Development and application of nsp5-ELISA for the detection of antibody to infectious bronchitis virus

Abstract

Infectious bronchitis virus (IBV) continues to be one of the most important poultry pathogens worldwide. The current commercially available enzyme-linked immunosorbent assay (ELISA) kits for IBV specific antibody detection are mostly based on the whole virion, and few serological tests based on nonstructural proteins of IBV have been developed. Herein, an alternative indirect ELISA for detection of IBV antibody was developed with IBV nonstructural protein 5 (nsp5) produced by Escherichia coli. Using an indirect immunofluorescence assay (IFA) and a commercial ELISA kit as reference, we optimized the nsp5-ELISA and determined its cut-off as 0.12. The diagnostic sensitivity (DSN), specificity (DSP) and accuracy of the nsp5-ELISA were 93.11%, 95.38% and 93.33%, respectively, compared with IFA in 660 field serum samples, and were 98.11%, 95.00% and 97.62%, respectively, compared with the commercial IBV ELISA kit (IDEXX) in 126 field sera samples. Furthermore, a time course of IBV specific antibody level detected by nsp5-ELISA following IBV infection and vaccination is consistent with that of IBV antibody detected by the commercial ELISA kit. The results presented in this study indicate that nsp5-ELISA has the potential to serve as a rapid, reliable and cost-effective method for IBV antibody detection. This study is the first to report the development of an nsp-based ELISA to detect an antibody to IBV.

Keywords: Enzyme-linked immunosorbent assay; Ibv; Nonstructural protein 5 (nsp5).

Fig. 1

(A) Expression and purification of recombinant GST-fused nsp5 analyzed by SDS-PAGE. M, standard protein marker; lane 1, uninduced E.coli transformed with PGEX-4T-1/nsp5; lane 2, induced E.coli transformed with PGEX-4T-1/nsp5; lane 3, uninduced E.coli transformed with PGEX-4T-1; lane 4, induced E. coli transformed with PGEX-4T-1; lane 5, purified GST-nsp5 protein; lane 6, purified GST. (B) Western blotting analysis of purified GST-nsp5. Positive serum to IBV SC021202 were used as the primary antibody. M, standard protein marker; lane 1, purified GST-nsp5; lane 2, GST.

Fig. 2

Optimization of ELISA working conditions. (a) Concentration of coating antigen. (b) Dilution of serum sample. (c) Dilution of conjugates. (d) Coating buffers. H2O, distilled water; NS, physiological saline (0.9% NaCl); PBS, 0.01 M phosphate-buffered saline pH 7.4; TB, 0.05 M Tris–HCl buffer, pH 8.5; CB, 0.05 M carbonate–bicarbonate buffer pH 9.6; NaOH, 0.1 M NaOH solution, pH 13. (e) Blocking buffers (PBS dissolved). SM1, 2% skim milk; SM2, 5% skim milk; BSA1, 0.1% BSA; BSA2, 0.5% BSA; BSA3, 1% BSA; NBS1, 5% new-born calf serum; NBS2, 10% new-born calf serum. (f) Serum diluent buffer (PBS dissolved). PBST1, 0.05% Tween-20; PBST2, 0.1% Tween-20; BSA, 0.1% BSA; BSAT, 0.1% BSA and 0.05% Tween-20; SM, 5% skim milk; SMT, 5% skim milk and 0.05% Tween-20. (g) Exposure time of serum sample. (h)Conjugate exposure time. The vertical dotted lines indicate selected condition. Ab, antibody/serum sample.

Fig. 3

Frequency plots of the number of IFA positive (n = 595) and negative (n = 65) samples of field sera and the natural log of S/P ratios obtained from the nsp5 ELISA demonstrating two overlapping populations. The number in parentheses is the S/P ratio. The negative–positive cut-off was defined as 0.12 with an OD₄₅₀ value of a positive serum control close to 1.5 for the nsp5 ELISA, at which the DSN and DSP of the assay were greater than 90%.

Fig. 4

Comparative detection of antibodies to IBV in SC021202 infected (A), M41 inactivated vaccine (B) and H52 attenuated vaccine (C) immunized chickens by nsp5 ELISA and IDEXX IBV ELISA. The blue dotted lines indicate the cut-off of nsp5 ELISA; the black dotted lines indicate the cut-off of IDEXX IBV ELISA. In the IBV SC021202 infected group, 21 days post infection, only one chicken survived; therefore, there is no standard deviation indicated after 21 d.p.i. in A. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)