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A Novel Conformation-Dependent Monoclonal Antibody Specific to the Native Structure of ß-Lactoglobulin and Its Application

W. L. Chen^*, W. T. Liu^*, M. C. Yang^*, M. T. Hwang^*, J. H. Tsao and S. J. T. Mao^*,1

^* Research Institute of Biochemical Engineering, Department of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taiwan, Republic of China
Yong Rong Dairy, Chyayi, Taiwan, Republic of China

¹ Corresponding author: mao1010{at}ms7.hinet.net

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

Key Words: ß-lactoglobulin structure • immunoassay • native monoclonal antibody • thermal denaturation

This article has been cited by other articles:

				H. C. Liu, W. L. Chen, and S. J. T. Mao Antioxidant Nature of Bovine Milk {beta}-Lactoglobulin J Dairy Sci, February 1, 2007; 90(2): 547 - 555. [Abstract] [Full Text] [PDF]