Tracheal carbohydrate antigens identified by monoclonal antibodies.
Tracheal carbohydrate antigens identified by monoclonal antibodies.
复制标题
通过单克隆抗体鉴定气管碳水化合物抗原。
DOI：
10.1016/0003-9861(86)90012-3
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发表时间：
1986
影响因子：
3.9
通讯作者：
Forsberg,LS
中科院分区：
文献类型：
--
作者：
Basbaum,CB;Chow,A;Macher,BA;Finkbeiner,WE;Veissiere,D;Forsberg,LS
在以前的研究中，我们描述了一个家族的单克隆抗体针对气管抗原具有多种细胞和亚细胞分布。在本研究中，我们已经扩展了我们的研究结果的四个代表性的抗体，以确定高碘酸盐的敏感性，糖苷酶的敏感性，和表观分子量的相应抗原。由于轻度高碘酸盐氧化选择性切割碳水化合物部分，使氨基酸保持完整，因此该处理后抗原性的丧失表明抗原决定簇中涉及糖残基。这可以通过测试抗原对特异性糖苷酶的敏感性来证实。通过酶联免疫吸附试验（ELISA），发现所有四种抗体对通过Bio-Gel A15 m色谱法分离的总气管分泌物的空隙体积组分具有最高的亲和力。在还原条件下通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳对该空隙体积材料进行进一步分析，然后进行免疫印迹分析，结果显示所有抗原均由高分子量物质（> 200，000）携带，这些物质为高碘酸盐-希夫碱阳性，但与考马斯蓝反应较差。在使用（a）免疫荧光和（B）ELISA的平行实验中，在对照条件下和在不同强度（10 mmIO 4 −，10 min，4 °C; 50 mmIO 4 −，1 h，4 °C; 100 mmIO 4 −，12 h，20 °C）下高碘酸盐处理抗原后，比较抗体结合。用这两种方法获得了类似的结果，表明在最温和的高碘酸盐处理后四种抗原之一的抗原性部分丧失，并且在两种延长处理中的每一种处理后所有四种抗原的抗原性全部丧失。所有四种抗原均对混合外切糖苷酶消化显示出显著的敏感性，并且三种抗原也对内切-β-半乳糖苷酶消化敏感。在与软骨素酶ABC、肝素酶或肝素酶孵育期间，抗原性未降低。气管组织切片的免疫荧光分析表明，这四种抗体在不同的位置，包括腺体和杯状细胞的细胞质颗粒和顶端上皮膜识别的决定因素。所有抗体的特征性免疫荧光模式通过气管切片的高碘酸盐孵育被消除。因此，这四种抗体似乎识别高分子量糖蛋白携带的碳水化合物抗原，每种糖蛋白具有不同的细胞来源。
In a previous study we described a family of monoclonal antibodies directed against tracheal antigens having a variety of cellular and subcellular distributions. In the present study, we have extended our findings on four representative antibodies to determine the periodate sensitivity, glycosidase sensitivity, and apparent molecular weight of the corresponding antigens. Since mild periodate oxidation selectively cleaves carbohydrate moiety, leaving amino acids intact, loss of antigenicity following this treatment suggests the involvement of sugar residues in the antigenic determinant. This can be confirmed by testing the sensitivity of the antigens to specific glycosidases. By enzyme-linked immunosorbent assay (ELISA), all four antibodies were found to have highest affinity for void volume components isolated by Bio-Gel A15m chromatography of the total tracheal secretion. Further analysis of this void volume material by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions followed by immunoblot analysis revealed that all antigens were carried by high-molecular-weight species (>200,000) which were periodate-Schiff positive but reacted poorly with Coomassie blue. In parallel experiments using (a) immunofluorescence and (b) ELISA, antibody binding was compared under control conditions and following periodate treatment of antigens under varying intensities (10 mmIO4−, 10 min, 4 °C; 50 mmIO4−, 1 h, 4 °C; 100 mmIO4−, 12 h, 20 °C). Similar results were obtained with the two methods, indicating a partial loss of antigenicity for one of the four antigens following the mildest periodate treatment, and total loss of antigenicity for all four antigens following each of the two prolonged treatments. All four antigens showed marked sensitivity to digestion with mixed exoglycosidases, and three antigens were also susceptible to endo-β-galactosidase digestion. Antigenicity was not decreased during incubation with chondroitinase ABC, heparitinase, or heparinase. Immunofluorescence analysis of tracheal tissue sections showed that the four antibodies recognized determinants in different locations, including gland and goblet cell cytoplasmic granules and the apical epithelial membrane. The characteristic immunofluorescence patterns of all antibodies were abolished by periodate incubation of the tracheal sections. Thus, the four antibodies appear to recognize carbohydrate antigens carried by high-molecular-weight glycoproteins, each with different cellular origins.