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@#Objective To develop and verify a double antibody sandwich ELISA method for quantitative detection of TrypLE.Methods The optimal concentration of capture antibody and detection antibody were determined by orthogonal experiments to develop TrypLE double antibody sandwich ELISA quantitative detection method,which was verified for linear range,specificity,limit of detection(LOD),limit of quantitation(LOQ),accuracy and reproducibility.A variety of biological products were detected by the developed method to verify the applicability.Results The TrypLE double antibody sandwich ELISA quantitative detection method was established by using 3 μg/mL capture antibody and 15 000 times dilution of detection antibody,with a linear range of 0.41 ～ 40.00 ng/mL,a LOD of 0.258 ng/mL,a LOQ of 0.5 ng/mL.The measurement deviation was less than 5% and the CV of reproducibility verification was less than 5% when detecting standards and samples.The recovery rates of different types of samples were within 80% ～ 120%.Conclusion The established TrypLE double antibody sandwich ELISA quantitative detection method accurately,effectively and quickly detected residual amount of TrypLE in various types of biological products with good specificity,accuracy and reproducibility.
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@#Objective To develop and verify a double antibody sandwich ELISA detection method for the determination of ovalbumin(OVA),in order to determine the OVA content in influenza vaccines.Methods The rabbit anti-OVA polyclonal antibody was used as coating antibody and HRP labeled rabbit anti-OVA polyclonal antibody as detection antibody to develop a double antibody sandwich ELISA for OVA.The antibody concentration(2,1,0.5 and 0.25 pg/mL) for coating,enzyme-labeled antibody concentration(0.5,0.25 and 0.125 μg/mL),and kinds of blocking reagent(blocking with1% BSA,blocking with 2% BSA,blocking with 1% BSA and 1% sucrose,and blocking with 2% BSA and 2% sucrose,using nonblocking as control) were optimized,and the Cut-off value was determined as the judgment standard.The developed method was verified for the linear range and detection limit,specificity,repeatability and accuracy.Results The optimum detection conditions were as follows:the concentration of coating antibody was 1 μg/mL,the concentration of enzyme-labeled antibody was 0.25 μg/mL;The blocking reagent was 2% BSA and 2% sucrose;The Cut-off value was 0.051 66.The linear range of the method was 5～0.313 ng/mL,and the detection limit was 0.078 ng/mL;It did not react with influenza virus,bovine serum albumin(BSA) and Vero cell supernatant;The intraplate and interplate coefficient of variation(CV) were between 2.562%～13.887% and 4.000%～16.497% respectively;The coincidence rate between the results of this method and the Germany SERAMUN OVA quantitative test kit ranged from 93.79% to 107.05%.Conclusion The developed OVA double antibody sandwich ELISA has good specificity,repeatability,linear range and accuracy with convenience and lower cost,which might be used for the quantitative detection of OVA.
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Objective:To establish a double antibody sandwich ELISA for detecting the specific antigen of Seoul virus (SEOV) L99 strain and to provide a means for antigen detection in the development, production and verification of vaccine against hemorrhagic fever with renal syndrome (HFRS).Methods:Monoclonal antibodies (McAbs) aganist L99 virus were induced in mice using four hybridoma cell lines and purified by Protein-A affinity chromatography. The purity, titer and specificity of McAbs were determined by SDS-PAGE, indirect ELISA and Western blot, respectively. Four McAbs were paired with each other and the additivity indices of paired McAbs were analyzed. After labeling McAbs with horseradish peroxidase (HRP), the concentrations of the coated and labeled antibodies were optimized by orthogonal test, and then a double antibody sandwich ELISA for virus antigen detection was established. Type Ⅱ HFRS inactivated vaccine standard was used as a quantitative standard to verify the sensitivity, linearity, specificity, accuracy and precision of the developed method. The applicability of the method was verified by testing three batches of vaccine stock solutions.Results:Four McAbs were at titers of greater than 1∶10 6 and their purity was all greater than 98%. The McAbs secreted by 1D5, 3A4 and 5B7 cells could specifically recognize the nucleocapsid protein of SEOV L99. There was cross-reaction between McAb secreted by 1D5 cells and Hantaan virus PS-6. The McAbs secreted by 3A4 and 1D5 were used as coating and labeling antibodies based on the results of antibody pairs. The working concentrations of the coating antibody and the horseradish peroxidase (HRP)-labeled antibody were 20 μg/ml and 1∶4 000, respectively. The minimum detection limit of the established method for the detection of SEOV L99 antigen was 0.078 1 μg/ml, and the linear range was 0.078 1-2.500 0 μg/ml with a R2 value of more than 0.99. There was no cross reaction with other HFRS vaccine. The virus antigen recovery rate was between 95.8% and 108.7%, and the coefficients of variation of precision was less than 10%. Three batches of Type II HFRS inactivated vaccine stocks were detected by this method and the results was dose-dependent. Conclusions:This study successfully established a double antibody sandwich ELISA method for specific detection of SEOV L99 strain antigen in the production of bivalent HFRS vaccines produced from hamster kidney cells.
