DBC Luteinizing Hormone (hLH) Elisa

PRINCIPLE OF THE TEST 
The principle of the following enzyme immunoassay test follows a typical two-step capture or ‘sandwich’ type assay. The assay makes use of two highly specifi c monoclonal antibodies: A monoclonal antibody specifi c for LH is immobilized onto the microplate and another monoclonal antibody specifi c for a different region of LH is conjugated to horse radish peroxidase (HRP). LH from the sample and standards are allowed to bind to the plate, washed, and subsequently incubated with the HRP conjugate. After a second 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 LH in the sample. 
A set of standards is used to plot a standard curve from which the amount of LH in patient samples and controls can be directly read.

CLINICAL APPLICATIONS 
Human luteinizing hormone (hLH) is a glycoprotein synthesized by the anterior lobe of the pituitary gland. This hormone consists of two subunits: α and β. The α subunit of LH is similar to the α subunit found in both the FSH and TSH glycoprotein hormones (which are also synthesized by the pituitary gland) as well as the α subunit of hCG (produced by the placenta). However, the β subunit of each of these hormones are unique. Therefore, the specifi city of these four hormones are due to the β peptide chains. It is to be noted that the α chain by itself has no biological activity. 
The hypothalamic decapeptide, namely the gonadotropin releasing hormone (GnRH), stimulates the release of LH. Both the LH and FSH hormones in men act on the testis, which have two functions: Leydig cells secrete androgens while sperm are formed by the seminiferous tubules. The secretion of testosterone and dihydrotestosterone by the Leydig cells is under the direct control of LH.

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 100 IU/L, 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.