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

Abstract

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.

β-Lactoglobulin Influences Human Immunity and Promotes Cell Proliferation

“…Then, the supernatant was carefully removed and the remaining fraction (whey protein) was saturated with 30% ammonium sulfate. LG was purified from the top fraction using a G-150 chromatography column as described previously [5, 36, 37]. HLG is LG that was heated at 95°C for 5 min.…”

An improved method for haptoglobin 1-1, 2-1, and 2-2 purification using monoclonal antibody affinity chromatography in the presence of sodium dodecyl sulfate☆

“…Monoclonal antibodies (mAb) against human Hp were produced using procedures previously described in our laboratory [12,13]. Rabbit anti-Goat IgG was purchased from Chemicon (Temecula, CA).…”

“…Monoclonal antibodies prepared against human Hp were characterized according to the standard procedures established in our laboratory [12,13]. MAb 3H8 prepared against ␣-chain of Hp, which possessed the highest binding affinity to Hp, was selected for preparation of the affinity column.…”

Rational Design for Crystallization of β-Lactoglobulin and Vitamin D3 Complex: Revealing a Secondary Binding Site

“…10 In a previous study, we first conducted a ligand binding assay using the fluorescence changes by retinol, palmitic LG was 2:1, whereas that of retinol or palmitic acid was 1:1 (Table 1), 26 suggesting that there was another binding site for the vitamin D 3 molecule. Second, we manipulated the binding capability by switching off the gate of calyx at low pH (2)(3)(4)(5)(6) 15 to further substantiate the "two site hypothesis". As expected, the binding capability of retinol and palmitic acid diminished under this condition, but it retained the vitamin D binding with a vitamin D 3 to LG stoichiometry of 1:1 (Table 1).…”

“…1 Because of its thermally unstable and molten-globule nature, LG has been studied extensively for its physical and biochemical properties. [2][3][4][5][6][7] Some essential functions of LG including hypocholesterolemic effect, 8 retinol transport, 9,10 and antioxidant activity [11][12][13][14] have been reported. According to the crystal structure, LG comprises predominantly a -sheet configuration containing nine antiparallel -strands from A to I (Figure 1a).…”