CAV (CIA89) was provided by Professor Yi Yang Lein of the National Pingtung University of Science and Technology (Taiwan). Two 1 day old specific pathogen free (SPF) hybrid white leghorn chickens purchased from the Animal Health Research Institute of the Council of Agriculture (Taiwan) were used to propagate the virus by passaging 20% liver homogenates (0.1 mL per bird). The phosphate buffered saline was used to dilute the homogenates containing virus particles. These animals were inoculated orally with CIA-89 containing titers of 10^7.5TCID₅₀. At 10-days post-infection, the chicken sera were collected and used to confirm the virus infection in terms of the performance of anti-CAV antibody using commercial CAV test kit (IDEXX, Netherlands). Then the chicken was sacrificed. The individual livers of the sacrificed chickens were removed, collected, immersed in formaldehyde, and then stored at room temperature until required.

The pGEX-6P-1-VP1 plasmid derived from pGEX-6P-1 plasmid (GE Healthcare, USA), which contains cDNA encoding the VP1 genes, was provided from Professor Yi-Yang Lien of National Pingtung University of Science and Technology (Taiwan) and initially used as the PCR template. To amplify the VP1 gene lacking the coding region for the first 129 amino acids from the full-length VP1 gene, two PCR primers, VP1-388FE and VP1-RHX, were designed and used as described in a previous study [8]. Using pGEX-6P-1-VP1 as the template, PCR reactions were performed at 95℃ for 5 min, 95℃ for 45 sec, 59℃ for 50 sec, and 72℃ for 1 min for 30 cycles. The last PCR cycle was carried out with a final elongation step of 10 min at 72℃. The amplified DNA fragments were digested with XhoI and EcoRI (Takara, Japan), and then cloned into the prokaryotic expression vector pET28a (Merck, Germany). The constructed recombinant plasmid, pET28a-VP1Nd129, was used to transform One Shot Top10 cells (Invitrogen, USA) and BL-21 (DE3) competent E. coli for maintaining the recombinant plasmids and for protein expression, respectively. Transformants with the correct gene size were identified by PCR and screened using restriction enzyme digestion and sequencing. The above PCR was performed in a 25 µL reaction mixture containing 0.4 mM of dNTPs, 5 pmole each of VP1-388FE and VP1-RHX, 1U Pro-taq DNA polymerase (Protech, Taiwan) and 1× Pro-taq buffer (10 mM Tri-HCl, 50 mM KCl, 0.01% gelatin, 1.5 mM MgCl₂, 0.1% Triton X-100, pH 9.0). The PCR conditions was 95℃ for 5 min, followed by 35 cycles of 95℃ for 1 min, 57.7℃ for 1 min, and 72℃ for 1 min, and a final extension cycle at 72℃ for 10 min. Digestion was carried out at 37℃ for 1 h in a 50 µL reaction mixture containing 50 mM of potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 1 mM DTT, 100 µg/mL BSA, 5U XhoI and 5U EcoRI. The resulting restriction digestion product was analyzed by 1% agarose electrophoresis and visualized using ethidium bromide staining and UV.

Recombinant BL-21 (DE3) E. coli containing pET28a-VP1Nd129 were used for protein induction and expression. The recombinant strains were grown overnight in Luria-Bertani (LB) medium (BD-Difco, USA) in the presence of kanamycin (50 µg/mL; Amresco, USA) at 37℃. Next, 0.5 mL of the overnight culture was used to inoculate 50 mL LB medium. The culture was grown at 37℃ for around 3 h by which time the optical density had reached 0.5. At this point, 0.1 mM isopropyl-β-D-thiogalactopyronoside (IPTG) (Amresco, USA) was added to the culture to induce protein expression, which continued for 6 h. The presence of expressed VP1Nd129 protein was confirmed by 12.5% SDS-PAGE followed by Western blotting using a monoclonal anti-His antibody (Invitrogen, USA), as described in previously work [8].

