Expression and purification of recombinant ASFV p54 protein
To prepare the antigen used for camel immunization, a pET-30 prokaryotic expression system was used to express recombinant ASFV p54 protein. Following the construction of the recombinant plasmid, pET-30-p54-His, the plasmids were sequenced, and the correct plasmids were transferred into Transetta (DE3) expression competent cells (TransGen Biotech, Beijing, China). A single clone was selected and treated with 1.0 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 37˚C for 12 h. Following ultrasonication, the lysates were centrifuged at 12,000 x g for 30 min at 4˚C, and precipitates and supernatants were subjected to SDS-PAGE and western blot analysis, respectively.
The ASFV p54 protein was purified using cOmplete His-Tag Purification Resin (Roche, Basel, Switzerland). Before protein purification, the resin was equilibrated with 10 times column volumes of buffer A (50 mM NaH2PO4 pH=8, 300 mM NaCl). Then the contaminated proteins were eluted with buffer A containing 20 mM imidazole, and the p54 protein was eluted with buffer A containing 250 mM imidazole. The eluted product was collected and analyzed by SDS-PAGE.
Immunization and construction of the Nb library
A Bactrian camel was first immunized with a mixture of 5 ml p54 protein (5 mg) and an equal volume of Freund’s complete adjuvant (Sigma-Aldrich, Merck KGaA, St. Louis, MO, USA). For the subsequent immunizations, p54 protein was mixed with an equal volume of Freund’s incomplete adjuvant (Sigma-Aldrich, Merck KGaA, St. Louis, MO, USA); immunizations were performed at 2-week intervals, four times. A week after the forth immunization, 300 ml whole blood was collected, and peripheral blood lymphocytes (PBLs) were separated from 200 ml whole blood using Ficoll-Paque PLUS (Cytiva) with Leucosep™ tubes (Greiner Bio-One GmbH). The remaining 100 ml whole blood was used for serum isolation and immunization titer detection. All camel experiments were performed according to guidelines approved by the Animal Care and Use Committee of Henan Agricultural University (Zhengzhou, China).
For library construction, total RNA from PBLs was extracted using an RNeasy® Plus Mini kit (Qiagen AB). cDNA was reverse transcribed using a SuperScript III First-Strand Synthesis system (Thermo Fisher Scientific, Inc.). An ~700-bp target band spanning the VHH-CH2 exons was cloned during the first round of PCR. The VHH encoding sequences (~400 bp) were amplified using the products from the first PCR as a template and then purified using agarose gel electrophoresis. Following digestion with PstI and NotI, the target segments were cloned into the phage display vector pCANTAB-5E (Cytiva) and then electro-transformed into freshly prepared E. coli TG1 competent cells. The transformation products were cultured in solid 2X YT medium containing 100 μg/ml ampicillin and 2% (w/v) glucose overnight at 37˚C. The colonies were scraped from the plates, placed into 3 ml liquid Luria-Bertani (LB) medium (Oxoid) supplemented with 20% (v/v) glycerol and stored at -80˚C. Following a gradient dilution, the capacity of the constructed library was detected by counting the number of colonies.
Library screening using phage display
The specific Nbs against ASFV p54 protein were screened via three consecutive rounds of biopanning with the p54 protein. Briefly, ~1x1010 TG1 cells (Beyotime Biotech, Shanghai, China) from the library stock were recovered and cultured in 2X YT medium containing 100 μg/ml ampicillin and 2% (w/v) glucose for 2 h at 37˚C. Then, the TG1 cells were infected with M13K07 Helper Phage (New England Biolabs, lpswich, MA, USA) (1.8x1013 pfu/ml) and incubated at 37˚C for 1 h without shaking. Cells were collected using centrifugation at 3,000 x g for 10 min at room temperature, followed by resuspending into 2X YT medium supplemented with 50 μg/ml kanamycin and 100 μg/ml ampicillin, which were cultured overnight at 37˚C at 220 rpm. The phages in the supernatant were precipitated using PEG 6000/NaCl for 3 h on ice, and were then centrifuged at 12,000 x g for 30 min at 4˚C and resuspended in sterile PBS. The phages were quantified using phage titration. For every round of biopanning, ~5x1011 pfu/ml phages were incubated in 96-well plates (Thermo Fisher Scientific, Inc.) coated with p54 protein (10 μg/well). The enrichment of specific phage particles was monitored using an anti-M13 HRP-conjugated antibody (Sino Biological, 1:2000) for ELISA and phage titration, as previously reported (30). After three consecutive rounds of biopanning, the enrichment of specific phage particles was calculated, and 96 individual colonies were randomly selected and treated with 1.0 mM IPTG. The positive clones expressing E-Tag p54-specific Nbs were identified using periplasmic extract ELISA (PE-ELISA) with an anti-E-Tag antibody (GenScript, 1:2000). If the absorbance in the antigen-coated well was >3-fold higher than that of the well containing PBS, the colony was regarded as positive. The identified positive clones were then sequenced, and the amino acid sequences of Nbs were analyzed and classified into different groups, based on their sequence diversity in third complementarity-determining (CDR3) regions.
