A comparison of two dissimilar monoclonal antibodies that are directed to a common epitope in the reduced and carboxymethylated chicken riboflavin carrier protein.
Two monoclonal antibodies, D2A1 and D5G3 were elicited by immunization with a preparation of chicken egg riboflavin carrier protein which had been reduced and carboxymethylated. Epitopes recognised by the monoclonal antibodies were mapped using the Pepscan method. Epitopic determinants for D2A1 as well as D5G3 were identified within a region spanning 13 amino acids (residues 170-182) in the primary sequence of riboflavin carrier protein. Interestingly, these monoclonal antibodies, despite sharing a common epitope exhibited a marked difference in their binding to native (folded) riboflavin carrier protein versus reduced carboxymethylated (unfolded) riboflavin carrier protein. Both monoclonal antibodies bound reduced carboxymethylated riboflavin carrier protein to comparable extents in solid phase (ELISA and immunoblots) and liquid phase (radioimmunoassay) assays. However, while D5G3 could bind native riboflavin carrier protein in solid and liquid phase assays, D2A1 showed negligible binding to the native structure. Alanine substituted peptide analogs of the epitope in question defined the crucial amino acids of the epitope needed for binding to the two antibodies.
Lobo
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Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore.
Karande
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Adiga
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D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D002264:Carboxylic Acids / Q000737:chemistry; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D002645:Chickens; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry / Q000235:genetics; D026901:Membrane Transport Proteins; D008745:Methylation; D051379:Mice; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D012256:Riboflavin / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D002264:Carboxylic Acids; D002352:Carrier Proteins; D000939:Epitopes; D026901:Membrane Transport Proteins; C019079:riboflavin-binding protein; D012256:Riboflavin