The CD2v protein extracellular region (GenBank: MK128995.1, CD2v-ex, 1–588 bp) gene sequence was optimized according to the CHO expression system and a 6 × His-tag was introduced at the carboxyl terminus. The recombinant CD2v-ex gene sequence was then cloned into the pTRIP-Pure vector, pTRIP-Pure-CD2v-ex (GenScript, Nanjing, China). Adherent human embryonic kidney (HEK) 293T (ATCC, Manassas, VA, USA) cells were maintained in high glucose Dulbecco's modified Eagle's medium (DMEM; Solarbio, Beijing, China). At 40–50% confluence, HEK 293T cells were seeded in 6-well plates and co-transfected with pTRIP-Pure-CD2v-ex (0.65 μg), psPAX (0.9 μg) and pMDG.2 (0.5 μg) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA); a blank pTRIP-puro vector was used as a control 6–8 h after transfection, the medium was replaced with maintenance medium (DMEM containing 2% FBS). Supernatant containing the pseudotyped LVs was collected at 72 h post-transfection, centrifuged at 4,000 rpm for 10 min at 4°C and filtered through a 0.22 μm filter. The supernatant was then used fresh in experiments or stored at −80°C as previously described (15).

CHO-S cells (Thermo Fisher Scientific, Waltham, MA, USA) were passaged in 125-mL vent-cap cell shaker flasks (NEST, Wuxi, China) containing 25 mL ExpiCHO Expression Medium (Gibco, Grand Island, NY, USA) on an orbital shaker platform (100 rpm). When the cell viability exceeded 95%, 2 × 10⁶ cells were mixed with the LV suspension at a ratio of 1:1 (16) in the presence of 5 μg/mL polybrene (Beyotime, Shanghai, China). After 24 h of culture in an orbital shaker at 37°C, the cells were collected by centrifugation at 500 rpm for 10 min at 25°C and resuspended in fresh ExpiCHO culture medium containing puromycin (Beyotime).

The process for screening of a stable cell clone was proceed as follows. Briefly, the transfected CHO-S cells were passaged at a density of 0.5 × 10⁶ viable cells/mL in ExpiCHO culture medium containing 7.5 μg/mL puromycin. When the cell viability exceeded 90%, a second round of screening was performed in ExpiCHO Stable Production Medium (Gibco) containing 22.5 μg/mL puromycin. After 12 h, expression of CD2v-ex by cell clones was evaluated by western blot analysis. Briefly, cell culture supernatants and cell fragmentation supernatants were denatured at 98°C for 10 minand proteins were separated by 7.5% SDS-PAGE before transfer to a polyvinylidene fluoride (PVDF) membrane (Merck Millipore, Billerica, MA, Germany). After blocking for 1 h with 5% skimmed milk (m/v, SM) in tris buffered saline containing 0.05% Tween-20 (TBST, v/v), the membrane was incubated at room temperature (RT) for 1 h with anti-His-tag antibody (1:5,000 in 5% SM, Proteintech, catlog number 66005-1-Ig, Wuhan, China) or standard ASFV-positive serum (1:2,000 in 5% SM, CVCC, Beijing, China). After washing with TBST, the membrane was incubated at 37°C for 1 h with the corresponding secondary antibodies [HRP-conjugated goat anti-mouse IgG (1:5,000 in 5% SM, Proteintech, catlog number SA00001-2) or mouse anti-pig IgG (1:5000 in 5% SM, Immunoway, catlog number RS030232, Plano, TX, USA)]. The antibody-reactive bands were detected using beyoECL star solution (Beyotime) and visualized with a multifunctional imaging system (GE Amersham Imager 600, Boston, Massachusetts, USA).

After two rounds of drug screening, stably transduced cell lines were generated by limiting dilution cloning. The cells were seeded in 96-well plates (0.5 cell per well) with ExpiCHO Expression Medium containing 6 mM L-glutamine (Gibco) and statically incubated at 37°C. Clones expressing high levels of the transfected protein were screened in 24-well and 6-well plates. Finally, the clones expressing the highest levels of the transfected protein was selected in fed-batch culture in 125-mL flasks by feeding with 5, 10, 15 g/L glucose (Gibco) and 2% (v/v) GluMAX-1 (100 ×, Gibco) on days 3, 5, 7 at 37°C. Dot blot and western blot analysis were used to selecting the high-expressing clones (17).

