Abstract:
Porcine epidemic diarrhea is caused by porcine epidemic diarrhea virus (PEDV), which causes swine watery diarrhea and higher mortality rates in piglets less than one week of age. Outbreaks of PED affect to economic loss, especially in a swine industrial country. Many strategies were used for preventing the outbreak and infection of PEDV, especially maternal antibody stimulation in pigs and then transfer to piglet through colostrum or milk. Thus, the investigation of antibody level for the situation on the farm is required. The method which convenient and rapid than viral neutralization (VN) test is an enzyme-linked immunosorbent assay (ELISA). The purpose of this study was to develop ELISA test kits to detect antibodies to PEDV from colostrum and milk of pig by preparing antigens of recombinant protein from the spike gene of PEDV and then a validation test of the ELISA test kit was performed from 250 VN positive and 250 VN negative samples. The results of the study showed that ability to produce the protein called S12. The protein is specific to neutralizing antibodies of PEDV and produced from a conserve region of the PEDV gene. S12 protein is 42 kDa of protein size, can be purified either by purification of glutathione S-transferase (GST) gene fusion proteins or histidine-tag protein. Protein S12 can be used as antigen to coat ELISA plate at 10 µg/ml and 20 µg/ml for immunoglobulin (Ig) G and IgA investigation, respectively. In addition, suitable formulations for the test kit were prepared. The precision was verified by the repeatability test, coefficient of variation is less than 10% and has high reproducibility of inter-laboratory results. The correlation between the standardized and ELISA showed strong correlation coefficients from both IgG and IgA. Thus, it can be said that the result is consistent with protection. The cutoff point of optimal density is 0.5, DSN, DSP and accuracy of IgG and IgA were 96.0, 95.6, 95.8 and 94.8, 95.6, 95.2, respectively. As for the cutoff level of the sample to positive ratio (S/P ratio) is 0.4, DSN, DSP and accuracy of IgG and IgA were 95.2, 90.8, 93.0 and 91.6, 93.2, 92.0, respectively. Long term storage of the ELISA kit up to 6 months. In addition, the test kit was used to detect antibodies from feedback and the results show that the ELISA test kit can be used. According to all this study, it can conclude that this ELISA kit can be an alternative possibility for detecting antibody response against PEDV in colostrum and milk.
