H7 avian influenza viruses(AIVs) normally circulated among birds before. From 1996 to 2012, human infections with H7 AIVs(H7 N2, H7 N3, and H7 N7) were reported in Canada, Italy, Mexico, the Netherlands, the United Ki...H7 avian influenza viruses(AIVs) normally circulated among birds before. From 1996 to 2012, human infections with H7 AIVs(H7 N2, H7 N3, and H7 N7) were reported in Canada, Italy, Mexico, the Netherlands, the United Kingdom and the USA. Until March 2013, human infections with H7 N9 AIVs were reported in China. Since then, H7 N9 AIVs have continued to circulate in both humans and birds. Therefore, the detection of antibodies against the H7 subtype of AIVs has become an important topic. In this study, a competitive enzyme-linked immunosorbent assay(cELISA)method for the detection of antibody against H7 AIVs was established. The optimal concentration of antigen coating was 5 μg mL^(-1), serum dilution was 1/10, and enzyme-labeled antibody was 1/3 000. To determine the cut-off value of cELISA, percent inhibition(PI) was determined by using receiver operating characteristic(ROC) curve analysis in 178 AIVs negative samples and 368 AIVs positive serum samples(n=546). When PI was set at 40%, the specificity and sensitivity of cELISA were 99.4 and 98.9%, respectively. This method could detect the antibodies against H7 Nx(N1–N4, N7–N9) AIVs, and showed no reaction with AIVs of H1–H6 and H8–H15 subtypes or common avian viruses such as Newcastle disease virus(NDV), Infectious bronchitis virus(IBV) and Infectious bursal disease virus(IBDV), exhibiting good specificity. This method showed a coincidence rate of 98.56% with hemagglutinin inhibition(HI) test. And the repeatability experiment revealed that the coefficients of variation(CV) of intra-and inter-batch repetition were all less than 12%. The data indicated that the cELISA antibody-detection method established in this study provided a simple and accurate technical support for the detection of a large number of antibody samples of H7-AIV.展开更多
Lumpy skin disease(LSD)is a highly contagious disease caused by lumpy skin disease virus(LSDV)in bovines.Rapid and accurate diagnosis is very important to controll it.However,current commercial detection kits need to ...Lumpy skin disease(LSD)is a highly contagious disease caused by lumpy skin disease virus(LSDV)in bovines.Rapid and accurate diagnosis is very important to controll it.However,current commercial detection kits need to be improved in terms of sensitivity or specificity.This study aimed to develop a novel diagnostic competitive enzyme-linked immunosorbent assay(cELISA)based on the newly identified antigen gene LSDV034.The rLSDV034 protein was identified as a potential diagnostic antigen,and it was expressed,purified,and used to immunize BALB/c mice.Using laboratory-prepared indirect ELISA(iELISA),the positive cell lines were screened,and their blocking activity was further verified by competitive ELISA(cELISA).The cell line,1H7,was chosen to produce mouse ascites,which were purified for a monoclonal antibody(mAb,5.395 mg/mL).The heavy chain type of the 1H7 mAb was identified as IgG1a,and its light chain subtype was identified as κ.Furthermore,cELISA was developed to detect bovine serum antibodies,with rLSDV034(4μg/mL)as the coating antigen and HRP-1H7 mAb(1:300)as the competitive antibody.The cutoff value of cELISA was 55%,based on 32 negative bovine serum samples.The analytical sensitivity was 1:8,and no cross-reaction was detected with bovine viral diarrhea virus(BVDV),infectious bovine rhinotracheitis virus(IBRV),Pasteurella multocida(P.multocida),or Mycoplasma bovis(M.bovis)from the serum samples.The diagnostic sensitivity and specificity of cELISA were 98.46%(95%confidence interval,CI:91.7–100)and 100%(95%CI:89.1–100),respectively,based on the analysis of 30 LSDV-infected bovine serum samples,35 GTPV-vaccinated samples,and 32 negative samples.The overall coincidence of the cELISA with the virus neutralization test(VNT)reached 98.97%(95%CI:94.4–100).Furthermore,we used cELISA to analyze 230 clinical bovine serum samples(including 59 infected and 171 vaccinated samples)and found that the serum positivity rates of the immunized samples(on d 60 postimmunization)and infected samples were 77.78%(95%CI:70.8–83.8%)and 71.19%(95%CI:57.9–82.2),respectively.These results indicate that the developed cELISA is promising for detecting serum antibodies in naturally infected or vaccinated cattle.展开更多
