Human serum samples
There were 121 human brucellosis sera (gifted by the School of Public Health of Jilin University), 90 control sera, including 50 healthy sera and 40 patient sera (confirmed by blood culture to be infected with other pathogens, collected by the infection department of the First Clinical Hospital of Jilin University; information on the patients is shown in Table S1).
Selection of the outer membrane proteins of Brucella
The antigenicity of the outer membrane proteins of Brucella was investigated by consulting the literature (https://www.ncbi.nlm.nih.gov/protein/). The amino acid sequence was obtained, and the conservation of the amino acid sequence was analyzed by BLAST.
B cell epitope prediction and peptide synthesis
The conserved amino acid sequence of the Brucella outer membrane protein was used to predict B cell epitopes by using the B cell epitope prediction tool bepipred linear epitope prediction 2.0 in IEDB (http://tools.iedb.org/bcell/). The predicted B cell epitopes were delivered to SGS and coupled with keyhole limpet hemocyanin (KLH). The predicted B cell epitope was handed over to Sangon Biotech (Shanghai, China) for synthesis and coupled with keyhole limpet hemocyanin (KLH).
Screening of peptide epitopes
To screen effective peptide epitopes, we verified the antigenicity of predicted epitopes by indirect ELISA. The experimental procedure was as follows: peptides coupled to KLH were diluted with carbonate buffer (pH = 9.6) to final concentrations of 30 μg/mL and 100 μL/well in a 96-well plate (Corning, USA) and incubated overnight at 4°C. Next, 300 µL/well of blocking solution (PBS containing 5% skimmed milk powder) was incubated at 37°C for 1 h, and the cells were washed 3 times with PBST (PBS containing 0.05% Tween 20). Afterwards, 100 µL/well of 1:400 serum was added and incubated at 37℃ for 1 h followed by washing 3 times with PBST. Next, 1:5000 diluted HRP-labeled protein G (Thermo, USA) was added; it was reacted at room temperature for 15 min and washed again with PBST 3 times. Next, 100 µL of TMB substrate solution was added to each well and reacted for 15 min at room temperature followed by the addition of 50 µL of stop solution (2 M H2SO4). The optical density was measured at 450 nm (OD450) using an ELISA plate reader (BioTek, USA). At the same time, KLH (30 μg/mL) and lipopolysaccharide (LPS, 1 μg/mL, provided by China Animal Health and Epidemiology Center (Qingdao, China)) were used as blank carriers and positive antigen controls to detect serum.
Fusion protein preparation
The selected effective peptides are connected in series, and the adjacent two peptide chains are connected with the linker 'GGGS'. The plasmid was constructed by full gene synthesis, subcloned into the expression vector pET-21a (Sangon Biotech, Shanghai, China) and further transformed into E. coli competent BL21(DE3) cells (Sangon Biotech, Shanghai, China). The cells were cultured, IPTG was used to induce expression, bacteria were collected, the protein was purified, and the target protein was verified by SDS-PAGE and Western blotting. The specific steps are described below.
After transferring the recombinant plasmid into BL21(DE3), 800 μL of nonresistant LB medium was added, followed by incubation at 37°C for 45 min and centrifugation at 5000 rpm for 3 min. Most of the supernatant was discarded (leave approximately 100-150 μL), the bacteria were resuspended, the LB plate with corresponding resistance was selected, and it was coated. After air-drying, it was inverted and cultured overnight in a 37°C incubator. The monoclonal colonies on the plate were chosen, placed into 10 mL of LB liquid medium and incubated at 37°C and 200 rpm. The cultured bacterial solution was transferred to 750 mL of LB liquid medium at 37°C and 200 rpm, cultured to OD600=0.6-0.8 with IPTG (0.5 mM) at 16°C and induced overnight. Then, the cells were centrifuged at 6000 rpm for 5 min, the supernatant was discarded, and the bacteria were collected. Bacteria were blown away with 20-30 mL 10 mM Tris-HCl (pH = 8.0) solution and ultrasonically broken (500 W, 60 times, 10 s each time, 15 s interval). After sonication, 100 μL of the bacterial suspension was centrifuged at 12000 rpm for 10 min, and 50 μL of supernatant was transferred to another EP tube. After the supernatant was removed, the precipitate was blown away with 50 μL of 10 mM Tris-HCl (pH = 8.0) solution. SDS-PAGE and Western blotting were used to detect protein expression. A nickel column (Ni Sepharose 6 Fast Flow, GE Healthcare) for affinity chromatography was used for protein purification. Taking 5 mL of Ni-NTA, the equilibrium column was washed with 5 times the column bed volume of binding buffer at a flow rate of 5 mL/min. The crude protein was incubated with the equilibrated column packing for 1 h; the incubated product was loaded onto the column and the effluent liquid was collected; the equilibrium column was washed with binding buffer; the column was washed with washing buffer, and the effluent liquid was collected; with the column was eluted with elution buffer, and the effluent liquid was collected; and the crude protein was treated, washed with effluent and eluted with effluent separately, followed by sample preparation and, SDS-PAGE and WB detection. The concentrated protein was divided into 1 mL/tube and stored at -80°C.
