[Objectives]This study was conducted to explore rapid and large-scale screening and detection of peste des petits ruminants(PPR),so as to provide important technical means for prevention,control and purification of PP...[Objectives]This study was conducted to explore rapid and large-scale screening and detection of peste des petits ruminants(PPR),so as to provide important technical means for prevention,control and purification of PPR.[Methods]Soluble N protein and NH fusion protein were successfully obtained in an Escherichia coli expression system by optimizing E.coli codon and expression conditions.Furthermore,based on purified soluble N protein and NH fusion protein,a double-antigen sandwich time-resolved fluorescence immunoassay method for detection of peste des petits ruminants virus(PPRV)was established.[Results]The method has high sensitivity and specificity and can specifically detect the antibody against PPRV in sheep serum,and it has no cross reaction with other related diseases.The method was used to detect 292 clinical samples,and compared with French IDVET competition ELISA kit.The coincidence rates of positive samples and negative samples from the two kinds of test kits were 92.47%and 97.26%,respectively,and the overall coincidence rate was 94.86%.The intra-group and inter-group coefficients of variation in the repeatability test were less than 10%.[Conclusions]Compared with the traditional ELISA method,the double-antigen sandwich time-resolved fluorescence immunoassay for detection of PPRV has equivalent sensitivity and specificity,and simple and rapid operation,and thus high application and popularization value.展开更多
The effect of the chelating agent 4,7-bis（chlorosulfophenyl）-1,10-phenanthroline-2,9-dicarboxylic acid （BCPDA） labeled hepatitis B surface antibodies（HBsAb） on time-resolved fluoroimmunoassays（TRFIA） was inves...The effect of the chelating agent 4,7-bis（chlorosulfophenyl）-1,10-phenanthroline-2,9-dicarboxylic acid （BCPDA） labeled hepatitis B surface antibodies（HBsAb） on time-resolved fluoroimmunoassays（TRFIA） was investigated. The labeling detection method was established. The biotin-streptavidin（BAS） system was introduced, and the multi-stage amplification effect of the BAS system was analyzed. It is found that the BCPDA-Eu^3＋-HBsAb-BAS fluorescent marker can emit strong fluorescence. Compared to BCPDA-Eu^3＋-HBsAb, BCPDA-Eu^3＋-HBsAb-BAS demonstrates an enhanced fluorescence intensity by over 10 times. This study provides a guidance for the next generation non-radio immunoassays in clinical diagnosis,展开更多
基金Supported by National Key R&D Program for the Prevention and Control of Major Exotic Animal Diseases(2022YFD1800500)National Mutton Sheep Industrial Technology System(CARS39)+2 种基金Key Research and Development Program of Shandong Province(Major Science and Technology Innovation Project)(2021CXGC011306)Scientific Research Project of General Administration of Customs(2024HK033)Scientific Research Project of Jinan Customs(2023JK005).
文摘[Objectives]This study was conducted to explore rapid and large-scale screening and detection of peste des petits ruminants(PPR),so as to provide important technical means for prevention,control and purification of PPR.[Methods]Soluble N protein and NH fusion protein were successfully obtained in an Escherichia coli expression system by optimizing E.coli codon and expression conditions.Furthermore,based on purified soluble N protein and NH fusion protein,a double-antigen sandwich time-resolved fluorescence immunoassay method for detection of peste des petits ruminants virus(PPRV)was established.[Results]The method has high sensitivity and specificity and can specifically detect the antibody against PPRV in sheep serum,and it has no cross reaction with other related diseases.The method was used to detect 292 clinical samples,and compared with French IDVET competition ELISA kit.The coincidence rates of positive samples and negative samples from the two kinds of test kits were 92.47%and 97.26%,respectively,and the overall coincidence rate was 94.86%.The intra-group and inter-group coefficients of variation in the repeatability test were less than 10%.[Conclusions]Compared with the traditional ELISA method,the double-antigen sandwich time-resolved fluorescence immunoassay for detection of PPRV has equivalent sensitivity and specificity,and simple and rapid operation,and thus high application and popularization value.
基金Supported by the National Natural Science Foundation of China（No. 81572082） and the Natural Science Foundation of Jilin Province, China（Nos.2011713, 20150414015GH, 20150204010YY）.
文摘The effect of the chelating agent 4,7-bis（chlorosulfophenyl）-1,10-phenanthroline-2,9-dicarboxylic acid （BCPDA） labeled hepatitis B surface antibodies（HBsAb） on time-resolved fluoroimmunoassays（TRFIA） was investigated. The labeling detection method was established. The biotin-streptavidin（BAS） system was introduced, and the multi-stage amplification effect of the BAS system was analyzed. It is found that the BCPDA-Eu^3＋-HBsAb-BAS fluorescent marker can emit strong fluorescence. Compared to BCPDA-Eu^3＋-HBsAb, BCPDA-Eu^3＋-HBsAb-BAS demonstrates an enhanced fluorescence intensity by over 10 times. This study provides a guidance for the next generation non-radio immunoassays in clinical diagnosis,
