Fluorescence quenching of heat‐treated immunoglobulins and determination of their biological activity by PCFIA
作者:
JackN. Losso,
Shuryo Nakai,
期刊:
Food and Agricultural Immunology
(Taylor Available online 1994)
卷期:
Volume 6,
issue 3
页码: 287-295
ISSN:0954-0105
年代: 1994
DOI:10.1080/09540109409354840
出版商: Taylor & Francis Group
关键词: Bovine IgG;chicken IgY;fluorescence quenching;PCFIA
数据来源: Taylor
摘要:
Samples of bovine milk, from cows vaccinated and non‐vaccinated against a wide range of pathogenic bacteria includingSalmonella 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 fromS. 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 immuno globulin 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, kqincreased 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‐labeled antibody bound to the antigen. The results are then similar to trends observed using ELISA.
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