A pan-genotypic indirect competitive ELISA for serological detection of pigeon circovirus antibodies

Abstract

Pigeon circovirus (PiCV) infection, which causes young pigeon disease syndrome (YPDS) and immunosuppression, significantly impacts both the meat and racing pigeon industries. Currently, no inactivated vaccine exists for PiCV prevention, primarily due to the challenges associated with isolating the PiCV virion, except for some gene subunit vaccines express the Cap protein of PiCV. The development of detection techniques is crucial for the diagnosis of PiCV. This study aimed to develop and validate a specific, sensitive indirect competitive enzyme-linked immunosorbent assay (icELISA) for detecting PiCV antibodies in pigeons. We identified the cap gene from a group C PiCV strain (PiCV/Shaanxi/China/10/2021, SX10) isolated from racing pigeons. The Cap of SX10, an immunogen, can self-assemble into virus-like particles (VLPs). A mouse monoclonal antibody (mAb) against Cap, 1G6-4C4, was selected to establish an icELISA. This mAb could identify the PiCV Cap of the strains in groups A to E. The pan-genotypic reactivity of mAb 1G6-4C4 might target a conserved conformational epitope, overcoming limitations of PCR and prior serological assays. The icELISA method exhibited no cross-reactivity with antibodies against other common pigeon pathogens, such as pigeon paramyxovirus type 1 (PPMV-1), avian influenza (H9N2), avian adenovirus type 4 (FAdV-4) or rotavirus (RV). Compared with indirect ELISA (iELISA), icELISA demonstrated comparable performance, as testing of 29 clinical serum samples revealed antibody-positive rates of 51.72% (icELISA) and 44.82% (iELISA), with a 93.10% concordance rate. To a certain extent, icELISA has demonstrated good specificity and sensitivity for detecting PiCV-specific antibodies in pigeons. The developed icELISA provides a robust, specific, and sensitive tool for the serological detection of PiCV infection. Complementary to PCR test, icELISA enhances the comprehensive detection of PICV in epidemiological studies by offering a more practical and sensitive alternative for field applications. Its utility for large-scale epidemiological surveillance in PiCV-endemic regions is validated, highlighting its potential to inform targeted biosecurity and control interventions.

Keywords: Cap; indirect competitive ELISA; monoclonal antibody; pigeon circovirus; virus-like particles.

Figure 1

Geographical distribution of PiCV and phylogenetic and homology analyses of PiCV. All the PiCV strains from GenBank were analyzed for geographical distribution worldwide (A) and in China (B). (C) Phylogenetic trees based on the amino acids of Cap. The trees were aligned with MAFFT v7.525 and constructed via IQ-TREE v2.3.6. (D) Pairwise identity matrices calculated with the ClustalW method comparing the percentage of amino acid identity among SX10 and the 296 PiCV Cap. The isolates used in this study are indicated by a red solid arrow. SX10, PiCV/Shaanxi/China/10/2021.

Figure 2

Purification of the PiCV Cap. (A) SDS-PAGE assay of the expression and purification of the PiCV Cap. (B) The purified PiCV Cap protein was detected using a Western blotting with a mouse anti-His-tag mAb. (C) VLPs self-assembled from Cap, as observed via TEM (red arrows).

Figure 3

Production, screening and characterization of Cap mAbs. (A) The serum antibody titers of five BALB/c mice immunized with the PiCV Cap protein were detected via iELISA. NC, negative control. (B) The splenocytes of immunized mice were fused with SP2/0 cells to form grape-string cells (red arrows). IFA assay and Western blotting of hybridoma cells (C,D) and monoclonal cells (E,F). A mouse anti-DDDDK-Tag mAb was used as a positive control, and the supernatant of SP2/0 cells was used as a negative control. (G) Ascites mAb titers were determined by indirect ELISA. All representative data from three independent experiments (mean ± SD) were analyzed with a two-tailed Student’s t-test. IFA assay (H) and Western blotting (I) for mAbs. (J) The subclass of the mAb 1G6-4C4 was determined with a mouse mAb isotyping kit.

Figure 4

Flowchart of icELISA establishment.

Figure 5

Establishment and optimization of icELISA. OD_450nm (A) and PI values (B) of serum at different dilutions. PI values with different concentration of skim milk in blocking buffer (C), reaction time with serum (D), incubation time with mAbs (E), dilution of secondary antibodies (F), incubation time with secondary antibodies (G) and reaction time with TMB (H). OD_450nm (I) and PI (J) of PiCV positive serum samples at different dilutions. OD_450nm (K) and PI (L) of different viral serum-positive antibodies. Representative data from three independent experiments (mean ± SD) were analyzed with a two-tailed Student’s t-test.

Figure 6

Establishment and optimization of iELISA. The ratio of the OD_450nm of the positive serum (P) to that of the negative serum (N) with different concentrations of skim milk used for blocking (A), the reaction time with the serum (B), the dilution of the secondary antibody (C), the incubation time with the secondary antibody (D) and the TMB reaction time (E). (F) OD_450nm values of serum samples at different dilutions. (G) OD_450nm values of different virus-positive serum samples. All representative data from three independent experiments (mean ± SD) were analyzed with a two-tailed Student’s t-test.