Browsing by Author "Chen, WL"

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A novel conformation-dependent monoclonal antibody specific to the native structure of beta-lactoglobulin and its application
(2006-03-01)
Chen, WL
;
Liu, WT
;
Yang, MC
;
Hwang, MT
;
Tsao, JH
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 similar to 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 = 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.
Analyses of beta-lactoglobulin as a thermal marker in raw and commercially processed hulk using a native gel electrophoresis and circular dichroic spectrum
(2002-03-22)
Chen, WL
;
Mao, SJT
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生物科技學系
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Department of Biological Science and Technology
beta-Lactoglobulin is a sensitive thermal maker in dry milk as identified by monoclonal antibody approach
(2003-03-17)
Chen, WL
;
Huang, MT
;
Liu, HC
;
Li, CW
;
Mao, SJT
beta-Lactoglobulin is a thermal marker in processed milk as studied by electrophoresis and circular dichroic spectra
(2005-05-01)
Chen, WL
;
Hwang, MT
;
Liau, CY
;
Ho, JC
;
Hong, KC
As much of the sterilization process involves heat treatment during the preparation of milk on an industrial scale, the unpredictable measures of the process are an essential issue in determining the quality of the milk. The purpose of the present study was to investigate the major protein change(s) of whey proteins in processed milk and extend the knowledge for future reference in the dairy industry. Using a native polyacrylamide gel electrophoresis, we showed almost a 90% loss and denaturation of beta-lactoglobulin (LG), but not alpha-lactalbumin ( LA), in some brands of the processed and dry milks. Immunochemical analysis using Western blotting revealed that part of the loss was attributed to the formation of large multiple forms of LG in the processed product. Such denaturation was presumably associated with the heating procedure used in the process. Essentially, LG was the only major fraction converted to aggregates in milk heated at 95 degrees C for 30 min on 2-dimensional PAGE. The detailed thermal denaturation of purified LG and LA at various temperatures ( 50 to 95 degrees C) and time ( 5 to 960 s) were investigated using a circular dichroic analysis. The maximal changes of ellipticity at 205 nm ( converting beta-structure to disordered structure) were correlated to heating temperature and time. There were no significant conformational changes of LG at temperatures below 70 degrees C for as long as 480 s. Pronounced and rapid changes occurred between 80 to 95 degrees C in a time-dependent manner. Fifty percent of the maximal changes could be reached within 15 s. In conclusion, the unique chemical and immunochemical loss and conformational changes made LG a superior marker for evaluating the thermal processing of milk. The detailed thermal denaturation curves of LG constructed with its time and temperature in this study provide a valuable reference for the dairy industry. We postulate that heat treatment over 80 degrees C in 15 s may induce a significant denaturation of milk LG.
beta-lactoglobulin plays a provocative role in lymphocyte proliferation
(2006-03-07)
Chen, WL
;
Liu, WT
;
Ko, LP
;
Mao, SJT
;
生物科技學系
Distinction between dry and raw milk using monoclonal antibodies prepared against dry milk proteins
(2004-08-01)
Chen, WL
;
Huang, MT
;
Liu, HC
;
Li, CW
;
Mao, SJT
It is well established that the heating process during the preparation of dry milk (DMLK) causes structural changes in some milk proteins. However, because such changes are subtle, whether they can be detected by an immunochemical approach remains questionable. The present study attempted to develop a sensitive mAb that might distinguish the DMLK from freshly prepared raw milk. To test this possibility, we immunized mice with commercially prepared DMLK and produced a panel of mAb. From 900 hybridomas screened using an ELISA, 4 clones were found to be specific to DMLK; the other 68 clones recognized both DMLK and raw milk. In contrast to polyclonal antibodies, only the specific mAb could detect the DMLK spiked into the raw milk at as low as 5% in concentration (vol/vol). Western blot analysis shows that these specific mAb were all directed against beta-lactoglobulin (LG) and LG-milk protein conjugates. These mAb reacted with raw milk heated at 95 for 15 min; the reaction with LG-conjugates, however, was abolished when treated with reducing reagent. Thus, results suggests that a new antigenic epitope was exposed in a heating process, and the thio group of LG cross linked with other protein moiety played a provocative role in mAb recognition. A hypothetical model with respect to the interaction between the mAb and DMLK is proposed and discussed.
