An enhanced immunochromatographic strip test using colloidal gold nanoparticle-labeled dual-type N proteins for detection of antibodies to PRRS virus

Abstract

Porcine reproductive and respiratory syndrome (PRRS) is recognized as one of the most important infectious diseases causing serious economic loss in the swine industry worldwide. Due to its increasing genetic diversity, a rapid and accurate diagnosis is critical for PRRS control. The immunochromatographic strip test (ICST) is a rapid and convenient type of immunoassay. In this study, an on-site immunochromatographic assay-based diagnostic method was developed for detection of PRRS virus (PRRSV)-specific antibodies. The method utilized colloidal gold nanoparticle-labeled dual-type nucleocapsid proteins encoded by open reading frame 7. We evaluated 991 field samples from pig farms and 66 serum samples from experimentally PRRSV-inoculated pigs. Based on true PRRSV-specific antibody-positive or -negative sera determined by immunofluorescence assay and IgM enzyme-linked immunosorbent assay (ELISA), the specificity and sensitivity of the ICST were 97.5% and 91.1%, respectively, similar to those of a commercial ELISA (IDEXX PRRS X3 Ab). More importantly, the ICST was completed within 15 min and could detect the PRRSV-specific antibody at an earlier stage of infection (3-7 days) than that of ELISA (7+ days). The results demonstrate that the developed ICST has great potential as an on-farm diagnostic method, providing excellent diagnostic performance in a quick and convenient manner.

Keywords: immunochromatographic assay; on-farm detection; porcine reproductive and respiratory syndrome virus.

Fig. 1. Schematic diagram of immunochromatographic strip test (ICST) developed for porcine reproductive and respiratory syndrome virus (PRRSV)-specific antibodies. The strip is composed of sample pad, gold conjugate pad, nitrocellulose membrane, and absorption pad. If the serum contains PRRSV-specific antibodies, those antibodies are captured by recombinant nucleocapsid (rN) gold conjugates in the gold conjugate pad. The rN gold conjugate-antibody complex is then captured by the rN protein on the test line (T), producing a red visible band, while unbound rabbit IgG gold conjugates move through the nitrocellulose membrane and are captured by anti-rabbit IgG on the control line (C) to form another visible band.

Fig. 2. Examples of the developed immunochromatographic strip test results. (A) A positive result for diluted porcine reproductive and respiratory syndrome virus (PRRSV)-positive serum from experimentally PRRSV-infected pigs. (B) A negative result for diluted PRRSV-negative control serum from a 3-month-old pig purchased from a PRRSV-negative farm. C, control line; T, test line.

Fig. 3. Detection of porcine reproductive and respiratory syndrome virus (PRRSV)-specific antibodies in swine sera from 6 experimentally inoculated pigs by the developed immunochromatographic strip test (ICST) and commercial enzyme-linked immunosorbent assay (ELISA) (IDEXX PRRS X3 Ab; IDEXX Laboratories, USA). Six pigs in three groups (two pigs in each group) were inoculated with three types of PRRSVs including LMY, PL97-1, and E38, and sera were obtained at 0, 7, 14, 28, 39, and 52 days post-infection (dpi). The ICST detected PRRSV-specific antibodies in all 6 pigs as early as 7 dpi, while the commercial ELISA detected PRRSV-specific antibody in 4 of 6 pigs at 7 dpi. Samples with time-resolved fluorescence reader test intensities ≥ 40 were considered positive; samples with sample-to-positive (S/P) ratios ≥ 0.4 were considered positive.

Fig. 4. Porcine reproductive and respiratory syndrome virus (PRRSV)-specific antibody responses in immunochromatographic strip test (ICST) and IgM enzyme-linked immunosorbent assay (ELISA) (in-house) for swine sera from 3 experimentally inoculated pigs. The serum samples of pigs 36, 37, and 39 (one pig in each group) were applied to the in-house IgM ELISA and the results compared with the ICST results. The PRRSV-specific antibody profile showed a marked peak at 7 days post-infection (dpi). There were similar trends in antibody profiles of both ICST and IgM ELISA during the early infection period. OD, optical density.

Fig. 5. Detection of porcine reproductive and respiratory syndrome virus (PRRSV)-specific antibodies at different times after infection. Six pigs in two groups (three pigs each) were inoculated with two prototype PRRSVs (VR2332 and Lelystad virus [LV]) and sera were collected at 0, 1, 3, 5, 7, and 14 days post-infection (dpi). The immunochromatographic strip test (ICST) could detect PRRSV-specific antibody in 1 of 6 pigs at 3 dpi, 2 of 6 pigs at 5 dpi, and all pigs at 7 dpi, while the commercial enzyme-linked immunosorbent assay (ELISA) detected PRRSV-specific antibody in only 1 of 6 pigs (Pig 3) at 7 dpi. All pigs were positive for PRRSV-specific antibodies in both ELISA and ICST results from 14 dpi onwards (data not shown). Samples with time-resolved fluorescence reader test intensities ≥ 40 were considered positive; samples with sample-to-positive (S/P) ratios ≥ 0.4 were considered positive.