Ultra-rapid detection of nuclear protein of severe fever with thrombocytopenia syndrome virus by colloidal gold immunochromatography assay

Animals

The experimental mice, aged 7 weeks, were all Balb/C mice and purchased from Charles River Co., Ltd (Beijing, China). Mice were maintained and bred in specific pathogen-free conditions at Zhejiang Provincial Center for Disease Control and Prevention. Animal Ethics Committee of Zhejiang Provincial Center for Disease Control and Prevention provided full approval for this research (approval number: 2023-002-01).

Virus, cells, and clinical samples

SFTSV strain (SFTSV-2018-ZJ10), Chikungunya virus (Chik Vero 2P 12.8.6), and Zika virus (KU866423 | China | 2016) were preserved in Zhejiang Provincial Center for Disease Control and Prevention. Vero cells (ATCC CCL-81; Sinovac Biotech, Beijing, China) were cultured in minimum essential medium (MEM; Gibco, Grand Island, NY, USA). The Vero cell medium was supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA), 1% penicillin-streptomycin, 25 mM HEPES, and 2 mM L-glutamine and all cells were passaged every 2–3 days using trypsin-EDTA (0.25%, Gibco, Grand Island, NY, USA). Human sera from clinical samples were provided by Zhejiang Provincial Center for Disease Control and Prevention.This study was approved by the Ethical Review Committee of Zhejiang Provincial Center for Disease Control and Prevention, China (2023-002-01).

Main materials

Anti-His IgG, anti-Mouse IgG, and Goat Anti-Mouse IgG H&L (Alexa Fluor^® 488) were purchased from Abcam (Cambridge, MA, USA). The mice used produce the monoclonal antibodies from hybridomas were purchased from Charles River Laboratories (Wilmington, MA, USA). The E. coli competent cells were purchased from Shanghai Weidi Biotechnology Co., Ltd. (Shanghai, China). QuickAntibody-Mouse and Freund’s adjuvant were purchased from Biodragon (Douglasville, GA, USA). SupraAjuvant was given by Zhimou, Yang (College of Life Sciences, Synergetic Innovation Center of Chemical Science and Engineering, and National Institute of Functional Materials, Nankai University).

Preparation of recombinant immunogens

The sequence information for the SFTSV-NP protein was obtained from NCBI. The sequence was optimized for expression in E. coli and synthesized by GenScript (Nanjing, China). Primers were designed to clone the target sequence into the pET-30a(+) expression vector (Primer F: 5′-GGGAATTCCATATGATGAGTGAATGGTCAAGGATAGCTG-3′ (Nde I); Primer R: 5′-AAATATCTCGAGCAGGTTGCGGTATGCA-3′ (Xho I). The ligation product was transformed into competent E. coli DH5α cells using heat shock. The recombinant plasmids were then transferred into the expression strain BL21(DE3). Expression of the SFTSV-NP recombinant protein was induced by adding isopropyl β-D-thiogalactoside (IPTG) at a concentration of 200 μg/mL at 25 °C with agitation. The bacterial cells were harvested, and the SFTSV-NP protein was purified using Ni²⁺ NTA resin affinity chromatography after cell lysis by ultrasonication at 200 rpm for 5 h.

The bacterial cells were resuspended in Binding Buffer (10 mM KH₂PO₄, 20 mM Na₂HPO₄·2H₂O, 500 mM NaCl, 50 mM imidazole, pH 8.0) and lysed using an ultrasonic cell disruptor (SCIENTZ-IID, Scientz, Ningbo, China). After sonication, the lysate was centrifuged at 12,000–13,000 rpm for 10 min at 4 °C. The supernatant was collected, filtered through a 1.2 μm filter using a sterile syringe, and set aside for purification.

A Ni-NTA affinity chromatography column was equilibrated with 10 column volumes of Binding Buffer. The sample was loaded onto the column at a flow rate of 2–4 mL/min (with a peristaltic pump at 3–6 rpm). The column was washed with Washing Buffer (10 mM KH₂PO₄, 20 mM Na₂HPO₄·2H₂O, 500 mM NaCl, 100 mM imidazole, pH 8.0) at 3–5 mL/min until the absorbance stabilized. The target protein was eluted using Elution Buffer (10 mM KH₂PO₄, 20 mM Na₂HPO₄·2H₂O, 500 mM NaCl, 500 mM imidazole, pH 8.0), and the fractions containing the protein were collected at the same flow rate. After use, the Ni-NTA column was washed with Washing Buffer, deionized water, and 20% ethanol before storage at 2–8 °C.

The eluate from the Ni-NTA column was dialyzed against 20 mM phosphate buffer (pH 7.4) and subjected to further purification using a DEAE column to optimize purity. The eluate from the DEAE column was dialyzed against phosphate buffer, sterile-filtered, sampled, and the final product was labeled and stored at −20 °C.

