Method establishment and preliminary application of sandwich ELISA for detecting Cryptosporidium parvum antigen / 中国人兽共患病学报

ABSTRACT

We established the method of Sandwich ELISA to detect Cryptosporidium parvum antigen.Purified anti-Cryp-tosporidium IgG and IgY were used as a capture antibody and detection antibody respectively to develop sandwich ELISA.A checkerboard titration study was carried out to determine the optimal conditions of ELISA.The PCR based on 18SrRNA was used to evaluate the pre-treatment effect of three methods (saturated sucrose solution floating,saturated salt water floating and PBST detergent washing).The optimum concentration of coated antibody,antigen,detection antibody and enzyme-labelled an-tibody were 1800,2.5 μg/mL,1100 and 15 000 respectively.The coating condition,antigen antibody reaction,opti-mum reaction time of enzyme-labelled antibody were 4 ℃ through the night after 37 ℃,incubated at 37 ℃ for 30 min and 45 min respectively;the optimal termination condition was 2 mol/L H2SO4,50 μL/well;TMB developed 10 minutes at room temperature.The developed sandwich ELISA has no cross reaction with the eggs/oocyts of Nematode,Coccidium and Asca-rid;coefficient of variation of intra-assay and inter-assay were all less than 10%.The results showed that the total coincidence rate of the three pre-treatment methods with nested PCR were 95.83%,91.67% and 83.33%,respectively,and the Saturated Sucrose Floatation method was the best one among the three methods,the sensitivity of the method was lower than the Cry p-tosporidium detection kit of IDEXX(6×103/mL),and whole test process was longer than the kit.While,its specificity and reproducibility were consistent with that of IDEXX kit,and the developed method was more economical.The method is simple,rapid,sensitive,and can be used for clinical epidemio-logical investigation of Cryptosporidiosis or pathogen detection.