Cells, vectors, and sera
Human embryonic kidney cells (HEK-293 T) were cultured in Dulbecco's Modified Eagle's Medium (DMEM; Solarbio, China) supplemented with 10% fetal bovine serum (FBS, Gibco, Carlsbad, CA, USA) and 1% antibiotic–antimycotic (Life Technologies Corp) at 37 °C under 5% CO2. ASFV-K205R protein was expressed using the pET-30a prokaryotic expression vector (Novagen, USA). The VHH library was constructed using the pCANTAB 5E vector (GE healthcare Life Science, Pittsburgh, USA), and the Nb-HRP fusion protein platform was constructed using the pCAGGS-HA vector as the backbone plasmid.
One hundred forty-seven ASFV antibody-negative serums were collected from ASF decontamination pig farms, all serums were all confirmed ASFV antigen- and antibody-negative using RT-qPCR and commercial ELISA kit detection (Beijing JinnuoBaitai Biotechnology Co., Ltd.). At the same time, 285 clinical serum samples collected from different farms in Henan Province were detected using the developed cELISA and commercial ELISA kit. It is clearly stated that the pig serum used in this experiment is inactivated at 60 °C for 30 min.
Expression and purification of recombinant K205R Protein
The K205R gene was amplified using PCR with primer pairs K205R-F and K205R-R (Table S1) using the synthesized K205R gene as the template (GENEWIZ Company, Jiangsu, China). Subsequently, the K205R gene was digested by enzymes BamH I and Xho I and then cloned into the pET-30a vector. The correctly sequenced plasmid was named pET-30a-K205R-His. Next, the pET-30a-K205R-His plasmids were transformed into E.coli BL21 (DE3) (TransGen Biotech, Beijing, China), a single clone was selected and treated with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 37˚C for 12 h. After sonication and centrifugation, K205R proteins in inclusion bodies were dissolved in 8 M urea and then purified using a Ni–NTA resin (Roche, Mannheim, Germany). The expression and purification of K205R protein was identified by SDS-PAGE, the antigenicity was analyzed by Western blot and iELISA.
Bactrian camel immunization and library construction
Based on previously reported procedures, a adult male Bactrian camel was immunized five times subcutaneously using the purified K205R protein [27, 28]. Briefly described as following, 2 ml K205R protein (2 mg) was mixed with an equal volume of Freund's complete adjuvant (Sigma-Aldrich, Merck KGaA, USA) for the first immunization. For the subsequent immunizations, 2 ml K205R protein (2 mg) was mixed with equal volume of Freund's incomplete adjuvant (Sigma-Aldrich). The camel was immunized once every 2 weeks. To determine the success of immunization, after the fourth immunization, camel serum sample was isolated and iELISA was performed to determine the antibody titer against K205R protein.
One week after the fifth immunization, 200 ml of camel whole blood was collected and peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque PLUS (Cytiva) with Leucosep™ tubes (Greiner Bio-One GmbH). Total RNA was extracted with 1 × 107 PBMCs and then reverse transcribed into cDNA. Nested PCR was performed to amplify the VHH gene using the cDNA as template. The first round of PCR products (~ 700 bp) were amplified with the VHH-F1 and VHH-R1 primers (Table S1), using the first round PCR products as template, the second round of PCR (~ 400 bp) were amplified with primers VHH-F2 and VHH-R2. Finally, the VHH gene was inserted into the phage display vector pCANTAB 5E with Not I and Pst I enzyme sites (NEB, Ipswich, MA, USA), and transformed into fresh E. coli TG1 competent cells using an electroporation machine.
Cells were incubated overnight at 37 °C on LB agar plates containing 100 mg/ml ampicillin and 2% glucose. The colonies on the plates were scraped and resuspended in 3 ml of LB medium containing 20% (v/v) glycerol to prepare a VHH phage library against K205R protein, the library was stored at -80 °C.
Screening and identification of ASFV K205R Nanobodies
As previously reported, phage display technology was used to screen anti-ASFV K205R protein specific Nbs by three rounds of bio-panning [26], and the M13K07 helper phage were utilized to rescue the VHH phage libraries. In bio-panning, 4 µg/well ASFV K205R protein was used as the coating antigen. The specific phage particles were significantly enriched after three rounds of bio-panning using anti-M13/HRP conjugate phage ELISA. Then, 96 single clones were randomly selected and induced to express soluble VHHs with E tag using 1 mM IPTG. Then, all the obtained soluble VHHs proteins were evaluated for their binding ability to ASFV K205R protein with iELISA using anti-E-Tag monoclonal antibody (GenScript, China). Finally, the positive clones identified by iELISA were sequenced and classified according to the sequence diversity in the third complementarity determining region (CDR3). Besides, the specificity and affinity of nanobodies screened were evaluated by iELISA using periplasmic extracts at different dilutions as primary antibodies.
Expression of Nb-HRP against ASFV K205R protein
To develop a cELISA using Nb-HRP fusion protein as a probe, the Nb-HRP fusion protein was prepared according to the previously reported method with appropriately modifications [26]. The VHH target gene was amplified using pCANTAB 5E-VHH plasmid as a template, after digesting with EcoR I and Nhe I (NEB, Ipswich, MA, USA), the VHH gene was then inserted into the pCAGGS-HA-HRP vector. The recombinant plasmid was named pCAGGS-Nbs-HRP, which contained codon-optimized HRP gene sequence, His tag, multiple cloning sites and secretion signal sequence. The sequencing correct plasmids were transfected into HEK-293 T cells using polyetherimide reagents (PEI; Polysciences Inc., Warrington, PA. USA). At 48 h post transfection, cells and culture supernatants were collected for detection of Nbs-HRP fusion protein expression using indirect fluorescence assay (IFA) or direct ELISA. At the same time, the specificity and affinity of the Nbs-HRP fusion protein expressed by HEK-293 T cells to ASFV K205R protein were detected by direct ELISA.
