Browsing by Author "Mao, SJT"

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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.
Accuracy of the EasyTouch blood glucose self-monitoring system: a study of 516 cases
(2004-11-01)
Dai, KS
;
Tai, DY
;
Ho, P
;
Chen, CC
;
Peng, WC
Background: Self-monitoring blood glucose device is an important toot for diabetes patients to efficiently control their blood glucose concentrations. We evaluated the accuracy of EasyTouch glucose monitoring system. Methods: Capillary blood glucose concentrations measured using EasyTouch and the reference values obtained from Yellow Springs Instruments (YSI) 2300 STAT were performed in the Department of Laboratory Medicine, Wei-Gong Memorial Hospital. Results were evaluated using (1) linear regression analysis, (2) Clarke Error Grid analysis, (3) percentage of readings within a defined range of deviation from the reference value, (4) bias plots, and (5) coefficients of variation (CVs) calculated from 60 measurements in series. Results: The window of the 516 EasyTouch readings covered a range from 42 to 555 mg/dl. Linear regression analysis yielded a regression slope 0.9972, intercept 1.899 mg/dl, r(2) 0.9571, and Syx 14.89 mg/dl. A Clarke Error Grid analysis showed 100% of the EasyTouch readings in clinically acceptable zones A and B. Of the EasyTouch readings, 98.3%, 91.9%, 78.3% and 46.9% were found within +/-20%, +/-15%, +/-10%, and +/-5%, respectively, of the reference values. Further analysis showed that the percentage of EasyTouch readings within the defined intervals was similar in three glucose ranges (less than or equal to100, 101-200, and greater than or equal to201 mg/dl). The CVs for the four lots of strips (lot 1 to lot 4) ranged from 3.5 to 5.5%, 2.1 to 4.8%, 1.8 to 3.6%, and 3.0 to 5.7%, respectively. Conclusions: EasyTouch provides high accurate and precise glucose readings over a wide range of glucose concentrations. (C) 2004 Elsevier B.V. All rights reserved.
An evaluation of clinical accuracy of the EasyTouch blood uric acid self-monitoring system
(2005-03-01)
Dai, KS
;
Tai, DY
;
Ho, P
;
Chen, CC
;
Peng, WC
Objectives: Self-monitoring blood uric acid device is an important tool for patients to efficiently monitor their blood uric acid concentrations. The objective of the present study was to evaluate the accuracy of EasyTouch uric acid monitoring system. Design and methods: Capillary blood uric acid concentrations measured using EasyTouch and the reference values obtained from COBAS MIRA were performed in the Department of Laboratory Medicine, Wei-Gong Memorial Hospital. Results were evaluated using (1) linear regression analysis, (2) percentage of readings within a defined range of deviation from the reference value, and (3) coefficients of variation (CVs) calculated from 60 measurements in series. Results: The window of the 177 EasyTouch readings covered a wide range from 0.1785 to 0.6367 mmol/L (3-10.7 mg/dL). Linear regression analysis yielded a regression slope of 0.975, an intercept of 0.0118 mmol/L and an R-2 of 0.8966. Of the EasyTouch readings, 64 (36.2%), 61 (34.5%), 34 (19.2%), 9 (5.08%), and 9 (5.08%) were within the intervals of <5%, 5-10%, 10-15%, 15-17%, and >17%, respectively, of the reference values. Further analysis for the performance of each lot of strips showed that both coefficients of correlation and the percentages of readings within the CLIA's criterion (+/-17%), respectively, were in a narrow range from 0.8777 to 0.9541 and from 92.1% to 100%. The CVs for the seven lots of strips (lot I to lot 7) ranged from 2.93% to 6.33%, 3.2% to 5.9%, 3.64% to 7.0%, 2.84% to 7.6%, 2.68% to 5.42%, 3.03% to 6.93%, and 3.18% to 5.17%, respectively. Conclusion: EasyTouch is an acceptable diagnostic device which provides accurate uric acid measurements. (C) 2004 The Canadian Society of Clinical Chemists. All rights reserved.
