A novel conformation-dependent monoclonal antibody specific to the native structure of β-lactoglobulin and its application

Wen-Liang Chen, W. T. Liu, M. C. Yang, M. T. Hwang, J. H. Tsao, S. J.T. Mao*

*Corresponding author for this work

Research output: Contribution to journalArticle

18 Scopus citations

Abstract

Molten globules are thought to be general intermediates in protein folding and unfolding. β-lactoglobulin (β-LG) is one of the major bovine whey proteins, constituting ∼10 to 15% of total milk proteins. We have recently identified β-LG as a superior marker for evaluating thermally processed milk. Strand D of β-LG participates in irreversible thermal unfolding as probed by a monoclonal antibody (mAb) specific to thermally denatured β-LG. In the present study, we used native β-LG as an immunogen to test the hypothesis that a specific mAb against the native β-LG could be established. As result, a mAb (4H11E8) directed against the native structure of β-LG was made. The antibody did not recognize the heat-denatured form of β-LG, such as its dimer and aggregates. Immunoassay using this "native" mAb showed that the stability of β-LG was at temperatures ≤70°C. β-Lactoglobulin began to deteriorate between 70 and 80°C over time. The denaturation was correlated with the transition temperature of β-LG. Further chemical modification of Cys (carboxymethylation) or positively charged residues (acetylation) of β-LG totally abolished its immunoreactivity, confirming the conformation-dependent nature of this mAb. Using competitive ELISA, the 4H11E8 mAb could determine the native β-LG content in commercially processed milks. Concentrations of native β-LG varied significantly among the local brands tested. From a technological standpoint, the mAb prepared in this study is relevant to the design and operation of appropriate processes for thermal sanitation of milk and of other dairy products.

Original languageEnglish
Pages (from-to)912-921
Number of pages10
JournalJournal of Dairy Science
Volume89
Issue number3
DOIs
StatePublished - 1 Jan 2006

Keywords

  • Immunoassay
  • Native monoclonal antibody
  • Thermal denaturation
  • β-lactoglobulin structure

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