. C-14 Phosphatidyl Choline (PC) is combined with cold PC, the solvents evaporated carefully under N2 stream, reconstituted in Tris buffer (pH 8) and sonicated to create radio-labeled substrate. 15 uL of standards, QC preparations or serum is pipetted to each tube followed by 20 uL C-14 labeled phospholipid/deoxycholate micellar substrate. Incubation of test sera with 14C-labeled phospholipid/deoxycholate micellar substrate in Tris buffer is followed by Dole solvent extraction to separate the released C-14. The free labeled fatty acid extracts into the heptane layer while the majority of the unhydrolyzed phospholipid and lysophospholipid remain in the aqueous layer. A silicic acid treatment of the heptane layer further extracts residual phospholipid from the sample before scintillation counting. The detected counts are corrected for the spontaneous lysis control, normalized in relation to the total label added, and calculated as % hydrolysis by determining the ratio to the total DPM added per vial expressed as percent.

sPLA2 Mass more info +

The secretory phospholipase A₂ mass assay (sPLA2) is a plate assay obtained from Cayman Chemical (Ann Arbor, MI) modified according to protocol provided by Marin Biologic Laboratories, Inc. (Marin, CA) which employs a quantitative sandwich enzyme immunoassay technique using a monoclonal antibody specific for sPLA2 Type IIa which has been pre-coated onto a microplate. Standards, QC ’s and samples are pipetted into the wells and any sPLA2 present is bound by the immobilized antibody during incubation. After washing away any unbound substances, an acetylcholinesterase:Fab ’ conjugate (AChE:Fab ’) binds selectively to a different epitope on the sPLA2 molecule. After washing, the concentration of analyte is determined by measuring the enzymatic activity of the AChE by adding Ellmen ’s reagent. The product is measured at 412nm and produces a standard curve with a range of 0.06-6.0 ng.mL. The limit of detection is 0.06 ng/mL. The intensity of the color produced is directly proportional to the amount of bound conjugate and the concentration of sPLA2. We report the following coefficients of variation. Inter-assay CV: 25.7% at 0.49 ng/mL, 8.3% at 0.85 ng/mL and 18.9%% at 1.85 ng/mL. Intra-assay CV: 6.7% at 0.49 ng/mL, 9.2% at 0.85 ng/mL and 6.8%% at 1.85 ng/mL.

T3 more info +

The ADVIA Centaur T3 assay is a competitive immunoassay using direct chemiluminescent technology. T3 in the patient sample competes with a T3 analog, which is covalently coupled to paramagnetic particles in the Solid Phase for a limited amount of acridinium ester-labeled monoclonal mouse anti-T3 antibody in the Lite Reagent. The system automatically performs the following steps: dispenses 50 uL of sample and 50 uL ofT31T4/VB12 Ancillary Reagent into a cuvette dispenses 100 uL of Lite Reagent and 300 uL of Solid Phase and incubates for 7.5 minutes dispenses 300 uL each of Acid Reagent and Base Reagent to initiate the chemiluminescent reaction reports results according to the selected option, as described in the system operating instructions or in the online help system. An inverse relationship exists between the amount of T3 present in the patient sample and the amount of relative light units (RLUs) detected by the system at 37°C. separates, aspirates, and washes the cuvettes with reagent water.

T4 more info +

The serum Thyroxine (T4) assay is a competitive immunoassay using direct, chemiluminescent technology. T4 in the serum sample competes with T4 covalently coupled to paramagnetic particles with acridinum ester-labeled mouse anti-T4 in the lite reagent. A sample of 25μL of serum is required for the assay that is withdrawn by the instrument, aspirated to the sample cuvette, and the appropriate reagents are added. Free label is separated from bound by means of a magnetic separation step and the residual trace measured. Relative light units (RLU) are inversely proportional the concentration of T4 in the sample. The reporting range for the T4 assay is 0.5 ug/dL (6.4 nmol) to 30 ug/dL (387 nmol). T4 standards are traceable to a Bayer Diagnostics internal standard manufactured using U.S.P material. The manufacturer reports the following Intra-assay coefficient of variation: 5.5% (6.47 ug/dL), 3.9% (9.0 ug/dL) 3.8% (16.62 ug/dL). The manufacturer reports the following expected results: hypothyroid 0.0-71.0 ug/dL, hyperthyroid, 139.3-246.4 ug/dL.