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Clostridium difficile is an important zoonotic intestinal pathogen, which is widely present in humans and a variety of animals. The ST11 type C. difficile is one of the most widespread and harmful subtypes in the world. As a large country in pig farming, China lacks efficient methods for detecting C. difficile of porcine origin, leaving hidden dangers for the prevention and control of C. difficile. The aim of this study was to develop a specific and sensitive double-antibody sandwich ELISA for the epidemiological investigation of ST11 type C. difficile of porcine origin. Firstly, a 97 kDa receptor binding domain (RBD) was expressed in a prokaryotic host and purified. A hybridoma cell line AE2D3 capable of stably secreting monoclonal antibody targeting the RBD was screened, and the antibody subtype was determined to be IgG2b (κ). Secondly, a double antibody sandwich ELISA method was developed, where the monoclonal antibody targeting the RBD was used as a detection antibody, and the rabbit polyclonal antibody was used as a capture antibody. The chessboard method was used to determine the matching concentration of the capture antibody and the detection antibody, the antigen coating conditions, the blocking conditions, the incubation conditions for detection antibody and samples to be tested, as well as the reaction conditions of HRP-conjugated and reaction conditions of TMB chromogenic solution. The negative cutoff OD450 was 0.152, and no cross-reaction with 13 strains of non-ST11 type C. difficile was found. The minimum detection concentration of RBD was 8.83 ng/mL. This specific and sensitive double-antibody sandwich ELISA provides a reliable serological detection method for epidemiological investigation of the ST11 type C. difficile in pig industry.
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Animals , Antibodies, Monoclonal , Bacterial Proteins/genetics , Bacterial Toxins , Clostridioides difficile , Enzyme-Linked Immunosorbent Assay , Hybridomas , SwineABSTRACT
Objective@#To establish a method for detection of chikungunya virus(CHIKV) antigen.@*Methods@#CHIKV virus like particle(VLP), that contains all structural proteins, was prepared by baculovirus expression system. Mice and rabbits were immunized with the VLP to develop antibodies against CHIKV. A double antibody sandwich ELISA was established for detection of CHIKV antigens. The concentrations of the antibodies used and the reaction conditions were optimized. The detection limit and repeatability of the ELISA was evaluated.@*Results@#The sensitivity and specificity was estimated by 10 mimicking CHIKV sera, 90 health person sera, 40 other virus infected sera. It was show that the specificity of DAS-ELISA was 100%, the detection limit was 10 TCID50, the coefficients of variation (CV) within plate was <5%, the CV of different plates was <10%.@*Conclusions@#The double antibody sandwich ELISA established in this study can be used to detect the CHIKV antigen in acute phase serum of patient and provide a method for detection of CHIKV.
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Objective To establish a double-antibody sandwich ELISA for the rapid detection of shiga toxin typeⅡ ( StxⅡ) in shiga toxin-producing Escherichia coli ( STEC) infection. Methods A pool of murine hybridomas was used to screen out the optimal antibody pair for the establishment of double-anti-body sandwich ELISA. The established ELISA system was used to detect StxⅡin the culture supernatants of 16 clinical strains of STEC. Specificity and sensitivity of the established ELISA system were also evaluated. Results Two antibodies, S2D8 and S2C6, were successfully screened out, based on which the double-anti-body sandwich ELISA was set up. StxⅡand its variants rather than StxⅠwas detected in the culture super-natants of STEC with a lowest detection limit of 4 ng/ml. Its performance was consistent with that of commer-cial colloidal gold test kit, indicating the characteristics of good specificity and sensitivity. Conclusion The S2D8/S2C6-based ELISA laid a foundation for researches which designates the shiga toxin as a potential can-didate on the diagnosis and therapy of STEC infection.
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Objective:To establish the detection method of double-antibody sandwich ELISA about CYFRA 21-1 in human ser-um.Methods:The paired antibody were screened among four strains mAbs of CYFRA 21-1, which was marked by sodium periodate method.The detecting method of double antibody sandwich ELISA was optimizted , and evaluated by specificity , stability and sensitivi-ty.Results:The results showed that a paired of antibody , which was 2F9 as the coated antibody and 6F11 as the labeled antibody, was selected from four mAbs .It was the optimum condition of double antibody sandwich ELISA that the coating antigen concentration of 2F9 was 0.50 μg/ml, while the labeled antibody of 6F11 was diluted 6 000 times.The linear range of standard curve was 0.7-25 ng/ml with r2 =0.990 8, while the limit of detection was 0.666 8 ng/ml, the recovery rate was 98.14%.The cross-reactions with the oth-er analogues in serum were less than 0.1%.The coefficient of variation in group (n=10) was 6.8%, whereas coefficient of variation among group(n=5) was 11.4%.The correlation compared with other foreign ELISA kit was 91.42%.Conclusion:In brief, we suc-cessfully established the method of double antibody sandwich ELISA detecting CYFRA 21-1 level in human serum , laying the foundation for the production of CYFRA21-1 ELISA kit.
