Application of an ELISA-elution assay as a screening tool for dissociation of yolk antibody-antigen complexes.
A modified enzyme-linked immunosorbent assay termed ELISA-elution assay was used as a screening tool to compare the efficiency of eluents for the dissociation of hen yolk immunoglobulin IgY bovine IgG complexes. The potential denaturing effects of the eluents were also monitored. Different buffers (pH 2.3-7.5), containing various types and concentrations of salts (NaCl, (NH4)2SO4 and MgCl2) as well as polyols (ethylene glycol (EG) and glycerol) were compared to the commonly reported glycine x HCl (pH 2.8) buffer and to a commercially available eluent, Actisep. Acidic pH buffers, Actisep and MgCl2 (3.5 M with EG or 4 M without EG) all successfully dissociated IgY from immobilized IgG. However, some denaturation was apparent using MgCl, and, to a lesser extent, Actisep. Furthermore, these same eluents demonstrated a diminished ability for liberating IgG from immobilized IgY(IgG). Information on eluent efficacy obtained by the ELISA-elution assays was applied to selectively isolate lower affinity antibodies for immunoaffinity column chromatography.
Kummer
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University of British Columbia, Department of Food Science, Vancouver, Canada.
Li-Chan
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D000645:Ammonium Sulfate; D000818:Animals; D000936:Antigen-Antibody Complex / Q000276:immunology*; D002021:Buffers; D002417:Cattle; D002645:Chickens; D002846:Chromatography, Affinity; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D007074:Immunoglobulin G / Q000276:immunology*; D007136:Immunoglobulins / Q000276:immunology*; D015636:Magnesium Chloride; D012965:Sodium Chloride
D000936:Antigen-Antibody Complex; D002021:Buffers; C016512:IgY; D007074:Immunoglobulin G; D007136:Immunoglobulins; D015636:Magnesium Chloride; D012965:Sodium Chloride; D000645:Ammonium Sulfate