The ADVIA Centaur T4 assay is a competitive immunoassay using direct chemiluminescent technology. T4 in the patient sample competes with T4, which is covalently coupled to paramagnetic particles in the Solid Phase, for a limited amount of acridinium ester-labeled monoclonal mouse anti-T4 antibody in the Lite Reagent. The system automatically performs the following steps: dispenses 25 uL of sample and 50 uL ofT3/T4/VB12 Ancillary Reagent into a cuvette dispenses 250 uL of Solid Phase and 100 uL of Lite Reagent and incubates for 7.5 minutes at 37°C separates, aspirates, and washes the cuvettes with reagent water5 dispenses 300 uL each of Acid Reagent and Base Reagent to initiate the chemiluminescent reaction reports results according to the selected option, as described in the system operating instructions or in the online help system An inverse relationship exists between the amount of T4 present in the patient sample and the amount of relative light units (RLUs) detected by the system.

Testosterone, free (fT) more info +

We will be using a commercially available enzyme-linked immunosorbent assay (ELISA) from Diagnostic Systems Laboratories (DSL) for the in vitro quantitative measurement of free testosterone (fT) in human serum. The DSL free testosterone assay is a direct assay which uses a highly specific antiserum and competitive binding scheme formulated to eliminate the need for laborious sample preparation schemes. There is no detectable cross-reactivity with closely related compounds. The expected values in females range from 1.2 to 6.6 pg/mL in women and 6.2 to 28.1 pg/mL in males. This method rwquires 100 ul of sample for duplicate analysis and features a standard range of 0.25 to 100 pg/mL, and a minimum detectable concentration of 0.19 pg/mL. Inter-assay coefficient of variation provided by the manufacturer is 2.2% at 2.87 pg/mL, 1.28% at 5.02 pg/mL and 6.18% at 67.73 pg/mL (mean = 2.6%); intra-assay coefficient of variation is 7.5% at 5.55 pg/mL, 5.4% at 8.71 pg/mL and 6.8% at 10.20 pg/mL (mean= 6.6%)

Testosterone, total more info +

Total testosterone was desired as a combined measure of changes in testosterone production derived from ovarian stromal, adrenal and peripheral conversion of precursors. The commercially available ACS:180 assay was adequate in terms of specificity and accuracy We modified the ACS:180 total testosterone assay to measure with greater precision samples in the low ranges found in women in the peri- and postmenopause(5). To accomplish this we increased the sample volume while evaluating the consequences of this change on volumes of subsequent reagents. The resulting chemiluminescent immunoassay involves the competitive binding of a DMAE-labeled testosterone derivative to a high titered, rabbit polyclonal anti-testosterone antibody which is premixed with a monoclonal anti rabbit IgG antibody immobilized on the solid phase paramagnetic particles. A non-detectable releasing agent (dihydrotestosterone) is included to dissociate testosterone from binding proteins. The limit of detection of this assay is <5.15 ng/dL. The limit of quantification (lowest reported value) is set at the lowest standard, 5.15 ng/dL. The assay range is 5.15 - 577 ng/dL (0.052 - 5.77 ng/mL). Closely related steroids show little cross reactivity in the assay indicating high specificity. The assay demonstrates acceptable parallelism of a diluted serum sample and recovery of added exogenous hormone. The expected values are 14-76 ng/dL in females. The intra assay coefficient of variation is 11.78% (24.4 ng/dL, n=30), 4.6% (191.2 ng/dL, n=30) and 9.1% (414.2 ng/dL, n=30). The inter assay coefficients of variation are 11.34% (53.3 ng/dL, n=37) and 9.7% (250.2 ng/dL, n=37).

Testosterone, total Centaur more info +

Total testosterone was desired as a combined measure of changes in testosterone production derived from ovarian stromal, adrenal and peripheral conversion of precursors. The commercially available ACS:180 assay was adequate in terms of specificity and accuracy We modified the ACS:180 total testosterone assay to measure with greater precision samples in the low ranges found in women in the peri- and postmenopause(5). To accomplish this we increased the sample volume while evaluating the consequences of this change on volumes of subsequent reagents. The resulting chemiluminescent immunoassay involves the competitive binding of a DMAE-labeled testosterone derivative to a high titered, rabbit polyclonal anti-testosterone antibody which is premixed with a monoclonal anti rabbit IgG antibody immobilized on the solid phase paramagnetic particles. A non-detectable releasing agent (dihydrotestosterone) is included to dissociate testosterone from binding proteins. The limit of detection of this assay is <5.15 ng/dL. The limit of quantification (lowest reported value) is set at the lowest standard, 5.15 ng/dL. The assay range is 5.15 - 577 ng/dL (0.052 - 5.77 ng/mL). Closely related steroids show little cross reactivity in the assay indicating high specificity. The assay demonstrates acceptable parallelism of a diluted serum sample and recovery of added exogenous hormone. The expected values are 14-76 ng/dL in females. The intra assay coefficient of variation is 11.78% (24.4 ng/dL, n=30), 4.6% (191.2 ng/dL, n=30) and 9.1% (414.2 ng/dL, n=30). The inter assay coefficients of variation are 11.34% (53.3 ng/dL, n=37) and 9.7% (250.2 ng/dL, n=37).

