Place J D, Thompson S G, Burd J F, Molinaro C, Ott R A, Jensen F C
Hybridoma. 1984 Summer;3(2):187-93. doi: 10.1089/hyb.1984.3.187.
Splenocytes from BALB/c mice immunized with a gentamicin-hemocyanin conjugate were fused with X63-Ag8.653 murine myeloma cells to produce hybridomas that secreted monoclonal antibody to gentamicin. Sixteen positive clones were obtained. The monoclonal antibody chosen for a gentamicin immunoassay has been characterized with respect to class, subclass, type of light chain, electrophoretic homogeneity, and binding affinity. Gentamicin monoclonal antibody purified from mouse ascites fluid was analyzed by immunoelectrophoresis, double immunodiffusion, enzyme-linked immunosorbent assay (ELISA), and polyacrylamide gel electrophoresis (PAGE). The results show that the antibody is an IgG2a (kappa). Two bands were detected when the purified antibody was electrophoresed on polyacrylamide gels: a major band and a less mobile minor component (5.6% of the major band). Both were IgG2a (kappa). The major band contained antibody which bound 2.1 moles of the substrate-labeled gentamicin derivative, beta-galactosyl-umbelliferone sisomicin, per mole of IgG, whereas one mole of the minor band bound only 0.095 moles of the substrate-labeled conjugate. The antibody has an affinity constant of 1.22 X 10(10) M-1.
用庆大霉素-血蓝蛋白偶联物免疫的BALB/c小鼠的脾细胞与X63-Ag8.653鼠骨髓瘤细胞融合，以产生分泌抗庆大霉素单克隆抗体的杂交瘤。获得了16个阳性克隆。所选择用于庆大霉素免疫测定的单克隆抗体已在类别、亚类、轻链类型、电泳均一性和结合亲和力方面进行了表征。对从小鼠腹水中纯化的庆大霉素单克隆抗体进行了免疫电泳、双向免疫扩散、酶联免疫吸附测定（ELISA）和聚丙烯酰胺凝胶电泳（PAGE）分析。结果表明该抗体为IgG2a（κ）。当纯化的抗体在聚丙烯酰胺凝胶上进行电泳时检测到两条带：一条主带和一条迁移较慢的次要组分（为主带的5.6%）。两者均为IgG2a（κ）。主带中的抗体每摩尔IgG结合2.1摩尔底物标记的庆大霉素衍生物β-半乳糖基伞形酮西索米星，而次要带每摩尔仅结合0.095摩尔底物标记的偶联物。该抗体的亲和常数为1.22×10¹⁰ M⁻¹。
