A Semiautomated Luciferase Immunoprecipitation Assay for Rapid and Easy Detection of African Swine Fever Virus Antibody

Abstract

African swine fever (ASF) is a highly contagious viral disease of domestic pigs and wild boars. For disease surveillance and control, we developed a rapid and easy luciferase immunoprecipitation assay (MB-LIPS) to detect ASF virus (ASFV) antibody. The MB-LIPS is based on magnetic beads modified with protein A/G and the recombinant fusion protein of ASFV p30 and luciferase, where p30 functioned as the recognition element and luciferase as the signal component. Incubation and washing could be finished automatically on a machine with magnetic rods. Under optimal conditions, the MB-LIPS showed 96.3% agreement to a commercial enzyme-linked immunosorbent assay (ELISA) kit for detecting ASFV antibody in swine sera. Analyzing serial dilutions of a swine serum sample showed that the MP-LIPS assay was 4 times more sensitive than the ELISA kit. The whole run of the MB-LIPS could be completed within 30 min. With its high sensitivity and simple operation, the MB-LIPS platform has great potential to be used for the detection of ASFV antibody and ASF control in small labs and farms.

Keywords: African swine fever virus; luciferase immunoprecipitation system; magnetic beads; rapid; sensitivity.

FIG 1

Schemes of the principle (A) and the process (B) of the MB-LIPS assay for ASFV antibody detection.

FIG 2

SDS-PAGE analysis (A), Western blot analysis (B), and luciferase activity (C) of the purified p30-Luc. (A) Lane 1, protein marker; lane 2, purified p30-Luc with a molecular weight of 85 kDa. (B) Lane 1, protein marker; lane 2, recombinant p30-Luc hybridized with anti-p30 mouse serum. (C) Luminescence intensity versus the concentration of p30-Luc. Data are shown as the mean from three repeats. Error bars were small enough not to be shown in the plot.

FIG 3

Optimization of the MB-LIPS assay for ASFV p30 antibody detection. (A) S/N ratios of an ASFV-positive serum and a negative serum versus p30-Luc concentrations. (B) S/N ratios of an ASFV-positive serum and a negative serum versus the amount of protein A/G-modified magnetic beads. (C) S/N ratios of an ASFV positive serum and a negative serum versus the dilution folds of sera. Data are shown with means ± SD. Error bars represent the standard deviations from triplicates.

FIG 4

Determination of the cutoff value of the MB-LIPS for ASFV antibody detection through ROC. (A) S/N ratios obtained by the MB-LIPS assay from a panel of the sera that are classified as positive or negative by the ELISA kit. (B) ROC curve based on the data obtained.

FIG 5

Sensitivity comparison between the MB-LIPS (blue line with squares) and the ELISA kit (red line with triangles) for ASFV antibody detection. Two ASFV (28 and 29)-positive sera are diluted to different folds. (A and B) The plots of the inset line charts show the signals under high dilutions, with dotted lines as the cutoff value for positive results. Data are shown with means ± SD. Error bars represent the standard deviations from triplicates.