TNF-α more info +

The Human TNF-α Chemiluminescent Immunoassay (R&D Systems, QTA00B) is a plate assay which employs the quantitative sandwich enzyme immunoassay technique using a monoclonal antibody specific for TNF-α which has been pre-coated onto a microplate. Standards, QC ’s and samples are pipetted into the wells and any TNF-α present is bound by the immobilized antibody during a 3 hr incubation on an orbital shaker at RT. After washing away any unbound substances, an enzyme-linked polyclonal antibody specific for TNF-αis added to the wells and incubated for 2 hrs on an orbital shaker at RT. Following a wash to remove any unbound antibody-enzyme reagent, an enhanced luminol/peroxide substrate solution is added to the wells, incubated for 5-20 min and then read in a microplate luminometer (ICN Titertek Luminoscan set to read for 2.0 seconds) which measures the intensity of the light emitted. Light is produced in proportion to the amount of TNF-α bound in the initial step. The counts are transferred to a computer program (StatLia) for analysis with a range of 1.1 - 560 pg/mL. We report the following coefficients of varation. Inter-assay CV: 8.3% at 116 pg/mL, 8.8% at 35.6 pg/mL, 9.6% at 58.3 pg/mL, 11.3% at 162 pg/mL. Intra-assay 2.6% at 116 pg/mL, 2.1% at 35.6 pg/mL, 3.5% at 58.3 pg/mL, 2.6% at 162 pg/mL

TNFα R2 more info +

The Human sTNF RII immunoassay from R&D Systems (DRT200) uses the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for sTNF RII has been pre-coated onto a microtiterplate. Standards and samples are pipetted into the wells and any sTNF RII present is bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked polyclonal antibody specific for sTNF RII is added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of sTNF RII bound in the initial step. The color development is stopped and the intensity of the color is measured. The assay range is 7.8 - 500 pg/mL.. We report the following coefficients of varation. Inter-assay CV: 9.1% at 33.8 pg/mL, 5.7% at 220 pg/mL, 3.2% at 414 pg/mL. Intra-assay 3.0% at 33.8 pg/mL, 1.7% at 220 pg/mL, 3.6% at 414 pg/mL.

tPA more info +

Introduction Tissue-type plasminogen activator (tPA) is a serine protease that converts the zymogen plasminogen into the active serine protease plasmin, the primary enzyme responsible for the removal of fibrin deposits (1). tPA is a 68 kDa glycoprotein that is synthesized by endothelial cells in normal blood vessels and displays relatively high affinity for fibrin, suggesting that it functions predominately in physiological thrombolysis in vivo ( 2). A high level of tPA is a good prognostic marker for breast cancer (3, 4). tPA may minimize the formation of metastasis by preventing tumor cell adherence at sites of trauma (5). On the other hand, gastrointestinal cancer is accompanied by a decrease in tPA (6).

The AssayMax Human tPA ELISA kit is designed for detection of human tPA in plasma, serum, urine, saliva, milk, cell culture supernatants, and tissue extract. This assay employs a quantitative sandwich enzyme immunoassay technique that measures tPA in less than 4 hours. A polyclonal antibody specific for tPA has been pre-coated onto a microplate. tPA in standards and samples is sandwiched by the immobilized antibody and a biotinylated antibody specific for tPA, which is recognized by a streptavidin-peroxidase conjugate. All unbound material is then washed away and a peroxidase enzyme substrate is added. The color development is stopped and the intensity of the color is measured. This method requires 100 ul of sample for duplicate analysis and features a standard range of 0.031 to 2.0 ng/mL, and a minimum detectable concentration of ~0.03 ng/mL. Inter-assay coefficient of variation provided by the manufacturer is 7%, intra-assay coefficient of variation is 4.9.

Triglycerides more info +

Triglycerides in serum are hydrolized by lipase to form glycerol and free fatty acids. In the presence of adenosine triphosphate and glycerol kinase the glycerol is converted to glycerol-1-phosphate and the adenosine triphosphate to adenosine diphosphate. Glycerol-1-phosphate is oxidized by glycerol phosphate oxidase to yield hydrogen peroxide which then acts to oxidatively couple p-hydroxybenzioc acid (catalyzed by peroxidase) and 4-aminoantipyrine producing the red quinoneimine chromophore which absorbes light strongly at 505 nm. The ACE triglyceride assay is linear from 6 to 1000 mg/dL. The MDL is 6 mg/dL (2 SD greater than the 0 Standard). The manufacturer provides the following Intra-assay precision expressed as CV: 1.7 % (73 mg/dL), 1.2% (142 mg/dL) and 1.2% (203 mg/dL). The manufacturer provides the following Inter-assay precision expressed as CV: 1.9 % (73 mg/dL), 1.8% (142 mg/dL) and 2.0% (203 mg/dL).