Production of Nb-HRP recombinant protein against the ASFV p54 protein
HRP-conjugated recombinant Nb was prepared, according to previous reports with some modifications (38). Specific DNA sequences, including a signal sequence for protein secretion derived from the human Ig kappa chain, which promotes the extracellular secretion of fusion proteins, and a codon-optimized HRP gene sequence, were synthesized and fused with the Nb gene amplified from the recombinant pCANTAB-5E vector using overlap extension PCR. Following digestion with EcoRI and NheI, the fusion fragments were inserted into the multiple cloning sites of the pCAGGS-hemagglutinin (HA) eukaryotic expression vector, termed pCAGGS-Nb-HRP in the following experiment. Following sequencing, the recombinant plasmid was transfected into 293T cells using polyetherimide reagents (Polysciences Inc.). At 48 h following transfection, cells and supernatants containing the Nb-HRP recombinant protein were harvested and analyzed using western blotting or an indirect fluorescence assay (IFA) to determine the Nb-HRP recombinant protein expression. Supernatants were filtered using a 0.22-μm filter membrane for further use.
For western blotting, the 293T cells were harvested 60 h post-transfection for analysis using mouse anti-HA mAb (Beyotime Institute of Biotechnology, Shanghai, China; 1:1000) as the primary antibody and HRP-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc.; 1:2000) as the secondary antibody. For IFA, at 36 h post-transfection, the 293T cells were fixed with -20˚C pre-cooled 70% alcohol and probed with mouse anti-HA mAb, followed by incubation with Alexa Fluor 594-conjugated goat anti-mouse IgG (H&L). The cells were then observed under a fluorescence microscope (Leica AF6000; Leica Microsystems GmbH, 200x). For ELISA analysis of the Nb-HRP recombinant protein in the supernatant, 100 and 200 µl supernatant with or without plasmid transfection, respectively was directly incubated in 96-well ELISA plates at 4˚C overnight. Following washing with PBS Tween 20 (PBST) three times, 100 μl tetramethylbenzidine (TMB) was added to each well and incubated at 37˚C for 15 min in the dark. Next, 50 μl/well of 3 M H2SO4 was added to stop the reaction, and the absorbance of the plate was measured at OD450 nm using a spectrophotometer (PerkinElmer, Inc.).
Specificity analysis of Nb-HRP recombinant protein against p54 protein
For specificity analysis, 96-well plates were coated with ASFV p54 protein, porcine reproductive and respiratory syndrome virus (PRRSV) N protein, PEDV N protein and ASFV p30 protein at a density of 200 ng/well at 4˚C overnight. Following blocking with 2.5% dried milk at 37˚C for 1 h, supernatants collected from pCAGGS-Nb-HRP-transfected 293T cells were added (100 μl/well) and incubated at 37˚C for 1 h, followed by washing with PBST three times. A total of 100 μl/well TMB was added and incubated at 37˚C for 15 min in the dark. Finally, 50 μl/well 3 M H2SO4 was added to stop the reaction and absorbance was measured at 450 nm (PerkinElmer, Inc.).
Analysis of the affinity of Nb-HRP recombinant protein based on the OD450 value
The kinetic characteristics of Nb-HRP in cell culture supernatants bound to recombinant p54 protein were determined using indirect ELISA. Briefly, 96-well plates coated with the purified recombinant p54 protein (200 ng/well) were incubated at 4˚C overnight. The plates were washed with PBST three times and incubated with serial dilutions of Nb-HRP supernatants (1:5, 1:10, 1:20, 1:40, 1:80 and 1:160) for 1 h at 37˚C. After washing three times with PBST, 100 μl/well TMB was added, followed by incubation at 37˚C for 15 min in the dark. The reaction was stopped by the addition of 50 µl/well of 3 M H2SO4 and absorbance was measured at OD450 nm. The affinity of specific Nb-HRP was analyzed based on the OD450 value.
Development of the blocking ELISA using Nb-HRP recombinant protein as a probe
Based on the specific Nb-HRP recombinant protein, a blocking ELISA was established for the detection of ASFV p54 antibodies in the serum. To optimize the detection effect, the optimal concentration of p54 protein and dilution ratio of supernatant Nb-HRP were first determined using direct ELISA via the checkerboard method. A serial dilution of p54 protein (10, 20, 40, 80, 160, 320 and 640 ng/well) and supernatant Nb-HRP (supernatant stock, 1:20, 1:50, 1:100, 1:200, 1:400 and 1:800) were performed simultaneously. The combination at an OD450 of 1.0 was determined to be the optimal antigen coating amount and Nb-HRP dilution ratio.