Antigenicity identification of fusion protein
Indirect ELISA was used to verify the antigenicity of the fusion protein. The procedure was as follows: 96-well microtiter plates were coated with 1 μg/well fusion protein at 4°C overnight, and then, 5% skimmed milk powder was blocked at 37°C for 2 h. Serum was diluted 1:400 and added at 100 μL per well, followed by incubation at 37°C for 1 h. HRP-labeled protein G was diluted with 1:8000, added at 100 μL/well, incubated at 37°C for 1 h, and finally developed with TMB substrate solution for 15 min. For termination, 2 M H2SO4 was used, and the OD450 was measured. After each step, the cells were washed with PBST (0.05% Tween-20 in PBS) 3 times.
Synthesis of nano-ZnO and paper modification
ZnO nanorods were synthesized on Whatman No. 1 filter paper by a hydrothermal method. The steps were as follows: Whatman filter paper was soaked in 100 mm zinc acetate solution for 60 s and then annealed at 100 ℃ for 1 h to form a seed layer (seed layer). Then, the filter paper with a seed layer was transferred to a hydrothermal reaction vessel containing an equimolar solution (100 mm, pH = 6.5) of hexamethylenetetramine (HMTA, Sigma) and zinc nitrate (Zn(NO3)2.6H2O, Sigma). ZnO nanorods were synthesized at 90 ℃ for 5 h. Next, Whatman filter paper with ZnO nanorods was immersed in anhydrous toluene solution (Sigma) with 1% APTES (Sigma) for 5 min, heated and dried at 100 ℃ for 15 min, and silanized. Scanning electron microscopy (SEM, JSM-7500F), X-ray diffraction (XRD, Bruker D8) and X-ray photoelectron spectroscopy (XPS, Escalable250Xi) were used to characterize the structure and surface of the paper. Whatman filter paper modified with ZnO nanorods was punched into circular paper pieces with a diameter of 10 mm by a punch, and A4 plastic packaging paper was punched into small holes with a diameter of 6 mm by a punch. The 10 mm filter paper pieces were placed in the center of the 6 mm holes of the plastic packaging paper, fixed by a plastic packaging machine, and cut into small strips with 3 holes for standby.
Establishment of p-ELISA
Five microliters of fusion protein solution was placed in each well (30 μg/mL in PBS), incubated at room temperature for 30 min, washed with 20 μL of deionized water 3 times, and blocked with 20 μL of 5% skimmed milk powder at room temperature for 15 min; PBST was used for washing 3 times, and 5 μL of serum was added (diluted with 1:400); PBST was used for washing 3 times, and 5 μL of HRP labeled protein G was added (diluted with 1:8000), followed by incubation at room temperature for 210 s; and PBST was used for washing 3 times, 5 μL of TMB substrate solution was developed for 10 min, and an HP Laser Jet Pro MFP M227 was used for scanning to obtain the image. ImageJ software carries out gray intensity analysis for quantitative analysis.
To compare with the nanomodified p-ELISA (nano-p-ELISA) method, we also performed the traditional p-ELISA (tra-p-ELISA) method. The specific steps have been described in the literature. Five microliters of chitosan was added to deionized water (0.25 mg/mL) and placed onto Whatman No.1 filter paper followed by drying at room temperature; then 5 μL of 2.5% glutaraldehyde solution (PBS) was added, followed by resting at room temperature for 2 h and washing with 20 μL of deionized water twice. The remaining steps are same as described in section 2.8.
Dot plot and receiver operating characteristic (ROC) curve analyses were performed using GraphPad Prism version 6.05 for Windows. The OD450 and gray intensity were determined by Student's t-test (unpaired t-test). P-values < 0.05 were considered to indicate significant differences.