Maéno M, Shimizu K, Shikata T
Department of Pathology, Nishon University School of Medicine, Tokyo.
Microbiol Immunol. 1988;32(12):1245-52. doi: 10.1111/j.1348-0421.1988.tb01488.x.
Two immunization techniques that enable production of mouse monoclonal antibodies were evaluated in terms of small quantities of antigen. Various amounts of purified influenza A virus particles were applied either for in vitro sensitization in cultured splenocytes or for intrasplenic immunization, followed by hybridization of the immunized cells with mouse myeloma cells. Hybridomas producing specific antibodies for influenza viral proteins were detected by enzyme-linked immunosorbent assay when more than 50 micrograms of antigens was used for the in vitro immunization method, and at least 5 micrograms was necessary for a single intrasplenic immunization. On the other hand, as little as 60 ng of antigen administered in two intrasplenic injections was sufficient to produce specific hybridomas. Two out of six randomly selected monoclonal antibodies obtained using the repeated intrasplenic immunization method were IgG and the other four were IgM. Immunoprecipitation analyses revealed that the recognized antigens involved a viral inner protein (nucleocapsid protein), as well as an envelope glycoprotein (hemagglutinin). We conclude that immunization by two direct injections of antigen into the spleen is the most effective method for sensitization with nanogram quantities of insoluble antigen such as influenza viruses.
针对少量抗原，评估了两种能够制备小鼠单克隆抗体的免疫技术。将不同量的纯化甲型流感病毒颗粒用于培养脾细胞的体外致敏或脾内免疫，然后将免疫细胞与小鼠骨髓瘤细胞杂交。当超过50微克抗原用于体外免疫方法时，通过酶联免疫吸附测定法检测到产生针对流感病毒蛋白的特异性抗体的杂交瘤，而单次脾内免疫至少需要5微克抗原。另一方面，两次脾内注射中给予低至60纳克的抗原就足以产生特异性杂交瘤。使用重复脾内免疫方法获得的六株随机选择的单克隆抗体中，有两株是IgG，另外四株是IgM。免疫沉淀分析表明，识别的抗原涉及病毒内部蛋白（核衣壳蛋白）以及包膜糖蛋白（血凝素）。我们得出结论，通过将抗原直接注射到脾脏两次进行免疫，是用纳克量的不溶性抗原（如流感病毒）进行致敏的最有效方法。
