Affinity of anti-peptide antibodies measured by resonance energy transfer.
Because of the increasing use of monoclonal anti-peptide antibodies we have undertaken to formulate a general method for the measurement of intrinsic association constants characterizing complex formation between peptide and antibody. The method is based on the phenomenon of resonance energy transfer between tryptophan-excited antibody and an appropriate fluorophor conjugated to the amino terminus of the peptide. The fluorophor we have employed is 8-(2-N-succinylaminoethylamino)-1-naphthalene-sulfonic acid with an absorption maximum at 344 nm and an emission maximum at 500 nm. The model peptide used was the sequence corresponding to residues 48-60 of the regulatory subunit of aspartyltranscarbamoylase. Three IgG and two IgM affinity-purified monoclonal anti-peptide antibodies were used in the fluorescence titration experiments. A maximum value of 2.1 X 10(6) M-1 was found for the IgG antibodies and a maximum of 2.7 X 10(4) M-1 for the IgM antibodies. These limited results suggest similar behavior for the anti-peptide B cell response with respect to affinity maturation as observed for other specificities. In particular it is likely that the IgM affinities are restricted to the potential available in the germline repertoire of variable region genes and, therefore, express only germline affinities.
Karush
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Department of Microbiology, School of Medicine, University of Pennsylvania, Philadelphia 19104.
Tang
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D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis / Q000096:biosynthesis; D000915:Antibody Affinity; D001221:Aspartate Carbamoyltransferase; D004735:Energy Transfer; D005456:Fluorescent Dyes; D006825:Hybridomas / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D009097:Multienzyme Complexes; D009842:Oligopeptides / Q000138:chemical synthesis / Q000276:immunology*; D011869:Radioligand Assay / Q000379:methods; D013050:Spectrometry, Fluorescence / Q000379:methods*
D000911:Antibodies, Monoclonal; D005456:Fluorescent Dyes; D009097:Multienzyme Complexes; D009842:Oligopeptides; D001221:Aspartate Carbamoyltransferase