Published online Apr 15, 2002. doi: 10.3748/wjg.v8.i2.276
Revised: October 22, 2001
Accepted: October 29, 2001
Published online: April 15, 2002
AIM: To establish a convenient immunoassay method based on recombinant antigen preS1(21-119 aa) to detect anti-preS1 antibodies and evaluate the clinical significance of antibodies in hepatitis B.
METHODS: The expression plasmid pET-28a-preS1 was constructed, and a large quantity of preS1(21-119 aa) fragment of the large HBsAg protein was obtained. The preS1 fragment purified by Ni2+-IDA affinity chromatography was used as coated antigen to establish the indirect ELISA based on streptavidin-biotin system for detection of the anti-preS1 antibodies in sera from HBV-infected patients. For follow-up study, serial sera were collected during the clinical course of 21 HBV-infected patients and anti-preS1 antibodies, preS1 antigen, HBV-DNA and other serological HBV markers were analyzed.
RESULTS: preS1(21-119 aa) fragment was highly expressed from the plasmid pET-28a-preS1 in a soluble form in E. coli (30 mg•L⁻¹), and easily purified to high purity over 90% by one step of Ni2+-IDA-sepharose 6B affinity chromatography. The purity and antigenicity of the purified preS1(21-119 aa) protein was determined by 150 g•L⁻¹ SDS-PAGE, Western blot and a direct ELISA. Recombinant preS1(21-119 aa) protein was successfully applied in the immunoassay which could sensitively detect the anti-preS1 antibodies in serum specimens of acute or chronic hepatitis B patients. Results showed that more than half of 19 acute hepatitis B patients produced anti-preS1 antibodies during recovery of the disease, however, the response was only found in a few of chronic patients. In the clinical follow-up study of 11 patients with anti-preS1 positive serological profile, HBsAg and HBV-DNA clearance occurred in 6 of 10 acute hepatitis B patients in 5-6 mo, and seroconversion of HBeAg and disappearance of HBV-DNA occurred in 1 chronic patients treated with lavumidine, a antiviral agent.
CONCLUSION: The high-purity preS1(21-119 aa) coated antigen was successfully prepared by gene expression and affinity chromatography. Using this antigen, a conveniently detective system of anti-preS1 antibodies in sera was established. Preliminarily clinical trial the occurrence of anti-preS1 antibodies in acute hepatitis B patients suggests the clearance of HBV from serum in a short-term time, and anti-preS1 positive in chronic patients means health improvement or recovery from the disease.