基于排阻高效液相色谱法的体外评估总毒液-抗毒液免疫复合物的形成和与抗毒液保护毒液毒性和致死性相关的结合参数。

In vitro evaluation of total venom-antivenin immune complex formation and binding parameters relevant to antivenin protection against venom toxicity and lethality based on size-exclusion high-performance liquid chromatography.

机构信息

Department of Biochemistry & Microbiology, Oklahoma State University Center for Health Sciences, 1111 W. 17th Street, Tulsa, OK 74107, USA.

出版信息

Toxicon. 2011 May;57(6):871-81. doi: 10.1016/j.toxicon.2011.03.003. Epub 2011 Mar 15.

DOI:10.1016/j.toxicon.2011.03.003

PMID:21392522

Abstract

Total venom-antivenin immune complex formation and binding parameters relevant to antivenin protection against venom toxicity and lethality can be evaluated using size-exclusion high-performance liquid chromatography (SE-HPLC). Simple integration of regions within SE-HPLC elution profiles was used to compare binding characteristics of Crotalidae Polyvalent Immune Fab (Ovine) antivenin (FabAV) and Crotalus atrox (western diamondback rattlesnake; C. atrox), C. varidis varidis (prairie rattlesnake; C. v. v.), Agkistrodon contortrix contortrix (southern copperhead; A. c. c.), and A. piscivorus leukostoma (western cottonmouth; A. p. l.) venom. Areas associated with bound venom and antivenin ({Area(bnd)}) were evaluated using a logistic dose-response equation to estimate EC(50) and {Area(bnd)}(max). The relative magnitudes of EC(50), which inversely reflect venom-antivenin binding affinity, were C. atrox > C. v. v. > A. c. c. > A. p. l. Less than 50% of FabAV appeared to be reactive with each of the venoms based on {Area(bnd)}(max). Data was also consistent with FabAV binding to multiple sites on polyvalent antigens within the venoms. Evaluation of immune complex formation using SE-HPLC was compared to neutralization of phospholipase A(2) (PLA(2)) activity of C. atrox, A. c. c., and A. p. l. venom by FabAV as reported in the literature. Maximum neutralization of PLA(2) activity occurred, in general, prior to maximum immune complex formation. Venom-antivenin binding at EC(50) determined via SE-HPLC appeared to be greater than binding associated with neutralization of venom lethality in mice based on LD(50) and ED(50) reported by others. SE-HPLC analysis of venom-antivenin binding could provide a priori information, relevant to reducing the use of animals in evaluating antivenin protection against venom-induced toxicity and lethality.

摘要

使用尺寸排阻高效液相色谱法（SE-HPLC）可以评估总毒液-抗毒液免疫复合物的形成和与抗毒液保护毒液毒性和致死性相关的结合参数。通过简单地整合 SE-HPLC 洗脱曲线中的区域，比较了 Crotalidae 多价免疫 Fab（羊）抗毒液（FabAV）与 Crotalus atrox（西部菱斑响尾蛇；C.atrox）、Crotalus varidis varidis（草原响尾蛇；C.v.v.）、Agkistrodon contortrix contortrix（南方铜头蛇；A.c.c.）和 Agkistrodon piscivorus leucostoma（西部棉口蛇；A.p.l.）毒液的结合特性。使用逻辑剂量反应方程评估与结合毒液和抗毒液相关的区域（{Area(bnd)}}），以估计 EC(50)和{Area(bnd)}(max)。EC(50)的相对大小，其反过来说明毒液-抗毒液结合亲和力，为 C.atrox>C.v.v.>A.c.c.>A.p.l. 基于{Area(bnd)}(max)，FabAV 似乎与每种毒液的反应性都不到 50%。数据也与 FabAV 结合到毒液中的多价抗原上的多个位点一致。使用 SE-HPLC 评估免疫复合物的形成，并与文献中报道的 FabAV 中和 C.atrox、A.c.c.和 A.p.l.毒液中的磷脂酶 A(2)（PLA(2)）活性进行比较。一般来说，最大 PLA(2)活性的中和先于最大免疫复合物的形成。通过 SE-HPLC 确定的 EC(50)处的毒液-抗毒液结合似乎大于其他人根据 LD(50)和 ED(50)报道的与中和毒液致死性相关的结合。基于 SE-HPLC 分析毒液-抗毒液结合可以提供先验信息，这与减少在评估抗毒液对毒液诱导的毒性和致死性的保护作用中使用动物有关。