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

Charles G. Sanny

doi:10.1016/j.toxicon.2011.03.003

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

Charles G. Sanny

Biochemistry & Microbiology

Research output: Contribution to journal › Article › peer-review

10 Scopus citations

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 EC₅₀ and {Area_bnd}_max. The relative magnitudes of EC₅₀, 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 A₂ (PLA₂) activity of C. atrox, A. c. c., and A. p. l. venom by FabAV as reported in the literature. Maximum neutralization of PLA₂ activity occurred, in general, prior to maximum immune complex formation. Venom-antivenin binding at EC₅₀ determined via SE-HPLC appeared to be greater than binding associated with neutralization of venom lethality in mice based on LD₅₀ and ED₅₀ 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.

Original language	English
Pages (from-to)	871-881
Number of pages	11
Journal	Toxicon
Volume	57
Issue number	6
DOIs	https://doi.org/10.1016/j.toxicon.2011.03.003
State	Published - May 2011

Keywords

Agkistrodon
Antivenin
Antivenom
Crotalidae polyvalent immune fab (ovine)
Crotalus
HPLC
Immune complex

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@article{53727729d9c34affb4b6a50e49992bdb,

title = "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",

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 ({Areabnd}) were evaluated using a logistic dose-response equation to estimate EC50 and {Areabnd}max. The relative magnitudes of EC50, 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 {Areabnd}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 A2 (PLA2) activity of C. atrox, A. c. c., and A. p. l. venom by FabAV as reported in the literature. Maximum neutralization of PLA2 activity occurred, in general, prior to maximum immune complex formation. Venom-antivenin binding at EC50 determined via SE-HPLC appeared to be greater than binding associated with neutralization of venom lethality in mice based on LD50 and ED50 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.",

keywords = "Agkistrodon, Antivenin, Antivenom, Crotalidae polyvalent immune fab (ovine), Crotalus, HPLC, Immune complex",

author = "Sanny, {Charles G.}",

note = "Funding Information: The author wishes to thank the Office of Research and the Department of Biochemistry and Microbiology of Oklahoma State University Center for Health Sciences for financial support, ProTherics, Inc., for their generous gift of Crotalidae Polyvalent Immune Fab (Ovine) antivenin, Randall E. Benton for his initial work in southern copperhead ( A . c . c .) venom and antivenin binding, and Willy A. Bustinza for his critical evaluation of the literature and suggestions during the preparation of this manuscript.",

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doi = "10.1016/j.toxicon.2011.03.003",

language = "English",

volume = "57",

pages = "871--881",

journal = "Toxicon",

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AU - Sanny, Charles G.

N1 - Funding Information: The author wishes to thank the Office of Research and the Department of Biochemistry and Microbiology of Oklahoma State University Center for Health Sciences for financial support, ProTherics, Inc., for their generous gift of Crotalidae Polyvalent Immune Fab (Ovine) antivenin, Randall E. Benton for his initial work in southern copperhead ( A . c . c .) venom and antivenin binding, and Willy A. Bustinza for his critical evaluation of the literature and suggestions during the preparation of this manuscript.

PY - 2011/5

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N2 - 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 ({Areabnd}) were evaluated using a logistic dose-response equation to estimate EC50 and {Areabnd}max. The relative magnitudes of EC50, 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 {Areabnd}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 A2 (PLA2) activity of C. atrox, A. c. c., and A. p. l. venom by FabAV as reported in the literature. Maximum neutralization of PLA2 activity occurred, in general, prior to maximum immune complex formation. Venom-antivenin binding at EC50 determined via SE-HPLC appeared to be greater than binding associated with neutralization of venom lethality in mice based on LD50 and ED50 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.

AB - 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 ({Areabnd}) were evaluated using a logistic dose-response equation to estimate EC50 and {Areabnd}max. The relative magnitudes of EC50, 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 {Areabnd}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 A2 (PLA2) activity of C. atrox, A. c. c., and A. p. l. venom by FabAV as reported in the literature. Maximum neutralization of PLA2 activity occurred, in general, prior to maximum immune complex formation. Venom-antivenin binding at EC50 determined via SE-HPLC appeared to be greater than binding associated with neutralization of venom lethality in mice based on LD50 and ED50 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.

KW - Agkistrodon

KW - Antivenin

KW - Antivenom

KW - Crotalidae polyvalent immune fab (ovine)

KW - Crotalus

KW - HPLC

KW - Immune complex

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U2 - 10.1016/j.toxicon.2011.03.003

DO - 10.1016/j.toxicon.2011.03.003

M3 - Article

C2 - 21392522

AN - SCOPUS:79955034114

SN - 0041-0101

VL - 57

SP - 871

EP - 881

JO - Toxicon

JF - Toxicon

IS - 6

ER -

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

Abstract

Keywords

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