Establishment and preliminary application of antibody-sandwich ELISA for detection of Mycoplasma ovipneumoniae antigen
In order to establish a rapid quantitative antigen detection method for Mycoplasma ovipneumoniae(MO),a monoclonal antibody-based sandwich ELISA method for detecting MO antigen was established through monoclonal antibody screening and condition optimization.The optimal conditions of the sandwich ELISA method are as follows:monoclonal antibody 1A11 was incubated at 37 ℃ for 1 h at 105 µg/mL and coated at 4 ℃ overnight.Sealed with l0g/L BSA at 37 ℃ for 1 h;act with ultrasonic treat-ed samples for 1.5 h;The antibody concentration was 3.81 μg/mL and treated at 37 ℃ for 2 h.The enzyme-la-beled antibody was diluted at 1:4 000 and reacted at 37 ℃ for 1.5 h.Add substrate and react at 37 ℃without light for 10 min.The coefficient of variation between and within batches were lower than 10％.The method showed good specificity,repeatabilityand sensitivity.And the MO antigen content detected by this method was positive correlation with its titer(CCU/mL).This study provided a new method for the quan-titative detection of MO antigen,which can be used as an alternatives to CCU(Color changing unit)test of MO cultures in the laboratory and in the production process of MO vaccine.
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