HEPATITIS B SURFACE ANTIGEN (HBsAg)
HBsAg Rapid test is a lateral flow chromatographic immunoassay for the qualitative detection of Hepatitis B surface antigen (HBsAg) in human serum or plasma at the level equal or higher than 2mg/ml. It is intended to be used as a screening test and as an aid in the diagnosis of infection with Hepatitis B virus (HBV).
Hepatitis B virus is the most common cause of persistent viremia and the most important cause of chronic liver disease. Clinically apparent HBV infection may have been extant for several millennia. It is estimated that there are 300 million chronic carriers of HBV in the world. The carrier rates vary from as little as 0.3% (western countries) to 20% (Asia, Africa).
HBV is a hepatotropic DNA virus. The core of the virus contains a DNA polymerase, the core antigen (HBcAg) and the e antigen (HBeAg). The core of HBV is enclosed in a coat that contains lipid, protein and carbohydrate and expresses an antigen terms hepatitis B surface antigen.
HBsAg is the first marker to appear in the blood in acute hepatitis B, being detected 1week to 2 months after exposure and 2 weeks to 2 months before the onset of acute hepatitis almost half of the patients will still be positive for HBsAg.
In the chronic carrier state, the HBsAg persist for long periods (6-12 months) with no seroconversion to the corresponding antibodies. Therefore, screening for HBsAg is highly desirable for all donors, pregnant women and people in high-risk groups.
Hepatitis B Test Principle
The HBsAg as described above is a lateral chromatographic immunoassay. The test strip consists of:
1) A burgundy colored conjugate pad containing anti-HBsAg antibody conjugated with colloid gold (HBsAg conjugates)
2) A nitrocellulose membrane strip containing a test band (T band) and a control band (C band). The T band is pre-coated with non-conjugated anti-HBsAg antibody, and the C band is pre-coated with goat anti-mouse lgG antibody.
When an adequate volume of test specimen is dispensed into the sample pad of the strip, the specimen migrates by capillary action across the strip. HBsAg if present in the specimen will bind to the HBsAg conjugates. The immunocomplex is then captured on the membrane by the pre-coated anti-HBsAg antibody, forming a burgundy colored T band, indicating an HBsAg positive test result.
ZAbsence of the Test band suggests a negative result. The test contains an internal control (Control band) which should exhibit a burgundy colored band of immunocomplex of goat anti mouse lg-gold conjugate regardless of the presence of coloured Test band. Otherwise, the test result is invalid and the specimen must be retested with another device.
Specimen Collection and Handling in Hepatitis B
Consider any materials of human origin as infectious and handle them using standard bio safety procedures.
- Collect blood sample into a lavender, blue or green top collection tube by vein puncture.
- Separate the plasma by centrifugation.
- Carefully withdraw the plasma into a new pre- labelled tube.
- Collect blood specimen into a red top collection tube (containing no anticoagulant in vecutainer) by vein puncture.
- Allow the blood to clot.
- Separate the serum by centrifugation.
- Carefully withdraw the serum into a new pre-labelled tube.
Test specimens as soon as possible after collecting. Store specimens at 2oC to 8oC if not tested immediately.
Store specimens at 2oC to 8oC up to 5 days. The specimens should be frozen at 20oC for longer storage.
- Bring the specimen and test components to room temperature if centrifuged or frozen. Mix the specimen well prior to assay once thawed.
- Collect at least 150-200nl or 3-4 drops of serum or plasma in a sample container.
- When ready to test, open the pouch at the notch and remove the test strip.
- Dip the strip into the specimen for at least 10 seconds; don’t allow the specimen reach above the level indicated by the arrows on the strip.
- Remove the strip from the specimen, and place it on a flat, dry surface.
- Read the test result in 15 minutes, positive result could be visible as short as 1 minute.
Interpretation of results
- Interpretation of Negative Result
If only the C band is developed, the test indicates that the level of HBsAg in the specimen is undetectable (lower than 2ng/ml)
- Interpretation of Positive Result
If both C and T bands are developed, the test indicates that the specimen contains HBsAg at the level equal or higher than 2ng/ml
- Interpretation of Invalid Result