CHO-CD2v cell lines were verified by measurement of genomic DNA and RNA expression. For DNA verification, genomic DNA was extracted from 1 × 10⁷ stably transduced monoclonal cells using the DNAiso Reagent (Takara, Tokyo, Japan) and identified by PCR amplification. The identification primers were designed based on the optimized gene sequence (CD2v-ex-F: 5'-ATGATCATCCTGATCTTCCTGATC-3', CD2v-ex-R: 5'-TCAGCTGGACAGTGTCAGGTA-3', Tsingke, Beijing, China). The PCR mixture (final volume 20 μL) contained 10 μL 2 × PrimeSTAR MAX Premix (Takara), 1 μL genomic DNA, 1 μL of each primer (10 μM), and 7 μL ddH₂O. The PCR conditions were set as follows: 98°C pre-denaturation for 30 s; 30 cycles of denaturation at 98°C for 10 s, annealing of 58.5°C for 30 s and extension at 72°C for 30 s; final extension at 72°C for 8 min. The PCR products were identified by 1% (m/v) agarose and sequenced by Beijing Tsingke (Tsingke). For RNA identification, total RNA was isolated using RNAiso Plus (Takara) and cDNA was obtained by reverse transcription using HiScript II One Step qRT-PCR Probe Kit (Vazyme, Shanghai, China). The following PCR operations were the same as the above mentioned.

To prepare purified CD2v-ex protein for subsequent tests, the selected CHO-CD2v cell line was cultured for 7–10 days in a shaking incubator (100 rpm) and fed with 5 g/L glucose (Gibco) and 2% (v/v) GlutaMax-1 (100 ×, Gibco) every 2 days. After centrifugation with 12,000 × g for 10 min, the supernatant was collected and CD2v-ex protein was purified from the cell culture supernatant using HisTrap excel (Cytiva, Sweden). The purified protein was analyzed by SDS-PAGE.

CD2v-ex protein was diluted with carbonate bicarbonate buffer (CBS buffer, pH 9.6) and used to coat 96-well plates (Beaver, Suzhou, China) at 4°C overnight. The plates were then washed five times with TBST and patted dry. After the plates were blocked for 1 h at RT with 5% SM (in TBST) and washed as above, serum samples were diluted with 1% bovine serum albumin (1% BSA, m/v) in 1 × phosphate buffered saline (PBS) and then added to plates (100 μL/well) and incubated for 30 min at 37°C. Subsequently, the plates were washed as above and incubated with HRP-conjected monoclonal mouse anti-pig IgG (Immunoway) diluted to 1:10,000 with 5% SM. After incubation for 40 min at 37°C and washing, the plates were incubated with 3,3′,5,5′-tetramethylbenzidine (TMB, Solarbio) at RT. The reactions were stopped after 10 min by adding 2 mol/L H₂SO₄ (50 μL/well). The optical density (OD) of each well was measured at 450 nm (OD₄₅₀) using a Multimode Microplate Reader (Tecan 10M, Switzerland).

The antigen and serum concentrations were optimized by checkerboard titration. Briefly, 96-well plates were coated with the recombinant antigen titrated to concentrations of 0.5–4 μg/mL (50–400 ng/well). ASFV-positive (ASFV⁺) and ASFV-negative (ASFV⁻) standard sera were serially diluted 1:10–1:320. Every combination was evaluated in duplicate.

The CD2v-iELISA reaction conditions were optimized according to the optimal antigen coating concentration and serum dilution determined as previously described. The optimal assay conditions were identified as those that yielded the highest OD₄₅₀ ratio between the ASFV⁺ and ASFV⁻ serum samples (P/N value) as previously described (18). Every sample was evaluated in triplicate.

The status of 82 swine ASFV⁻ serum samples stored in our laboratory was first confirmed using the P30-iELISA kit (Kernal, US) according to the manufacturer's instructions. These 82 serum samples were then analyzed using the optimized CD2v-iELISA method. The cut-off value was defined as the mean OD₄₅₀ value + 3 × the standard deviation (SD), and samples above this cut-off value were considered to be ASFV⁺ (19).

The specificity of the established CD2v-iELISA method was determined against the following serum samples: ASFV⁺ (positive control), ASFV⁺ with CD2v deleted (CD2v⁻), porcine reproductive and respiratory syndrome virus positive (PRRSV⁺), classical swine fever virus positive (CSFV⁺), porcine circovirus positive (PCV⁺), porcine pseudorabies virus positive (PRV⁺), swine foot and mouth disease virus positive (FMDV⁺) and porcine epidemic diarrhea virus positive (PEDV⁺). The OD₄₅₀ of each pathogenic serum sample was compared with the cut-off value and samples above this cut-off value indicated a cross-reaction.

The sensitivity of the CD2v-iELISA was evaluated using three clinical ASFV⁺ and three ASFV⁻ serum samples diluted from 1:160 to 1:5,120. The assay sensitivity correlated with the highest dilution of serum detectable according to the cut-off value.

Eight ASFV⁺ positive and/or ASFV⁻ serum samples were randomly selected. Triplicate samples were assayed in one batch to evaluate intra-assay variation and in three different batches were assayed separately to evaluate inter-assay variation expressed as the coefficient of variation (CV).

A total of 179 clinical swine ASFV⁺ (n = 18), ASFV⁻ (n = 112) and ASFV⁺ (CD2v⁻) (n = 49) serum samples determined by real-time quantitative PCR (qPCR) and HAD tests were kindly provided by Lanzhou Veterinary Research Institute and analyzed using the established CD2v-ex-iELISA method to determine the compliance rate.

