Specifications

Enzyme-linked immunosorbent assay for the detection of antibody to Hepatitis B Virus surface antigen in serum or plasma.

HBsAb ELISA Kit

Catalog No.:BE102A

Enzyme-linked immunosorbent assay for the detection of antibody to Hepatitis B Virus surface antigen in serum or plasma.

                                                                                              

 

HBsAb ELISA kit uses the “One-Step Incubation”, “Double Antigen Sandwich Principle” a solid phase enzyme-linked immunoassay technique and is used for the qualitative determination of antibody to Hepatitis B Virus surface antigen (HBsAb) in human serum or plasma. It is intended for use in medical laboratories for diagnosis and management of patients related to infection with hepatitis B virus.

 

 

Hepatitis B is an infectious illness caused by hepatitis B virus (HBV) which infects the liver of hominoidea, including humans, and causes an inflammationcalled hepatitis. HBV is excreted in body fluids such as semen, saliva, blood and urine in persons with acute or chronic infection. The hepatitis B virus is known as a blood-borne virus because it is transmitted from one person to another via blood or fluids contaminated with blood. Classification of a hepatitis B infection requires the identification of several serological markers expressed during three phases (incubation, acute and convalescent) of the infection.

Hepatitis B surface antigen (HBsAg) is an important viral envelope protein, which appears shortly after infection and is a key serological marker for detection and diagnosis of HBV.Clearance during treatment shows recovery and development of neutralizing antibodies (anti-HBs) occurs in 90% of the patients. During the acute phase of the infection, strong immunological response develops and increasing titers of HBsAg neutralizing antibodies (anti-HBs) are marker for recovery.

Due to the introduction of hepatitis B vaccination programs, the detection of anti-HBs has become important method for monitoring of recipients upon vaccination with synthetic and natural HBsAg. The absence of anti-HBs indicates susceptibility to HBV infection.

The serological detection of anti-HBs has become important method for the follow up of patients infected by HBV, prospective prevalence studies, and the monitoring of recipients upon vaccination with synthetic and natural HBsAg based vaccines.

 

PRINCIPLE OF THE ASSAY

The Bioneovan HBsAb ELISA kit uses antigen “sandwich” ELISA method where polystyrene microwell strips are pre-coated with recombinant HBsAg. Serum or plasma sample is added to the micro wells together with a second HBsAg conjugated to Horseradish Peroxidase (HRP-Conjugate). When antibodies to HBsAg are present in the sample, the pre-coated and conjugated antigens will be bound to the two variable domains of the antibody; the specific immunocomplex formed is captured on the solid phase during incubation. Non-reactive antibodies and unbound HRP-Conjugates are removed with the wash buffer. 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 anti-HBs. The enzyme reaction is stopped by the addition of sulphuric acid. 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. No color or very pale blue color indicates a negative reaction.

 

REAGENTS

Materials provided with the kits:

Item	Description	96T	480T
1	Micro titer Well	1	5
2	Negative Control	0.5ml	2.5ml
3	Positive Control	0.5ml	2.5ml
4	Enzyme Conjugate	6ml	30ml
5	Wash Buffer Concentrate (20x)	20ml	100ml
6	Substrate Solution A	6ml	30ml
7	Substrate Solution B	6ml	30ml
8	Stop Solution	6ml	30ml
9	User manual	1 copy	5 copy

 

Materials required but not provided:

1. Precision pipettes: 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 with 450nm (or 450 nm/630 nm)wavelength
8. Timer

 

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.

 

PRECAUTIONS FOR USERS

 

1. For in-vitro diagnostic use only.
2. Do not use kit beyond expiration date.
3. Do not mix components from kits with different lot number.
4. Avoid microbial contamination of reagents.
5. Do not pipet reagent by mouth and no smoking or eating while performing assays.
6. Wear gloves during the whole process and avoid reagents or specimen spilling-out.
7. Wipe up the spills using 5% hypochlorite solution.
8. Decontaminate all liquids or solid wastes before deposing.

 

STORAGE OF TEST KITS

 

Unopened test kits should be stored at 2-8C. DO NOT FREEZE KIT COMPONNETS. 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.

 

WORKING WASH BUFFER

 

Dilute the 20X wash buffer concentrate with deionized or distilled water 1:20. For example, 5 ml of wash buffer concentrate should be diluted to a total volume of 100 mL with deionized or distilled water.

 

ASSAY PROCEDURE:

  

It is strongly advised to analyze each specimen and controls in duplicate. All the reagents should equilibrate to room temperature before use.

1. For each test, set one blank well as background control, two positive and two negative controls.
2. Add 50μl positive control, Negative control, and Specimen into their respective wells. Neither samples nor HRP-Conjugate should be added into the Blank well.
3. Add 50μl Enzyme Conjugant to each well except the Blank. Mix it gently by swirling the micro titer plate on flat bench for 1 minute. Do not add Enzyme Conjugate to the blank well.
4. Cover wells with seal paper,Place the micro titer plates into a humidified box, and incubate at 37°C for 30 minutes.
5. 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.
6. Add one drop (50ul) of Substrate Solution a (HRP-substrate) to each well, then add one drop (50ul) of Substrate Solution B (TMB) to each well. Mix gently and incubate at 37°C for 15 minutes. .
7. Add 1 drop (50ul) 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)