Wheat dwarf disease caused by wheat dwarf virus(WDV) is currently present in wheat growing regions in China and causes serious losses in wheat yield. To develop reliable and effective serological detection methods for...Wheat dwarf disease caused by wheat dwarf virus(WDV) is currently present in wheat growing regions in China and causes serious losses in wheat yield. To develop reliable and effective serological detection methods for WDV, the coat protein(CP) gene of WDV was cloned and expressed in Escherichia coli. The purified recombinant CP protein was immunized to BALB/c mice, and four hybridoma cell lines(i.e. 18G10, 9G4, 23F4 and 22A10) secreting anti-WDV monoclonal antibodies(MAbs) were obtained through the hybridoma technique. Using the prepared MAbs, an antigencoated-plate enzyme-linked immunosorbent assay(ACP-ELISA) and a dot-ELISA were established for detecting WDV in wheat samples. The most sensitive ACP-ELISA based on MAb 23F4 or 22A10 was able to detect WDV in 1:163,840(w/v,g/mL) diluted WDV-infected wheat plant crude extracts. The dot-ELISA based on MAb 23F4 was the most sensitive and able to detect the virus in 1:5,120(w/v, g/mL) diluted wheat plant crude extracts. A total of 128 wheat samples were collected from wheat growing regions in the Shaanxi and Qinghai provinces, China, and were screened for the presence of WDV using two developed serological assays. Results from the survey showed that approximately 62% of the samples were infected with WDV. PCR followed by DNA sequencing and sequence alignment validated the results from the two serological assays. Therefore, we consider that these two serological detection methods can be significantly useful for the control of WDV in China.展开更多
Wheat high molecular weight glutenin subunits(HMW-GS)1Bx14 and 1By15 isolated by preparative SDS-PAGE are used as antigen to immunize BALB/c mice.Subcutaneous inoculation of the antigen is performed.The intra-peritone...Wheat high molecular weight glutenin subunits(HMW-GS)1Bx14 and 1By15 isolated by preparative SDS-PAGE are used as antigen to immunize BALB/c mice.Subcutaneous inoculation of the antigen is performed.The intra-peritoneal injection is completed 3 days before fusion with myeloma cell(SP2/0)via PEG-1500.The fusion cells are selected by indirect enzyme-linked immuno-sorbent assay(ELISA).Positive hybrid cells are further verified three times by limit dilution of the culture cells.A hybridoma cell line is successfully obtained.The monoclonal antibody belongs to IgG1 subclass.In immunoblotting,the antibody binds to all HMW-GS of T.aestivum cultivars,but does not bind to other storage proteins in seeds of wheat.This result is consisting with the high homology in amino acid sequences among the HMW glutenin subunits in wheat.The antibody also binds to HMW-GS storage proteins in Aegilops squarrosa and T.durum(durum wheat).Furthermore,it also binds to HMW storage proteins in Secale cereale(rye),Hordeum vulgare(barley).However,it never binds seed storage proteins in other cereals such as maize,oat,rice,foxtail millet,sorghum etc.The antigen determinant recognized by the antibody has been located within hexapeptide[PGQGQQ]or/and nonapeptide[GYYPTSPQQ]in the central repetitive region of HMW-GS.展开更多
Herbicide residues in agricultural products can have adverse effects on the environment and human health,therefore,there is an urgent need to establish a sensitive,rapid,and wide-ranging detection method.In this study...Herbicide residues in agricultural products can have adverse effects on the environment and human health,therefore,there is an urgent need to establish a sensitive,rapid,and wide-ranging detection method.In this study,haptens of phenylurea herbicides(PUs)and sulfonylurea herbicides(SUs)were analyzed and designed based on computational simulation techniques,and two high-performance broad-spectrum monoclonal antibodies against PUs and SUs were prepared.On this basis,a multi-colloidal gold immunochromatography assay(multi-CGIA)was developed to simultaneously detect 13 herbicides in wheat.The visual limit of detection(vLOD)for PUs including diuron,chlortoluron,neburon,chlorbromuron,and linuron was 1-2μg/kg.The vLOD for SUs including metsulfuron methyl,ethametsulfuron-methyl,sulfometuron-methyl,tribenuron methyl,cinosulfuron,triasulfuron,chlorimuron-ethyl,and chlorsulfuron was 2-10μg/kg.The results of real sample determination indicated that the multi-CGIA is accurate,stable,and reliable,and adaptable to on-site preliminary screening of actual samples.展开更多