To purify the recombinant VP1Nd129 protein, a cell pellet was spun down using 5,000 × g, 15 min, at 4℃ from 50 mL of the culture supernatant and resuspended in denaturing binding buffer (20 mM NaH₂PO₄, 0.5 M NaCl, and 8 M urea, pH 7.8). The mixture was then sonicated on ice three times for 3 min with a 20% pulsed activity cycle (Sonics & Materials, USA), and then centrifuged for 10 min at 13,300 × g to remove cell debris. The resulting cell lysate was poured into an Enco-column (Bio-Rad, USA) with 2 mL of Ni²⁺-NTA agarose and the resin was allowed to settle by gravity. The packed resin in column was washed by gravity with three volumes of denaturing binding buffer and then a similar volume of wash buffer (20 mM NaH₂PO₄, 0.5 M NaCl, and 8 M urea, pH 6.3). Finally, the bound proteins were eluted with elution buffer (20 mM NaH₂PO₄, 0.5 M NaCl, and 8 M urea, pH 4). For each fraction, 2 mL of elute was collected. The fractions were monitored at OD₂₈₀ using a U-2001 spectrophotometer (Hitachi, Japan) with wash buffer as a blank. Putative peaks corresponding to the recombinant CAV viral protein were identified and the eluate was collected for analysis. The total protein concentration of each fraction was determined using a Micro BCA kit (Pierce, USA) with bovine serum albumin as the reference protein. The purity of the protein samples was analyzed using aliquots of the concentrated fraction; this was done by 12.5% SDS-PAGE and Coomassie brilliant blue staining.

SPF BALB/c mice (Charles River, MA) were immunized by subcutaneous injection of 20 µg purified VP1Nd129 protein emulsified with complete Freund's adjuvant (Sigma-Aldrich, USA). After immunization, the BALB/c mice were sacrificed, the spleens were removed, and the 2 × 10⁸ splenocytes were fused with 2 × 10⁷ SP2/0 myeloma cells (ATCC CRL1581) by treatment with polyethylene glycol [6]. After 2 weeks of growth, the resultant hybridomas were examined the presence of antibodies against VP1Nd129 protein. Antibodies secreted from the various hybridomas were screened by ELISA. They were subcloned three times using the limiting dilution technique [20], and then ascitic fluid containing monoclonal antibodies was produced by introducing the cloned hybridomas into Pristane-primed mice (Charles River, USA). The immunoglobulin class of the hybridoma antibodies was determined by ELISA with a Mab kit (Zymed Laboratories, USA) using rabbit antisera against mouse immunoglobulin (Ig)M, IgG1, IgG2a, IgG3, and IgA, and goat anti-rabbit IgG serum conjugated with horseradish peroxidase (HRP).

To evaluate the specificity and reactivity of the mAb against CAV, an ELISA with the antibody was performed with purified VP1Nd129 protein or lysates from tissues infected with CAV as the antigen. After this, polyvinyl chloride 96-well plates were coated with recombinant VP1Nd129 or tissue lysates containing CAV. The antigen was buffered and diluted with carbonate-bicarbonate buffer (pH 9.6). Varying amounts of antigen from 1 to 500 ng were added to the 96-well strip plate and incubating overnight at 4℃. After antigen coating, the plates were washed twice with phosphate-buffered saline (PBS), containing 0.05% Tween 20 (PBS-T), and then 0.2 mL of blocking reagent (PBS containing 5% skim milk) was added to each well. After 30 min of incubation at room temperature, the plates were washed twice with PBS. One hundred µL of the hybridoma supernatants were then added to the plate wells coated with CAV antigen or recombinant antigen and incubated at 37℃ for 1 h. The bound mAbs were detected using HRP-conjugated rabbit anti-mouse IgG (Jackson Immuno Research, USA) and the color was developed with o-phenylenediamine dihydrochloride (Sigma-Aldrich, USA).

