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| Development of PRRSV Antibody Detection Method with xMAP Technology |
| XIA Li-Ye1, JIANG Yan2, SHAN Hu1,3, LI Gui-Mei1,3,* |
1 Animal Medical School, Qingdao Agricultural University, Qingdao 266109, China;
2 Nanjing Customs, Nanjing 210001, China;
3 Shandong Provincial Key Laboratory of Preventive Veterinary Medicine, Qingdao 266109, China |
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Abstract Porcine reproductive and respiratory syndrome (PRRS) is one of the most serious viral diseases affecting pig (Sus scrofa) industry. In order to develop a high-throughput serological detection method for Porcine reproductive and respiratory syndrome virus (PRRSV), PRRSV antibody detection was developed by multi analyze profiling solve for unknown x (xMAP) technology. Firstly, PRRSV nucleocapsid (N) protein was coupled with carboxylated fluorescent microspheres. The coupling efficiency was verified. Microspheres was then reacted with serum samples and biotinylated detection antibody thereafter, the fluorescent signal of microspheres was detected with Luminex 200 System. Next, in order to determine the detection threshold, 32 serum samples of special pathogen free (SPF) pigs was detected at 1∶100 dilution. The threshold was calculated based on the median fluorescence intensity (MFI) and standard deviation. The optimal amount of binding protein was investigated. The repeatability of the method was verified, and the sensitivity and consistency of the method were studied compared with commercial kits. To verify the specificity of the method, the positive sera of Vesicular stomatitis virus (VSV) and Pseudorabies virus (PRV) were used. Finally, 83 clinical pig serum samples were detected by enzyme linked immunosorbent assay (ELISA) kit and liquid chip analysis, respectively, to verify the consistency of the 2 methods. The results showed that the MFI detection threshold was 103.22, and the optimal concentration of binding protein was 8 μg/1.25×106 beads; The coefficient of variation between and within batches was within 8%, suggesting a good repeatability. Compared with ELISA kit, the sensitivity of liquid chip detection method was higher. Neither VSV nor PRV positive sera showed positive by liquid chip analysis, indicated that the established liquid chip detection method had good specificity. When 83 clinical pig serum samples were detected by ELISA and liquid chip detection technology, results showed that the consistency between 2 methods were 91.57%. It showed that the high-throughput detection method of PRRSV using liquid chip detection technology had been successfully established. This study provides a new method for the serological detection of PRRSV and a reference for the detection of other pathogens.
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Received: 02 April 2020
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Corresponding Authors:
*ligm@qau.edu.cn
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