Analysis of antibody-antigen interactions in mixtures containing reactive and nonreactive components using size-exclusion high-performance (pressure) liquid chromatography

Charles G. Sanny; Joseph A. Price

doi:10.1006/abio.2001.5204

Analysis of antibody-antigen interactions in mixtures containing reactive and nonreactive components using size-exclusion high-performance (pressure) liquid chromatography

Charles G. Sanny, Joseph A. Price

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

7 Scopus citations

Abstract

Analysis of antibody-antigen interactions using size-exclusion high-performance (pressure) liquid chromatography was applied to a polyvalent system composed of both reactive and nonreactive components. Mixtures containing varying concentrations of antivenin (Crotalidae) polyvalent (equine origin) and either Crotalus atrox (Western diamondback rattlesnake) venom (CV) or isolated C. atrox phospholipase A₂ (PLA₂) were separated using a Bio-Sil SEC 250-5 size-exclusion column (300 × 7.8 mm). Major regions containing high-molecular-weight aggregates, antivenin IgG, and CV components or PLA₂ could be identified from the elution profiles. Changes in elution profile areas could be modeled by equations derived from the law of mass action that included values for the maximum fraction of reactivity, antigen valence, apparent binding constants, profile area proportionality constants, and nonreactive profile area. The analysis was simple, fast, and readily interpretable and may be applicable to a variety of situations in which stable antigen-antibody complexes are formed in the presence of nonreactive components.

Original language	English
Pages (from-to)	57-65
Number of pages	9
Journal	Analytical Biochemistry
Volume	295
Issue number	1
DOIs	https://doi.org/10.1006/abio.2001.5204
State	Published - 1 Aug 2001

Keywords

Binding assays
Chromatography
HPLC
Immune complexes
Immunoglobulins

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10.1006/abio.2001.5204

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title = "Analysis of antibody-antigen interactions in mixtures containing reactive and nonreactive components using size-exclusion high-performance (pressure) liquid chromatography",

abstract = "Analysis of antibody-antigen interactions using size-exclusion high-performance (pressure) liquid chromatography was applied to a polyvalent system composed of both reactive and nonreactive components. Mixtures containing varying concentrations of antivenin (Crotalidae) polyvalent (equine origin) and either Crotalus atrox (Western diamondback rattlesnake) venom (CV) or isolated C. atrox phospholipase A2 (PLA2) were separated using a Bio-Sil SEC 250-5 size-exclusion column (300 × 7.8 mm). Major regions containing high-molecular-weight aggregates, antivenin IgG, and CV components or PLA2 could be identified from the elution profiles. Changes in elution profile areas could be modeled by equations derived from the law of mass action that included values for the maximum fraction of reactivity, antigen valence, apparent binding constants, profile area proportionality constants, and nonreactive profile area. The analysis was simple, fast, and readily interpretable and may be applicable to a variety of situations in which stable antigen-antibody complexes are formed in the presence of nonreactive components.",

keywords = "Binding assays, Chromatography, HPLC, Immune complexes, Immunoglobulins",

author = "Sanny, {Charles G.} and Price, {Joseph A.}",

note = "Funding Information: The authors thank Oza M. McClain for her technical support in this study. Financial support was provided by the Office of Research and the Department of Biochemistry/Microbiology of the Oklahoma State University College of Osteopathic Medicine and NIH Grant NIH R15 GM46117-01A1. We also thank C. Ownby for generously supplying the C. atrox venom.",

year = "2001",

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doi = "10.1006/abio.2001.5204",

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N2 - Analysis of antibody-antigen interactions using size-exclusion high-performance (pressure) liquid chromatography was applied to a polyvalent system composed of both reactive and nonreactive components. Mixtures containing varying concentrations of antivenin (Crotalidae) polyvalent (equine origin) and either Crotalus atrox (Western diamondback rattlesnake) venom (CV) or isolated C. atrox phospholipase A2 (PLA2) were separated using a Bio-Sil SEC 250-5 size-exclusion column (300 × 7.8 mm). Major regions containing high-molecular-weight aggregates, antivenin IgG, and CV components or PLA2 could be identified from the elution profiles. Changes in elution profile areas could be modeled by equations derived from the law of mass action that included values for the maximum fraction of reactivity, antigen valence, apparent binding constants, profile area proportionality constants, and nonreactive profile area. The analysis was simple, fast, and readily interpretable and may be applicable to a variety of situations in which stable antigen-antibody complexes are formed in the presence of nonreactive components.

AB - Analysis of antibody-antigen interactions using size-exclusion high-performance (pressure) liquid chromatography was applied to a polyvalent system composed of both reactive and nonreactive components. Mixtures containing varying concentrations of antivenin (Crotalidae) polyvalent (equine origin) and either Crotalus atrox (Western diamondback rattlesnake) venom (CV) or isolated C. atrox phospholipase A2 (PLA2) were separated using a Bio-Sil SEC 250-5 size-exclusion column (300 × 7.8 mm). Major regions containing high-molecular-weight aggregates, antivenin IgG, and CV components or PLA2 could be identified from the elution profiles. Changes in elution profile areas could be modeled by equations derived from the law of mass action that included values for the maximum fraction of reactivity, antigen valence, apparent binding constants, profile area proportionality constants, and nonreactive profile area. The analysis was simple, fast, and readily interpretable and may be applicable to a variety of situations in which stable antigen-antibody complexes are formed in the presence of nonreactive components.

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KW - Chromatography

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Analysis of antibody-antigen interactions in mixtures containing reactive and nonreactive components using size-exclusion high-performance (pressure) liquid chromatography

Abstract

Keywords

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