EBV-VCA IgA Ab ELISA Kit Catalog No.: BE501A
Enzyme immunoassay for the detection of IgA antibody to the viral capsid antigen (VCA) of Epstein-Barr Virus (EBV) .
 
1. PRINCIPLE OF THE ASSAY
The EBV-VCA IgA Ab ELISA Kit utilizes a detection system where microplate wells are coated with recombinant antigen corresponding to a highly antigenic segment EBV-VCA. Serum or plasma specimens, controls are added to the wells. During incubation, IgA antibodies specific for EBV-VCA present in the specimen will bind to the recombinant antigen fixed onto the microplate wells. The wells are washed to remove unbound materials, and anti-human IgA peroxidase conjugate will bind to the antigen-antibody complex and excess unbound enzyme conjugates are again removed by washing. The enzyme substrate, tetramethylbenzidine (TMB), is added upon incubation the substrate will be hydrolyzed by the bound enzyme and a blue or blue-green colour develops in wells containing EBV-VCA IgA antibodies. The enzyme reaction is stopped by the addition of sulphuric acid. The intensity of colour developed is read spectrophotometrically at 450nm (450 nm/630 nm) and is proportional to the amount of antibodies present in the specimen.
REAGENTS
 
2. Materials provided with the kits:

 
3. ASSAY PROCEDURE

1. Bring ELISA Kit (all reagents), and Specimens to room temperature before use (approximately 30 minutes).
2. Dilute concentrated wash buffer 1:19 with ddH2O. For example, 5 ml of wash buffer concentrate should be diluted to a total volume of 100 mL with deionized or distilled water.
3. For each test, set one blank well as background control, two positive and three negative controls. Dispense 100ml positive control and negative control duplicate into individual wells. Neither samples nor HRP-Conjugate should be added into the Blank well.
4. Dispense 100ml of Sample Dilution into individual test wells except the controls.
5. Add 10ml of each test sample into the test wells; vortex to mix.
6. Incubate for 30 minutes at 37°C
7. Wash each well 5 times by filling each well with diluted wash buffer, then inverting the plate vigorously to get all water out and blocking the rim of wells on absorbent paper for a few seconds.
8. Add 100ml of Enzyme Conjugate to each well. Mix it gently by swirling the microtiter plate on flat bench for 1 minutes. Do not add Enzyme Conjugate to the blank well.
9. Incubate for 20 minutes at 37°C
10. Wash the plate 5 times as step 7.
11. Add one drop (50ml) of Substrate Solution A (HRP-substrate) to each well, then add one drop (50ml) of Substrate Solution B (TMB) to each well. Mix gently and incubate at 37°C for 30 minutes. .
12. Add one drop (50ml) of Stop Solution to each well to stop the color reaction. Read the OD value at 450 nm/630 nm with dual filter plate reader. It is option to read the OD value at 450 nm with single filter plate reader. (using the OD value of the blank well to correct all the OD reading from all wells)