Smyth C J, Friedman-Kien A E, Salton M R
Infect Immun. 1976 Apr;13(4):1273-88. doi: 10.1128/iai.13.4.1273-1288.1976.
Crossed immunoelectrophoresis was used to study two complex antigenic preparations from Neisseria gonorrhoeae, one of cytoplasmic origin and the other derived by Triton X-100 extraction of isolated washed gonococcal envelopes, with the aim of developing suitable reference antigen-antibody systems that could be subsequently used to investigate the immune response to gonococcal infection and to monitor envelope preparations for cytoplasmic contamination. A number of parameters were investigated to optimized and standardize antigen preparation, e.g., harvesting and washing of gonococci, methods of bacterial disruption, and washing of envelopes. The effects of Triton X-100 concentration, initial total envelope protein concentration, and the composition, pH, and concentration of buffer on cell envelope extractability were studied to obviate the need to concentrate material before use in crossed immunoelectrophoresis. The electroendoosmotic properties of agarose were a major determining factor in resolving envelope antigens. From 25 to 30 immunoprecipitates were revealed in the envelope antigen-antibody system; 75 to 80 were revealed in the cytoplasmic sytem. Envelope immunoprecipitates with reduced nicotinamide adenine dinucleotide and lactate dehydrogenase activities were identified. Crossed immunoelectrophoresis with intermediate gels revealed the presence of antibodies in a preimmune rabbit antiserum pool to a distinctive fact-moving component in both the envelope and cytoplasmic antigen preparations. The intermediate gel technique also demonstrated that extensive washing of envelope preparations with buffer did not remove cytoplasmic ontamination completely. The method provides a much more sensitive means of monitoring the purity of envelope fractions than the use of single enzy,e markers as indexes of such contamination. The use of rabbit antisera raised to formolized gonococci in intermediate gels indicated that both reference antigen-antibody systems were of potential use in screening immune responses to N. gonorrhoeae.
采用交叉免疫电泳法研究了淋病奈瑟菌的两种复合抗原制剂，一种来自细胞质，另一种是通过用Triton X - 100提取分离并洗涤过的淋球菌包膜获得，目的是建立合适的参考抗原 - 抗体系统，随后用于研究对淋球菌感染的免疫反应以及监测包膜制剂是否受到细胞质污染。研究了许多参数以优化和标准化抗原制备，例如淋球菌的收获和洗涤、细菌裂解方法以及包膜的洗涤。研究了Triton X - 100浓度、初始包膜总蛋白浓度以及缓冲液的组成、pH和浓度对细胞膜提取物的影响，以避免在交叉免疫电泳中使用前对材料进行浓缩。琼脂糖的电内渗特性是分离包膜抗原的主要决定因素。在包膜抗原 - 抗体系统中发现了25至30种免疫沉淀物；在细胞质系统中发现了75至80种。鉴定出了烟酰胺腺嘌呤二核苷酸还原酶和乳酸脱氢酶活性降低的包膜免疫沉淀物。用中间凝胶进行的交叉免疫电泳显示，在免疫前兔抗血清池中存在针对包膜和细胞质抗原制剂中一种独特的快速移动成分的抗体。中间凝胶技术还表明，用缓冲液大量洗涤包膜制剂并不能完全去除细胞质污染。与使用单一酶标记作为此类污染指标相比，该方法提供了一种更灵敏的监测包膜级分纯度的手段。在中间凝胶中使用针对甲醛固定的淋球菌产生的兔抗血清表明，两种参考抗原 - 抗体系统都有可能用于筛选对淋病奈瑟菌的免疫反应。
