Second generation assays for the detection of antibody to HBsAg using recombinant DNA-derived HBsAg

Mimms, Larry; Goetze, Andrew M.; Swanson, Suzanne M.; Floreani, Marco; Edwards, Brook; Macioszek, Jerzy; Okasinski, Greg; Kiang, Willie

doi:10.1016/0166-0934(89)90034-7

Cited by 19 publications

(9 citation statements)

References 16 publications

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“…Rabbit anti-alkaline phosphatase and sheep anti-M13 were purchased from 5 Prime-3 Prime, Inc. Donkey anti-sheep horseradish peroxidase and goat anti-rabbit horseradish peroxidase conjugates were obtained from Jackson ImmunoResearch Labs, Inc. Recombinant HBsAg (rHBsAg) was prepared as described in ref. 11 (12). We conclude that the epitope is a loop formed by residues 121-124 held together by a disulfide bond, consistent with previous epitope mapping studies (7,12,13).…”

Section: Methodsmentioning

confidence: 99%

“…The immunogen was human plasma-derived HBsAg particles, subtype adw2, ayw3, or ayw2 (11). Rabbit anti-alkaline phosphatase and sheep anti-M13 were purchased from 5 Prime-3 Prime, Inc. Donkey anti-sheep horseradish peroxidase and goat anti-rabbit horseradish peroxidase conjugates were obtained from Jackson ImmunoResearch Labs, Inc. Recombinant HBsAg (rHBsAg) was prepared as described in ref.…”

Section: Methodsmentioning

confidence: 99%

“…The recognition sequences of these antibodies were unknown since they could not be mapped by using synthetic peptides. The fourth antibody, H166, was known to interact with a continuous epitope (11). We report here the sequences of epitopes recognized by these antibodies and propose a partial map of the surface of HBsAg and a topological model for its structure.…”

mentioning

confidence: 99%

“…The four mAbs were chosen because they represent four distinct, nonoverlapping epitopes within the "a" group-specific determinant located on the S gene product, and all four react with high affinity to all known HBV subtypes. Three of four mAbs, namely H5, H35, and H53 (10,11), were thought to bind to unknown discontinuous epitopes since they did not cross-react with denatured HBsAg samples in Western blotting experiments. The recognition sequences of these antibodies were unknown since they could not be mapped by using synthetic peptides.…”

mentioning

confidence: 99%

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Discontinuous epitopes of hepatitis B surface antigen derived from a filamentous phage peptide library.

Chen

Delbrook

Dealwis

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

130

105

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The structure of the small hepatitis B virus surface antigen (HBsAg) was investigated by epitope mapping of four anti-HBsAg monoclonal antibodies (mAbs). Amino acid sequences of epitopes were derived from affinity-enrichment experiments (biopanning) using a filamentous phage peptide library. The library consists of 109 different clones bearing a 30-residue peptide fused to gene IlI. Sequence homologies between peptides obtained from panning the library against the antibodies and the native HBsAg sequence allowed for precise description of the binding regions. Three of four mAbs were found to bind to distinct discontinuous epitopes between amino acid residues 101 and 207 of HBsAg. The fourth mAb was demonstrated to bind to residues 121-124. The sequence data are supported by ELISA assays demonstrating the binding of the HBsAg-specific peptides on filamentous phage to mAbs. The sequence data were used to map the surface of HBsAg and to derive a topological model for the a-carbon trace of the 101-207 region of HBsAg. The approach should be useful for other proteins for which the crystal structure is not available but a representative set of mAbs can be obtained.Elucidation of epitope structure is an integral part of studying antigen-antibody interactions. Peptide libraries on filamentous phage have recently been used for rapid epitope mapping of antibodies. The procedure entails affinity screening the library against the antibody, isolating the phage that bind, and determining the peptide sequence of the binding phage through DNA sequencing of the relevant portion of the phage genome. By using this procedure, a large number of peptides can be screened for binding to an antibody in a relatively short time (1, 2).A filamentous phage in a typical library carries on its surface 3 to 5 random peptides, 6-38 residues long, fused to the gene III protein. The peptide library used in this paper contains 109 different phages, each displaying a 30-residue peptide fused to the gene III protein (3). The library was originally applied to select peptides that bind to monoclonal antibodies (mAbs) raised against human immunodeficiency virus gpl2O or hepatitis C virus core proteins. In this study, we applied the phage library approach to mapping the surface antigen of the hepatitis B virus (HBV). The antigen is the diagnostic marker for the HBV. The virus causes major endemic illness throughout the world and is associated with a greatly increased frequency of primary hepatoma, a major cancer in the world (4).The envelope of the hepatitis B virion consists of three proteins, termed small, middle, and large HBV surface proteins, and their glycosylated derivatives. The small HBV surface antigen (HBsAg) is the major component of the envelope of the virion and is mainly present in the plasma of HBV-infected individuals as 22-nm spherical particles com-

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Discontinuous epitopes of hepatitis B surface antigen derived from a filamentous phage peptide library.

Chen

Delbrook

Dealwis

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

130

105

View full text Add to dashboard Cite

show abstract

“…The bridging ELISA takes advantage of the two antigen binding sites on each Ab molecule (Ab bivalency) that allows Abs to form a bridge between (i.e., cross-link) drug immobilized on plastic wells with enzyme-labeled drug that is added in the detection step (61). In this assay, the unlabeled drug is first immobilized onto the plastic wells, and optimally diluted Abs in a test serum sample are allowed to bind, followed by the usual washing step to remove unbound Abs.…”

Section: Elisamentioning

confidence: 99%

Current Methods for Detecting Antibodies against Erythropoietin and Other Recombinant Proteins

Thorpe

Swanson

2005

Clin Vaccine Immunol

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Hepatitis B virus with antigenically altered hepatitis B surface antigen is selected by high-dose hepatitis B immune globulin after liver transplantation

et al. 1998

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Second generation assays for the detection of antibody to HBsAg using recombinant DNA-derived HBsAg

Cited by 19 publications

References 16 publications

Discontinuous epitopes of hepatitis B surface antigen derived from a filamentous phage peptide library.

Discontinuous epitopes of hepatitis B surface antigen derived from a filamentous phage peptide library.

Current Methods for Detecting Antibodies against Erythropoietin and Other Recombinant Proteins

Hepatitis B virus with antigenically altered hepatitis B surface antigen is selected by high-dose hepatitis B immune globulin after liver transplantation

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