Abstract:

【Background】 Foot-and-mouth disease (FMD) is an acute, febrile and virulent infectious disease caused by foot-and-mouth disease virus (FMDV), and vaccination is an effective measure to prevent the spread of FMD. The level of immune antibody monitoring is an important basis of evaluating the effectiveness of vaccine immunization, and developing immunization procedures. It is an indispensable step in the prevention and control of FMD. Therefore, to establish an efficient, rapid and fully automated antibody detection method is of great significance. 【Objective】The aim of this study was to establish a novel, fully-automatic and quantitative antibody detection method for FMD virus type O based on the chemiluminescence immunoassay (CLIA) technology of magnetic particles (MPs), in order to provide the technical support for the immunization monitoring of FMD and epidemic prevention and control.【Method】In this study, the magnetic nanoparticles were used for solid-phase carriers and separation carriers to encapsulate the captured antibody, the detection antibodies was labelled by using alkaline phosphatase (ALP), and AMPPD was used for the luminescent substrate. Finally, a new magnetic particle Chemiluminescence CLIA method (MP-CLIA) was established after optimization of conditions. In this method, firstly, the magnetic particle-polyclonal antibodies (MPs-pAbs), the samples to be tested, and the antigen of FMDV type O were added to a reaction system and incubated at 37 ℃, then the appropriate amount of enzyme-labeled antibodies (ALP-pAbs) was added and incubated at 37 ℃, finally, the chemiluminescence substrate AMPPD was added to detect the relative light unit (RLU). In this study, a standard curve was fitted by testing the standards, and thereceiver operating characteristic (ROC) curve was applied to determine the determination criteria of the assay. The quality control samples were utilized for methodological evaluation, and the field samples were also tested and compared with the liquid phase blocking ELISA (LPB-ELISA) to validate the assay results. 【Result】 The optimized reaction conditions were 0.25 mg·mL^-1 of magnetic particles, 1:1 000 dilution of FMD O antigen, 1:2 000 dilution of enzyme-labeled antibody, and 20 µL of spiked sample volume. The entire detection process was completed in a fully automated chemiluminescence immunoassay analysis apparatus, with a reaction time of 20 min, The quantitative detection could be conducted within the range of antibody content of 0-1 280 U (potency of 0-1:2 048), and the standard curve R²>0.99. The sensitivity of assay was 94.66%, the specificity was 97.10%, and there was no cross reactivity with the antibodies of six different pathogens which include SVV, PRRSV, BEFV, PCV2, QRFV, and PPRV. The repeatability of the assay showed the coefficient of variation less than 10%. The detection of field samples demonstrated the accordance rate was 94.69% between CLIA and LPB-ELISA, and the result of quantitative detection showed a correlation (R²=0.8473, P<0.0001) during two method. 【Conclusion】 The established MP-CLIA method was time-consuming, easy to operate, and could be fully automated by matching with the domestic automatic chemiluminescence instrument, which was a new and highly efficient method for the quantitative detection of antibody to FMD virus type O, and had a high value for clinical application.

Key words: magnetic particle chemiluminescence immunoassay (MP-CLIA), foot and mouth disease (FMD), micromagnetic particles, fully-automated