Epitope mapping of a monoclonal antibody specific to bovine dry milk - Involvement of residues 66-76 of strand D in thermal denatured beta-lactoglobulin
(2005-02-04)
Song, CY
;
Chen, WL
;
Yang, MC
;
Huang, JP
;
Mao, SJT
beta-Lactoglobulin (beta-LG) is a bovine milk protein sensitive to thermal denaturation. Previously, we demonstrated that such structural change can be detected by a monoclonal antibody (mAb) specific to denatured beta-LG. In the present study, we show a dramatic increase in beta-LG immunoreactivity when heating raw milk between 70 and 80 degreesC. To map out the specific epitope of beta-LG recognized by this mAb, we used a combined strategy including tryptic and CNBr fragments, chemical modifications (acetylation and carboxymethylation), peptide array containing in situ synthesized peptides, and a synthetic soluble peptide for immunoassays. The antigenic determinant we defined was exactly located within the D strand (residues 66-76) of beta-LG. Circular dichroic spectral analysis shows that carboxymethylation on beta-LG not only resulted in a substantial loss of beta-configuration but also exerted a 10 times increase in immunoreactivity as compared with heated beta-LG. The result suggests that a further disordered structure occurred in beta-LG and thus rendered the mAb recognition. Mutations on each charged residue (three Lys and one Glu) revealed that Lys-69 and Glu-74 were extremely essential in maintaining the antigenic structure. We also show an inverse relationship between the immunoreactivity in heated beta-LG and its binding to retinol or palmitic acid. Most interestingly, pH 9-10, which neutralizes the Lys groups of beta-LG, not only reduced its immunoreactivity but also its binding to palmitic acid implicating a role of Lys-69. Taken together, we concluded that strand D of beta-LG participated in the thermal denaturation between 70 and 80 degreesC and the binding to retinol and palmitic acid. The antigenic and biochemical roles of mAb specific to D strand are discussed in detail.
Modeling non-uniform-sediment fluvial process by characteristics method - Closure
(1996-04-01)
Yeh, KC
;
Li, SJ
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Chen, WL
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交大名義發表
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土木工程學系
Purification of human plasma haptoglobin by hemoglobin-affinity column chromatography
(2003-06-25)
Liau, CY
;
Chang, TM
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Pan, JP
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Chen, WL
;
Mao, SJT
Haptoglobin (Hp) is an acute-phase protein; its plasma levels increase consistently in response to infection and inflammation. The concentration of human plasma Up is ranged between 1 and 1.5 mg/ml. Similar to blood type, individual human Hp is classified as Hp 1-1, 2-1, or 2-2. The structural and functional analysis of the Hp, however, has not been studied in detail due to its difficult isolation procedure. Previously, we reported a single step for the purification of porcine Hp. In this study, we established a purification method using a high capacity hemoglobin-affinity column. Briefly, DEAE-purified human hemoglobin was first coupled to Sepharose 4B to prepare an affinity column in a 15-ml bed volume. Following a flow through of human plasma and an extensive wash, the bound material was eluted with a solution of 0.15 M NaCl, pH 11 (adjusted by ammonium), to remove low-affinity bound proteins. The high-affinity bound Hp was then eluted with 0.15 M NaCl containing 5 M urea, pH 11, and collected in tubes containing 100 mul of 1 M Tris buffer, pH 7.0. The biological activity of dialyzed Hp was retained as it formed a complex with hemoglobin on, a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Using this procedure, approximately 10 mg of Hp 1-1, with homogeneity greater than 96%, was obtained from 15 nil of human plasma. Affinity purified Up 2-1 or 2-2, however, contained trace amounts of apoA-I with the similar approach. The Hp could be further purified by HPLC using a Superose 12 gel-permeation chromatography, if desired, to achieve 100% purity. All the phenotypes of purified Hp consisted of alpha and beta chains on SDS-PAGE in the presence of a reducing reagent, further confirmed by a Western blot analysis. We conclude that human hemoglobin-affinity column was most suitable for the isolation of Hp 1-1 in large quantities. Whereas, one additional step using a gel-permeation was necessary for that of Hp 2-1 and 2-2. (C) 2003 Elsevier Science B.V. All rights reserved.
Thermal denaturation of beta-lactoglobulin as probed by an monoclomal antibody
(2004-03-23)
Chen, WL
;
Huang, MT
;
Li, CW
;
Liu, HC
;
Mao, SJT