Greenlee A R, Magnuson N S, Smith C, Butt B M, McKiernan A J, Davis W C
Department of Veterinary Microbiology and Pathology, Washington State University, Pullman 99164-7040.
Vet Immunol Immunopathol. 1990 Nov;26(3):267-83. doi: 10.1016/0165-2427(90)90096-b.
Production of porcine monoclonal antibodies for use in research and immunotherapy has been hampered by the lack of suitable fusion partners which promote high efficiencies of hybridoma out-growth and immunoglobulin synthesis. To overcome these obstacles, five heteromyeloma fusion partners (HM-1,2,3,4 and 5) were constructed by successively fusing porcine lymphocytes with murine myeloma cells or murine x bovine heteromyeloma cells. Following section of hypoxanthine/aminopterin/thymidine (HAT)-sensitive mutants, karyotypes, growth rates and surface phenotypes of the heteromyelomas were determined. Karyotyping revealed an increase in the mean number of chromosomes present in HM-1,4 and 5 cells. Peak doubling times of the parental and HM cells ranged between 12.2 and 17.4 h. Uisng flow microfluorimetry and monoclonal antibodies specific for class I/II major histocompatability antigens, it was determined that the surface phenotype of HM-1,2,3,4 and 5 resembled that of the parental murine X63 myeloma cells. HM 1,2,3,4 and 5 were evaluated for their abilities to serve as fusion partners. Highest percentages of hybrid outgrowth (37%) and immunoglobulin synthesis (52%) were observed when HM-1 was fused with procine lymphocytes. When cloned, percentage of outgrowth and immunoglobulin synthesis increased if HM-1 and HM-2 were used as fusion partners. Cryopreservation of HM-1 and HM-2 did not adversely affect their abilities to promote hybrid outgrowth or immunoglobulin synthesis. During the first week following fusion of porcine lymphocytes with heteromyelomas, murine thymocytes were found to be essential for survival of the nascent hybrids. To confirm that immunoglobulin secreted by hybridomas was of porcine and not murine or bovine origin, culture supernates were subjected to SDS gel electrophoresis, electroblotted and identified. using species-specific isotyping reagents. Two of four cell lines tested secreted porcine light chains and one of four cell lines secreted whole IgM molecules. This paper is the first to describe porcine heteromyelomas for use as fusion partners. Similar to findings of human and bovine studies, our data suggest that heteromyeloma fusion partners perform better than rodent myelomas for creating hybridomas synthesizing porcine immunoglobulin.
用于研究和免疫治疗的猪单克隆抗体的生产一直受到缺乏合适融合伙伴的阻碍，这些融合伙伴能够促进杂交瘤的高效生长和免疫球蛋白的合成。为了克服这些障碍，通过将猪淋巴细胞与鼠骨髓瘤细胞或鼠×牛杂种骨髓瘤细胞依次融合，构建了五种杂种骨髓瘤融合伙伴（HM-1、2、3、4和5）。在筛选出次黄嘌呤/氨基蝶呤/胸腺嘧啶核苷（HAT）敏感突变体后，测定了杂种骨髓瘤的核型、生长速率和表面表型。核型分析显示，HM-1、4和5细胞中染色体的平均数量增加。亲本细胞和HM细胞的倍增时间峰值在12.2至17.4小时之间。使用流式细胞荧光测定法和针对I/II类主要组织相容性抗原的单克隆抗体，确定HM-1、2、3、4和5的表面表型与亲本鼠X63骨髓瘤细胞相似。对HM 1、2、3、4和5作为融合伙伴的能力进行了评估。当HM-1与猪淋巴细胞融合时，观察到杂交瘤生长（37%）和免疫球蛋白合成（52%）的最高百分比。克隆时，如果使用HM-1和HM-2作为融合伙伴，生长和免疫球蛋白合成的百分比会增加。HM-1和HM-2的冷冻保存对它们促进杂交瘤生长或免疫球蛋白合成的能力没有不利影响。在猪淋巴细胞与杂种骨髓瘤融合后的第一周，发现鼠胸腺细胞对新生杂交瘤的存活至关重要。为了确认杂交瘤分泌的免疫球蛋白是猪源的，而非鼠源或牛源的，对培养上清液进行了SDS凝胶电泳、电印迹并使用种属特异性分型试剂进行鉴定。测试的四个细胞系中有两个分泌猪轻链，四个细胞系中有一个分泌完整的IgM分子。本文首次描述了用作融合伙伴的猪杂种骨髓瘤。与人类和牛的研究结果相似，我们的数据表明，杂种骨髓瘤融合伙伴在创建合成猪免疫球蛋白的杂交瘤方面比啮齿动物骨髓瘤表现更好。