For IFA, at 24 h post-transfection, the HEK-293 T cells were fixed with -20 °C pre-cold 70% alcohol and blocked with 1% BSA for 2 h at 37 °C. Subsequently, the cells were incubated overnight with mouse anti-HA monoclonal antibody as the first antibody at 4 °C and followed by washing three times with PBS. Then the cells were incubated with Alexa Fluor 594-conjugated goat anti-mouse IgG (H&L) for 1 h at 37 °C in the dark. After washing three times with PBS, nuclei were stained with DAPI for 5 min in the dark. Images were obtained using a fluorescence microscope (Leica AF6000; Leica Microsystems GmbH).
For direct ELISA, the ELISA plate was coated with K205R protein (400 ng/well) and incubated overnight at 4 °C. After blocking with 2.5% dry milk at room temperature (RT) for 1 h (200 μl/well), the plate was washed three times with PBS'T. 100 and 200 µL cell supernatants containing the Nbs-HRP fusion protein were incubated in ELISA plate for 1 h at 37 °C. Following washing three times with PBS'T, 100 μl/well TMB was added 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 each sample was detected at 450 nm using a spectrophotometer (BioTek Instruments, Winooski, VT, USA).
Establishment of a competitive ELISA based on Nb-HRP probe
First, the optimal antigen coating concentration and dilution of Nb-HRP fusion protein were analyzed using a checkerboard titration assay by direct ELISA. Different concentrations of K205R protein (10, 20, 40, 80, 160, 320, and 640 ng/well) were coated into ELISA plates, and then the OD450 value was calculated when the Nb-HRP supernatants was diluted at ratios of 1:10, 1:100, 1:1000 and 1:10,000. When the OD450 absorbance value reached 1.0, the combination was determined as the optimal antigen coating concentration and the dilution ratio of Nb-HRP fusion protein. Three separate positive and negative pig serum were diluted at 1:5, 1:10, and 1:20 and detected using cELISA to determine the optimal dilution ratio of pig serum. According to the minimum ratio of OD450 value of positive serum and negative serum (P/N), the optimal serum dilution was determined. The optimal reaction time of the mixtures containing the Nb-HRP probe and the positive or negative serum with K205R protein were measured at 10, 30, and 50 min, respectively, followed with TMB imcubation for 10 and 15 min. Using a checkerboard titration method, the optimal incubation and colorimetric reaction time were determined when the minimum ratio of P/N value was obtained.
A novel cELISA was established after optimizing different reaction conditions: (1) ELISA plates were coated with the optimal concentration of K205R protein and incubated overnight at 4 °C. (2) After washing three times with PBS'T, the ELISA plates were blocked with 2.5% dried milk in PBS'T (200 µl/well) at RT for 1 h. (3) After washing three times with PBS'T, 100 µl of the optimal dilutions of serum sample and Nb-HRP supernatants mixtures in PBS'T was added to each well and incubated at RT for optimal times. (4) After washing three times with PBS'T, 100 µl/well of fresh TMB was added and incubated at 37 °C in the dark for the optimal duration. (5) 50 μl/well of 3 M H2SO4 was used as stop solution, and the absorbance value of each sample was detected at 450 nm.
Cut-off value, sensitivity, specificity and repeatability analysis of the developed cELISA
The PI values were determined using the following formula: PI (%) = [1-(OD450nm value of testing pig serum sample/ OD450nm value of negative pig serum sample)] × 100%. The cut-off value was determined using 154 ASFV antibody-negative pig serum samples. To ensure a 99% confidence level of negative porcine serum samples within this range, the cut-off value was calculated using the mean PI value of the negative samples plus 3 standard deviation (SD) after testing the above 154 serum samples using the established cELISA.
To assess the sensitivity of the developed cELISA, 5 inactivated ASFV antibody-positive pig serum samples were doubling diluted (from 1:8 to 1:1024) and detected using the cELISA to determine the lowest detection dilution. At the same time, 89 inactivated ASFV-clinical positive sera were detected to analyze PI distribution as well.
The specificity of cELISA was assessed via analyzing PI distribution by testing 320 ASFV-clinical negative sera. Meanwhile, the cross-reacted assay was evaluated between the Nb-HRP and serum antibodies of some other common swine viruses, such as porcine reproductive and respiratory syndrome virus (PRRSV), porcine epidemic diarrhea virus (PEDV), porcine pseudorabies virus (PRV), classical swine fever virus (CSFV) and porcine circovirus type 2 (PCV2).To evaluate the repeatability of the developed cELISA, 8 separate ASFV antibody-positive and -negative pig serum were detected and the inter-polate (between plates) and intra-plate (within a plate) variabilities were analyzed. The coefficient of variation (CV) was used to evaluate inter-plate variation and intra-plate variation. To calculate the inter-plate CV, each serum sample was detected using three different ELISA plates, while for the intra-plate CV, three replicates within each ELISA plate were detected.
Evaluation of agreement between the developed cELISA and commercial ELISA kits
Two hundred eighty-five clinical pig serum samples were detected using the developed cELISA and commercial ELISA kit, separately, and analyzed using SPSS software to evaluate the clinical agreement of the two detection method.
Statistical analysis
Statistical analysis was performed using GraphPad Prism version 5.0 (GraphPad Software, San Diego, CA, USA). Data are expressed as the mean ± SD. Repeatability was assessed using CV (CV = SD/mean), where a CV value less than 15% for the intra-plate assay was considered an acceptable repeatability level for the assay. Kappa values were calculated using SPSS software version 20 (IBM Corp.; http://www.spss.com.cn).