Detection of anti-preS1 antibodies for recovery of hepatitis B patients by immunoassay.
OBJECTIVE
To establish a convenient immunoassay method based on recombinant antigen preS1(21-119aa) 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(21-119aa) fragment of the large HBsAg protein was obtained. The preS1 fragment purified by Ni(2+)-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(21-119aa) fragment was highly expressed from the plasmid pET-28a-preS1 in a soluble form in E.Coli (30mg.L(-1)), and easily purified to high purity over 90% by one step of Ni(2+)-IDA-sepharose 6B affinity chromatography. The purity and antigenicity of the purified preS1(21-119aa) protein was determined by 150g.L(-1) SDS-PAGE, Western blot and a direct ELISA. Recombinant preS1(21-119aa) 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 months, and seroconversion of HBeAg and disappearance of HBV-DNA occurred in 1 chronic patients treated with lavumidine, a antiviral agent.


CONCLUSIONS
The high-purity preS1(21-119aa) 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.
Wei
Jun
J
Institute of Biochemsitry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
Wang
Yu-Qin
YQ
Lu
Zhi-Meng
ZM
Li
Guang-Di
GD
Wang
Yuan
Y
Zhang
Zu-Chuan
ZC
D000595:Amino Acid Sequence; D000906:Antibodies / Q000097:blood; D001483:Base Sequence; D005822:Genetic Vectors; D006509:Hepatitis B / Q000097:blood / Q000276:immunology; D006514:Hepatitis B Surface Antigens / Q000235:genetics / Q000276:immunology*; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism; D011498:Protein Precursors / Q000235:genetics / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000235:genetics / Q000276:immunology*
D000906:Antibodies; D006514:Hepatitis B Surface Antigens; D010446:Peptide Fragments; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C050806:presurface protein 1, hepatitis B surface antigen