Molecular interaction of growth hormone with two monoclonal antibodies recognizing distinct epitopes.
Two monoclonal antibodies (mAb), designated PS-7.6 and PS-11.2, were generated against recombinant porcine growth hormone (pGH) and shown to enhance the hormonal activity in promoting the growth of animals. The mAb were compared and their functional relationship investigated. It was demonstrated by radioimmunoassay that PS-11.2 did not compete, but rather enhanced binding of 125I-pGH tracer to PS-7.6, suggesting that both mAb recognized distinct epitopes and also were additive in their antigen bindings. Surface plasmon resonance analysis using optical BIAcore technology (Pharmacia Biosensor, Piscataway NJ, USA) provided additional data to support this idea because pGH, after being captured by PS-11.2, remained capable of interacting with PS-7.6. An anti-idiotypic mAb was employed and shown to interact with PS-7.6 but not PS-11.2, implying that the differences in the Fab variable regions of these two mAb might contribute to their epitope specificity. Binding kinetics were determined by the BIAcore and the individual affinities of PS-7.6 and PS-11.2 to pGH were 6.8 x 10(-8) and 1.2 x 10(-9) mol/L, respectively. When these mAb were sequentially subjected to the BIAcore, however, their affinities decreased by approximately 100-fold. Therefore, binding of pGH with one mAb significantly impaired a subsequent interaction with another mAb despite the fact that both mAb targeted different epitopes. Hypophysectomized rats were used for functional analysis and pGH was active in promoting growth of these GH-deficient animals. The hormonal effect was further improved by incubating pGH with either PS-7.6 or PS-11.2 prior to administration. However, enhancement by individual mAb was completely abolished when pGH was treated with both mAb together, indicating their unpredictable biological interference with each other. Therefore, the present findings clearly demonstrate that although PS-7.6 and PS-11.2 recognize separate epitopes, their individual interactions with pGH are closely interrelated both immunologically and biologically.
Wang
B S
BS
Laboratory of Immunoendocrinology, Fort Dodge Animal Health, Cyanamid Agricultural Research Center, Princeton, New Jersey 08543-0400, USA.
Lumanglas
A L
AL
Zhang
R J
RJ
Tian
Z G
ZG
D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000378:metabolism; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions / Q000502:physiology; D015374:Biosensing Techniques; D000939:Epitopes / Q000032:analysis / Q000276:immunology*; D005260:Female; D013006:Growth Hormone / Q000276:immunology*; D007700:Kinetics; D011863:Radioimmunoassay; D051381:Rats; D017207:Rats, Sprague-Dawley; D013552:Swine
D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D000939:Epitopes; D013006:Growth Hormone