Chemiluminescence Immunoassay for the Detection of Antibodies against the 2C and 3ABC Nonstructural Proteins Induced by Infecting Pigs with Foot-and-Mouth Disease Virus

Abstract

The potential diagnostic value of chemiluminescence immunoassays (CLIAs) has been accepted in recent years, although their use for foot-and-mouth disease (FMD) diagnostics has not been reported. Full-length 3ABC and 2C proteins were expressed in bacteria and purified by affinity chromatography to develop a rapid and accurate approach to distinguish pigs infected with foot-and-mouth disease virus (FMDV) from vaccinated pigs. The recombinant proteins were then used as antigens to develop two CLIAs for the detection of antibodies against nonstructural viral proteins. The diagnostic performance of the two assays was compared by analyzing serum from pigs (naive pigs, n = 63; vaccinated, uninfected pigs, n = 532; naive, infected pigs, n = 117) with a known infection status. The 3ABC-2C CLIA had a higher accuracy rate, with a diagnostic sensitivity of 100% and a diagnostic specificity of 96.5%, than the 3ABC CLIA, which had a diagnostic sensitivity of 95.7% and a diagnostic specificity of 96.0%. The results of the 3ABC-2C CLIA also had a high rate of concordance with those of two commercial FMDV enzyme-linked immunosorbent assay (ELISA) kits used to assess serum collected from 962 pigs in the field (96.2% and 97.8%, respectively). The 3ABC-2C CLIA detected infection in serum samples from infected pigs earlier than the commercial ELISA kits. In addition, the 3ABC-2C CLIA produced results within 15 min. On the basis of these findings, the 3ABC-2C CLIA could serve as the foundation for the development of penside FMD diagnostics and offers an alternative method to detect FMDV infections.

Keywords: 2C; 3ABC; chemiluminescence immunoassay; diagnosis; foot-and-mouth disease virus.

FIG 1

(A) SDS-PAGE profile of the expressed rmu3ABC protein. Lane M, prestained protein ladder (Thermo Scientific, USA); lane 1, uninduced E. coli cell extract; lane 2, soluble protein fraction; lane 3, insoluble protein fraction. (B) SDS-PAGE profile of the expressed r2C protein. Lane M, prestained protein ladder; lane 1, soluble protein fraction; lane 2, insoluble protein fraction; lane 3, uninduced E. coli cell extract. (C) SDS-PAGE profile of the purified rmu3ABC protein determined using nickel affinity chromatography. Lane M, prestained protein ladder; lane 1, the proteins after washes with IB washing buffer; lane 2, unbound protein; lanes 3 and 4, the eluted purified protein. (D) SDS-PAGE profile showing the r2C protein affinity purified using Ni-NTA resin. Lane M, prestained protein ladder; lanes 1 and 2, the eluted purified 2C protein. (E) Western blots showing the reactivity of rmu3ABC and r2C proteins with sera from FMDV-infected animals. Lane M, prestained protein ladder; lane 1, affinity-purified rmu3ABC protein; lane 2, affinity-purified r2C protein.

FIG 2

Results of checkerboard titration method used to optimize the rmu3ABC protein concentration and serum dilution. Standard positive and negative serum samples were serially diluted 2-fold, and the coating concentration was varied. An rmu3ABC protein coating concentration of 25 ng/well and a serum dilution of 1:40 were considered the best conditions. P, positive serum sample; N, negative serum sample.

FIG 3

Determination of the cutoff values of the 3ABC CLIA and the 3ABC-2C CLIA. (A and B) Each point on the ROC plot represents a sensitivity-specificity pair corresponding to a particular decision threshold. (C and D) Plot-versus-criterion values. The 95% confidence intervals of Dsn and Dsp are plotted against the different criterion values. (E and F) Interactive dot diagram. 0, NSP-negative serum samples; 1, NSP-positive serum samples.

FIG 4

The Ase of the 3ABC-2C CLIA was determined using 2-fold serially diluted standard positive pig serum samples and compared to that of the 3ABC monoclonal antibody blocking ELISA and the PrioCHECK FMDV NSP ELISA.

FIG 5

Comparison of the diagnostic performance in the early FMDV infection phase. (A) Results for a naive pig challenged with the A/GDMM/CHA/2013 FMDV strain; (B) results for a naive pig challenged with the O/China/99 FMDV strain.