DiaMetra b-HCG Elisa Kit

CLINICAL SIGNIFICANCE
Human chorionic gonadotropin (HCG) is a glycoprotein hormone secreted in pregnancy, that is made by the embryo soon after conception and later by syncytiotrophoblasts (part of the placenta). Its role is to prevent the disintegration of the corpus luteum of the ovary and thereby maintain progesterone production that is critical for a pregnancy in humans. HCG may have additional functions, for instance it is thought that it affects the immune tolerance of the pregnancy.
Pregnancy tests measure the levels of HCG in the blood or urine to indicate the presence or absence of an implanted embryo. Pregnancy tests employ an antibody that is specific to the β-subunit of HCG (β-HCG). This is important so that tests do not make false positives by confusing HCG with LH and FSH.
β-HCG is also secreted by some cancers including teratomas and choriocarcinomas. Elevated levels cannot prove the presence of a tumour and low levels do not rule it out.

PRINCIPLE OF THE METHOD
b-HCG ELISA is based on the simultaneous capture of HCG by a monoclonal antibody immobilised on the microplate and directed against the β-HCG fraction and another monoclonal antibody conjugated with peroxidase horseradish (HRP) and directed against the fraction β -HCG.
After the incubation, the bound/free separation is performed by a simple solid phase washing. Then, the enzyme HRP in the bound fraction reacts with the Substrate (H2O2) and the TMB Substrate and develops a blue colour that changes into yellow when the Stop Solution (H2SO4) is added. The colour intensity is proportional to the β-HCG concentration in the sample.
β-HCG concentration in the sample is calculated through a calibration curve.

REAGENT STORAGE AND STABILITY
Store the kit at 2 – 8°C in the dark.
The kit is stable at 2 – 8°C until the expiry date stated on the external kit label.
Once opened, the calibrators are stable at 2 – 8°C for 6 months*.
The diluted wash solution is stable for 30 days at 2 – 8°C.
Important note: open the bag containing the Coated Microplate only when it is at room temperature and close it immediately after use.

QUALITY CONTROL
Good Laboratory Practice (GLP) requires the use of quality control specimens in each series of assays in order to check the performance of the assay. Controls should be treated as unknown samples, and the results analysed with appropriate statistical methods.
The kit controls provided in the kit should be tested as unknown and is intended to assist in assessing the validity of results obtained with each assay plate.
The mean concentration of the control level is documented in the QC report included with each kit. These mean concentration levels are determined over several assays which are run in duplicate in multiple locations across each plate.

CALCULATION OF RESULTS
A variety of data reduction software packages are available, which may be employed to generate the mean calibration curve and to calculate the mean concentrations of unknown samples and controls. A 4-parameter logistic (4PL) curve fit or Sigmoidal, including Calibrator 0 is required. Other curve fitting algorithms are not recommended.
Alternatively, a calibration curve may be prepared on semilog graph paper by plotting mean absorbance on the Y-axis against concentration of analyte on the X-axis. Calibrator 0 should be included in the calibration curve. Read the mean absorbance value of each unknown sample off the curve.
In order for the assay results to be considered valid the kit calibrators and control must fall within the specifications detailed in the lot specific certificate of analysis.
If a control is out of its specified range, the associated test results are invalid and samples must be retested.

NOTE: If the absorbance of a sample exceeds 400 mIU/mL, the sample should be diluted in incubation buffer and reanalysed.