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
;
生物科技學系
;
Department of Biological Science and Technology
Antioxidant role of human haptoglobin
(2004-08-01)
Tseng, CF
;
Lin, CC
;
Huang, HY
;
Liu, HC
;
Mao, SJT
Human plasma haptoglobin (Hp) is classified according to three phenotypes: Hp 1-1, 2-1, and 2-2 attributed by their two common alleles 1 and 2. Clinically, the 2-2 phenotype is associated with the risk of cardiovascular diseases and diabetes mellitus in patients. In this study, we demonstrate that Hp is an extremely potent antioxidant, which directly protects low density lipoprotein from Cu2+-induced oxidation. Its potency was markedly superior to probucol (one of the most potent antioxidants). Ranking of the IC50 of antioxidant activity was as follows: Hp 1-1 > Hp 2-1 > Hp 2-2 > probucol > vitamin E. Blockage of disulfide linkages between Hp subunits, not only abolished the alpha-helical content but also diminished the ability of Hp to form a complex with hemoglobin. The modified Hp subunits exerted almost 4 times greater antioxidant activity than that of native Hp. To investigate the antioxidant role of Hp on the cellular level, the cDNA of Hp 1-1 was cloned, introduced into the pcDNA3.0 vector which contains the cytomega lovirus promoter and transfected into chinese hamster ovary (CHO)-K1 cells. Following transfection, CHO cells were able to express Hp 1-1 protein and significantly (p < 0.001) elevated cell tolerance against oxidative stress. Transfected cells showed 2-fold higher resistance to hydrogen peroxide exposure for 24 h compared to control cells. Thus, Hp plays a provocative antioxidant role as demonstrated by our in vitro and ex vivo studies.
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
Cloning and expression of human haptoglobin subunits in Escherichia coli: The potential use of mini-haptoglobin as an antioxidant
(2006-03-07)
Lai, IH
;
Tsai, TI
;
Lai, WY
;
Lin, HH
;
Mao, SJT
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.
Epidemiology of haptoglobin phenotypes in chronic diseases
(2003-03-17)
Cheng, TM
;
Pan, JP
;
Wang, JS
;
Mao, SJT
;
生物科技學系
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.
Expression, characterization, and purification of recombinant porcine lactoferrin in Pichia pastoris
(2002-06-01)
Wang, SH
;
Yang, TS
;
Lin, SM
;
Tsai, MS
;
Wu, SC
Recombinant porcine lactoferrin (rPLF) was synthesized in Pichia pastoris using a constitutive promoter from the glyceraldehyde-3-phosphate dehydrogenase gene. Strains expressing rPLF with its own signal sequence or with that from the yeast alpha-mating factor (alpha-MF) were able to produce and secrete rPLF, but levels were consistently higher using alpha-MF constructs. In contrast, P. pastoris strains that expressed rPLF without a signal sequence produced the protein in an insoluble intracellular form. Increasing the initial pH of shake-flask culture medium from 6.0 to 7.0 or adding ferric ions to the medium (to 100 muM) resulted irk significant improvements in expression of rPLF from P. pastoris. Expression levels (approximately 12 mg/L) were much higher than those observed from Saccharomyces cerevisiae strains (1-2 mg/L). P pastoris-secreted rPLF was isolated and purified via a one-step simple procedure using a heparin column. The molecular size (78 kDa), isoelectric point (8.8-9.0), N-terminal amino acid sequence, and iron-binding capability of rPLF were each similar to that of native milk PLF. (C) 2002 Elsevier Science (USA).
Expression, characterization, and purification of recombinant porcine lactoferrin in Pichia pastoris
(2002-03-20)
Mao, SJT
;
Wang, SH
;
生物科技學系
;
Department of Biological Science and Technology
Expression, characterization, and purification of recombinant porcine lactoferrin in Pichia pastoris
(2002-03-20)
Mao, SJT
;
Wang, SH
;
生物科技學系
;
Department of Biological Science and Technology
Identification of Atractylodes plants in Chinese herbs and formulations by random amplified polymorphic DNA
(2001-06-01)
Chen, KT
;
Su, YC
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Lin, JG
;
Hsin, LH
;
Su, YP
AIM: An efficient, precise, and sensitive method for identifying Atractylodes plants has been established and will contribute significantly to quality control and scientific analysis in Chinese traditional medicine. METHODS: Twenty primers were applied for setting up the RAPD (randomly amplified polymorphic DNA) markers of Atractylodes plants, Atractylodes lancea DC (A lancea MG:), Atractylodes japonica Koidz (A japonica K), and Atractylodes ovata DC (A ovata DC). The primer OPF03, OPF05, and OPF14 could discriminate them successfully. The results were also able to apply on the Chinese formulations with Atractylodes purchased from local markets. RESULTS: RAPD was used to investigate phylogenetic relationships among and within closely related species. RAPD analysis reflects heritable changes in the nucleotides sequence in both the coding and noncoding regions, because it is conducted directly from the DNA level. This work first conducted RAPD analysis of Atractylodes plants to establish their RAPD makers. CONCLUSION: The RAPD markers could be applied extensively in the Chinese herbal formulations.
