A novel conformation-dependent monoclonal antibody specific to the native structure of beta-lactoglobulin and its application.
Molten globules are thought to be general intermediates in protein folding and unfolding. beta-lactoglobulin (beta-LG) is one of the major bovine whey proteins, constituting approximately 10 to 15% of total milk proteins. We have recently identified beta-LG as a superior marker for evaluating thermally processed milk. Strand D of beta-LG participates in irreversible thermal unfolding as probed by a monoclonal antibody (mAb) specific to thermally denatured beta-LG. In the present study, we used native beta-LG as an immunogen to test the hypothesis that a specific mAb against the native beta-LG could be established. As result, a mAb (4H11E8) directed against the native structure of beta-LG was made. The antibody did not recognize the heat-denatured form of beta-LG, such as its dimer and aggregates. Immunoassay using this "native" mAb showed that the stability of beta-LG was at temperatures < or =70 degrees C. beta-Lactoglobulin began to deteriorate between 70 and 80 degrees C over time. The denaturation was correlated with the transition temperature of beta-LG. Further chemical modification of Cys (carboxymethylation) or positively charged residues (acetylation) of beta-LG totally abolished its immunoreactivity, confirming the conformation-dependent nature of this mAb. Using competitive ELISA, the 4H11E8 mAb could determine the native beta-LG content in commercially processed milks. Concentrations of native beta-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.
Chen
W L
WL
Research Institute of Biochemical Engineering, Department of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taiwan, Republic of China.
Liu
W T
WT
Yang
M C
MC
Hwang
M T
MT
Tsao
J H
JH
Mao
S J T
SJ
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D015153:Blotting, Western; D003488:Cyanogen Bromide; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005511:Food Handling / Q000379:methods; D006358:Hot Temperature; D007782:Lactoglobulins / Q000032:analysis / Q000737:chemistry* / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008892:Milk / Q000737:chemistry*; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D014357:Trypsin / Q000378:metabolism
D000911:Antibodies, Monoclonal; D007782:Lactoglobulins; D010446:Peptide Fragments; D014357:Trypsin; D003488:Cyanogen Bromide