Browsing by Author "Inouve, S."

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Comparison of recombinant protein and cell lysate antigens for detection of anti-chikungunya (CHIK) IgM antibody
(Sri Lanka College of Microbiologists, 2011) Athapaththu, A.M.M.H.; Khanna, N.; Inouve, S.; Gunasena, S.; Abeyewickreme, W.; Hapugoda, M.D.
INTRODUCTION: Chikungunya (CHIK) virus specific antigen which has high specificity and low cross reactivity with other related diseases is required for laboratory confirmation. OBJECTIVE: To compare two antigens for detection of anti-CHIK antibody. DESIGN, SETTING AND METHODS: In this study, two antigens {viral cell lysate and recombinant protein) were evaluated for detection of anti-1 CH IK antibody by using IgM ELISA. A novel recombinant | protein antigen was designed based on envelope domain, a critical antigenic region of the major structural protein, I This protein was expressed in Escherichia coli and resultant protein was affinity purified and ~10mg with >95% of purity per liter of culture was obtained. Cell lysate antigen was prepared using a crude culture fluid. Two antigens were evaluated separately using a panel of well characterized serum samples obtained from the Dept. of Virology (WHO Reference Centre for Viral Reference and Research), Institute of Tropical Medicine,] Nagasaki University. RESULTS: Atotal of 64 serum samples confirmed as positives andl 22 confirmed as negatives were used to evaluate the antigens. Specificity and sensitivity of the recombinant protein antigen was 48% and 90% respectively. Specificity and sensitivity of the viral lysate antigen was] 17% and 100% respectively. Conclusion Viral lysate antigens can cause biohazard risk, high production cost and cross reactivity with other organisms of the same genus/family. Recombinant protein antigen which shows high specificity and sensitivity used in this study is important to overcome problems associated with viral lysate antigen. Testing of a large number of samples is needed to reconfirm this finding. ACKNOWLEDGMENT: Financial assistance and technical co-operation by International Center for Genetic Engineering and Biotechnology (ICGEB CRP SRL 08/02), National Science Foundation (NSF/RG/2009/BT/01) and International Atomic Energy Authority (IAEA/SRL/5/042) is acknowledged.
Comparison of recombinant protein and cell lysate antigens for detection of anti-chikungunya (CHIK) IgM antibody
(University of Kelaniya, 2011) Athapaththu, A.M.M.; Abeyewickreme, W.; Hapugoda, M.; Khanna, N.; Inouve, S.; Tun, M.M.N.; Gunasena, S.
Chikungunya (CHIK) virus specific antigen which has high specificity and low cross reactivity with other related diseases is required for laboratory confirmation. The objective of this study is to compare two antigens for detection of anti-CHIK antibody. In this study, two antigens (viral cell lysate and recombinant protein) were evaluated for detection of anti-CHIK antibody by using IgM ELISA. A novel recombinant protein antigen was designed based on envelope domain, a critical antigenic region of the major structural protein. This protein was expressed in Escherichia coli and resultant protein was affinity purified and 10mg with >95% of purity per liter of culture was obtained. Cell lysate antigen was prepared using a crude culture fluid. Two antigens were evaluated separately using a panel of well characterized serum samples obtained from the Dept. of Virology (WHO Reference Centre for Viral Reference and Research), Institute of Tropical Medicine, Nagasaki University. A total of 64 serum samples confirmed as positives and 22 confirmed as negatives were used to evaluate the antigens. Specificity and sensitivity of the recombinant protein antigen was 48% and 90% respectively. Specificity and sensitivity of the viral lysate antigen was 17% and 100% respectively. Viral lysate antigens can cause biohazard risk, high production cost and cross reactivity with other organisms of the same genus/family. Recombinant protein antigen which shows high specificity and sensitivity used in this study is important to overcome problems associated with viral lysate antigen. Testing of a large number of samples is needed to reconfirm this finding. Acknowledgment: Financial assistance and technical co-operation by International Center for Genetic Engineering and Biotechnology (ICGEB CRP SRL 08/02), National Science Foundation (NSF/RG/2009/BT/01) and International Atomic Energy Authority (IAEA/SRL/5/042) is acknowledged.
Development of recombinant protein antigens using a bacterial expression system for the detection of anti-Chikungunya (CHIK) antibodies
(Sri Lanka College of Microbiologists, 2013) Athapaththu, A.M.M.H.; Khanna, N.; Inouve, S.; Gunasena, S.; Abeyewickreme, W.; Hapugoda, M.
INTRODUCTION AND OBJECTIVE: Laboratory confirmation of Chikungunya (CHIK) virus is very useful as clinical symptoms of CHIK can overlap with other diseases. Chikungunya virus specific antigen, which shows high specificity, sensitivity and low cross reactivity with other related diseases, is required for laboratory confirmation. Objective of this study was to develop and compare two recombinant protein antigens for detection of anti-CHIK antibodies. DESIGN, SETTING AND METHODS: Recombinant CHIK protein antigens were prepared using Envelope (E1 and E2) regions of the CHIK virus. The genes were custom designed and chemically synthesized with a 6X His tag. Bacterial expression systems [BL21 (DE3)] were used to clone and express the recombinant proteins. The recombinant proteins were purified with >95% of purity per liter of culture using Ni-NTA columns under denature conditions. In this study, two antigens were evaluated for detection of anti-CHIK antibody by using novel optimized in-house IgM and IgG ELISAs, using a panel of well characterized serum samples obtained from the Dept. of Virology (WHO Reference Center for Viral Reference and Research) Institute of Tropical Medicine, Nagasaki University, Japan. RESULTS: Atotal of 55 serum samples confirmed as positives and 186 confirmed as negatives by HA! test, IgM capture ELISA and indirect IgG ELISA using the purified CHIK antigen were used to evaluate the antigens using novel IgM ELISA. A total of 78 serum samples confirmed as positives and 148 (E1) or 227 (E2) (148 + extra 79) confirmed ac negatives were used to evaluate the antigens using novel IgG ELISA. The E1 recombinant protein showed 5% (3/ 55) sensitivity and 99% (184/186) specificity for IgM ELISA and 60% (47/78) sensitivity and 63% (94/148) specificity for IgG ELISA. The E2 recombinant protein showed 65% (36/55) sensitivity and 70% (131/186) specificity for IgM ELISA and 83% (65/78) sensitivity and 86% (195/227) specificity for IgG ELISA. CONCLUSION: Recombinant CHIK-E2 protein antigen showed higher specificity and sensitivity in detection of both IgM and IgG antibodies. Thus the E2 recombinant protein antigen used in this study could be expressed in an eukaryotic expression system to achieve much higher results. ACKNOWLEDGMENT: International Center for Genetic Engineering and Biotechnology (ICGEB CRP SRL 08/02), National Science Foundation (NSF/RG/2009/BT/01) and International Atomic Energy Authority (lAEA/SRL/5/042) are gratefully acknowledged.