TSH more info +

The serum Thyroid Stimulating Hormone (TSH) assay is a sandwich immunoassay using direct, chemiluminescent technology. The first antibody is a acridinum ester-labeled mouse monoclonal anti-TSH in the lite reagent. The second antibody is a polyclonal sheep anti TSH antibody covalently coupled to paramagnetic particles. . Separation, aspiration, and deionized water wash steps separate bound from free. A direct relationship exists between the amount of TSH in the sample and the relative light units detected by the luminometer (photomultiplier tube). The test results are determined from a calibration curve derived from standard which is calibrates against the WHO 2^nd IRP 80/558. The manufacturer reports the following a limit of detection of 0.01 uIU/mL; Intra-assay coefficient of variation: 4.7% (0.3 uIU/mL), 4.0% (4.7 uIU/mL), 3.6% (15.85 uIU/mL); and Interassay coefficient of variation 6.6% (0.3 uIU/mL), 5.3% (4.7 uIU/mL) and 4.9% (15.85 uIU/mL) The manufacturer reports the following expected results: Euthyriod 0.35-5.50 uIU/mL, hypothyroid >5.50 uIU/mL, hyperthyroid <0.35 uIU/mL.

Undercarboxylated Osteocalcin (Glu-OC) EIA Kit more info +

Osteocalcin (OC), also known as bone gla protein (BGP), is a vitamin K-dependent Ca2+-binding protein of molecular weight 5900, consisting of 49 amino acid residues. It carries three γ-carboxylated glutamic acid residues (Gla) at positions 17, 21, and 24 which are known to mediate strong binding of OC to hydroxyapatite. The synthesis of OC depends on both the vitamin D and vitamin K. Vitamin D induces directly the OC synthesis, and vitamin K stimulates the γ-carboxylation of glutamic acid residues. For resulting these enzymes deficiency or osteoclastic bone resorption, undercarboxylated osteocalcin (Glu-OC) decreases binding to the bone and increases circulating in blood stream and urine.P.D. Delmas and his co-worker have previously shown that undercarboxylated OC which does not bind to hydroxyapatite, is significantly increased in elderly woen, suggesting an age dependent impairment of the γ-carboxylation of OC. Further they suggest that serum undercarboxylated OC reflects some change in bone matrix associated with increased fragility. Until now, measurement of undercarboxylated OC in serum sample is usually performed by hydroxyapatite combination radioimmunoassay. We raised the monoclonal antibody specific to undercarboxylate osteocalcin and constructed ELISA system. This Undercarboxylated Osteocalcin (Glu-OC) EIA Kit utilizes a set of monoclonal antibodies highly reactive to the decarboxylated osteocalcin (Glu-OC) and less reactive to carboxylated at positions 17, 21, 24 form. Direct measurement of Glu-OC by this EIA Kit provides as useful leads of clinical information of bone metabolism as Gla-Type Osteocalcin (Gla-OC) EIA Kit (Cat. #MK111).

We will be using a commercially available enzyme-linked immunosorbent assay (ELISA) from Takara Bio, Inc for the in vitro quantitative measurement of Undercarboxylated Osteocalcin (Glu-OC) in human serum. The Takara Undercarboxylated Osteocalcin in human Coated Well ELISA kit Features a direct competitive immunoassay with no required sample extraction or hydrolysis. There is no detectable cross-reactivity with closely related compounds. This kit specifically measures Glu-OC with 5.0% crossreactivity with human bone OC (probably Gla type). Normal values are expected to be 1.24 +- 0.57 ng/ml (mean age =29). The assay measures analyte concentrations from 0.25 - 8 ng/ml with a minimum detectable concentration of 0.25 ng/mL. The inter assay coefficient of variation provided by the manufacturer is 5.7% at 6.5 ng/mL, 9.5% at 1.6 ng/mL and 9.9% at 0.7 ng/mL. The intra assay coefficient of variation provided by the manufacturer is 4.6% at 6.9 ng/mL, 4.4% at 1.8 ng/mL and 6.7% at 0.8 ng/mL.