All statistical analysis was conducted using GraphPad Prism 8.2 software (Graph Pad Prism Inc., California, USA). Data were presented as the mean + SD. Significant differences between samples were assessed using Student's t-test. P < 0.05 was set as the threshold for statistical significance.

To establish a stable CD2v-ex protein expressing CHO-S cell line, CHO-S cells were first transduced with packaged LV suspensions to generate CHO-S cells expressing CD2v-ex (CD2v-ex) and CD2v-ex expression was verified by western blot analysis. A specific diffuse band (70–95 kD) was detected in CHO-CD2v cell culture supernatant (Figures 1A,B), consistent with eukaryotic expressed glycosylated proteins' characteristics (16, 20), suggesting that CHO-CD2v cells successfully secreted express glycosylated CD2v-ex. Furthermore, the protein was recognized by anti-His-tag antibody and ASFV⁺ serum. To select a stable CD2v-expressing CHO cell line, monoclonal cells were obtained by limiting dilution cloning and then successively selected and expanded from 96-well plates (n = 92), to 24-well plates (n = 26) and finally, to 6-well plates (n = 8, 1C6, 2D2, 3B3, 3G4, 4D12, 5C5, 6E4, 7B5). Three high-expression CHO-CD2v cell clones (1C6, 6E4, 7B5) were then selected (Figure 1C), and the highest expression monoclonal cell line in fed-batch culture was identified as 1C6 by western blot analysis (Figure 1D). In addition, viable cells monitoring revealed that 1C6 showed a comparable growth curve compared with blank CHO-S cells in fed-batch culture (Figure 1E), suggesting that 1C6 was a high-expressing CHO-CD2v clone with growth characteristics similar to those of the parent line.

For further verification of the selected CHO-CD2v clone (1C6), we extracted its genomic DNA and RNA, respectively. Integration of the CD2v-ex gene sequence into CHO-S cell genomic DNA was confirmed by PCR and transcription into mRNA for protein expression was verified by RT-PCR, as evidenced by detection of a specific band of the expected size (588 bp) (Figures 2A,B), and the sequencing results were correct. The CD2v-ex protein was purified from cell culture supernatant using HisTrap excel (Cytiva) in binding buffer (20 mM Tris-HCl,300 mM NaCl, pH 8.0) to purified it from cell culture supernatant. CD2v-ex was eluted from the column with the same binding buffer supplemented with 75 mM imidazole and SDS-PAGE confirmed the presence of the purified CD2v-ex protein (Figure 2C).

To determine the optimal reaction conditions of CD2v-iELISA, we first identified the optimal coating antigen concentration and serum dilution factor by checkerboard titration, with the coating antigen concentration at 0.5–4 μg/mL and serum dilution at 1:10–1:320. The maximum positive/negative (P/N) value was obtained when the concentration of CD2v-ex protein was 2 μg/mL (200 ng/well) and the serum dilution was 1:160 (Table 1). Using the same criterion of the conditions that yield the maximum P/N value, the remaining reaction conditions were successfully optimized (Figures 3A–J) as described in Table 2. The cut-off value was determined using the optimal CD2v-iELISA method to analyze 82 ASFV⁻ serum samples. The mean OD₄₅₀ value was 0.1017 and the SD was 0.0616 (Figure 3K), resulting in a cut-off value of 0.2865 (0.287, mean + 3 × SD). Therefore, only samples with OD₄₅₀ values ≥0.287 were considered to be ASFV⁺; all others were classified as ASFV⁻. negative.

To determine the CD2v-iELISA method specificity, we analyzed ASFV⁺(CD2v⁻), PRRSV⁺, CSFV⁺, PCV⁺, PRV⁺, FMDV⁺ and PEDV⁺ serum samples; ASFV⁺ was used as the positive control. All pathogen positive sera tested negative with the exception of the ASFV⁺ control (Figure 4A), indicating excellent assay specificity.

To determine the CD2v-iELISA sensitivity, we analyzed three clinical ASFV⁺ and three ASFV⁻ serum samples (diluted from 1:160 to 1:5,120) using the determined cut-off value. The highest dilution of the ASFV⁺ sample that was detected in the assay was 1:2,560 (Figure 4B), indicating an assay sensitivity up to 1:2,560.

The reproducibility of the CD2v-iELISA was determined based on intra- and inter-assay CV values calculated by analyzing eight randomly selected ASFV⁺ and/or ASFV⁻ serum samples in a single batch and three different batches, respectively. As shown in Table 3, the intra- and inter-assay values were both <10%, suggesting a high repeatability and low variability.

To determine the CD2v-iELISA compliance, was analyzed ASFV⁺ (n = 18), ASFV⁻ (n = 112), and ASFV⁺ (CD2v⁻) (n = 49) serum samples. The assay showed 100% sensitivity and specificity in identification of ASFV⁺ and ASFV⁺ (CD2v⁻) serum samples, and the total compliance rate was 99.4% (Figure 4C).