Optimum dissociating condition for immunoaffinity and preferential isolation of antibodies with high specific activity.
Using an immunoaffinity model consisting of a high performance matrix (Affi-prep-10) with normal human IgG as ligand, and hyperimmune goat anti-human IgG (heavy and light chain active) antibodies, we compared the efficacies of 13 elution reagents. Efficacy was considered in terms of specific activity and total quantitative recovery of the eluted antibody. The optimum, general-purpose dissociation reagent for this immunoaffinity system is 3.0 M MgCl2.6H2O, 0.075 M Hepes/NaOH, with 25% ethylene glycol pH 7.20. The antibodies recovered from diluted (1/2) goat serum with this dissociation reagent have a SpAct of 1.87 times and a total recovery of 6.33 times that of a comparable experiment using the usual eluant of 1.0 M glycine/HCl, pH 2.00. We also demonstrated that the SpAct of antibodies recovered from immunoaffinity procedures performed under antibody excess and antigen limiting conditions is 2.36-8.00 times higher than that produced by antigen excess and antibody limiting configurations.
Tsang
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Parasitic Diseases Branch, Centers for Disease Control, U.S. Department of Health and Human Services, Atlanta, GA 30333.
Wilkins
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D000818:Animals; D000937:Antigen-Antibody Reactions; D002021:Buffers; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D006041:Goats; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000302:isolation & purification*; D007202:Indicators and Reagents; D008024:Ligands
D002021:Buffers; D007074:Immunoglobulin G; D007202:Indicators and Reagents; D008024:Ligands