An immuno-precipitation assay for determining specific interactions between antibodies and phage selected from random peptide expression libraries.
Libraries of random peptides displayed by bacteriophage can be screened to select phage expressing peptides that specifically bind antibodies, so that the peptide sequence motifs expressed by the phage can help to define the epitopes of the antibodies. It is often desirable to screen antibody-selected phage for binding of the selecting antibody in an immunoassay in order to verify the specificity of the interaction. Enzyme-linked immunosorbent assays (ELISAs) are commonly used for this purpose. However, for many antibodies, the best techniques for measuring specific, high affinity interactions are immuno-precipitation assays. Immuno-precipitation was therefore investigated as a means of measuring interactions between antibodies and phage clones selected from random peptide display libraries. Three mouse monoclonal antibodies specific for glutamic acid decarboxylase were used to select peptides as 9-mers on T7 phage, linear 12-mers on pIII of M13 phage, or constrained 15-mers on pVIII of M13 phage. Following the cloning and sequencing of selected phage, mixtures of antibody and phage were incubated in solution and the immune complexes were precipitated with Protein G bound to Sepharose beads. In order to detect and quantitate the phage that had formed immune complexes and been precipitated, advantage was taken of the biological properties of the phage by inducing infection of Escherichia coli by the precipitated phage. The aim was to quantitate the phage precipitated by determining the number of plaques produced, which would therefore be proportional to the degree of interaction between the phage and the antibody in solution. The results presented here indicate that this method of measuring monoclonal antibody interactions with phage selected for expression of peptides recognised by the monoclonal antibody is highly specific and sensitive.
Division of Molecular and Clinical Immunology, School of Clinical Laboratory Sciences, University of Nottingham, A Floor West Block, Queen's Medical Centre, Nottingham, UK.
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Al-bukhari
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Tighe
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Todd
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D000918:Antibody Specificity; D017104:Bacteriophage M13 / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D017123:Bacteriophage T7 / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D001666:Binding Sites, Antibody; D002999:Clone Cells; D004797:Enzyme-Linked Immunosorbent Assay; D005968:Glutamate Decarboxylase / Q000276:immunology / Q000378:metabolism; D007527:Isoenzymes / Q000276:immunology / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D019151:Peptide Library; D011233:Precipitin Tests / Q000379:methods*; D020539:Sequence Analysis, Protein; D010948:Viral Plaque Assay
D000911:Antibodies, Monoclonal; D007527:Isoenzymes; D010446:Peptide Fragments; D019151:Peptide Library; D005968:Glutamate Decarboxylase; C401141:glutamate decarboxylase 2