The Glu-OC EIA Kit is a solid phase EIA based on a sandwich method that utilizes two mouse monoclonal antibodies to detect Glu-OC by two-step procedure. One of the mouse monoclonal antibodies, anti-undercarboxylated OC, is immobilized onto the microtiter plate and blocked against non-specific binding. Samples and standars are added to each well and incubated. The second step is to wash the plate and to add the second anti-OC labelled with peroxidase (POD). During this incubation, Glu-OC is bound to anti-undercarboxylated OC (solid phase) on one side and tagged on the other by POD-anti OC. The reaction between POD and substrate (3, 3', 5, 5' tetramethylbenzidine) results in color development with intensities proportional to the amount of Gla-OC present in samples and standards. The amount of Glu-OC can be quantitated by measuring the absorbance using an EIA plate reader. Accurate sample concentrations of Glu-OC can be determined by comparing their specifc absorbances with those obtained for the standards plotted on a standard curve

VCAM more info +

This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for sVCAM-1has been pre-coated onto a microplate. Standards, samples, controls, and conjugate are pipetted into the wells and any sVCAM-1 present is sandwiched by the immobilized antibody and the enzyme-linked monoclonal antibody specific for sVCAM-1. Following a wash to remove any unbound substances and/or antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of sVCAM-1 bound. The color development is stopped and the intensity of the color is measured.

WHOLE BLOOD ASSAYS

Hemoglobin A_1c (HbA1c) NEEDS UPDATE-NEW METHOD more info +

Hemoglobin A1c is performed on the Alfa-Wasserman ACE analyzer. Whole blood (K₂-EDTA or NH₄-heparin) samples require a pretreatment step in which the red cells are lysed by a hemoglobin denaturant and the hemoglobin chain is hydrolyzed. For determination of HbA1c a latex agglutination inhibition assay is used. In the absence of HbA1c I the sample, the agglutinator (a synthetic polymer containing the immunoreactive portion of HbA1c) in the HbA1c Agglutinator Reagent and the antibody coated microparticles in the HbA1c Antibody Reagent will agglutinate. The presence of HbA1c in the sample competes for antibody binding sites and inhibits agglutination. The increase in absorbance, monitored at 592 nm, is inversely proportional to the HbA1c present in the sample. For the determination of total hemoglobin, all hemoglobin derivatives in the sample are converted to alkaline hematin. The reaction produces a green colored solution, which is measured bichromatically at 573 and 692 nm. The intensity of color is directly proportional to the total hemoglobin concentration in the sample. Expected values for HbA1c are 4.0-5.2% of total Hb. The manufacturer provides the following Intra-assay precision expressed as CV: 2.2% (6.0% HbA1c), 1.6% (8.8% HbA1c) and 1.5% (11.7 % HbA1c). The manufacturer provides the following Inter-assay precision expressed as CV: 3.1% (6.0 % HbA1c), 3.3% (8.8 % HbA1c) and 2.8% (11.7 % HbA1c).

SALIVARY ASSAYS

Salivary Cortisol more info +

The Salivary Cortisol assay is a competitive immunoassay run on the Siemen Centaur automated analyzer using chemiluminescent technology. Cortisol in the patient sample competes with acridinium ester-labeled cortisol in the Lite Reagent for binding to polyclonal rabbit anti-cortisol antibody in the Solid Phase Reagent. The polyclonal rabbit anti-cortisol antibody is bound to monoclonal mouse anti-rabbit antibody, which is covalently coupled to paramagnetic particles in the Solid Phase. 20 uL of saliva is required for the assay in addition to sufficient dead volume for aspiration and repeat analysis. The assay range is 0.07 - 7.84 ug/dL and the assay is standardized analytically and confirmed by gas chromatography- mass spectroscopy. We report the following coefficients of variation. Inter-assay CV: 12.4 % at 0.7 ug/dL, 5.2 % at 5.2 ug/dL. Intra-assay CV: 3.6 % at 0.7 ug/dL, 4.2 % at 5.2 ug/dL.

Salivary CRP more info +

The Salimetrics^TM CRP kit is an enzyme-linked immunoassay specifically designed and validated for the quantitative measurement of salivary CRP. A microtitre plate is coated with mouse antibodies to human CRP. CRP in standards and unknowns and goat anti-human CRP antibodies linked to horseradish peroxidase are added. A “sandwich” is formed with the precoated antibody on the bottom, the CRP in the middle, and the antibody linked to horseradish peroxidase on the top. After incubation, unbound components are washed away. Bound CRP peroxidase is measured by the reaction of the peroxidase enzyme on the substrate tetramethylbenzidine (TMB). This reaction produces a blue color. A yellow color is formed after stopping the reaction with 2-molar sulfuric acid. Optical density is read on a standard microplate reader at 450 nm. The amount of CRP peroxidase detected is directly proportional to the amount of CRP present. Intra assay coefficient of variation, provided by the manufacturer, is 5.9% (178 pg/mL) and 1.9% (1993 pg/mL). Inter assay coefficient of variation, provided by the manufacturer, is 11.2% (238 pg/mL) and 3.7% (2167 pg/mL). The lower limit of sensitivity is 10 pg/mL.