Molecular properties of human haptoglobin as probed by monoclonal antibodies
(2004-03-24)
Mao, SJT
;
Yang, YT
;
Lai, SM
;
Wu, CF
;
Chang, TM
Purification of human haptoglobin 1-1, 2-1, and 2-2 using monoclonal antibody affinity chromatography
(2004-02-01)
Tseng, CF
;
Huang, HY
;
Yang, YT
;
Mao, SJT
;
生物科技學系
Similar to blood type, human plasma haptoglobin (Hp) is classified as 3 phenotypes: Hp 1-1, 2-1, or 2-2. The structural and functional relationship between the phenotypes, however, has not been studied in detail due to the complicated and difficult isolation procedures. This report provides a simple protocol that can be used to purify each Hp, phenotype. Plasma was first passed through an affinity column coupled with a high affinity Hp monoclonal antibody. The bound material was washed with a buffer containing 0.2 M NaCl and 0.02 M phosphate, pH 7.4, eluted at pH 11, and collected in tubes containing 1 M Tris-HCl, pH 6.8. The crude Hp fraction was then chromatographed on a HPLC Superose 12 column in 0.05 M ammonium bicarbonate at a flow rate of 0.5ml/min. The homogeneity of purified Hp 1-1, 2-1, or 2-2 was greater than 95% as judged by SDS-polyacrylamide gel electrophoresis. Essentially, each Hp isolated was not contaminated with hemoglobin and apolipoprotein A-I as that reported from the other methods, and was able to bind hemoglobin. Neuraminidase treatment demonstrated that the purified Hp possessed a carbohydrate moiety, while Western blot analysis confirmed alpha and beta chains corresponding to each Hp 1-1, 2-1, and 2-2 phenotype. The procedures described here represent a significant improvement in current purification methods for the isolation of Hp phenotypes. Circular dichroic spectra showed that the alpha-helical content of Hp 1-1 (29%) was higher than that of Hp 2-1 (22%), and 2-2 (21%). The structural difference with respect to its clinical relevance is discussed. (C) 2003 Elsevier Inc. All rights reserved.
Purification of human haptoglobin 1-1, 2-1, and 2-2 using monoclonal antibody affinity chromatography
(2004-03-24)
Tseng, CF
;
Huang, HY
;
Yang, YT
;
Mao, SJT
;
生物科技學院
Purification of human haptoglobin 1-1, 2-1, and 2-2 using monoclonal antibody affinity chromatography
(2004-02-01)
Tseng, CF
;
Huang, HY
;
Yang, YT
;
Mao, SJT
;
生物科技學系
Similar to blood type, human plasma haptoglobin (Hp) is classified as 3 phenotypes: Hp 1-1, 2-1, or 2-2. The structural and functional relationship between the phenotypes, however, has not been studied in detail due to the complicated and difficult isolation procedures. This report provides a simple protocol that can be used to purify each Hp, phenotype. Plasma was first passed through an affinity column coupled with a high affinity Hp monoclonal antibody. The bound material was washed with a buffer containing 0.2 M NaCl and 0.02 M phosphate, pH 7.4, eluted at pH 11, and collected in tubes containing 1 M Tris-HCl, pH 6.8. The crude Hp fraction was then chromatographed on a HPLC Superose 12 column in 0.05 M ammonium bicarbonate at a flow rate of 0.5ml/min. The homogeneity of purified Hp 1-1, 2-1, or 2-2 was greater than 95% as judged by SDS-polyacrylamide gel electrophoresis. Essentially, each Hp isolated was not contaminated with hemoglobin and apolipoprotein A-I as that reported from the other methods, and was able to bind hemoglobin. Neuraminidase treatment demonstrated that the purified Hp possessed a carbohydrate moiety, while Western blot analysis confirmed alpha and beta chains corresponding to each Hp 1-1, 2-1, and 2-2 phenotype. The procedures described here represent a significant improvement in current purification methods for the isolation of Hp phenotypes. Circular dichroic spectra showed that the alpha-helical content of Hp 1-1 (29%) was higher than that of Hp 2-1 (22%), and 2-2 (21%). The structural difference with respect to its clinical relevance is discussed. (C) 2003 Elsevier Inc. All rights reserved.