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

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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 languageEnglish
Pages (from-to)57-65
Number of pages9
JournalAnalytical Biochemistry
Volume295
Issue number1
DOIs
StatePublished - 1 Aug 2001

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Antivenins
Phospholipases A2
Liquid chromatography
Crotalid Venoms
High Pressure Liquid Chromatography
Crotalus
Antigens
Antibodies
Antigen-Antibody Complex
Horses
Immunoglobulin G
Molecular Weight
Molecular weight

Keywords

  • Binding assays
  • Chromatography
  • HPLC
  • Immune complexes
  • Immunoglobulins

Cite this

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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.}",
year = "2001",
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language = "English",
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T1 - Analysis of antibody-antigen interactions in mixtures containing reactive and nonreactive components using size-exclusion high-performance (pressure) liquid chromatography

AU - Sanny, Charles G.

AU - Price, Joseph A.

PY - 2001/8/1

Y1 - 2001/8/1

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.

KW - Binding assays

KW - Chromatography

KW - HPLC

KW - Immune complexes

KW - Immunoglobulins

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DO - 10.1006/abio.2001.5204

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