DBC Chorionic Gonadotropin (hCG) Elisa

PRINCIPLE OF THE TEST 
The principle of the following enzyme immunoassay test follows a typical one-step capture or ‘sandwich’ type assay. The assay makes use of two highly specific monoclonal antibodies: A monoclonal antibody specifi c for hCG is immobilized onto the microplate and another monoclonal antibody specific for a different region of hCG is conjugated to horse radish peroxidase (HRP). hCG from the sample and standards are allowed to bind simultaneously to the plate and to the HRP conjugate. The washing and decanting steps remove any unbound HRP conjugate. After the washing step, the enzyme substrate is added. The enzymatic reaction is terminated by addition of the stopping solution. The absorbance is measured on a microtiter plate reader. The intensity of the colour formed by the enzymatic reaction is directly proportional to the concentration of hCG in the sample. 
A set of standards is used to plot a standard curve from which the amount of hCG in patient samples and controls can be directly read. 
Please note that a two-step procedure is included and is to be used for assaying serum from pregnant women.

CLINICAL APPLICATIONS 
Human chorionic gonadotropin (hCG) is a glycoprotein hormone produced by the placenta. During normal pregnancy the level of this hormone in serum and urine gradually increases up to about the eighth week of pregnancy. hCG has two subunits, namely α and β. The α subunit is similar to the α subunit of the anterior pituitary gland glycoprotein hormones, namely, LH, TSH and FSH. However, the β subunits of these hormones are different. Therefore, this uniqueness distinguishes one hormone from the other, hence specifi city. In raising this monoclonal antibody, the immunogen used was βhCG, which helps to make the DBC assay system very specifi c and sensitive. 
In normal pregnancy the increase in hCG starts at about the 5th day after conception and continues to rise until it reaches a maximum at about the eighth week. In some pathological conditions the level of hCG in serum and/or urine is increased. It is a well known fact that hCG is also a tumour marker which is very important in the diagnosis of choriocarcinoma. In the case of hydatiform mole, hCG is also elevated. In about 50% of patients with testicular teratomas the level of hCG is elevated. It is also relevant to note that hCG is a good indicator, in order to follow the response to treatment.

SPECIMEN COLLECTION AND STORAGE 
Approximately 0.1 mL of serum is required per duplicate determination. Collect 4–5 mL of blood into an appropriately labelled tube and allow it to clot. Centrifuge and carefully remove the serum layer. Store at 4°C for up to 24 hours or at -10°C or lower if the analyses are to be done at a later date. Consider all human specimens as possible biohazardous materials and take appropriate precautions when handling.

CALCULATIONS 
1. Calculate the mean optical density of each calibrator duplicate. 
2. Calculate the mean optical density of each unknown duplicate. 
3. Subtract the mean absorbance value of the “0” calibrator from the mean absorbance values of the calibrators, controls and serum samples. 
4. Draw a calibrator curve on log-log paper with the mean optical densities on the Y-axis and the calibrator concentrations on the X-axis. If immunoassay software is being used, a 4-parameter or 5-parameter curve is recommended. 
5. Read the values of the unknowns directly off the calibrator curve. 
6. If a sample reads more than 500 IU/L then dilute it with calibrator A at a dilution of no more than 1:20. The result obtained should be multiplied by the dilution factor