Audette M, Buchegger F, Schreyer M, Mach J P
Ludwig Institute for Cancer Research, University of Lausanne, Epalinges, Switzerland.
Mol Immunol. 1987 Nov;24(11):1177-86. doi: 10.1016/0161-5890(87)90164-7.
Two new forms of non-specific crossreacting antigens (NCAs) were identified in the Nonidet P40 (NP-40) extracts of normal granulocytes by precipitation with the monoclonal antibody (MAb) 192 directed against carcinoembryonic antigen (CEA) and already known to crossreact with the perchloric acid soluble NCA-55. The NP-40 soluble NCAs recognized by MAb 192 have apparent mol. wts of 90,000 and 160,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Both NCAs appear to consist of a single monomeric polypeptide chain, since they have the same electrophoretic mobility in SDS-PAGE under reduced and non-reduced conditions. When granulocytes were extracted with perchloric acid instead of NP-40, only the 55,000 mol. wt antigen, corresponding to the previously described NCA-55, was precipitated by MAb 192. Furthermore, it was shown that NCA-55 is not a degradation product of NCA-90 or NCA-160 due to the perchloric acid treatment because exposure to perchloric acid of NCA preparations purified from NP-40 extracts did not change their apparent mol. wts in SDS-PAGE. It was also shown that NCA-160 is not a granulocytic form of CEA because it was not precipitated by the MAb 35 reacting exclusively with CEA. Immunocytochemical studies of granulocytes and macrophages showed that MAb 192 stained both types of cells whereas MAb 47 stained only the granulocytes and MAb 35 none of these cells. In granulocytes both MAbs reacted with antigens associated with granules and also present at the periphery of the nucleus as well as in the Golgi apparatus. The NCA-90 identified by MAb 192 was found by sequential immunodepletion to be antigenically distinct from the NCA-95 precipitated by MAb 47. The epitope recognized by MAb 192 on CEA and NCA molecules appears to be on the peptidic moiety because the antigens deglycosylated by the enzyme Endo F were still precipitated by this MAb. Taken together, the results indicate that MAb 192 identifies two novel forms of NCA (NCA-90 and NCA-160) in NP-40 extracts of granulocytes, which are distinct from CEA and the previously described NCA-55 and NCA-95 identified by MAbs 192 and 47, respectively, in perchloric acid extracts of granulocytes.
通过用针对癌胚抗原（CEA）的单克隆抗体（MAb）192进行沉淀，在正常粒细胞的Nonidet P40（NP - 40）提取物中鉴定出两种新形式的非特异性交叉反应抗原（NCA），已知该抗体与高氯酸可溶性NCA - 55发生交叉反应。MAb 192识别的NP - 40可溶性NCA在十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS - PAGE）中的表观分子量分别为90,000和160,000。两种NCA似乎都由单一的单体多肽链组成，因为它们在还原和非还原条件下的SDS - PAGE中具有相同的电泳迁移率。当用高氯酸而非NP - 40提取粒细胞时，MAb 192仅沉淀出分子量为55,000的抗原，对应于先前描述的NCA - 55。此外，研究表明NCA - 55不是高氯酸处理导致的NCA - 90或NCA - 160的降解产物，因为从NP - 40提取物中纯化的NCA制剂经高氯酸处理后，其在SDS - PAGE中的表观分子量并未改变。还表明NCA - 160不是CEA的粒细胞形式，因为它不能被仅与CEA反应的MAb 35沉淀。粒细胞和巨噬细胞的免疫细胞化学研究表明，MAb 192可对两种细胞进行染色，而MAb 47仅对粒细胞染色，MAb 35对这些细胞均不染色。在粒细胞中，两种单克隆抗体均与颗粒相关抗原反应，这些抗原也存在于细胞核周边以及高尔基体中。通过连续免疫去除发现，MAb 192识别的NCA - 90与MAb 47沉淀的NCA - 95在抗原性上不同。MAb 192在CEA和NCA分子上识别的表位似乎位于肽部分，因为经内切糖苷酶F去糖基化的抗原仍可被该单克隆抗体沉淀。综上所述，结果表明MAb 192在粒细胞的NP - 40提取物中鉴定出两种新的NCA形式（NCA - 90和NCA - 160），它们与CEA以及先前分别在粒细胞高氯酸提取物中由MAb 192和47鉴定出的NCA - 55和NCA - 95不同。