基金supported by Public Science and Technology Research Funds Projects of Agriculture (201303021)the National Basic Research Program (973) of China (No.2014CB138400)
文摘Wheat dwarf disease caused by wheat dwarf virus(WDV) is currently present in wheat growing regions in China and causes serious losses in wheat yield. To develop reliable and effective serological detection methods for WDV, the coat protein(CP) gene of WDV was cloned and expressed in Escherichia coli. The purified recombinant CP protein was immunized to BALB/c mice, and four hybridoma cell lines(i.e. 18G10, 9G4, 23F4 and 22A10) secreting anti-WDV monoclonal antibodies(MAbs) were obtained through the hybridoma technique. Using the prepared MAbs, an antigencoated-plate enzyme-linked immunosorbent assay(ACP-ELISA) and a dot-ELISA were established for detecting WDV in wheat samples. The most sensitive ACP-ELISA based on MAb 23F4 or 22A10 was able to detect WDV in 1:163,840(w/v,g/mL) diluted WDV-infected wheat plant crude extracts. The dot-ELISA based on MAb 23F4 was the most sensitive and able to detect the virus in 1:5,120(w/v, g/mL) diluted wheat plant crude extracts. A total of 128 wheat samples were collected from wheat growing regions in the Shaanxi and Qinghai provinces, China, and were screened for the presence of WDV using two developed serological assays. Results from the survey showed that approximately 62% of the samples were infected with WDV. PCR followed by DNA sequencing and sequence alignment validated the results from the two serological assays. Therefore, we consider that these two serological detection methods can be significantly useful for the control of WDV in China.
基金This work was supported by the grants on transgenic plant(J99-A-018)of National 863 Plan(2003AA207090)of lhe Minisury of Science and Technology of China to 2hang Xueyong
文摘Wheat high molecular weight glutenin subunits(HMW-GS)1Bx14 and 1By15 isolated by preparative SDS-PAGE are used as antigen to immunize BALB/c mice.Subcutaneous inoculation of the antigen is performed.The intra-peritoneal injection is completed 3 days before fusion with myeloma cell(SP2/0)via PEG-1500.The fusion cells are selected by indirect enzyme-linked immuno-sorbent assay(ELISA).Positive hybrid cells are further verified three times by limit dilution of the culture cells.A hybridoma cell line is successfully obtained.The monoclonal antibody belongs to IgG1 subclass.In immunoblotting,the antibody binds to all HMW-GS of T.aestivum cultivars,but does not bind to other storage proteins in seeds of wheat.This result is consisting with the high homology in amino acid sequences among the HMW glutenin subunits in wheat.The antibody also binds to HMW-GS storage proteins in Aegilops squarrosa and T.durum(durum wheat).Furthermore,it also binds to HMW storage proteins in Secale cereale(rye),Hordeum vulgare(barley).However,it never binds seed storage proteins in other cereals such as maize,oat,rice,foxtail millet,sorghum etc.The antigen determinant recognized by the antibody has been located within hexapeptide[PGQGQQ]or/and nonapeptide[GYYPTSPQQ]in the central repetitive region of HMW-GS.
基金supported by the National Natural Science Foundation of China(Nos.22306074 and 222360020)the National Key Research and Development Program of China(Nos.2023YFF1105003 and 2022YFA1207300)the grants from Jiangsu province(Nos.CX(22)1013,BK20212014,and M20221006).
文摘Herbicide residues in agricultural products can have adverse effects on the environment and human health,therefore,there is an urgent need to establish a sensitive,rapid,and wide-ranging detection method.In this study,haptens of phenylurea herbicides(PUs)and sulfonylurea herbicides(SUs)were analyzed and designed based on computational simulation techniques,and two high-performance broad-spectrum monoclonal antibodies against PUs and SUs were prepared.On this basis,a multi-colloidal gold immunochromatography assay(multi-CGIA)was developed to simultaneously detect 13 herbicides in wheat.The visual limit of detection(vLOD)for PUs including diuron,chlortoluron,neburon,chlorbromuron,and linuron was 1-2μg/kg.The vLOD for SUs including metsulfuron methyl,ethametsulfuron-methyl,sulfometuron-methyl,tribenuron methyl,cinosulfuron,triasulfuron,chlorimuron-ethyl,and chlorsulfuron was 2-10μg/kg.The results of real sample determination indicated that the multi-CGIA is accurate,stable,and reliable,and adaptable to on-site preliminary screening of actual samples.
