| The S.boydii disease is a new infectious disease Against chickens, which found in recent years.The disease is an acute disease caused by Shigella.It can lead to lots of different breed and day-old chicken occurre diarrhea and died. The disease has possibility of cross-infection between human and birds,which has strong public health significance.In this study, an indirect ELISA method was established and applied to detecting serum antibody angainst inactivated chicken S.boydii vaccine.It provides an serological detection method for evaluating the immune effect of chicken S.boydii vaccine to develop the reasonable immunization procedure for preventing or controlling disease effectively. The results are as follows:1.The establishment of indirect ELISA method.In this study, Chicken S.boydii whole thallus was used as a coated antigen inactivated by formaldehyde.Rabbit anti-chicken IgG labeled by horseradish peroxidase (HRP) was used as the second antibody. The indirect ELISA method was established for the detection of inactivated vaccine immune antibody of chicken S.boydii, and the best work of the ELISA conditions were determined:the concentration of coated antigen was 108 cfu/ml,the best temperature and time for coating was at 4℃in 12h; the best conditions for closing was used 1% BSA at 37℃in 2h; the best dilution of serum was 1:160;the best dilution of second antibody was 1:4000, and the best working conditions was at 37℃in lh; the best working conditions of substrate was at 37℃in 20min. The cross-testing, specificity, sensitivity tests, and repeatability tests show that the method has good reproducibility, great specificity, high sensitivity. The coefficient of variation of one-plate was 0.36%～0.64%, and 1.05%～1.85% of inter-plate; the coated whole cell antigen and other diarrheal diseases had no cross-reactivity, the results of blocking were positive. The sensitivity of indirect ELISA method is 80-160 times as much as micro-agglutination test.The OD values of 20 different positive chicken serum in different dilution were detected and found that linear correlation existed between the OD value of positive serum and the logarithm of ELISA titer reciprocal of serum.The regression equation of log [ET countdown]= .776+1.921 x was determined under the proposed standard curve.The establishment of the regression equation provided a mathematical model for forecasting and analyzing antibody levels by the serum OD values after clinical immunity.2.Indirect ELISA used in chicken S.boydii inactivated vaccine serum antibody detection.In order to evaluate the humoral immune effects of chicken S.boydii inactivated vaccine, the indirect ELISA method were applied to testing the post-vaccination antibody. The results showed that the immune antibody titer of immunity class was significantly higher than non immunity class (p<0.05)in the same period, the antibody levels of the second immunization were significantly higher than the first immunization (p<0.05).The antibody levels of immunity class increased rapidly after immunization and reached a peak in the two weeks.Nine weeks after the second immunization was still higher than the first immunization (p<0.05).The antibody levels of the second immunization in the forth week or the sixth weeks after the first immunization didn't have obvious difference in the same period (p> 0.05).The results showed that chicken S.boydii inactivated vaccine can improve the body's humoral immune function significantly;The established indirect ELISA method can evaluate the effects of chicken S.boydii vaccine truly; the vaccine and evaluation methods provide a good technical for testing S.boydii vaccine antibody and establishing the rational immune program.It will provide a good technical for preventing or controlling the disease.It is suit for applying and popularizing in production cheaply. |
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