Chen W L, Liu W T, Yang M C, Hwang M T, Tsao J H, Mao S J T
Research Institute of Biochemical Engineering, Department of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taiwan, Republic of China.
J Dairy Sci. 2006 Mar;89(3):912-21. doi: 10.3168/jds.S0022-0302(06)72156-7.
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.
熔融球蛋白被认为是蛋白质折叠与解折叠过程中的一般中间体。β-乳球蛋白（β-LG）是主要的牛乳清蛋白之一，约占总乳蛋白的10%至15%。我们最近已确定β-LG是评估热处理牛奶的优良标志物。β-LG的D链参与不可逆的热解折叠过程，这一过程可通过针对热变性β-LG的单克隆抗体（mAb）进行检测。在本研究中，我们使用天然β-LG作为免疫原，以检验是否能够制备出针对天然β-LG的特异性单克隆抗体这一假设。结果，制备出了一种针对β-LG天然结构的单克隆抗体（4H11E8）。该抗体不识别β-LG的热变性形式，如其二聚体和聚集体。使用这种“天然”单克隆抗体进行的免疫分析表明，β-LG在温度≤70℃时具有稳定性。随着时间推移，β-乳球蛋白在70至80℃之间开始降解。变性与β-LG的转变温度相关。对β-LG的半胱氨酸（羧甲基化）或带正电荷残基（乙酰化）进行进一步化学修饰，完全消除了其免疫反应性，证实了该单克隆抗体的构象依赖性本质。使用竞争性酶联免疫吸附测定法，4H11E8单克隆抗体能够测定商业加工牛奶中天然β-LG的含量。在所测试的本地品牌中，天然β-LG的浓度差异显著。从技术角度来看，本研究中制备的单克隆抗体与牛奶及其他乳制品热消毒适宜工艺的设计与操作相关。