CAV-infected and uninfected liver and thymus tissues (positive and negative controls, respectively) were all from clinical cases of CAV infection confirmed by PCR using specific primers as previously described [8]. The liver and thymus samples were fixed using 30% neutral buffered formaldehyde and embedded in paraffin. The paraffin-embedded tissues were sectioned, mounted, and then stained with hematoxylin and eosin. IHC staining was carried out at room temperature. Tissue sections (5-µm thick) of the paraffin-embedded tissues were washed with phosphate saline buffer containing 0.3% hydrogen peroxide to inactivate endogenous peroxidase. After washing three times with phosphate saline buffer, antigen retrieval was carried out by incubating the tissue sections with a 0.1% of phosphate buffer diluted trypsin solution. The 250 fold diluted monoclonal antibody E3 mAb, which is specific for CAV VP1, was then applied as the primary antibody for 2 hours of the incubation. E3 mAb was recognized by HRP-conjugated goat anti-mouse IgG secondary antibody (Jackson ImmunoResearch, USA). 0.2 µg/mL of secondary antibody solution was used for 2 h of the incubation. After IHC staining, the sections were counterstained with hematoxylin and examined under light microscopy.

For IF microscopy, infected and un-infected MSB-1 cells on glass coverslips were fixed in 4% formaldehyde, washed with PBS, and permeabilized with 0.01% Triton X-100. After washing with PBS, samples were incubated with blocking solution (0.5% bovine serum albumin in PBS) for 1 h, followed by 1 h of incubation with 2 µg/mL of E3 mAb solution. 0.2 µg/mL of fluorescein isothiocyanate-conjugated goat anti-mouse IgG (Jackson Immuno-Research, USA) was used to detect E3 mAb binding. Cell nuclei were counterstained with 4',6-diamidino-2-phenylindole (Sigma-Aldrich, USA) and fluorescence images were captured using a laser scanning confocal microscope (Leica Microsystems, Germany).

To create an immunoaffinity column, cytoplasmic extracts from chicken liver tissues infected with CAV were prepared by centrifugation at 13,300 × g for 30 min at 44℃. Next, 100 µL of cell extract were mixed with 50 µL of ascitic fluid and incubated overnight at 4℃. Then, 50 µL of protein A agarose beads (Sigma-Aldrich, USA) were added and incubated overnight at 44℃ with gentle rotation. The agarose beads were washed three times with buffer containing 50 mM Tris-HCl (pH 7.5), 500 mM NaCl, 0.2% NP-40, and 0.05% sodium deoxycholate. Finally, viral protein and/or CAV virus particles were eluted from the protein A agarose beads by boiling with SDS-PAGE sample buffer (60 mM Tris-Cl pH 6.8, 2% SDS, 10% glycerol, 5% β-mercaptoethanol, 0.01% bromophenol blue) for 5 min. The extracted proteins were then subjected to SDS-PAGE followed by Western blotting using E3 mAb as primary antibody and HRP-conjugated goat anti-mouse IgG secondary antibody, respectively. In addition, CAV genomic DNA from the CAV virus particles was detected by PCR using CAV genome-specific primers. The sequences of the CAV VP1 gene PCR primers were VP1F: 5'-ATGGCAAGACGAGCTCGCAGACCGAGAGG-3' and VP1R: 5'-CTAACCATGGTGATGGTGATGGTGGGGCTGCGTCCCCCAGTA-3'. The sequences of the CAV VP3 primers were VP3F: 5'-CCGCTCGAGCAGTCTTATACACCTTCTTG-3' and VP3R: 5'-GCGAATTCATGAACGCTCTCCAAGAAGATAC-3'. The PCR conditions was 95℃ for 5 min, followed by 35 cycles of 95℃ for 1 min, 57.7℃ for 1 min, and 72℃ for 1 min, and a final extension cycle at 72℃ for 10 min.

To express the CAV VP1 as an antigen for immunization, the VP1Nd129 construct was created by PCR using VP1 cDNA as the template DNA. The VP1 cDNA partially overlapped partially the VP2 gene of the CAV genome, which had been previously cloned into the pGEX-6P-1 plasmid [4]. Using the primers VP1-388FE and VP1-RHX, the VP1Nd129 construct was amplified by PCR and cloned into the pET28 a vector at the EcoRI and XhoI sites, thereby creating a protein with an in-frame His-tag. This plasmid, pET28a-VP1Nd129, was used to transform E. coli BL-21 (DE3) cells. The E. coli were examined for protein expression after 4 h of induction with IPTG. VP1Nd129 protein was successfully expressed in E. coli, as noted by the correct size band on a gel stained with Coomassie blue gel and recognition by anti-His tag antibodies (Fig. 1). The estimated molecular weight of pET28a-VP1Nd129 was 40 kDa. VP1Nd129 in E. coli was expressed at a high level which reached 26.2 mg/L when 0.1 mM IPTG was added to the culture broth. After affinity chromatography purification, the purified VP1Nd129 protein was confirmed by Western blotting using anti-His and anti-CAV antibodies. The purity of VP1Nd129 approached homogenicity in elution fractions E1 to E7 at pH 4.0. Purification of His-tag-fused VP1Nd129 was therefore feasible using a Ni-NTA column. Additionally, the identity of the Ni-NTA-purified VP1Nd129 protein was confirmed by mass spectrometry (data not shown).

