Fluorescence Quenching of Heat-treated Immunoglobulins and Determination of Their Biological Activity by PCFIA

Document Type

Article

Publication Date

1-1-1994

Abstract

Samples of bovine milk, from cows vaccinated and non-vaccinated against a wide range of pathogenic bacteria including Salmonella enteritidis, previously heated to different temperatures between 37°C and 75°C, were examined for specificity and the ability of immunoglobulin G (IgG) to bind to antigen (lipopolysaccharide fraction from S. enteritidis) in a particle concentration fluorescence immunoassay (PCFIA), Antigen binding activity falsely increased with heating temperature. This trend was not observed when using enzyme-linked immunosorbent assays (ELISAs). In order to identify the factor(s) responsible for the increased fluorescence values when using PCFIA, purified bovine and chicken IgG molecules were heat treated and the fluorescence values recorded. The fluorescence of the purified immunoglobulins also increased with heating temperature. Tryptophan (trp) is present at the active binding site of IgG molecules. Quenching of trp residues with 0.6 M-acrylamide demonstrated that the increase in fluorescence observed with increased heating temperature was associated with the presence of trp residues in the immunoglobulin molecule (antigen binding site). When the Stern-Volmer equation was applied to the fluorescence data, the results indicated that none of the proteins studied gave an evident non-linear plot. The bimolecular quenching constant, kq increased from 21°C to 75°C, indicating increased exposure of trp residues to solvent. A method is proposed to use acrylamide as a trp fluorescence quencher in studies involving the determination of biological activity of previously heat-treated immunoglobulins by PCFIA. The measured fluorescence is then only associated with the concentration of fluorescein isothiocyanate-la-beled antibody bound to the antigen. The results are then similar to trends observed using ELISA. © 1994, Taylor & Francis Group, LLC. All rights reserved.

Publication Source (Journal or Book title)

Food and Agricultural Immunology

First Page

287

Last Page

295

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