Characterization of Monoclonal Antibodies against Nonstructural Protein 1 (NS1) of Avian Influenza Virus

碩士 === 國立臺灣大學 === 獸醫學研究所 === 102 === NS1 is an indictor Ag of influenza virus (IV) infection and it’s only produced during IV replication in very early stage of infection. It can be used in determining whether the chicken was infected or not, which also can be applied on early diagnosis of IV infect...

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Main Authors: Pin-Cheng Chien, 錢品澄
Other Authors: Ivan-Chen Cheng
Format: Others
Language:zh-TW
Published: 2014
Online Access:http://ndltd.ncl.edu.tw/handle/43455856100074850441
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spelling ndltd-TW-102NTU055410092016-03-09T04:24:03Z http://ndltd.ncl.edu.tw/handle/43455856100074850441 Characterization of Monoclonal Antibodies against Nonstructural Protein 1 (NS1) of Avian Influenza Virus 抗禽流感病毒非結構蛋白單源抗體之特性分析 Pin-Cheng Chien 錢品澄 碩士 國立臺灣大學 獸醫學研究所 102 NS1 is an indictor Ag of influenza virus (IV) infection and it’s only produced during IV replication in very early stage of infection. It can be used in determining whether the chicken was infected or not, which also can be applied on early diagnosis of IV infection. Therefore, my study aimed to characterize the anti-NS1 MAb (αNS1 MAb) by indirect ELISA (iELISA), indirect immunofluorescence assay (IFA), western blotting (WB), immunohistochemistry stain (IHC) and mapping the antibody binding site by eukaryotic expression system (EES). Subsequently, to develop a rapid, sensitive and specific diagnostic MAbs-based NS1 Ag sandwich ELISA that might be incorporated with NP and M Ag ELISA kit for detecting the IV infection. The parental hybridoma have been prepared by immunizing Balb/c mice with E. coli expressed recombinant nonstructural protein 1 (rNS1) oringinated from IV isolates of A/chicken/Taiwan/2838V/00 (H6N1/2838). 16 αNS1 MAbs have been characterized by WB, IFA and iELISA with NS1 Ag from several IV isolates such as A/chicken/Miaoli/2904/00 (H6N1/2904), A/chicken/Taiwan/1209/03 (H5N2/1209). Meanwhile, the epitope mapping was studied in EES. Expression of full length rNS1 (residues 1-230) were all positive and 4 αNS1 MAb (4M4, 4M6, 4N5, 4R3) were negative in C-terminal deletion (residues 1-207). Accroding to epitope mapping analysis results, which can divide 16 αNS1 MAb into four groups (A, B, C &; D). The predictive epitopes of 16 αNS1 MAb mainly recognize the effector domain (residues 74-230) and group B recognize the C-terminal tail (residues 202-230) of NS1 protein. MAb-based NS1 Ag sandwich ELISA was designed as sixteen MAb individually conjugated with HRP as the tracer Ab paired with each non-conjugated MAb acted as docking Ab respectively to compare the binding ability of those combinations with E. coli expressed rNS1. Current results indicated that MP-5 (4R11 – 4M2/HRP), MP-9 (4R11 – 4J12/HRP) and MP-10 (4M2 – 4M2/HRP) can detect NS1 Ag from 15 subtypes of IV (H1~H15). It is believed the three MP (MP-5, MP-9 &; MP-10) we studied have potential to be applied on detecting the IV NS1 Ag. Ivan-Chen Cheng 鄭益謙 2014 學位論文 ; thesis 134 zh-TW
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language zh-TW
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description 碩士 === 國立臺灣大學 === 獸醫學研究所 === 102 === NS1 is an indictor Ag of influenza virus (IV) infection and it’s only produced during IV replication in very early stage of infection. It can be used in determining whether the chicken was infected or not, which also can be applied on early diagnosis of IV infection. Therefore, my study aimed to characterize the anti-NS1 MAb (αNS1 MAb) by indirect ELISA (iELISA), indirect immunofluorescence assay (IFA), western blotting (WB), immunohistochemistry stain (IHC) and mapping the antibody binding site by eukaryotic expression system (EES). Subsequently, to develop a rapid, sensitive and specific diagnostic MAbs-based NS1 Ag sandwich ELISA that might be incorporated with NP and M Ag ELISA kit for detecting the IV infection. The parental hybridoma have been prepared by immunizing Balb/c mice with E. coli expressed recombinant nonstructural protein 1 (rNS1) oringinated from IV isolates of A/chicken/Taiwan/2838V/00 (H6N1/2838). 16 αNS1 MAbs have been characterized by WB, IFA and iELISA with NS1 Ag from several IV isolates such as A/chicken/Miaoli/2904/00 (H6N1/2904), A/chicken/Taiwan/1209/03 (H5N2/1209). Meanwhile, the epitope mapping was studied in EES. Expression of full length rNS1 (residues 1-230) were all positive and 4 αNS1 MAb (4M4, 4M6, 4N5, 4R3) were negative in C-terminal deletion (residues 1-207). Accroding to epitope mapping analysis results, which can divide 16 αNS1 MAb into four groups (A, B, C &; D). The predictive epitopes of 16 αNS1 MAb mainly recognize the effector domain (residues 74-230) and group B recognize the C-terminal tail (residues 202-230) of NS1 protein. MAb-based NS1 Ag sandwich ELISA was designed as sixteen MAb individually conjugated with HRP as the tracer Ab paired with each non-conjugated MAb acted as docking Ab respectively to compare the binding ability of those combinations with E. coli expressed rNS1. Current results indicated that MP-5 (4R11 – 4M2/HRP), MP-9 (4R11 – 4J12/HRP) and MP-10 (4M2 – 4M2/HRP) can detect NS1 Ag from 15 subtypes of IV (H1~H15). It is believed the three MP (MP-5, MP-9 &; MP-10) we studied have potential to be applied on detecting the IV NS1 Ag.
author2 Ivan-Chen Cheng
author_facet Ivan-Chen Cheng
Pin-Cheng Chien
錢品澄
author Pin-Cheng Chien
錢品澄
spellingShingle Pin-Cheng Chien
錢品澄
Characterization of Monoclonal Antibodies against Nonstructural Protein 1 (NS1) of Avian Influenza Virus
author_sort Pin-Cheng Chien
title Characterization of Monoclonal Antibodies against Nonstructural Protein 1 (NS1) of Avian Influenza Virus
title_short Characterization of Monoclonal Antibodies against Nonstructural Protein 1 (NS1) of Avian Influenza Virus
title_full Characterization of Monoclonal Antibodies against Nonstructural Protein 1 (NS1) of Avian Influenza Virus
title_fullStr Characterization of Monoclonal Antibodies against Nonstructural Protein 1 (NS1) of Avian Influenza Virus
title_full_unstemmed Characterization of Monoclonal Antibodies against Nonstructural Protein 1 (NS1) of Avian Influenza Virus
title_sort characterization of monoclonal antibodies against nonstructural protein 1 (ns1) of avian influenza virus
publishDate 2014
url http://ndltd.ncl.edu.tw/handle/43455856100074850441
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