A poly(dimethylsiloxane)-based solid-phase microchip platform for dual detection of Pseudorabies virus gD and gE antibodies

Abstract

Pseudorabies caused by pseudorabies virus (PRV) infection is still a major disease affecting the pig industry; its eradication depends on effective vaccination and antibody (Ab) detection. For a more rapid and accurate PRV detection method that is suitable for clinical application, here, we established a poly(dimethylsiloxane)-based (efficient removal of non-specific binding) solid-phase protein chip platform (blocking ELISA) for dual detection of PRV gD and gE Abs. The purified gD and gE proteins expressed in baculovirus were coated into the highly hydrophobic nanomembrane by an automatic spotter, and the gray values measured by a scanner were used for the S/N (sample/negative) value calculation (gD and gE Abs standard, positive: S/N value ≤0.6; negative: S/N value >0.7; suspicious: 0.6 < S/N ≤ 0.7). The method showed an equal sensitivity in the gD Ab test of immunized pig serum samples compared to the neutralization test and higher sensitivity in the gE Ab test compared to the commercial gE Ab detection kit. In the clinical evaluation, we found an agreement of 100% (122/122) in the gD Ab detection compared to the neutralization test and an agreement of 97.5% (119/122) in the gE Ab detection compared to the commercial PRV gE Ab detection kit. In summary, the protein chip platform for dual detection of PRV gD and gE Abs showed high sensitivity and specificity, which is suitable for PRV immune efficacy evaluation and epidemic monitoring.

Keywords: Pseudorabies virus; clinical evaluation; dual detection; poly(dimethylsiloxane); protein chip.

Figure 1

Schematic diagram of the design of the double Abs detection system. (A) Spotting design of detection points in the hole of the chip board. QC, quality control. (B) Schematic diagram of the steps of the blocking ELISA method for PRV gD and gE Abs detection. Ag: gD or gE coated in the membrane. Ab-gD/gE: PRV gD- or gE-specific Ab in serum samples. Ab-X: PRV gD- or gE-non-specific Ab in serum samples. HRP-Mab-gD/gE: HRP-labeled Mabs of PRV gD or gE in the blocking assay. (C) Screening process for the reaction conditions of the detection system, including the optimal spot volume, spot concentrations of gD, gE and goat anti-mouse IgG, and reaction speed and time.

Figure 2

Expression of PRV gD and gE and the Ab detection based on the PDMS membrane. (A) SDS-PAGE analysis of the purified gD and gE proteins expressed in Sf9 cells after size exclusion chromatography. (B) Scanning electron microscopy of the PDMS membrane. (C) Abs detection results of gD and gE in the nitrocellulose membrane. (D) Abs detection results of gD and gE in the PDMS membrane. mm, millimeter.

Figure 3

Frequency analysis of PRV gD and gE Abs-negative serum samples using the dual-detection chip platform. The S/N values of gD (A) and gE (B) Abs of 270 Ab-negative serum samples after the detection of the dual detection chip platform. The frequency distribution of the tested serum samples was constructed using GraphPad Prism 8.0.

Figure 4

Sensitivity and specificity evaluation of the chip detection system in gD and gE Abs. (A) The gD Ab of immunized pig serum samples (0–16 wpi) tested by the protein chip detection system. (B) The gD Ab of immunized pig serum samples (0–16 wpi) tested by neutralization assay. (C) The gE Ab of challenged pig serum samples (0–14 dpc) tested by the protein chip detection system and commercial gE Ab kit. (D) Specificity assay of the chip detection system in gD Ab detection using the positive serum samples of ASFV, PCV2, PPV, PRRSV, CSFV, PEDV, PDCoV, FMDV serotype O (pig), FMDV serotype A (pig), baculovirus, and PRV-gD (n = 5). (E) Specificity assay of the chip detection system in gD Ab detection using the positive serum samples of ASFV, PCV2, PPV, PRRSV, CSFV, PEDV, PDCoV, FMDV serotype O (pig), FMDV serotype A (pig), baculovirus, and PRV-gE (n = 5). S/N value > 0.7 is considered negative. The comparison of chip detection and commercial kit methods was analyzed by one-way ANOVA using GraphPad Prism 8.0. ns, not significant. *p < 0.05.