Browsing by Author "Chen, Wen-Hung"

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Characterization and epitope mapping of Dengue virus type 1 specific monoclonal antibodies
(2017-10-02)
Chen, Wen-Hung
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Chou, Feng-Pai
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Wang, Yu-Kuo
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Huang, Sheng-Cih
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Cheng, Chuan-Hung
Background: Dengue virus (DV) infection causes a spectrum of clinical diseases ranging from dengue fever to a life-threatening dengue hemorrhagic fever. Four distinct serotypes (DV1-4), which have similar genome sequences and envelope protein (E protein) antigenic properties, were divided. Among these 4 serotypes, DV1 usually causes predominant infections and fast diagnosis and effective treatments are urgently required to prevent further hospitalization and casualties. Methods: To develop antibodies specifically targeting and neutralizing DV1, we immunized mice with UV-inactivated DV1 viral particles and recombinant DV1 E protein from residue 1 to 395 (E395), and then generated 12 anti-E monoclonal antibodies (mAbs) as the candidates for a series of characterized assays such as ELISA, dot blot, immunofluorescence assay, western blot, and foci forming analyses. Results: Among the mAbs, 10 out of 12 showed cross-reactivity to four DV serotypes as well as Japanese encephalitis virus (JEV) in different cross-reactivity patterns. Two particular mAbs, DV1-E1 and DV1-E2, exhibited strong binding specificity and neutralizing activity against DV1 and showed no cross-reactivity to DV2, DV3, DV4 or JEV-infected cells as characterized by ELISA, dot blot, immunofluorescence assay, western blot, and foci forming analyses. Using peptide coated indirect ELISA, we localized the neutralizing determinants of the strongly inhibitory mAbs to a sequence-unique epitope on the later-ridge of domain III of the DV1 E protein, centered near residues T346 and D360 ((346)TQNGRLITANPIVTD(360)). Interestingly, the amino acid sequence of the epitope region is highly conserved among different genotypes of DV1 but diverse from DV2, DV3, DV4 serotypes and other flaviviruses. Conclusions: Our results showed two selected mAbs DV1-E1 and DV1-E2 can specifically target and significantly neutralize DV1. With further research these two mAbs might be applied in the development of DV1 specific serologic diagnosis and used as a feasible treatment option for DV1 infection. The identification of DV1 mAbs epitope with key residues can also provide vital information for vaccine design.
Gold nanoparticle-based inductively coupled plasma mass spectrometry amplification and magnetic separation for the sensitive detection of a virus-specific RNA sequence
(2011-04-08)
Hsu, I-Hsiang
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Chen, Wen-Hung
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Wu, Tung-Kung
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Sun, Yuh-Chang
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生物科技學系
We have developed a sensitive gold nanoparticle (AuNP)-based inductively coupled plasma mass spectrometry (ICP-MS) amplification and magnetic separation method for the detection of oligonucleotide sequences. The assay relies on (i) the sandwich-type binding of two designed probe sequences that specifically recognize the target oligonucleotide sequences, (ii) magnetic bead separation, and (iii) AuNP-based ICP-MS amplification detection. To enhance the analytical signal and minimize the background signal resulting from nonspecific binding, we performed a series of experiments to evaluate the effects of various parameters (the concentration of the capture probe; the time required for hybridization; the number of washings required to eliminate nonspecific binding) on the oligonucleotide detection. Under the optimized conditions, the detection limit was 80 zmol (corresponding to 1.6fM of the target sequence in a sample volume of 50 mu L). Moreover, it employs a shorter hybridization step and ICP-MS, this procedure is relatively simple and rapid (ca. 1.5 h). Based on the analytical results obtained using complementary and mismatched sequences, our method exhibits good performance in distinguishing complementary and random oligonucleotides. Compared with the "gold standard" methodology (plaque assay) for the quantification of dengue virus, our method has the capability to allow early detection of dengue virus in complicated and small-volume samples, with high specificity, good analytical sensitivity, and superior time-effectiveness. (C) 2011 Elsevier B.V. All rights reserved.
