Specifications

Enzyme immunoassay for the detection of IgA antibody to the viral capsid antigen (VCA) of Epstein - Barr virus (EBV).

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).

                                                                                

INTRODUCTION

 

Epstein-Barr virus, frequently referred to as EBV, is a member of the herpes virus family and one of the most common human viruses.The virus occurs worldwide, and most people become infected with EBV sometime during their lives. In developed countries, 80 - 90% of the adult population has been infected by the virus.

EBV is associated with several diseases state where it may act as the direct agent or as one of several cofactors. These diseases include: Infectious Mononucleosis(IM), Nasopharyngeal carcinoma(NPC), Burkitt's lymphoma(BL), Lymph proliferative disease and lymphoma in the immunosuppressed, X-linked lymph proliferative syndrome, Chronic infectious mononucleosis, Oral leukoplakia in AIDS patients, and Chronic interstitial pneumonitis in AIDS patients. EB virus enters the body through the mouth and establishes a productive infection in the pharyngeal epithelial cells. B-cells become infected and are disseminated throughout the body via the bloodstream. During acute primary infection, IgA, IgM and IgG to VCA as well as IgG to EA(D) and MA are induced. When infection with EBV occurs during adolescence or young adulthood, it causes infectious mononucleosis 35% to 50% of the time. NPC is a genetically restricted tumor; being most common in the Southern Chinese.100% of sera from undifferentiated NPC patients have high-titre antibodies to EB-viral antigens. As in BL, antibodies against VCA are at a 10 times geometric mean titre. IgG and IgA levels against EA (D) and VCA rise as the disease progress and may be used for screening and monitoring purposes. VCA and EA (D) IgA are also uniquely found in the saliva of NPC patients. This is an enzyme linked immunosorbent assay for the detection of EBV-VCA IgAantibodies in Human serum or plasma and is indicated as a screening test for serum or plasma.

 

PRINCIPLE OF THE ASSAY

 

The EBV-VCA IgA Ab ELISA Kit utilizes a detection system where micro plate 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 micro plate wells. The wells are washed to remove unbound materials, and anti-human IgA peroxides 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 color develops in wells containing EBV-VCA IgA antibodies. The enzyme reaction is stopped by the addition of sulphuricacid. The intensity of color developed is read spectrophotometrically at 450nm(450 nm/630 nm) and is proportional to the amount of antibodies present in the specimen.

 

REAGENTS

 

Materials provided with the kits:

Item	Description	96T	480T
1	Micro titer Well	1	5
2	Negative Control	1ml	5ml
3	Positive Control	1ml	5ml
4	Sample Dilution	12ml	60ml
5	Enzyme Conjugate	12ml	60ml
6	Wash Buffer Concentrate (20x)	30ml	150ml
7	Substrate Solution A	6ml	30ml
8	Substrate Solution B	6ml	30ml
9	Stop Solution	6ml	30ml
10	Plastic Bag	1	5
11	Seal Paper	3	15
12	Manual	1 copy	5 copy

 

 

 

 

 

 

 

Materials required but not provided:

1. Precision pipettes: 0.02, 0.05, 0.10, 0.15, 0.20, and 1.0 ml.
2. Disposable pipette tips.
3. Distilled water.
4. HumidifiedBox capable of maintaining 37°C
5. Absorbent paper or paper towel.
6. Micro titer plate or strip-well washer
7. Micro titer plate reader.

 

PRECAUTION FOR USERS

 

Because no test method can offer complete assurance of the absence of infectious agents, this product should be handled with caution.

1. Avoid contact of reagents with the eyes and skin. If that occurs, wash thoroughly with water.
2. Wear gloves.
3. Do not pipette by mouth.
4. Do not smoke.
5. Dispose all used materials in a suitable biohazard us waste container. Remains of samples, controls, aspirated reagents and pipette tips should be collected in a container for this purpose and autoclaved 1-hour at 121°C or treated with 10% sodium hypochlorite (final concentration) for 30 min before disposal. (Remains containing acid must be neutralized prior addition of sodium hypochlorite).
6. Adjust washer to the plate used (flat bottom) in order to wash properly.
7. Do not mix reagents from different lots.
8. Do not use reagents after expiration date.
9. Extreme care should be taken to avoid microbial contamination and cross contamination of reagents.
10. Use a new pipette tip for each specimen and each reagent.
11. Soaps and/or oxidizing agents remaining in containers used for the substrate-TMB solution can interfere with the reaction.

 

SPECIMEN COLLECTION AND PREPARATION

 

Serum should be prepared from a whole blood specimen obtained by acceptable medical techniques. Either serum or plasma can be used in this test. Remove serum or plasma from the clot or blood cells as soon as possible to avoid hemolysis. Specimen with extensive particulate should be clarified by centrifugation prior to use. Specimen frozen at -20°C or colder may be used. Avoid repeated freeze thaw.

 

STORAGE OF TEST KIT

 

Unopened test kits should be stored at 2-8°C upon receipt and the micro titer plate should be kept in a sealed bag to minimize exposure to damp air. Use up the reagents as soon as possible after the kit is unpacked.

 

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 demonized 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 100mlof Sample Dilution into individualtest wells except the controls.
5. Add 10ml of each test sample intothetest 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 micro titer 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)

 

 

EIA Reader at 450 nm (using the OD value of the blank well to correct all the OD reading from all wells or at 450 nm/630 nm with dual filter plate reader.

Cut-off Calculations:

0.10 + average OD values of Negative control:

             0.10 +NC x = Cut-off.

If the absorbance of negative controls is below 0.05, calculate it as 0.05.If the absorbance of negative controls is above 0.05, calculate it as its original value.

Positive   OD₄₅₀ of sample ≥Cut-off

NegativeOD450of sample <Cut-off

 

LIMITATIONS OF THE ASSAY

 

1.