Immunochemical quantification of myoglobin heat denaturation: Comparative studies with monoclonal and polyclonal antibodies
作者:
Didier Levieux,
Annie Levieux,
期刊:
Food and Agricultural Immunology
(Taylor Available online 1996)
卷期:
Volume 8,
issue 2
页码: 111-120
ISSN:0954-0105
年代: 1996
DOI:10.1080/09540109609354909
出版商: Taylor & Francis Group
关键词: Myoglobin;heat;denaturation;monoclonal antibodies;polyclonal antibodies;ELISA;immunodiffusion
数据来源: Taylor
摘要:
Solutions of bovine myoglobin in 0.1 M‐phosphate buffer, pH 6.0, were heated over the temperature range 55–85°C for 60 min. Their immunological reactivity was tested after 0.2 μm filtration using polyclonal antibodies in the single radial immunodiffusion assay (SRID) and six monoclonal antibodies (MAbs) in a competitive and a sandwich ELISA. Reproducible results were obtained in the SRID assay (coefficient of variation (CV) = 4.0%), with a mid‐point denaturation of 79.0 ± 0.4°C. Similar results were obtained with two MAbs (mid‐point denaturation at 79.4 ± 0.5°C and 78.3 ± 0.8°C for competitive and sandwich ELISAs respectively), but the CV% values were higher (8 and 13% respectively). With the remaining four MAbs, quantification was unreliable as immunoreactivity increased during heating with large inter‐assay variations. A careful control of MAb immunoreactivity in situations covering all possible denaturation states of the test protein is an absolute prerequisite to their use in ELISAs for the immunoquantification of protein heat denaturation.
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