Immunocapture couples with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for rapid detection of type 1 dengue virus
(2013-05-03)
Chen, Wen-Hung
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Hsu, I-Hsiang
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Sun, Yuh-Chang
;
Wang, Yu-Kuo
;
Wu, Tung-Kung
A facile method for accurate detection of type I dengue virus (DV1) infection from complex biological mixtures, using type specific immunocapture coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), was developed. The biological mixtures were treated with magnetic particles coated with a monoclonal antibody directly against type 1 dengue virus. After immunocapture purification, the DV1 was eluted with 30% acetic acid, directly spotted with seed-layer method, and analyzed by MALDI-TOF MS for DV1 capsid protein. The detection limit of the assay was similar to 10(5) pfu/mL by MALDI-TOF MS. The immunocapture could unambiguously differentiate the DV1 from other serotypes of the dengue viruses and Japanese encephalitis virus, and could be used as a specific probe to detect DV1 from complex biological mixtures. (C) 2013 Elsevier B.V. All rights reserved.
Purification, crystallization and preliminary X-ray analysis of a thermostable direct haemolysin from Grimontia hollisae
(2011-02-01)
Wang, Yu-Kuo
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Huang, Sheng-Cih
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Wu, Yi-Fang
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Chen, Yu-Ching
;
Chen, Wen-Hung
Vibrio hollisae, a halophilic species recently reclassified as Grimontia hollisae, is a causative agent of gastroenteritis and septicaemia. One important pathogenic Vibrio factor, thermostable direct haemolysin (TDH), has been purified and crystallized in two crystal forms using the vapour-diffusion method. The crystals belonged to an orthorhombic space group, with unit-cell parameters a = 104.8, b = 112.4, c = 61.3 angstrom and a = 122.9, b = 123.3, c = 89.8 angstrom. The crystals contained either four or eight molecules per asymmetric unit, with predicted solvent contents of 49.4 and 46.3% and Matthews coefficients (V(M)) of 2.4 and 2.3 angstrom(3) Da(-1), respectively. These crystals were suitable for structure determination, which would yield structural details related to the cytotoxicity and oligomeric structure of this pore-forming toxin.
Site-Directed Mutations of Thermostable Direct Hemolysin from Grimontia hollisae Alter Its Arrhenius Effect and Biophysical Properties
(2011)
Wang, Yu-Kuo
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Huang, Sheng-Cih
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Wu, Yi-Fang
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Chen, Yu-Ching
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Lin, Yen-Ling
Recombinant thermostable direct hemolysin from Grimontia hollisae (Gh-rTDH) exhibits paradoxical Arrhenius effect, where the hemolytic activity is inactivated by heating at 60 degrees C but is reactivated by additional heating above 80 degrees C. This study investigated individual or collective mutational effect of Tyr53, Thr59, and Ser63 positions of Gh-rTDH on hemolytic activity, Arrhenius effect, and biophysical properties. In contrast to the Gh-rTDH wild-type (Gh-rTDH(WT)) protein, a 2-fold decrease of hemolytic activity and alteration of Arrhenius effect could be detected from the Gh-rTDH(Y53H/T59I) and Gh-rTDH(T59I/S63T) double-mutants and the Gh-rTDH(Y53H/T59I/S63T) triple-mutant. Differential scanning calorimetry results showed that the Arrhenius effect-loss and -retaining mutants consistently exhibited higher and lower endothermic transition temperatures, respectively, than that of the Gh-rTDHWT. Circular dichroism measurements of Gh-rTDH(WT) and Gh-rTDH(mut) showed a conspicuous change from a beta-sheet to alpha-helix structure around the endothermic transition temperature. Consistent with the observation is the conformational change of the proteins from native globular form into fibrillar form, as determined by Congo red experiments and transmission electron microscopy.