Salivary DHEAS more info +

The Salimetrics^TM DHEA-S kit is a competitive immunoassay specifically designed for the quantitative measurement of salivary DHEA-S. A microtitre plate is coated with rabbit antibodies to DHEA-S. DHEA-S in standards and unknowns competes with DHEA-S linked to horseradish peroxidase for the antibody binding sites. After incubation, unbound components are washed away. Bound DHEA-S peroxidase is measured by the reaction of the peroxidase enzyme on the substrate tetramethylbenzidine (TMB). This reaction produces a blue color. A yellow color is formed after stopping the reaction with 3-molar sulfuric acid. Optical density is read on a standard plate reader at 450 nm. The amount of DHEA-S peroxidase detected is inversely proportional to the amount of DHEA-S present. Intra assay coefficient of variation, provided by the manufacturer, is 8.8% (526 pg/mL) and 5.7% (9635 pg/mL). Inter assay coefficient of variation, provided by the manufacturer, is 7.7% (519 pg/mL) and 7.4% (9368 pg/mL). The lower limit of sensitivity is <43 pg/mL.

Salivary Testosterone (SalTest) more info +

The Salimetrics^TM expanded range (ER) testosterone kit is a competitive immunoassay specifically designed and validated for the quantitative measurement of salivary testosterone. The protocol uses only 25 μL of saliva per test. No separation or extractions are necessary. A microtitre plate is coated with rabbit antibodies to testosterone. Testosterone in standards and unknowns competes with testosterone linked to horseradish peroxidase for the antibody binding sites. After incubation, unbound components are washed away. Bound testosterone peroxidase is measured by the reaction of the peroxidase enzyme on the substrate tetramethylbenzidine (TMB). This reaction produces a blue color. A yellow color is formed after stopping the reaction using 2-molar sulfuric acid. Optical density is read on a standard plate reader at 450 nm. The amount of testosterone peroxidase detected is inversely proportional to the amount of testosterone present.

URINARY ASSAYS

Urinary 8-Hydroxydeoxyguanosine (8-OHdG) more info +

The NWLSS^TM 8-OHdG ELISA assay uses a competitive format wherein a murine monoclonal antibody to 8-OHdG (Primary Antibody) and sample or standard are added to a microtiter plate which has been precoated with 8-OHdG. Sample or calibrator 8-OHdG competes with plate-bound 8-OHdG for binding with the antibody. Accordingly, higher concentrations of sample or calibrator lead to reduced binding of the antibody to the 8OHdG coated on the plate. A subsequent wash step removes any free 8-OHdG/antibody adduct leaving stationary plate bound 8-OHdG complexed to antibody for later detection. Anti-murine antibody conjugated to horse radish peroxidase (HRP-Conjugate) is then added to the plate. HRP-conjugate binds to remaining murine anti-8-OHdG and unbound HRP-conjugate is removed in another wash step. Addition of 3,3',5,5'tetramethylbenzidine (TMB Substrate) results in blue color development proportional to the amount of anti 8-OHdG antibody bound to the plate and inversely proportional to theconcentration 8-OHdG in original samples or calibrators applied to the plate. The reaction is terminated by addition of phosphoric acid (Stop Solution) producing yellow color with measurable absorbance at 450 nm.

Urinary Albumin more info +

Albumin reflects the major protein in human plasma (40-60%). It is synthesized in the liver depending on protein uptake. Albumin in fecal samples refers to an inflammation reaction combined with intestinal bleeding. Elevated levels of albumin and hemoglobin are stool is found not only by colorectal carcinoma, but also with polyps and during chronic inflammatory diseases (Morbus Crohn, Colitis Ulcerosa).

This Enzyme-Linked Immunosorbent Assay (ELISA) is a two step assay for the ultra sensitive determination of human albumin in stool and urine. A polyclonal rabbit antibody specific for human Albumin is immobilized on micro plates and a second anti-Albumin antibody is conjugated to peroxidase (POD). In a first incubation step, the albumin in the samples is bound to the immoblized anti-albumin antibodies. A washing step is carried out to remove all unboundsubstances, a POD-substrate, tetramethylbenzidine (TMB) is added. The enzymatic reaction is terminated by an acidic stop solution, whereby the color converts from blue to yellow. The intensity of the yellow color is directly proportional to the concentration of the albumin in the sample. A dose response curve of the absorbance unit (optical density, OD) vs. concentration is generated using the results obtained from the calibrators. Albumin in the sample isdetermined directly from this curve.

Intra-Assay The precision (intra-assay variation) was calculated from 20 replicate determinations on one sample.

