Alape-Girón A, Gustafsson B, Lomonte B, Thelestam M, Gutiérrez J M
Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San Jose.
Toxicon. 1994 Jun;32(6):695-712. doi: 10.1016/0041-0101(94)90339-5.
Eleven murine monoclonal antibodies (MAbs) against Micrurus nigrocinctus nigrocinctus venom were produced and partially characterized. When M. n. nigrocinctus venom proteins were separated by SDS-PAGE under non-reducing conditions four sharp and three diffuse bands were observed. The sharp bands had migration rates comparable to reduced standards of 10, 12, 50 and 72 kDa. The diffuse bands migrate in the range of reduced standards from 14.5 to 32 kDa. When venom proteins were separated under reducing conditions the same sharp bands and an additional prominent 14.5 kDa band were observed. Three antibodies (MAbs 4, 21 and 28) recognized the diffuse bands in western blots of non-reducing SDS-PAGE, whereas MAbs 7G, 22 and 26 reacted with only the 72 kDa protein. MAbs 21 and 28 reacted with the 14.5 kDa band whereas MAb 7G recognized the 72 kDa band in blots of reducing SDS-PAGE. Two M. nigrocinctus antivenoms cross-reacted by ELISA against nine neurotoxic snake venoms, as well as with gamma-toxin from Naja nigricollis and notexin. One antibody (MAb 9A) was used to affinity purify a fraction (called nigroxin) from M. n. nigrocinctus venom. Nigroxin showed phospholipase and myotoxic activities and appeared as a single 15 kDa band in SDS-PAGE under reducing conditions. However, three bands with slight differences in charge were resolved by urea-PAGE, representing isoforms named nigroxin a, b, and c. Nigroxin induced a dose-dependent release of peroxidase trapped in negatively charged liposomes. Nigroxin induced myonecrosis and increased the plasma creatine kinase levels in mice, when injected intramuscularly. The plasma membrane of cultured L6 myoblasts was permeabilized by nigroxin, as evidenced by the release of 3H-uridine nucleotides from prelabelled cells. This effect was completely abolished after preincubation with MAb 9A, although this antibody failed to neutralize the enzymatic activity of nigroxin. Nigroxin was also recognized by MAbs 4, 7H, 21, 27 and 28. Additionally, the epitope recognized by MAb 27 is also present in notexin and beta-bungarotoxin.
制备了11种针对黑带珊瑚蛇（Micrurus nigrocinctus nigrocinctus）毒液的鼠单克隆抗体（MAb），并对其进行了部分特性鉴定。当在非还原条件下通过SDS-PAGE分离黑带珊瑚蛇毒液蛋白时，观察到四条清晰条带和三条弥散条带。清晰条带的迁移率与10、12、50和72 kDa的还原标准品相当。弥散条带在14.5至32 kDa的还原标准品范围内迁移。当在还原条件下分离毒液蛋白时，观察到相同的清晰条带以及一条额外的14.5 kDa显著条带。三种抗体（单克隆抗体4、21和28）在非还原SDS-PAGE的蛋白质印迹中识别弥散条带，而单克隆抗体7G、22和26仅与72 kDa蛋白反应。单克隆抗体21和28与14.5 kDa条带反应，而单克隆抗体7G在还原SDS-PAGE的印迹中识别72 kDa条带。两种黑带珊瑚蛇抗蛇毒血清通过ELISA与九种神经毒性蛇毒以及眼镜蛇（Naja nigricollis）的γ-毒素和诺毒素交叉反应。一种抗体（单克隆抗体9A）用于从黑带珊瑚蛇毒液中亲和纯化一个组分（称为黑毒素）。黑毒素显示出磷脂酶和肌毒性活性，在还原条件下的SDS-PAGE中呈现为单一的15 kDa条带。然而，通过尿素-PAGE解析出三条电荷略有差异的条带，代表名为黑毒素a、b和c的同工型。黑毒素诱导被困在带负电荷脂质体中的过氧化物酶呈剂量依赖性释放。当肌肉注射时，黑毒素诱导小鼠发生肌坏死并增加血浆肌酸激酶水平。培养的L6成肌细胞的质膜被黑毒素通透化，这通过预标记细胞中3H-尿苷核苷酸的释放得以证明。在用单克隆抗体9A预孵育后，这种效应完全消除，尽管该抗体未能中和黑毒素的酶活性。黑毒素也被单克隆抗体4、7H、21、27和28识别。此外，单克隆抗体27识别的表位也存在于诺毒素和β-银环蛇毒素中。
