Experiment 3

ELISA test for b HCG 
 

Highlights:

This Exercise focuses on how to analyze some Biochemical assays. We will use ELISA (Enzyme linked immunosorbent assay) test to detect HCG in a control sample. 
 

Time: two to three periods, including discussion. 
 

Material (per class of 30, working in teams of 2) 
 

• Antibody coated microtiter –wells.
• Antibody – Enzyme conjugate in buffered solution containing horse- radish peroxidase and protein stabilizer.
• CRSB (substrate Reagent containing hydrogen peroxide in buffered solution + chromogen reagent containing tetramethyl benzidine in methanol).
• Negative Reference.
• Positive Reference.
• 15 Well holder tray.
• 15 Plastic pipettes.

Introduction: 
 

      The ELISA (Enzyme linked immunosorbent assay) test for HCG is used to detect whether a woman is pregnant or not using a sample of urine or blood.

The fertilized egg at about 4 days old begins to secrete hormone called human chorionic gonadotropin (HCG) which composed of two subunits Alpha (a)and Beta (b).(The b subunit of HCG confers both the biological and immunological specificity of the intact molecules, so the b – HCG specificity is a critical factor when low levels of HCG are measured in the specimen).

The enzyme–linked immunosorbent assay (ELISA) for HCG are highly sensitive and specific that involve an enzyme (a protein that catalyzes a Biochemical reaction). It also involves antibody or antigen (immunologic molecule) that allows quick & easy determination of the presence of even small quantities of the hormone in the blood or the urine. 
 

Principle of the test:

      In a positive test, HCG is bound by its Beta subunit to the antibodies on the well and the Antibody – Enzyme conjugate binds with the unbound portions of HCG.

The substrate and chromogen added are catalyzed by the enzyme to form a blue color. As shown in (Fig 3.1) In a negative test, absence of HCG prevents binding of the antibody enzyme conjugate via the HCG antigen bridge. Thus no enzyme is present to catalyze the reaction and the test is colorless. 
 
 
 
 
 

Fig 3.1 Binding of the antibody enzyme conjugate via the HCG antigen bridge.

Procedure: 
 

1. Put 1 drop (50μL) of the control, the references and patient samples into correspondingly numbered wells.
2. Add 1 drop (50μL) of Antibody – Enzyme conjugate into each well.
3. Shake gently to mix and keep at room temperature (15-30 ºC) for 5 minutes.
4. Wash the well 5 times with tap water.
5. Add 1 drop (50 μl) of CRSB  (Chromogen Reagent containing tetramethyl benzidine in methanol + substrate Reagent containing hydrogen peroxide in buffered solution) to each well.
6. Shake gently to mix and keep at room temperature for 5 minutes.

Longer incubation up to 20 minutes are acceptable.

7. Compare the color of the patient sample wells to the color of the positive and negative references and control wells.
8. Record your observation.