Urinary C2 C16 Isoprostanes more info +

2-OHE1 and 16α-OHE1 are assayed by enzyme immunoassay (ESTRAMET^TM) in triplicate (Klug 1994). Because urinary forms of 2-OHE1 and 16α-OHE1 are found as 3-glucuronides or 3,3,16-glucuronides, it is necessary to remove these sugars to achieve recognition sites for the monoclonal antibodies. Therefore, the estrogens are deconjugated from glucuronic acid and sulfate using a mixture of β-glucuronidase and arylsulphatase enzyme isolated from the snail Helix Pomatia. The assay range is 0.6 to 40.0 ng/ml. Samples are purified with F_2a-isprostane affinity columns according to the manufacturer ’s instructions (Cayman Chemical, Ann Arbor, MI). Samples are diluted 1:5 with 0.1 M phosphate buffer, applied to the column and eluted with 95% ethanol. Following vacuum centrifugation, samples are dissolved in enzyme assay buffer and assayed using the F_2a-isoprostane EIA kit (Cayman Chemical, Ann Arbor, MI). Samples are read at 415 nm in a 96-well microplate reader. The intra- and inter-assay coefficients of variation for 2-OHE1 and 16α-OHE1 were 11.2% (5.1ng/mL) and 14.5% (2.9 ng/mL).

Urinary Creatinine Assay more info +

Creatinine is a spectrophotometric assay that uses the Hamilton Microlab AT Plus to pipette standards, controls, unknowns, diluent, and reagents (picric acid and sodium hydroxide) into a microtiter plate. The plate is then incubated for 15 minutes at room temperature, the light absorbance read at 490 nm and the concentrations of the unknowns calculated using the SpectraMax 340 PC Plate Reader. 10 μL of urine is required for the assay in addition to sufficient dead volume for aspiration and repeat. The assay is standardized against Creatinine obtained from the Sigma Co. The SWAN reporting range for the creatinine assay is 0.05-1.15 mg/mL (actual assay range: 0.05-1.4 mg/mL). We report the following: Inter-assay CV: 11.4%, Intra-assay CV: 4.3%.

Urinary Estrone Conjugate Assay more info +

The Estrone-conjugate (E1C) assay is a competitive immunoassay with manual steps and an off-line incubation. Urine samples and quality, semi-automated control preparations are pre-diluted in potassium phosphate buffer. Standards, samples, and quality control preparations are pipeted into 12x75 polypropylene tubes. First antibody, rabbit anti-E1C antibody, and Estrone-glucuronide labeled with DMAE are added and then the tubes incubate for two hours at room temperature. All tubes are then placed on Siemens Centaur automated analyzer with a goat anti-rabbit antibody covalently coupled to PMP for analysis. 50 μL of urine is required for the assay in addition to sufficient dead volume for aspiration, repeat and creatinine correction. The reporting range for the Urine E1C assay is 5.10-408.0 ng/mL. The assay is standardized against a dual standard prepared with Estrone-glucuronide and Estrone-sulfate obtained from SIGMA Co. We report the following: Inter-assay CV: 11.00%, Intra-assay CV: 7.77 %.

Urinary Follicle Stimulating Hormone (FSH) more info +

Urinary Follicle Stimulating Hormone (FSH) concentrations will be measured with a two-site chemiluminescence (sandwich) immunoassay, which uses constant amounts of two antibodies that have a specificity for the intact FSH molecule; a polyclonal sheep anti-FSH antibody labeled with acridinium ester and a monoclonal mouse anti-FSH antibody covalently coupled to paramagnetic particles. Separation, aspiration, and deionized water wash steps separate bound from free. A direct relationship exists between the amount of FSH in the sample and the relative light units detected by the luminometer (photomultiplier tube). The test results are determined from a calibration curve derived from the standard, WHO 2nd IRP 78/549. The assay measures uFSH concentrations up to 152 mIU/mL with a minimum detectable concentration of 1.18 mIU/mL. Inter- and intra-assay coefficients of variation are 10.9% and 3.9%, respectively.

Urinary Luteinizing Hormone (LH) more info +

Urinary Luteinizing Hormone (LH) concentrations will also be measured with a two-site chemiluminescent immunoassay by Bayer diagnostics, using the same technique as the FSH assay with two monoclonal antibodies, One antibody is directed at the alpha subunit and the other at the beta subunit (one coupled to paramagnetic particles and the other labeled with DMAE) with specificity for intact LH. The expected values are for normally menstruating females is 0.5-75 mIU/mL, for postmenopausal females 16-54 mIU/mL is expected. The assay measures analyte concentrations to 200 mIU/mL with a minimum detectable concentration of 0.07 mIU/mL. For serum, inter- and intra-assay coefficients of variation for serum are projected to be 6.8% and 2.8 %, respectively and for urine, they are projected to be 10.7% and 4.8%, respectively.