Next, the optimal dilution ratio of the serum to be tested was determined. According to the determined optimal working concentration of p54 protein and Nb-HRP, three separated inactivated ASFV antibody-positive (No. 1-3) and three negative serum (No. 4-6) samples were selected and diluted at 1:5, 1:10, 1:20, 1:40, 1:80, 1:160 and 1:320 for blocking ELISA detection. They were then added to the wells at 37˚C for different durations (15, 30, 45, 60, 90 and 120 min). Then, the plate was washed three times with PBST, and Nb-HRP was added to the wells and incubated for different durations (30, 45, 60, 90 and 120 min). Following another three times washing with PBST, 100 µl freshly prepared TMB solution was added to the plate, which was incubated at 37˚C for 5, 10 or 15 min. The reaction was stopped by the addition of 50 µl 3 M H2SO4, and the absorbance was measured at OD450 nm using a microplate reader. If the percentage inhibition (PI) = 100 x [negative serum OD450 value - (test serum OD450 value / negative reference serum OD450 value)] was the highest and the absorbance of the negative serum was the closest to 1.0, the experimental conditions were considered optimal.
Establishment of blocking ELISA
Based on the optimized reaction conditions, a blocking ELISA was developed as follows: 96-well plates were coated with the optimal concentration of purified p54 protein overnight at 4˚C. After discarding the coating buffer and washing three times with PBST (300 µl/well), the plates were blocked with 2.5% dried milk (200 µl/well) for 1 h at 37˚C. Following washing three times with PBST (300 µl/well), serum samples to be tested were added to the wells at the optimal dilution ratio (100 μl/well) and incubated at 37˚C for the optimal duration. The plates were washed three times with PBST and Nb-HRP recombinant protein at the optimal dilution was added to the plates and incubated at 37˚C for the optimal duration. Following washing three times with PBST, 100 µl/well fresh TMB was added to the well plate and incubated at 37˚C for the optimal duration. Next, 50 μl/well 3 M H2SO4 was added to each well to stop the reaction, and the absorbance value was detected at OD450 nm. The blocking rate was calculated according to the serum OD450 value. The serum PI was then calculated using the aforementioned equation.
Determination of the cutoff value of blocking ELISA
A total of 152 serum samples confirmed as standard ASFV-negative using both RT-PCR and a commercial ELISA kit (Beijing Jinnuobaitai Biotechnology, Beijing, China) were used to determine the cutoff values for a positive and negative result for the blocking ELISA. The established blocking ELISA method was used to detect ASFV antibody-negative serum, in which the dilution of Nb-HRP and serum was determined. The serum blocking rate was then calculated. According to the statistical method, the test result was analyzed using the following formula: Negative-positive critical value = +3SD, where is the average value of the blocking rate of negative serum and SD is the SD of the blocking rate of negative serum. Serum samples with PI values greater than or equal to the negative-positive critical value were considered ASFV antibody-positive. Serum samples with PI values less than the negative-positive critical value were considered ASFV-antibody negative.
Specificity, sensitivity and repeatability of blocking ELISA
To determine the specificity of the blocking ELISA, PRRSV, pseudorabies virus (PRV), PEDV, porcine transmissible gastroenteritis virus (TGEV), porcine parvovirus (PPV), classical swine fever virus (CSFV) and inactivated ASFV antibody-positive pig serum samples, were confirmed using RT-PCR, a commercial ELISA kit and the developed blocking ELISA. Next, 96-well plates were coated with the optimal ASFV p54 protein concentration at 4˚C overnight, and the blocking ELISA was conducted as described previously.
To test the sensitivity of the blocking ELISA, three serum samples confirmed as ASFV antibody-positive using RT-PCR and the commercial ELISA kit were serially diluted (1:5, 1:10, 1:20, 1:40, 1:80, 1:160, 1:320, 1:640, 1:1,280, 1:2,560, 1:5,120, 1:10,240 and 1:20,480) and tested using blocking ELISA to determine the lowest limit detection.
The established blocking ELISA method was used to perform a repeatability test six times on the same test plate and different test plates from those used for the three inactivated ASFV antibody-negative serum samples and three inactivated ASFV antibody-positive serum samples. For the intra-assay repeatability test, the swine serum samples were detected using a blocking ELISA. The same batch of p54 protein-coated ELISA plates was tested three times in triplicate wells for each sample, and the OD450 value was detected. For the inter-assay repeatability test, different batches of plates were tested separately three times in triplicate wells for each sample, the OD450 value was detected and the PI value was calculated.
Detection of field serum samples
A total of 210 clinical porcine field serum samples collected from three farms in the Henan and Hebei provinces between 2019 and 2020 were tested using the developed blocking ELISA and commercial ELISA kit, as previously described and following the manufacturer’s instructions, respectively. The coincidence rates between the developed blocking ELISA and the commercial ELISA kit (Beijing Jinnuobaitai Biotechnology) were calculated using Microsoft Excel’s Correl function (Microsoft Corporation).
Statistical analysis
Statistical analysis was performed using GraphPad Prism version 6.0 software (GraphPad Software, Inc.). Data are expressed as the mean ± SD. Kappa values were calculated to estimate the coincidence between the developed blocking ELISA and the commercial ELISA kit using SPSS software version 20 (IBM Corp.; http://www.spss.com.cn).