GENERATION OF HETEROHYBRIDOMAS CAPABLE OF RELEASING SWINE MONOCLONAL-ANTIBODY SPECIFIC TO PORCINE GROWTH-HORMONE

An effort was made to generate stable swine hybridomas capable of rele asing monoclonal antibodies (MAb) with antigenic specificity. Crossbre d pigs were immunized with recombinant porcine growth hormone (r-pGH) and the splenic cells were harvested from these animals. B lymphocytes enriched by gradient centrifugation and nylon wool adherence were bri efly stimulated in vitro with r-pGH prior to hybridization with murine SP2/0 myeloma cells. The fused hybrids were screened for their abilit y to produce anti-pGH antibody and the positive ones were subcloned by a limiting dilution procedure. The stable cell lines were maintained by serial passages in cultures for further analysis. One such hybridom a, designated PM20/20, was found to secrete swine IgM. It recognized n ot only the immunizing r-pGH but also the native pGH extracted from th e swine pituitary glands, as demonstrated by Western analysis. It also recognized two smaller fragments with m.w. of 10 kD and 5 kD of r-pGH following trypsin digestion. In addition to pGH, PM20/20 immunoreacte d with several other GH species including bovine, chicken, and human o rigins, but not with ovine prolactin nor rat GH binding protein. The b inding association rate constant and dissociation rate constant of PM2 0/20 to pGH were 5.3 x 10(4) M(-1) s(-1) and 1.0 x 10(-4) s(-1), respe ctively, thus producing a dissociation constant of 1.9 x 10(-9) M. The refore, stable swine-mouse heterohybridoma lines have been established and shown to continuously release swine mAb in cultures. These mAb ma y serve as useful alternatives to murine mAb in certain areas of resea rch.