Urinary N-telopeptides (NTx) more info +

Osteomark NTx assay provides a quantitative measurement of the cross-linked N-telopeptides of bone type I collagen (NTx). NTx is a specific biochemical indicator of bone resorption that is generated as result of osteoclast activity on bone. The NTx molecule is specific to bone due to the unique amino acid sequence and orientation of the cross-linked alpha-2 (I) N teleopeptide. We use a commercially available enzyme-linked immunosorbent assay (ELISA) from Osteomark (Inverness Medical Professional Diagnostics, Princeton, New Jersey, 08540) for the in vitro quantitative measurement of N-telopeptides (NTx) in human urine. The Osteomark NTx assay is a competitive inhibition enzyme linked immunosorbent assays (ELISAs) that use microwells in strips as the solid phase onto which NTx has been absorbed. NTx in the patient sample competes with the NTx absorbed on the solid phase for binding sites on a horseradish peroxide labelled monoclonal antibody. The amount of antibody bound is inversely proportional to the amount of NTx in the sample. The NTx concentration in the patient sample is determined spectrophotometrically and calculated from a standard calibration curve. In urine, ELISA results are expressed in nM of bone collagen equivalents (nM BCE) per mM of creatinine. The assay requires 50 uL of urine for duplicate analysis. The reportable assay range (standard curve) is 20-3000 nM BCE. The intra-assay coefficient of variation reported by the manufacturer is 7.6%; the intra-assay coefficient of variation reported by the manufacturer is 4.0%. Reference Range (nM BCE/mM Creatinine) pre-menopausal women: 35 (mean), 5-65 (range); men: 33 (mean), 3-63 (range).

Urinary Osteoprotegerin (OPG) more info +

Osteoprotegerin (OPG) or Osteoclast inhibitory factor (OCIF) is a dimeric glycoprotein of the TNF receptor family with a molecular weight of 60 kD and 120 kD respectively. OPG acts as a soluble secreted receptor for RANKL. OPG prevents the binding of RANKL to RANK and thus inhibits RANK activation (osteoclast differentiation and activation receptor, ODAR) on the osteoclast surface. OPG is an inhibitor of osteoclast development. The Osteoprotegerin Immunoassay (ALPCo Diagnostics) is a two-site ELISA [Enzyme-Linked ImmunoSorbent Assay] for the measurement of Osteoprotegerin. A mouse monoclonal antibody to human OPG, purified by affinity chromatography, and a biotin-labeled goat polyclonal antibody to human OPG are specific for well defined regions on the OPG molecule. At the end of the assay incubation, the microwell is washed to remove unbound components and the enzyme bound to the solid phase is incubated with the substrate, tetramethylbenzidine (TMB). An acidic stopping solution is then added to stop the reaction and converts the color to yellow. The intensity of the yellow color is directly proportional to the concentration of OPG in the sample. A dose response curve of absorbance unit vs. concentration is generated using results obtained from the calibrators. Concentrations of OPG present in the controls and patient samples are determined directly from this curve. The assay requires 100 uL of serum for duplicate analysis. The reportable assay range (standard curve) is 0.37-30 pmol/L. The intra-assay coefficient of variation reported by the manufacturer is 10% (4.59 pmol/L) and 4% (10.76 pmol/L). The intra-assay coefficient of variation reported by the manufacturer is 7% (5.53 pmol/L) and 8% (10.1 pmol/L). Reference Range: 1.8 pmol/L (n=1134, age =19-96). Conversion factor: 1 pg/mL= 0.05pmol/L (MW 19.9 kDa).

Urinary Pregnanediol-glucuronide Assay more info +

The Pregnanediol-glucuronide (PDG) assay measured in urine is a competitive semi-automated immunoassay with manual steps and an off-line incubation. Urine samples and quality, control preparations are pre-diluted in potassium phosphate buffer. Standards, samples, and quality control preparations are pipeted into 12x75 polypropylene tubes. First antibody, rabbit anti-PDG antibody, and PDG labeled with DMAE are added and then the tubes incubate for two hours at room temperature. All tubes are then placed on Siemens Centur automated analyzer with goat anti-rabbit antibody covalently coupled to PMP for analysis. 50 μL of urine is required for the assay in addition to sufficient dead volume for aspiration, repeat, and creatinine correction. The SWAN reporting range for the Urine PDG assay is 0.005-25.5 ug/mL. The assay is standardized against PDG obtained from SIGMA. Samples are run in singleton. We report the following: Inter-assay CV: 12.31%, Intra-assay CV: 7.65 %.