基金supported by the National Key R&D Program of China(2016YFD0500800)。
文摘H7 avian influenza viruses(AIVs) normally circulated among birds before. From 1996 to 2012, human infections with H7 AIVs(H7 N2, H7 N3, and H7 N7) were reported in Canada, Italy, Mexico, the Netherlands, the United Kingdom and the USA. Until March 2013, human infections with H7 N9 AIVs were reported in China. Since then, H7 N9 AIVs have continued to circulate in both humans and birds. Therefore, the detection of antibodies against the H7 subtype of AIVs has become an important topic. In this study, a competitive enzyme-linked immunosorbent assay(cELISA)method for the detection of antibody against H7 AIVs was established. The optimal concentration of antigen coating was 5 μg mL^(-1), serum dilution was 1/10, and enzyme-labeled antibody was 1/3 000. To determine the cut-off value of cELISA, percent inhibition(PI) was determined by using receiver operating characteristic(ROC) curve analysis in 178 AIVs negative samples and 368 AIVs positive serum samples(n=546). When PI was set at 40%, the specificity and sensitivity of cELISA were 99.4 and 98.9%, respectively. This method could detect the antibodies against H7 Nx(N1–N4, N7–N9) AIVs, and showed no reaction with AIVs of H1–H6 and H8–H15 subtypes or common avian viruses such as Newcastle disease virus(NDV), Infectious bronchitis virus(IBV) and Infectious bursal disease virus(IBDV), exhibiting good specificity. This method showed a coincidence rate of 98.56% with hemagglutinin inhibition(HI) test. And the repeatability experiment revealed that the coefficients of variation(CV) of intra-and inter-batch repetition were all less than 12%. The data indicated that the cELISA antibody-detection method established in this study provided a simple and accurate technical support for the detection of a large number of antibody samples of H7-AIV.
基金financially supported by the National Key Research and Development Program of China(#2022YFD1800701)the Key Research and Development Program of the Ningxia Hui Autonomous Region(#2021BEF02028)the Chinese Agricultural Research System of MOF and MARA(#CARS-37).
文摘Lumpy skin disease(LSD)is a highly contagious disease caused by lumpy skin disease virus(LSDV)in bovines.Rapid and accurate diagnosis is very important to controll it.However,current commercial detection kits need to be improved in terms of sensitivity or specificity.This study aimed to develop a novel diagnostic competitive enzyme-linked immunosorbent assay(cELISA)based on the newly identified antigen gene LSDV034.The rLSDV034 protein was identified as a potential diagnostic antigen,and it was expressed,purified,and used to immunize BALB/c mice.Using laboratory-prepared indirect ELISA(iELISA),the positive cell lines were screened,and their blocking activity was further verified by competitive ELISA(cELISA).The cell line,1H7,was chosen to produce mouse ascites,which were purified for a monoclonal antibody(mAb,5.395 mg/mL).The heavy chain type of the 1H7 mAb was identified as IgG1a,and its light chain subtype was identified as κ.Furthermore,cELISA was developed to detect bovine serum antibodies,with rLSDV034(4μg/mL)as the coating antigen and HRP-1H7 mAb(1:300)as the competitive antibody.The cutoff value of cELISA was 55%,based on 32 negative bovine serum samples.The analytical sensitivity was 1:8,and no cross-reaction was detected with bovine viral diarrhea virus(BVDV),infectious bovine rhinotracheitis virus(IBRV),Pasteurella multocida(P.multocida),or Mycoplasma bovis(M.bovis)from the serum samples.The diagnostic sensitivity and specificity of cELISA were 98.46%(95%confidence interval,CI:91.7–100)and 100%(95%CI:89.1–100),respectively,based on the analysis of 30 LSDV-infected bovine serum samples,35 GTPV-vaccinated samples,and 32 negative samples.The overall coincidence of the cELISA with the virus neutralization test(VNT)reached 98.97%(95%CI:94.4–100).Furthermore,we used cELISA to analyze 230 clinical bovine serum samples(including 59 infected and 171 vaccinated samples)and found that the serum positivity rates of the immunized samples(on d 60 postimmunization)and infected samples were 77.78%(95%CI:70.8–83.8%)and 71.19%(95%CI:57.9–82.2),respectively.These results indicate that the developed cELISA is promising for detecting serum antibodies in naturally infected or vaccinated cattle.
