Doellgast G J, Triscott M X, Beard G A, Bottoms J D, Cheng T, Roh B H, Roman M G, Hall P A, Brown J E
Department of Biochemistry, Bowman Gray School of Medicine, Winston-Salem, North Carolina 27157.
J Clin Microbiol. 1993 Sep;31(9):2402-9. doi: 10.1128/jcm.31.9.2402-2409.1993.
A new immunoassay amplification method has been applied to the measurement of toxins A, B, and E from Clostridium botulinum. The technique is a modified enzyme-linked immunosorbent assay (ELISA) which relies on the detection of sandwich complexes on microtiter plates by a solid-phase coagulation assay known as ELCA, or enzyme-linked coagulation assay. In the method, a coagulation activating enzyme (RVV-XA) isolated from the venom of Russell's viper is conjugated to affinity-purified horse antibodies specific for toxin type A, B, or E. Plates are coated with affinity-purified antibodies, and standard captag (capture-tag) protocols using labeled antibody are employed to bind the toxin from solution. Complexes are detected by adding a modified plasma substrate which contains all the coagulation factors mixed with alkaline phosphatase-labeled fibrinogen and solid-phase fibrinogen; deposition of solid-phase, enzyme-labeled fibrin on the solid phase is then a reflection of formation of toxin-RVV-XA-antibody complexes on the solid phase. Because of the ability to detect RVV-XA by this coagulation assay at concentrations < 0.1 pg/ml, it was possible to measure C. botulinum toxins A, B, and E at mouse bioassay levels (< 10 pg/ml, or < 0.07 pM) for both purified neurotoxin and crude culture filtrates obtained from strains known to produce appropriate single toxins. ELISA-ELCA should be applicable to measurement of toxins in most of the materials (contaminated food, blood, and excreta) for which the comparably sensitive mouse bioassay is currently employed. This method has the potential of broad application to the measurement of low concentrations of any antigen for which appropriate immunochemical reagents are available, in a color test format.
一种新的免疫测定放大方法已应用于肉毒梭菌毒素A、B和E的检测。该技术是一种改良的酶联免疫吸附测定（ELISA），它依赖于通过一种称为ELCA（酶联凝血测定）的固相凝血测定法来检测微量滴定板上的夹心复合物。在该方法中，从罗素蝰蛇毒液中分离出的凝血激活酶（RVV-XA）与针对毒素A、B或E的亲和纯化马抗体结合。平板用亲和纯化抗体包被，并采用使用标记抗体的标准捕获标签（capture-tag）方案从溶液中结合毒素。通过添加一种改良的血浆底物来检测复合物，该底物包含与碱性磷酸酶标记的纤维蛋白原和固相纤维蛋白原混合的所有凝血因子；然后固相上酶标记的固相纤维蛋白的沉积反映了固相上毒素-RVV-XA-抗体复合物的形成。由于通过这种凝血测定法能够检测浓度<0.1 pg/ml的RVV-XA，因此对于纯化的神经毒素和从已知产生相应单一毒素的菌株获得粗培养滤液，有可能在小鼠生物测定水平（<10 pg/ml，或<0.07 pM）下测量肉毒梭菌毒素A、B和E。ELISA-ELCA应适用于目前采用灵敏度相当高的小鼠生物测定法的大多数材料（受污染的食物、血液和排泄物）中毒素的检测。这种方法有潜力广泛应用于以颜色测试形式测量任何有合适免疫化学试剂的低浓度抗原。
