Epitope mapping and analysis of a growth-enhancing monoclonal antibody by limited tryptic digestion of porcine GH.
In this study, the epitope of a murine PS-7.6 monoclonal antibody (mAb) which was raised against the recombinant porcine GH (pGH) and subsequently shown to enhance the growth-promoting activity of pGH in a hypophysectomized rat model, was mapped by the limited tryptic digestion of pGH. A pGH fragment corresponding to amino acid residues 70-95 was separated by reverse-phase HPLC and also immunoprecipitated by PS-7.6 mAb. This fragment was found in an RIA to compete with radiolabelled pGH for the binding of PS-7.6 mAb in a dose-dependent fashion. Several peptides covering this potential epitope region of pGH(70-95) were synthesized and assayed by competitive RIA. The results suggested that pGH(75-90) was the optimal sequence recognized by PS-7.6 mAb. Sequential alanine substitution of each residue of pGH(75-90) revealed that the side chains of Leu76, Ile83 and Leu87 were critical for binding to PS-7.6 mAb. Other residues could be replaced by alanine without substantially altering the binding affinity. The region of amino acids 75-95 comprises the C-terminal end of the second helix of pGH and the repeating pattern of i and i + 3 (i + 7) of the critical amino acids appears consistent with PS-7.6 mAb binding to the hydrophobic side of the helix. The sequence and the helical structure of the epitope of PS-7.6 mAb provide the basis for designing the effective peptide vaccines to enhance the growth performance of animals.
Shieh
H M
HM
Agricultural Research Division, American Cyanamid Company, Princeton, New Jersey 08543-0400, USA.
Bass
R T
RT
Wang
B S
BS
Corbett
M J
MJ
Buckwalter
B L
BL
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology*; D001667:Binding, Competitive; D018604:Epitope Mapping; D006128:Growth / Q000276:immunology*; D013006:Growth Hormone / Q000276:immunology*; D006728:Hormones / Q000276:immunology*; D010446:Peptide Fragments / Q000276:immunology*; D013552:Swine
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D006728:Hormones; D010446:Peptide Fragments; C094376:somatotropin (54-95); D013006:Growth Hormone