To establish monoclonal antibodies, spleen cell were removed from five mice immunized with Ni-NTA-purified recombinant VP1Nd129 protein and fused with SP2/0 myeloma cells. After 14 days, hybridoma cell lines secreting mAbs specific for VP1Nd129 were screened by ELISA using the Ni-NTA-purified VP1Nd129 protein as the coating antigen. Four mAbs were found active against VP1Nd129 (B1-1, B1-5, D4 and E3) were identified out of 64 clones; these were then screened by subcloning at least three times using the limiting dilution method. The mAb from the E3 hybridoma cells showed the greatest reactivity compared to the other mAbs from the B1-1, B1-5, and D4 hybridomas (data not shown). Even after the supernatant was diluted 50-fold, E3 still has recognition activity to react the antigen. In addition, the specificity and reactivity of the E3 mAb were also confirmed by Western blotting using purified recombinant VP1Nd129 and E. coli cell lysates. As illustrated in Fig. 2A, the E3 mAb showed good specificity and reactivity against the antigen and purified recombinant VP1Nd129 while showing no reaction against cell lysates from E. coli harboring the blank pET28a vector.

When the specificity of the E3 mAb against different amounts of antigens was evaluated, it was found to be constant and the reactivity of the E3 mAb increased with increasing amounts of antigen. There were very few background bands that reacted with E3 mAb in either the samples of purified VP1Nd129 or the E. coli cell lysate. Moreover, 1 ng of purified VP1Nd129 was sufficient to react with E3 mAb (Fig. 2B). Thus, E3 mAb has good specificity and reactivity against CAV antigen. Additionally, IgG subtype analysis showed that E3 mAb was IgG2b with a κ light chain. At this point, the E3 hydridoma was selected to produce mAbs in mice. Mouse ascitic fluid was then used for further diagnostic applications and characterization.

To evaluate the possible clinical application of E3 mAb for CAV diagnosis, the mAb was used for CAV detection by IHC and immunochromatographic assay. First, four CAV-infected chicken livers were analyzed using IHC; all of the paraffin-embedded tissues were positive. The positive control slides demonstrated strong immunoreactivity against CAV antigen (Fig. 3B). In addition, four CAV mock-infected chicken livers were analyzed as the negative control; these were all negative for immunoreactivity against CAV antigen, (Fig. 3A). Moreover, similar immunoreactivity results were found in the thymus tissues. The CAV-infected thymus shown in Fig. 4B was recognized by the E3 mAb using IHC in contrast to the negative sample (Fig. 4A).

In CAV-infected MSB-1 cells and CAV-infected MSB-1 showing a CPE, the presence of VP1 antigen in the cells was recognized by the E3 mAb using an IF assay (Fig. 5). These results indicated that E3 mAb was able to discriminate CAV-infected tissues or cells from uninfected tissue or cells in experimental samples using IHC staining and IF, respectively. Furthermore, E3 mAb was used to react with VP1 present in the lysate of CAV-infected liver tissue. As illustrated in Fig. 6A, VP1 with a molecular weight of 50 kDa was eluted from the protein A agarose beads. In contrast, no VP1 was detected from the eluted sample of CAV mock-infected liver lysate. Detection of the protein was performed by Western blotting using E3 mAb as the primary antibody. In addition, Fig. 6B shows that CAV-specific VP1 and VP3 genes with lengths of 1.35 kb and 348 bp, respectively, were amplified by PCR using the eluted fraction from the immunochromatographic column as a template. This suggests that the eluted fraction from the immunochromatographic column included the CAV VP1 capsid.