Howard R J, Barnwell J W
J Cell Biochem. 1984;24(3):297-306. doi: 10.1002/jcb.240240310.
Four detergents have been compared for identification of the Plasmodium knowlesi variant antigen on infected erythrocytes by immunoprecipitation analysis. Erythrocytes infected with late trophozoite and schizont forms of cloned asexual parasites were labeled by lactoperoxidase-catalyzed radioiodination and extracted either with the anionic detergents sodium dodecyl sulfate (SDS) or cholate, the neutral detergent Triton X-100, or the zwitterion 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS). After addition of Triton X-100 to SDS and cholate extracts, parallel immunoprecipitations of the four extracts were performed using rhesus monkey antisera of defined agglutinability. Identical results were obtained with clone Pk1(A+), which has 125I-variant antigens of Mr 210,000 and 190,000, and with clone Pk1(B+)1+, which has variant antigens of Mr 200,000-205,000. SDS yielded maximal levels of immunoprecipitated 125I-variant antigens. Variant-specific immunoprecipitation was detected in some experiments with Triton X-100 and cholic acid but with significantly lower recovery than with SDS. CHAPS extraction did not yield the variant antigens on immunoprecipitation. The variant antigens could also be identified in Triton X-100-insoluble material by subsequent extraction with SDS, indicating that failure to recover these proteins in the Triton X-100-soluble fraction is due to failure of this detergent to extract the variant antigens rather than to degradation during extraction. We suggest that the 125I-variant antigens either have a structure that renders them intrinsically insoluble in Triton X-100, cholate, or CHAPS, or that they are associated in some way with host cell membrane components that also resist solubilization by these detergents.
通过免疫沉淀分析，对四种去污剂进行了比较，以鉴定感染红细胞上的诺氏疟原虫变异抗原。用乳过氧化物酶催化的放射性碘化标记感染晚期滋养体和裂殖体形式的克隆无性寄生虫的红细胞，并用阴离子去污剂十二烷基硫酸钠（SDS）或胆酸盐、中性去污剂 Triton X - 100 或两性离子 3 - [(3 - 胆酰胺丙基)二甲基铵]-1 - 丙烷磺酸盐（CHAPS）进行提取。在 SDS 和胆酸盐提取物中加入 Triton X - 100 后，使用具有确定凝集性的恒河猴抗血清对四种提取物进行平行免疫沉淀。克隆 Pk1(A+)（具有分子量为 210,000 和 190,000 的 125I - 变异抗原）和克隆 Pk1(B+)1+（具有分子量为 200,000 - 205,000 的变异抗原）得到了相同的结果。SDS 产生了最高水平的免疫沉淀 125I - 变异抗原。在一些实验中，用 Triton X - 100 和胆酸检测到了变异特异性免疫沉淀，但回收率明显低于 SDS。CHAPS 提取在免疫沉淀时未产生变异抗原。通过随后用 SDS 提取，也可以在 Triton X - 100 不溶性物质中鉴定出变异抗原，这表明在 Triton X - 100 可溶部分未能回收这些蛋白质是由于这种去污剂未能提取变异抗原，而不是提取过程中的降解。我们认为，125I - 变异抗原要么具有使其本质上不溶于 Triton X - 100、胆酸盐或 CHAPS 的结构，要么它们以某种方式与宿主细胞膜成分相关联，而这些成分也抵抗这些去污剂的溶解作用。
