Woodard C S, Splawski J B, Goldblum R M, Denney R M
J Immunol. 1984 Oct;133(4):2116-25.
We have compared the epitopes present in various forms of human secretory component by using a panel of hybridoma-derived antibodies elicited by immunizing mice with free secretory component (FSC) or secretory IgA (sIgA). Enzyme-linked immunosorbent binding assays (ELISA) were used to assess antibody binding to FSC- and SC-containing antigens, including sIgA isolated from milk, reduced and alkylated sIgA, and sIgA assembled in vitro by incubating dimeric IgA with FSC. Immunofluorescence assays were also used to assess binding to a human epithelial tumor cell line (HT29) that expresses secretory component as an integral protein of the plasma membrane. The results can be summarized as follows. 1) Most antibodies from fusions in which sIgA was the immunizing antigen bound preferentially to sIgA. 2) Most antibodies from fusions in which FSC was the immunizing antigen bound preferentially to FSC. 3) Antibodies that bound preferentially to sIgA invariably bound sIgA assembled in vitro; antibodies that bound preferentially to FSC invariably did not. 4) Antibodies that bound readily to both sIgA and FSC were rare in all fusions. 5) The monoclonal antibodies defined at least six classes of epitopes on SC, including epitopes that were a) FSC specific and reduction sensitive, b) FSC specific and reduction insensitive, c) sIgA specific and reduction-sensitive, d) sIgA specific and reduction insensitive, e) shared by FSC and sIgA and reduction-sensitive, and f) shared by FSC and sIgA and reduction-insensitive. 6) Antibodies that mediated intense immunofluorescent staining of secretory component on HT29 cell membranes were rare and constituted a distinct subset of those which recognized epitopes shared by FSC, reduced and alkylated sIgA, and some preparations of native sIgA. Results of these antibody-binding studies indicate that most SC epitopes are not shared by FSC and sIgA. Most SC-related epitopes on sIgA appear to be generated by the physical interaction of SC with dimeric IgA, whereas most epitopes on FSC are masked or altered by this interaction. Finally, epitopes that are shared by membrane SC and FSC and/or sIgA represent a minor and immunochemically distinct subset of epitopes on SC. The high proportion of unique epitopes on the different physical forms of SC suggest that the epitopes of this molecule are highly sensitive to its molecular environment. The monoclonal reagents described here will be useful in studying the structure and function of SC; quantitating FSC, sIgA, and membrane SC; purifying various molecular forms of SC by immunoaffinity chromatography; and localizing SC in human tissues and cultured cells by immunocytochemical techniques.
我们使用了一组杂交瘤衍生抗体，通过用游离分泌成分（FSC）或分泌型IgA（sIgA）免疫小鼠来引发这些抗体，比较了各种形式的人分泌成分中存在的表位。酶联免疫吸附结合试验（ELISA）用于评估抗体与含FSC和SC的抗原的结合情况，这些抗原包括从牛奶中分离的sIgA、还原和烷基化的sIgA，以及通过将二聚体IgA与FSC孵育在体外组装的sIgA。免疫荧光试验也用于评估与人类上皮肿瘤细胞系（HT29）的结合，该细胞系将分泌成分表达为质膜的整合蛋白。结果总结如下。1）大多数以sIgA作为免疫抗原的融合产生的抗体优先结合sIgA。2）大多数以FSC作为免疫抗原的融合产生的抗体优先结合FSC。3）优先结合sIgA的抗体总是能结合体外组装的sIgA；优先结合FSC的抗体则不然。4）在所有融合中，能同时轻易结合sIgA和FSC的抗体很少见。5）单克隆抗体在SC上定义了至少六类表位，包括a）FSC特异性且对还原敏感的表位，b）FSC特异性且对还原不敏感的表位，c）sIgA特异性且对还原敏感的表位，d）sIgA特异性且对还原不敏感的表位，e）FSC和sIgA共有的且对还原敏感的表位，以及f）FSC和sIgA共有的且对还原不敏感的表位。6）介导HT29细胞膜上分泌成分强烈免疫荧光染色的抗体很少见，并且构成了识别FSC、还原和烷基化sIgA以及一些天然sIgA制剂共有的表位的抗体的一个独特亚群。这些抗体结合研究的结果表明，大多数SC表位不是FSC和sIgA共有的。sIgA上大多数与SC相关的表位似乎是由SC与二聚体IgA的物理相互作用产生的，而FSC上的大多数表位在这种相互作用中被掩盖或改变。最后，膜SC与FSC和/或sIgA共有的表位代表了SC上一个较小的且免疫化学上不同的表位亚群。SC不同物理形式上独特表位的高比例表明该分子的表位对其分子环境高度敏感。这里描述的单克隆试剂将有助于研究SC的结构和功能；定量FSC、sIgA和膜SC；通过免疫亲和色谱法纯化SC的各种分子形式；以及通过免疫细胞化学技术在人体组织和培养细胞中定位SC。
