Systematic fractionation of serum antibodies using multiple antigen homologous peptides as affinity ligands

Tribbick, Gordon; Triantafyllou, B; Lauricella, R.; Rodda, Stuart J.; Mason, Tom J.; Geysen, H. Mario

doi:10.1016/0022-1759(91)90185-i

Cited by 19 publications

(13 citation statements)

References 21 publications

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“…This technique has been modified from that described by Tribbick et al (1991). Peptides synthesized on rods were incubated in a 1 : 50 dilution of serum in PBS with 0.05 % Tween 20 in a wet box on a rocking tray at 4 °C overnight.…”

Section: Methodsmentioning

confidence: 99%

Antibodies are produced to the variable regions of the external envelope glycoprotein of human immunodeficiency virus type 1 in chimpanzees infected with the virus and baboons immunized with a candidate recombinant vaccine

Stephens

Eichberg

Haigwood³

et al. 1992

Journal of General Virology

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Section: Methodsmentioning

confidence: 99%

Antibodies are produced to the variable regions of the external envelope glycoprotein of human immunodeficiency virus type 1 in chimpanzees infected with the virus and baboons immunized with a candidate recombinant vaccine

Stephens

Eichberg

Haigwood³

et al. 1992

Journal of General Virology

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“…In its standard form, this technique involves synthesis of a number of peptides on acrylic-coated polyethylene pins and testing of these peptides for binding of anti-viral antibodies. While this method is extremely well suited for identificationof sequencedependent linear epitopes that are targets of diagnostic reagents, it has been argued that the antibodies binding to short peptides may not necessarily bind to whole antigen molecules (reviewed by Tribbick et al 1991) and thus may not in all cases represent epitopes present on intact antigen. A recently published modification (Tribbick et al 1991) of the standard technique overcomes this problem.…”

Section: Introductionmentioning

confidence: 99%

“…la). It has been suggested, in connection with a similar case (Tribbick et al 1991), that even low amounts of antibody, escaping detection when bound to peptide, may have after elution sufficient affinity to show significant binding to viral antigens. In the case of peptides 23-28, 36 and 55-56, but not in the case of peptides 37-38, 42-43 and 200-202, the amino acid substitutions (at positions 24, 26, 29, 31, 36 and 58; Robaglia et al 1989, van der Vlugt et al 1989) between peptides and the virus may have contributed to the weakness of binding of an anti-PVY antibody to a peptide.…”

mentioning

confidence: 99%

Mapping antigenic epitopes of potato virus Y with antibodies affinity-purified by using overlapping synthetic peptides

Vihinen‐Ranta

Sironen

Vuento

1994

AFSci

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Synthetic, overlapping peptides representing the entire amino acid sequence of potato virus Y (PVY) coat protein were used to affinity-purify antibodies from polyclonal antisera to PVY. In testing the binding of the purified antibodies to PVY particles, antigenic epitopes were identified. The N-terminal and C-terminal regions of the PVY coat protein were found to contain most of the antigenic epitopes. The results will facilitate the development of detection methods for PVY based on synthetic peptides.

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“…However, since all discontinuous epitopes contain continuous segments, the study of short peptides can give important information about more complex determinants (6). Indeed, recent studies using peptide-based assays have identified the antigenically important portions of such discontinuous sites (7)(8)(9)(10)(11)(12)(13), even to the resolution of single critical amino acids within these sites (14,15). Studies have also shown that antibodies (Abs) to pathogens, including human immunodeficiency virus (16) and foot-and-mouth-disease virus (17), can recognize or be induced by linear or sequence continuous epitopes.…”

mentioning

confidence: 99%

Altering the antigenicity of proteins.

Alexander¹,

Alexander²,

Getzoff³

et al. 1992

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

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To better understand the binding interaction between antigen and antibody we need to distinguish protein residues critical to the binding energy and mechanism from residues merely localized in the interface. By analyzing the binding ofmonoclonal antibodies to recombinant wild-type and mutant myohemerythrin (MHr) proteins, we were able to test the role ofindividual critical residues at the highly antigenic site , within the context of the folded protein. The results directly show the existence of antigenically critical residues, whose mutations significantly reduce antibody binding to the folded protein, thus verifying peptide-based assignments of these critical residues and demonstrating the ability of buried side chains to influence antigenicity. Taken together, these results (i) distinguish the antigenic surface from the solvent-exposed protein surface before binding, (ii) support a two-stage interaction mechanism allowing inducible changes in protein antigens by antibody binding, and (iii) show that protein antigenicity can be significantly reduced by alteration of single critical residues without destroying biological activity.Antigenic epitopes ofproteins are composed of continuous or discontinuous portions of the polypeptide chain. The discontinuous epitopes are formed by two or more noncontiguous segments brought into proximity by the folding ofthe protein.Although much attention has been paid to epitope mapping and characterization of epitopes, there is still no agreement on the precise definition and the make-up of an epitope or on the best ways to identify antigenic epitopes (1-3). It has been suggested that experimental approaches that rely on the linear sequence of the protein to locate and define antigenic determinants would miss many biologically significant sites (4, 5). However, since all discontinuous epitopes contain continuous segments, the study of short peptides can give important information about more complex determinants (6). Indeed, recent studies using peptide-based assays have identified the antigenically important portions of such discontinuous sites (7-13), even to the resolution of single critical amino acids within these sites (14,15). Studies have also shown that antibodies (Abs) to pathogens, including human immunodeficiency virus (16) and foot-and-mouth-disease virus (17), can recognize or be induced by linear or sequence continuous epitopes.In our studies of the myohemerythrin (MHr) protein, we have used synthetic peptides to identify a set of amino acid residues that are critical to the immune response, in that their omission or replacement in component peptides results in loss of reactivity with polyclonal and monoclonal anti-MHr Abs (15,18,19). However, there has been no direct experimental evidence, for MHr or any other protein antigen (Ag), to verify whether amino acid residues that are identified as antigenically critical by the peptide-based approach are indeed critical within the context of the folded protein. Here, we test directly the correlation between criti...

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Systematic fractionation of serum antibodies using multiple antigen homologous peptides as affinity ligands

Cited by 19 publications

References 21 publications

Antibodies are produced to the variable regions of the external envelope glycoprotein of human immunodeficiency virus type 1 in chimpanzees infected with the virus and baboons immunized with a candidate recombinant vaccine

Antibodies are produced to the variable regions of the external envelope glycoprotein of human immunodeficiency virus type 1 in chimpanzees infected with the virus and baboons immunized with a candidate recombinant vaccine

Mapping antigenic epitopes of potato virus Y with antibodies affinity-purified by using overlapping synthetic peptides

Altering the antigenicity of proteins.

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