| Foot-and-mouth disease(FMD)is a most highly contagious disease of cloven-hoofed animals caused by foot-and-mouth disease virus(FMDV),which has a great threat to livestock and international trade in animals.Detection of FMDV antibody is one of the main methods of FMDV surveillance,among which liquid-phase blocking ELISA(LPB-ELISA)is an important method for detection of FMDV type O antibody,which has been widely used in vaccine evaluation and animal quarantine.However,it has the shortcomings of low detection throughput,great time and energy consumption and so on.In this experiment,a high throughput liquid phase blocking CLIA method for detection of FMDV type O antibody(LPB-CLIA)was developed.The detection throughput was increased by 5-10 times as well as the accuracy of detection was preserved,and the stability of detection was optimized.It provides a new method for the prevention and control of foot-and-mouth disease in China.The key factor to increase the detection throughput is to reduce the dilutions of serum.In order to determine the best dilution of serum,8 sera with different titers were detected under different dilutions,and the effective detection range of each dilution was investigated.The results showed that the detection range was limited in 3-5 titers under all dilutions.Therefore,in order to ensure the accuracy and throughput optimization,the serum was divided into high titer serum(ELISA titer ≥ 1:128)and low titer serum(ELISA titer < 1:128),and determine the best dilution multiple,respectively.Through detecting 90 low titer serum and 54 high titer serum,it was determined that 8-fold dilution was suitable for low titer serum and 64-fold dilution was great for high titer serum.In order to interpret the results of test effectively and meaningfully,the percentage of inhibition was calculated after detecting a large amount of serum,and the best cut-off value of each titer was determined by ROC curve of PI.Thus the corresponding relationship between PI and ELISA titer was established,and the accurate assignment of serum was realized.Finally,the standards of judgment of the two detection systems were established,and the cross parts(titer of 1:90 and 1:180)were verified.The results showed that 79 out of 88 serum samples were consistent with each other.The detection results of 8-fold dilution in 9 abnormal sera were in better agreement,therefore,for the cross part of serum,take the 8-fold dilution test results as standard.The repeatability,sensitivity and specificity of the test were verified after the determination of the whole detection system.The results showed that the intro-batch CV of PI was less than 10%,and the inter-batch CV was under 15%.The mean standard deviation of 45 serums among 3 tests was 0.03,which showed great repeatability of test.The test sensitivity was 98.5% and the specificity was 96.3% by detecting 214 positive serum and 246 FMD negative serum.37 positive sera of FMD type A and 7 positive sera of FMD type Asia I were all negative,indicating that there was no cross reactivity in the test.The comparison between the results of LPB-ELISA and LPB-CLIA with 239 sera showed that the correlation coefficient of the two methods was 0.95,which implied they were highly correlated.In the end,the immune effects of 6 different vaccines were evaluated.The results showed that the change of antibody responds after immunized detected by LPB-CLIA was basically the same as that of LPB-ELISA test.Thus it can be used in the evaluation of vaccine quality and the development of vaccine immunization procedure.The high throughput liquid-phase blocking CLIA antibody detection method can be used to detect qualitatively by single dilution multiple and quantitatively by two dilutions.It improves detection throughput and efficiency greatly,while maintains the accuracy of LPBELISA.LPB-CLIA provides a better choice for mass detection of FMD. |
