Lumanglas A L, Sadeghi H, Wang B S
Laboratory of Immunoendocrinology, Agricultural Research Division, American Cyanamid Company, Princeton, New Jersey 08543-0400.
Hybridoma. 1994 Jun;13(3):237-40. doi: 10.1089/hyb.1994.13.237.
An effort was made to generate stable swine hybridomas capable of releasing monoclonal antibodies (MAb) with antigenic specificity. Crossbred 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 briefly stimulated in vitro with r-pGH prior to hybridization with murine SP2/0 myeloma cells. The fused hybrids were screened for their ability 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 hybridoma, designated PM20/20, was found to secrete swine IgM. It recognized not only the immunizing r-pGH but also the native pGH extracted from the 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 immunoreacted with several other GH species including bovine, chicken, and human origins, but not with ovine prolactin nor rat GH binding protein. The binding association rate constant and dissociation rate constant of PM20/20 to pGH were 5.3 x 10(4) M-1 s-1 and 1.0 x 10(-4) s-1, respectively, thus producing a dissociation constant of 1.9 x 10(-9) M. Therefore, stable swine-mouse heterohybridoma lines have been established and shown to continuously release swine mAb in cultures. These mAb may serve as useful alternatives to murine mAb in certain areas of research.
人们致力于培育能够分泌具有抗原特异性单克隆抗体（MAb）的稳定猪杂交瘤。用重组猪生长激素（r-pGH）免疫杂种猪，然后从这些动物身上采集脾细胞。通过梯度离心和尼龙棉黏附富集的B淋巴细胞在与鼠SP2/0骨髓瘤细胞杂交之前，先用r-pGH进行短暂的体外刺激。筛选融合的杂交瘤产生抗pGH抗体的能力，阳性杂交瘤通过有限稀释法进行亚克隆。稳定的细胞系通过在培养物中连续传代进行维持，以便进一步分析。发现一种这样的杂交瘤，命名为PM20/20，能分泌猪IgM。如Western分析所示，它不仅能识别免疫用的r-pGH，还能识别从猪垂体中提取的天然pGH。它还能识别胰蛋白酶消化后r-pGH的两个分子量分别为10 kD和5 kD的较小片段。除了pGH，PM20/20还能与包括牛、鸡和人来源的其他几种生长激素发生免疫反应，但不与绵羊催乳素或大鼠生长激素结合蛋白发生反应。PM20/20与pGH的结合缔合速率常数和解离速率常数分别为5.3×10⁴ M⁻¹ s⁻¹和1.0×10⁻⁴ s⁻¹，因此解离常数为1.9×10⁻⁹ M。因此，已经建立了稳定的猪-鼠异种杂交瘤系，并证明其在培养物中能持续分泌猪单克隆抗体。在某些研究领域，这些单克隆抗体可能是鼠单克隆抗体的有用替代品。
