Generation of epitope-specific antibodies to rat GHBP in the sheep using an interspecies switching strategy involving site-directed mutagenesis of ovine GHBP

Site-directed antibodies to the growth hormone receptor could be potentiall y useful as growth hormone mimics but, in previous attempts, we found that antisera generated using peptides derived from growth hormone receptor sequ ences failed to recognize the intact protein. As an alternative approach to this problem, we have now adopted a strategy of epitope-switching between rat and ovine growth hormone receptors to produce rat epitopes in the corre ct structural context. Using site-directed mutagenesis, we altered the two dominant linear epitopes in the ovine growth hormone binding protein to the analogous sequences in rat growth hormone binding protein. Site A, between Thr28 and Leu34. is equivalent to epitope 1 in ovine growth hormone bindin g protein and site B, between Ser121 and Asp124, corresponds to epitope 5. The wild-type ovine growth hormone binding protein and the two mutant prote ins were bacterially expressed, refolded and, following purification by met al-chelate affinity chromatography, used to raise antisera in sheep. We sho wed using RIA, in which wild-type ovine growth hormone binding protein acte d as a competitor for the binding of rat growth hormone binding protein, th at only the site A mutant protein elicited a specific anti-rat growth hormo ne binding protein response. This was confirmed in subsequent RIA studies u sing the antiserum to the site A mutant protein in which only peptides corr esponding to the site A sequences in mutant ovine growth hormone binding pr otein and rat growth hormone binding protein, but not that in wild-type ovi ne growth hormone binding protein, were able to act as competitors for rat growth hormone binding protein. Antibodies specific for rat growth hormone binding protein could be separated from the antiserum to the site A mutant protein by means of affinity chromatography using immobilized wild-type ovi ne growth hormone binding protein to remove antibodies which cross-reacted with the ovine protein. The work lays the foundations for further studies i n which the biological effects of these antibody fractions will be investig ated and demonstrates an approach with general applicability in the product ion of antibodies directed towards specific epitopes on protein molecules.