CroFabtrade mark total anti-venom activity measured by SE-HPLC, and anti-PLA(2) activity assayed in vitro at physiological pH.
One problem in the development and refinement of anti-venoms is ascertaining both overall anti-venom reactivity and which key toxins are neutralized. Here we show by SE-HPLC that the in vitro reaction of CroFab anti-venin with Crotalus atrox venom asymptotically nears completion (>95%) by 11 min at 4 degrees C by following the change in area under chromatographic peaks. The peaks for reactants decrease and the formation of high molecular weight complexes increases with time. To assay the large number of samples a new microplate format phospholipase A(2) (PLA(2)) assay at an initial pH of 7.5 was developed using phosphotidyl choline as the substrate. The change in absorbance is due to the pH change caused by release of fatty acids, and is linear with dilution of enzyme. This choice of substrate limits detection to PLA(2) and nonspecific esterase (if any) activities. The neutralization mixtures show a dose dependent (CroFab anti-venin) inactivation of C. atrox PLA(2) activity approaching a maximum of 85% neutralization. This approach of revealing antibody binding to venom components coupled with enzyme activity measurements is effective and may lead to greater in vitro assessment of antivenin activity in product development, and less routine use of mouse lethality assays.
Price
Joseph A
JA
Department of Pathology, Oklahoma State University, Center for Health Sciences, Tulsa, OK 74107-1898, USA. joseph.price@okstate.edu
Sanny
Charles G
CG
D000818:Animals; D000906:Antibodies / Q000276:immunology; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D000997:Antivenins / Q000737:chemistry / Q000494:pharmacology*; D002851:Chromatography, High Pressure Liquid; D003435:Crotalid Venoms / Q000276:immunology*; D006863:Hydrogen-Ion Concentration; D007140:Immunoglobulin Fab Fragments; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000494:pharmacology*; D066298:In Vitro Techniques; D009500:Neutralization Tests; D010741:Phospholipases A / Q000378:metabolism*; D017819:Viperidae
D000906:Antibodies; D000997:Antivenins; D003435:Crotalid Venoms; C431183:Crotalidae Polyvalent immune Fab; D007140:Immunoglobulin Fab Fragments; D007128:Immunoglobulin Fragments; D010741:Phospholipases A