DBC Progesterone Elisa

PRINCIPLE OF THE TEST 
The principle of the following enzyme immunoassay test follows the typical competitive binding scenario. Competition occurs between an unlabelled antigen (present in standards, controls and patient samples) and an enzyme-labelled antigen (conjugate) for a limited number of antibody binding sites on the microplate. The washing and decanting procedures remove unbound materials. 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 is inversely proportional to the concentration of progesterone in the sample. A set of standards is used to plot a standard curve from which the amount of progesterone in patient samples and controls can be directly read.

CLINICAL APPLICATIONS 
Progesterone is a C-21 female sex steroid hormone with a variety of physiological effects. In the follicular phase of the menstrual cycle, progesterone is produced in low levels. It increases to the LH peak and then sharply rises 3 to 4 days later to higher levels, remaining elevated through the 10th to 12th days after the LH peak. Then there is a sharp decline to the low levels of the follicular phase. It is responsible for the induction of the cyclic changes in the endometrium of the uterus allowing implantation and successful growth of the fertilized ovum and maintenance of pregnancy. 
Progesterone measurements are used in documenting ovulation and in the management of diffi culties during the fi rst trimester of pregnancy. Levels of progesterone may be useful in the evaluation of sterility due to luteal phase defects, prediction of impending abortion, and the diagnosis of ectopic pregnancy. 
Normal values of progesterone may be affected by drugs such as, oral contraceptives, superovulatory drugs, estrogen replacement therapy medication, and GnRH analogues. The removal of ovarian function following surgical oophorectomy or chemotherapy may infl uence serum progesterone values.

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. Draw a calibrator curve on semi-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. 
3. Calculate the mean optical density of each unknown duplicate. 
4. Read the values of the unknowns directly off the calibrator curve. 
5. If a sample reads more than 60 ng/mL then dilute it with calibrator A at a dilution of no more than 1:8. The result obtained should be multiplied by the dilution factor.