Chin J C, Scully C, Pang B
Res Vet Sci. 1986 Jul;41(1):102-7.
Ovine secretory IgA (sIgA) has been purified to relative homogeneity by ammonium sulphate precipitation (to 40 per cent w/v) of lung lavage fluid from 3- or 12-month-old lambs, followed by molecular sieve chromatography on a Sephacryl S300 matrix. Three peaks A, B and C with molecular sizes corresponding to 550,000, 400,000 and 165,000 respectively were eluted from the column. Immunoelectrophoresis, radial immunodiffusion and enzyme-linked immunosorbent assays (ELISAs) with class specific antiserum confirmed that peak B contained only IgA. Polyacrylamide gel electrophoresis of peak B under reducing conditions resolved three subunits corresponding to secretory component, heavy and light chains. Hybridomas generated by fusing spleen cells from IgA-primed Balb/C mice with murine myeloma (Sp2/0) were screened for IgA-specific monoclonal antibodies against a panel of ovine IgG2, IgG1, IgA and IgM. One particular clone, F3-4B4, identified as murine IgG1, was monospecific against ovine IgA with no cross reactivity against bovine immunoglobulins. This hybridoma was successfully tested as a serological probe by ELISA profiling to locate IgA containing fractions in the course of immunoglobulin purification from biological fluids.
通过对3月龄或12月龄羔羊的肺灌洗液进行硫酸铵沉淀（至40% w/v），然后在Sephacryl S300基质上进行分子筛色谱，已将绵羊分泌型IgA（sIgA）纯化至相对均一。从柱上洗脱了三个峰A、B和C，其分子大小分别对应于550,000、400,000和165,000。用类特异性抗血清进行免疫电泳、放射免疫扩散和酶联免疫吸附测定（ELISA）证实峰B仅含有IgA。在还原条件下对峰B进行聚丙烯酰胺凝胶电泳，解析出对应于分泌成分、重链和轻链的三个亚基。通过将来自用IgA免疫的Balb/C小鼠的脾细胞与鼠骨髓瘤细胞（Sp2/0）融合产生的杂交瘤，针对一组绵羊IgG2、IgG1、IgA和IgM筛选IgA特异性单克隆抗体。一个特定的克隆F3 - 4B4，鉴定为鼠IgG1，对绵羊IgA具有单特异性，对牛免疫球蛋白无交叉反应。通过ELISA分析，该杂交瘤作为一种血清学探针成功进行了测试，以在从生物流体中纯化免疫球蛋白的过程中定位含IgA的组分。
