title: Epitope mapping of a monoclonal antibody specific to bovine dry milk: involvement of residues 66-76 of strand D in thermal denatured beta-lactoglobulin.
abstract: beta-Lactoglobulin (beta-LG) is a bovine milk protein sensitive to thermal denaturation. Previously, we demonstrated that such structural change can be detected by a monoclonal antibody (mAb) specific to denatured beta-LG. In the present study, we show a dramatic increase in beta-LG immunoreactivity when heating raw milk between 70 and 80 degrees C. To map out the specific epitope of beta-LG recognized by this mAb, we used a combined strategy including tryptic and CNBr fragments, chemical modifications (acetylation and carboxymethylation), peptide array containing in situ synthesized peptides, and a synthetic soluble peptide for immunoassays. The antigenic determinant we defined was exactly located within the D strand (residues 66-76) of beta-LG. Circular dichroic spectral analysis shows that carboxymethylation on beta-LG not only resulted in a substantial loss of beta-configuration but also exerted a 10 times increase in immunoreactivity as compared with heated beta-LG. The result suggests that a further disordered structure occurred in beta-LG and thus rendered the mAb recognition. Mutations on each charged residue (three Lys and one Glu) revealed that Lys-69 and Glu-74 were extremely essential in maintaining the antigenic structure. We also show an inverse relationship between the immunoreactivity in heated beta-LG and its binding to retinol or palmitic acid. Most interestingly, pH 9-10, which neutralizes the Lys groups of beta-LG, not only reduced its immunoreactivity but also its binding to palmitic acid implicating a role of Lys-69. Taken together, we concluded that strand D of beta-LG participated in the thermal denaturation between 70 and 80 degrees C and the binding to retinol and palmitic acid. The antigenic and biochemical roles of mAb specific to D strand are discussed in detail.
authors.affiliation: Research Institute of Biochemical Engineering, Department of Biological Science and Technology, National Chiao Tung University, Hsinchu and Genesis Biotech Inc., 131, Lane 235, Bao-Chiao Road, Hsintien, Taipei, Taiwan, Republic of China.
authors.forename: Chun Ying
authors.initials: CY
authors.lastname: Song
authors.forename: Wen Liang
authors.initials: WL
authors.lastname: Chen
authors.forename: Ming Chi
authors.initials: MC
authors.lastname: Yang
authors.forename: Jen Pin
authors.initials: JP
authors.lastname: Huang
authors.forename: Simon J T
authors.initials: SJ
authors.lastname: Mao
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D002417:Cattle; D002942:Circular Dichroism; D003890:Desiccation; D018604:Epitope Mapping; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007782:Lactoglobulins / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D008892:Milk / Q000276:immunology*; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000276:immunology; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
chemical_list: D000911:Antibodies, Monoclonal; D007782:Lactoglobulins