Preparation of Artificial Antigen and ELISA Identification of Mouse Polyclonal Antiserum for the Detection of Doxycycline
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Abstract:
To prepare complete antigen of doxycycline (DC) and obtain mouse polyclonal antiserum with good immunological characteristics, carboxyl groups were introduced into DC molecules by succinate method in this work. A Complete antigens were prepared by coupling with carrier proteins using an improved succinic acid method. The coupling effect of complete antigen was identified by UV and SDS-PAGE. Four BALB/c mouse were immunized with 30 μg artificial antigen, and the antiserum immunological characteristics were identified by indirect ELISA and indirect competitive ELISA. The results showed that the coupling effect of artificial antigen was good, the titer of antiserum of all four mouse were higher than 1:12800, the serum of all four mouse had inhibitory effect. The antiserum produced by mouse number 4 was the best. The inhibitory concentration (IC50) was 24.75 ng/mL. The cross-reaction rates of OTC, CTC and TC of their homologues were 3.4%, 3.8% and 3.0%, respectively. The cross-reaction rate with other veterinary drugs and carrier protein was less than 0.3%, and had a good specificity. This study successfully synthesized DC artificial antigens and prepared DC antiserum with good immunological properties, which provided a good foundation for the preparation of monoclonal antibody and the establishment of immunology rapid detection method.
