dos Santos M C, D'Império Lima M R, Furtado G C, Colletto G M, Kipnis T L, Dias da Silva W
Fundação Ezequiel Dias, Universidade Federal de Minas Gerais, Brazil.
Toxicon. 1989;27(3):297-303. doi: 10.1016/0041-0101(89)90177-3.
Pooled horse plasma containing antibodies against Crotalus durissus terrificus whole venom were digested with pepsin at an enzyme-substrate ratio of 8:1, pH 3.1, for 40 min and the F(ab')2M fragments purified by adding 8.7% caprylic acid (pH 5.0). For comparison, F(ab')2B purified by precipitation with ammonium sulphate and uncleaved IgG purified with caprylic acid were also prepared. Fab' fragments were obtained by reduction and alkylation of F(ab')2B. The anti-whole C.d. terrificus venom titers, determined by Dot-Blot were 12,800 (IgG), 6400 [F(ab')2B], 4800 [F(ab')2M] and 3200 (Fab'B). Immunochemical analysis of these fragments by SDS gel electrophoresis, Western blot and by double immunodiffusion revealed that the solution containing F(ab')2M was free of IgG and of other plasma proteins, whereas that containing F(ab')2B was not. One milligram of either F(ab')2B, F(ab')2M or Fab'B was able to neutralize respectively 20.7 micrograms, 20.2 micrograms and 13.8 micrograms of C.d. terrificus venom.
将含有抗杜氏菱背响尾蛇全毒液抗体的马血浆混合液，以8:1的酶-底物比例，在pH 3.1条件下用胃蛋白酶消化40分钟，然后通过添加8.7%辛酸（pH 5.0）纯化F(ab')2M片段。作为对照，还制备了通过硫酸铵沉淀纯化的F(ab')2B和用辛酸纯化的未切割IgG。通过F(ab')2B的还原和烷基化获得Fab'片段。通过斑点印迹法测定的抗杜氏菱背响尾蛇全毒液效价分别为12,800（IgG）、6400 [F(ab')2B]、4800 [F(ab')2M]和3200（Fab'B）。通过SDS凝胶电泳、蛋白质印迹和双向免疫扩散对这些片段进行免疫化学分析表明，含有F(ab')2M的溶液不含IgG和其他血浆蛋白，而含有F(ab')2B的溶液则不然。1毫克F(ab')2B、F(ab')2M或Fab'B分别能够中和20.7微克、20.2微克和13.8微克的杜氏菱背响尾蛇毒液。