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ObjectiveTo develop an ELISA method for quantitative determination of enterovirus 71 (EV71) antigen.The method can be applied to detect EV71 antigen contents and analyze the correlation between immunogenicity and immunoprotection.It also can be used for tissue culture infective dose( TCID50 ) assay.MethodsA double antibody sandwich ELISA method was developed for quantitative determination of EV71 antigen on the basis of the high-affinity neutralizing monoclonal antibodies ( K8G2 and Y8H2-HRP).This method was compared with microscopic observation for the detection of EV71 TCID50.The correlation was analyzed between the specific activity of EV71 antigen and the EV71 neutralizing antibody titer in immune serum.ResultsThe linear range of this method was 0.125-4.0 U/ml and the R2 value was 0.9911.The reagent did not react with other antigens except EV71 antigen.The recovery ratio of this method was 0.89-1.16.The coefficient of variation was less than 15％.The heat recovery rate was above 85％ when the reagent was in 37℃ for 9 days.There was a good correlation in TCID50 of EV71 between this method and microscopic observation,r=0.990.The specific activity of EV71 antigen had positive correlation with the neutralization titer of immune serum in 21 EV71 strains,r=0.930.ConclusionThe quantitative ELISA method for EV71 antigen was developed,which could be used to detect EV71 antigen contents and analyze TCID50.The specific activity of EV71 antigen detected by the method could be used to evaluate the immunoprotection of the vaccine potency test.
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A double antibody sandwich ELISA for quantitative analysis of recombinant fusion protein IL-2-HSA was constructed using a polyclonal antibody to human IL-2 for capture and a monoclonal antibody to HSA with HRP-labeled conjugate for detection.The optimal concentration of the first coating antibody and detection antibody were 2 μg/mL and 0.5 μg/mL,respectively.Regression equation of the linear calibration curve was:y = 0.442 9 x-1.143 3 with a correlation coefficient of 0.996 6,and the linear detection ranged from 39.06 ng/mL to 1 250 ng/mL.Recovery from the supernatant of fermentation broth was 98.13% to 102.94%.The specificity assay indicated that it had little cross-reactions with IL-2 and HSA.The soundness analysis suggested that fermentation broth,mouse serum and dilution had no influence on the method.The present method can be used in the studies on fermentation,purification and clinical diagnosis.
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Objective To develop a double antibody sandwich ELISA for quantitatively detecting Japanese encephalitis virus(JEV) antigen.Methods The anti-JEV polyclonal antibodies were used to coat ELISA plates.Anti-JEV monoclonal antibodies were used as enzyme-labeled conjugate.A standard curve based on known amounts of JEP antigen was established by the ELISA.Various parameters of the assay were analyzed.Results The optimal linear range was 12.5～200 U/ml(r=0.9989).The quantitation limit was 12.5 U/ml.The recovery rate for the accuracy test was 85.0%～103.3%.The coefficients of variation for intra-assay and inter-assay precision were 4.3%and 5.5%respectively.No cross-reaction was observed with HAV vaccine,influenza vaccine,Vero cell Iysates,newborn bovine serum,or human albumin.Conclusions The data indicate that the ELISA developed in this study has high specificity,precision, accuracy,and stability.The assay should be suitable for quantitative determination of JEV antigen in various vaccine products.
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Objective To develop a double-antibody sandwich ELISA for determining the concentration of nucleoprotein (NP) of rabies virus in various products of rabies vaccine.Methods The purified rabies antibodies from a rabbit were used to coat microwell plates. Horseradish peroxidase-conjugated anti-NP monoclonal antibody was used to probe the NP bound to coated antibodies.The assay was used to quantitatively determine the concentration of NP of rabies virus.Results The results showed that the coefficient of linear correlation was higher than 0.97.The optimal linear range was 0.000625~0.01 IU/ml and the detection limit was 0.000625 IU/ml. The recovery rate was 102~109% and the coefficient of variation was only 7.2%~9.4%.No cross reactions were observed with bovine serum,bovine serum albumin,ovalbumin,refined solution of influenza vaccine,encephalitis B vaccine,and hepatitis A vaccine.Conclusions The results indicated that the assay is specific,sensitive,accurate,reproducible,and stable,and could be suitable for quantitative determination of different rabies vaccine's processes products.