Preparation The Antiserum Of Ureaplasma Urealyticum And Detection The Ureaplasma Urealyticum Through ELISA

ObjectiveWith ureaplasma urealyticum (Uu) serotype3(T3) and ureaplasma urealyticum serotype8(T8) all mycoprotein immune the New Zealand white rabbit, preparation of polyclonal antibody of Uu. In the preparation of polyclonal antibody to establish an indirect ELISA method for detection and identification of Uu.Methods1.Cultivation of T3and T8, by crush with ultrasonic, with protein mixed Freundadjuvant gained by the four times to more pionts of subcutaneous injection of New Zealand white rabbit, blood after immune four times and separation of serum.2.Will antiserum according to the latter,1:2000,1:4000,1:8000,1:16000,1:32000,1:64000,1:128000dilution proportion, at the same time to1:5000dilution of unimmune serum negative contrast, in western blot method, to determine the serum specificity and titer.3.According to the latter,1:1000,1:2000,1:8000,1:16000,1:32000,1:64000,1:128000diluted antiserum, indirect ELISA was carried out on the same antigen, and contrast with antigen did not join group.4. Analyze the results with Graphpad prism6.0software, and at times serum dilution degrees drawing, the results of the ELISA to determine the most suitable for ELISA of serum dilution degrees.5.The above step to determine the dilution degrees of serum respectively for101CCU to107CCU concentrations of the same species and the not same species of Uu to ELISA, to determine the sensitivity and specificity of the test.Results1.Western blot results in the figure, to a certain degree of diluting antiserum visible in the corresponding lane clear bands, the corresponding negative control without stripe, show that in the experiment of antibody has good specificity. T3antibody titer in for between1:16000to1:32000, T8antibody titer in between1:32000andl:64000. 2.In different concentration dilution of antiserum to the same antigen in ELISA, join antigen group compared with no antigen of the control group, there are differences between the two groups in statistical sense.3.The Graphpad prism6.0software analysis different dilution degrees of antiserum and antigen ELISA reactions OD value of the corresponding curve on the location of1:8000dilution degrees achieve relatively saturated, can determine the antiserum of1:8000diluted concentration is the most suitable for application of ELISA.4.Under the same concentration of antigen, polyclonal antibodies react with alloantigen result is significantly higher than the result of the reaction with heterologous antigen (P<0.05), and proved in this study to establish indirect ELISA method has good specificity.5.After the antiserum of diluted1:8000reaction to the bacteria of the concentration between101CCU and107CCU, in addition to the101CCU, the rest of the concentrations were significantly difference compared with negative control group (P <0.05), and proved that this method has better sensitivity.Conclusion1.At250μl of T3or T8mycoprotein immune New Zealand white rabbits, all may obtain good price of the polyclonal antibody specificity and more efficient.2.In this work, the preparation of polyclonal antibody to establish the detection of Uu ELISA method has high specificity and sensitivity, can achieve clinical use need, can consider to become a method of detection of Uu and Up.