Antibody to Hepatitis B Virus Core Antigen (HBcAb)

ELISA Test Kit

For in vitro diagnostic and professional use only.

PRODUCT NAME

Anti-HBc ELISA Test Kit (Serum/Plasma)

INTENDED USE

This anti-HBc ELISA Test Kit is a qualitative detection of antibodies to hepatitis B virus core antigen (HBcAb) in human serum/plasma. The reagent is suitable for clinical screening and diagnosis of hepatitis B virus infection in human serum/plasma.

TEST PRINCIPLE

Hepatitis B virus (HBV) is an envelope, double-stranded DNA virus belonging to the Hepadnaviridae family and is recognized as the major cause of blood transmitted hepatitis together with hepatitis C virus (HCV). Infection with HBV induces a spectrum of clinical manifestations ranging from mild, unapparent disease to fulminate hepatitis, severe chronic liver diseases, which in some cases can lead to cirrhosis and carcinoma of the liver. Classification of a hepatitis B infection requires the identification of several serological markers expressed during three phases (incubation, acute and convalescent) of the infection.

The main component of the virus is Hepatitis B core antigen (HBcAg). This core antigen is comprised of a single polypeptide of approximately 17kD that is discharged upon disaggregation of the core particles. At least one immunological determinant is present in the antigen.

Shortly after the onset of HBsAg, antibodies to HBcAg (anti-HBc total antibody and IgM) appear and never go away. In isolated cases, a Hepatitis B infection can be contracted without immunologically detectable anti-HBc. This is found usually in immunosuppressed patients. Screening for anti-HBc yields data on the prevalence of hepatitis B in various populations. This is because anti-HBc is a marker of acute, chronic, or resolved HBV infection. The identification of anti-HBc is vital when being diagnosed in a clinical setting. Together with other hepatitis B tests, the anti-HBc marker allows correct diagnosis and proper monitoring of progress of the virus. Anti-HBc may possibly be the only indicator of a hepatitis B infection (including other tests of HBsAg-negative patients).

The system of the HBcAb ELISA test is founded on the solid phase, one-step incubation competitive principle. When anti-HBc is present, it competes with monoclonal anti-HBc conjugated to horseradish peroxidase (HRP-Conjugate) for a fixed amount of purified HBcAg pre-coated in the microplate. If no anti- HBc is present, HRP-conjugated anti-HBc will be bound together with antigens inside the wells. In the course of washing, any unbound HRP-Conjugate is removed. After chromogen solutions A and B are added into the wells and during incubation, a blue-colored product appears. After the reaction is stopped with sulfuric acid, the blue color turns yellow. A presence of antibodies to HBcAg in the sample is indicated by low color, or no color present at all.

SAMPLE REQUIREMENTS

1. Specimen Collection: No special patient’s preparation required. Collect the specimen in accordance with the normal laboratory practice. Either fresh serum/plasma specimens can be used with this assay. Blood collected by venipuncture should be allowed to clot naturally and completely – the serum must be separated from the clot as early as possible as to avoid hemolysis of the RBC. Care should be taken to ensure that the serum/plasma specimens are clear and not contaminated by microorganisms.

2. Highly lipaemic, icteric, or hemolytic specimens should not be used as they can give false results in the assay. Do not heat inactivate specimens. This can cause deterioration of the target analyte. Samples with visible microbial contamination should never be used.

3. HBcAb ELISA is intended ONLY for testing of individual serum/plasma samples. Do not use the assay for testing of cadaver samples, saliva, urine or other body fluids, or pooled (mixed) blood.

4. Transportation and Storage: Store specimens at 2-8°C. Specimens not required for assaying within 3 days should be stored frozen (-20°C or lower). Multiple freeze-thaw cycles should be avoided. For shipment, samples should be packaged and labeled in accordance with the existing local and international regulations for transportation of clinical samples and ethological agents.

TEST PROCEDURE

1. All reagents should be allowed to reach room temperature for 15 minutes before use.

2. Dilute the wash buffer at the rate of 1:40 dilution with distilled water before use.

3. The sample should be corresponding to the number of microplate, each plate should be provided with negative control 3 wells, positive control 2 wells and blank control 1 well. (If detect with dual wavelength detection, setting no blank control well is allowed).

Note: Use a separate disposal pipette tip for each specimen, Negative and Positive Control as to avoid cross contamination.

4. Add 50μL sample in the corresponding well (When this kit is used for clinical auxiliary diagnosis, the sample to be tested should be diluted with normal saline at 1:30 for testing. When it is used for epidemiological investigation, the original sample can be detected ), add 50μL negative control and positive control to negative control wells and positive control wells. Respectively adding Enzyme Conjugate 50µL (Do not add in the blank well).

5. Shake for 30 seconds with an oscillator (This step is very important). Incubate at 37 °C for 30 minutes with the sealing plate membrane sealing the plate.

6. At the end of the incubation, remove and discard the plate cover. Take out, add wash buffer to each well for 20 seconds. Repeat 5 times. After the final washing cycle, turn the plate over onto blotting paper or clean towel, and tap it to remove any remainders.

7. Add Substrate A (50µL) and Substrate B (50µL) (Do not add in the blank well). Mix gently by shaking . Incubate at 37 °C for 15 minutes with the sealing plate membrane sealing the plate.

8. Add 50μL Stop Solution to each well (Do not add in the blank well). Mix gently by shaking, read the absorbance within 10 minutes after stopping the reaction. Calibrate the plate reader with the Blank well and read the absorbance at 450nm.If a dual filter instrument is used, set the reference wavelength at 630nm. Set no blank wells is allowed if use dual wavelength to detect. Calculate the Cut-off value and evaluate the results.