Biotecnologia Aplicada Elfos Scientiae ISSN: 0684-4551 Vol. 12, Num. 2, 1995, pp. 89-90

REPORTE CORTO/SHORT REPORT

Presented in the Congress Biotecnologia Habana 94. La Habana, Cuba, Nov. 28 - Dec. 3, 1994

MURINE MONOCLONAL ANTIBODIES SPECIFIC FOR THE HBsAg a DETERMINANT

Maria E. Fernandez-de-Cossio1, Tamara Diaz1, Minerva Sewer2, Olga Jorge2, G. Garcia1, Osvaldo Reyes1, Hilda Garay1, Karelia Cosme1, Isel Guerra1, Orlando Herrera1, Rodolfo Valdes1, Ricardo Pierrat2, R. Morales2, Isary Almagro1, Lilia Perez1, Alberto Agraz1, Jorge V. Gavilondo1.

1Center for Genetic Engineering & Biotechnology., P.O. Box 6162, Havana City, Cuba. 2National Center for Biorreagents, P.O. Box 6048, Bejucal, Havana, Cuba.

Code Number: BA95030
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INTRODUCTION

The glycoprotein known as the Hepatitis B surface antigen (HBsAg) is the major surface antigen of the 42 nm particles of the Hepatitis B virus (1). Serologically, HBsAg has one group- specific antigenic determinant a and two sets of mutually exclusive determinants, d or y, and w or r resulting in four major serotypes: adw, adr, ayw and ayr (2). Antibodies to the common a determinant of HBsAg confer protection against infection with Hepatitis B virus (HBV) (3). Perturbation of tertiary structure by chemical reduction, detergent treatment and some single point mutations of the a determinant drastically diminishes antigenicity (4). We have generated different monoclonal antibodies (Mabs) that bind to epitopes within the common a determinant of HBV. Two of them were used as immunoaffinity chromatography ligands allowing to obtain a highly pure recombinant HBsAg useful as a vaccine antigen.

EXPERIMENTAL PROCEDURES

Mouse monoclonal antibodies (Mabs) were obtained by the hybridization technique from mice immunized with a yeast recombinant HBsAg. They were early selected for their ability to bind to native HBsAg particles using an indirect ELISA. A competition assay was performed by incubating HBsAg-coated microtiter plates with biotinylated antibodies in the presence of unconjugated competitor Mab. A series of overlapping peptides covering the amino acid sequence 118-153 (118-TGPCKTCTTPA-128; 126-TPAQGNSMFPSCCCTK-141; 134-FPSCCCTKPTDGNCTCIPIP-153; 124- CTTPAQGNSMFPSC-137; 139-CTKPTDGNC-147) on HBsAg subtype adw were syntetized using the tea bag method. The antibodies were incubated with biotinylated antigen or synthetic peptides in solution. The performed antigen-antibody complexes were captured by polyclonal antibodies specific to mouse immunoglobulins, coated to microtiter plates. Biotinylated antigen of the antigen antibody complex was then detected by incubation with a streptavidin-HRP conjugate and a substrate chromogen. For immunoaffinity chromatography, a homogeneous preparation of Mabs was immobilized on cyanogen bromide activated Sepharose CL-4B (5). The purity of the recombinant HBsAg fraction recovered from the immunoadsorbents was estimated by SDS-PAGE.

RESULTS AND DISCUSSION

Twelve monoclonal antibodies (CBHep1 to CBHHep12) with specificity against the group specific a determinant of HBsAg were selected. All Mabs recognized both the recombinant and the native antigen on an ELISA system. Competition between antibodies was determined from the effect of increasing concentrations of each unlabelled antibody on the binding of labelled antibody. From these results we could identify three epitope groups. Mabs CBHep1 and CBHep3 did not compete inter each other, neither with other antibodies. The rest of the antibodies competed for antigen binding, and this group was further characterized using the representative CBHep4 Mab. The peptide sequence 134-153, containing the nonapeptide 139-147, was recognized by all (figure 1). Most of the studies with synthetic peptides have been focused on the region encompassing residues 110-150 of the major protein of HBsAg, against which protective immunity is directed. Recent studies indicated that the cyclic 139-147 peptide may represent a more immunodominant a epitope vis-a-vis the cyclic 127-137 peptide, in humans vaccinated with either the plasma or recombinant derived Hepatitis B vaccine (6). Our Mabs could be useFig. 1.- Peptide specific binding

Fig. 1.- Peptide specific binding

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2. LEVENE, C.; B. S. BLUMBERG (1969). Nature 221: 195-196.

3. JILG, W.; B. LORBEER; H. SCHMIDT; B. WILSKE; G. ZOULEK and F. DEINHARDT (1984). Lancet I: 1174-1175.

4. WATERS, J.A.; M. KENNEDY; P. VOET; P. HAUSER; J. PETRE; W. CAMMAN and H. C THOMAS (1992). J. Clin. Invest. 90: 2543-2547.

5. KOHN, J.; M. WILCHEK (1982). Biochem. Biophys. Res. Commun 107: 878.

6. HOWARD, C.; H. STIRK; A. BUCKLEY; S. BROWN and M. STEWARD (1989). In: Synthetic Peptides: Approaches to Biological Problems. J. P. Tam and E.T. Kaiser, eds. Alan R. Liss, New York, 211.

Copyright 1995 Sociedad Iberolatinoamericana de Biotecnologia Aplicada a la Salud

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