Preparation of monoclonal antibodies using hybridoma technology

Three adjuvants were chosen for this experiment: SupraAjuvant, QuickAntibody-Mouse, and Freund’s adjuvant. Ten mice were randomly assigned to each group. In this study, 7-week-old Balb/C mice were selected. Mouse immunization was performed using inactivated SFTSV cultures cross-immunized with recombinant SFTSV-NP, using four mice per round of each immunization method, at 2-week intervals. Immunized mice with antibody titers meeting the fusion requirements were selected for cell fusion using the PEG-mediated method, and semi-solid, limited dilution liquid culture was used to select the fusion cells. ELISA was used to screen the fusion cells. After obtaining positive clones, the clone was expanded to obtain the monoclonal antibodies. All mice were euthanized by cervical dislocation, whose spleen was taken for cell fusion. The two monoclonal antibody strains obtained were named SV01-13 and SV06-21.

Indirect enzyme-linked immunosorbent assay

After the fourth immunization, the titer of mouse serum was determined using an indirect ELISA. The recombinant SFTSV-NP protein was diluted to a concentration of 4 μg/mL. A volume of 100 μL of the diluted protein was added to each well of a 96-well plate, followed by incubation at 37 °C for 2 h in a constant temperature incubator. The coating solution was discarded, and the plate was washed five times with PBST (cwBio, Jiangsu, China). Following the washes, 200 μL of blocking buffer (cwBio, Jiangsu, China) was added to each well, and the plate was incubated at 37 °C for 1 h. The blocking buffer was then discarded, and the plate was washed five times with PBST. A total of 100 μL of diluted serum was added to each well, followed by incubation at 37 °C for 1 h. The liquid was discarded, the plate was washed five times with PBST, and 100 μL of HRP-labeled secondary antibody was added to each well. The plate was incubated at 37 °C for 1 h. After discarding the liquid and washing the plate five times with PBST, 50 μL of TMB (Solarbio Science & Technology Co., Ltd., Beijing, China) substrate was added to each well, followed by incubation at 37 °C for 10 min. The color development was stopped by adding 50 μL of stop solution (Solarbio Science & Technology Co., Ltd., Beijing, China) to each well. The OD450 value was then read using a SpectraMax Plus 384 microplate reader (Molecular Devices, Shanghai, China).

Western blot

We infected Vero cells with SFTSV virus, and after the cells showed lesions, samples of virus-infected cells were collected and lysed. The cell lysate was separated with sodium dodecyl sulfate–polyacry-lamide gel electrophoresis (SDS-PAGE), then transferred to polyvinylidene fluoride (PVDF) membranes. The blots were visualized with Western blot imaging equipment (Bio-Rad, Hercules, CA, USA). Briefly, the membranes were transferred to TBST (cwBio, Jiangsu, China) with 5% non-fat dry milk powder and shaken on a decolorizing shaker at room temperature for 1 h. SV01-13 and SV06-21 were diluted with antibody dilution buffer at 1:10,000 as primary antibodies. The membranes were removed from the sealing solution and incubated with the primary antibody at room temperature. The primary antibodies were shaken and incubated overnight on a decolorizing shaker at 4 °C. The blot was rinsed with TBST solution and incubated for 2 h with the secondary antibody (Vazyme, Nanjin China). After rinsing, the blots were incubated for 1 min with a LumiBest ECL Substrate solution kit, then observed in an imager.

Indirect immunofluorescence assay

Vero cells (3 × 106/well) were seeded into 12-well plates. After 48 h, the cells were infected with SFTSV. After 48 h, the medium was aspirated, the cells were washed once with PBS containing 2% BSA, and fixed for 15 min with methanol that had been pre-chilled for 15 min at −20 °C. After three washes with 2% BSA in PBS, the cells were permeabilized with 0.5% Triton X-100 (Solarbio Science & Technology Co., Ltd., Beijing, China) for 10 min. Then, the cells were blocked for 1 h with 2 mL 2% BSA, the blocking solution discarded, and the cells were washed three times with PBS. SV01-13 and SV06-21 were diluted with antibody dilution buffer at 1:10,000 as primary antibodies. Subsequently, 2 mL primary antibodies were added to each well separately and incubated at room temperature for 1 h or 4 °C overnight. Then, the sample was rinsed three times with 2% BSA for 5 min per rinse. Next, 2 mL secondary antibody was added to each well and incubated at room temperature for 1 h. Subsequently, the samples were rinsed three times with 2% BSA for 5 min per rinse. 4′,6-diamidino-2-phenylindole (DAPI) solution (2 mL, 1 mg/mL) was added to each well and reacted for 10 min in the dark. The analysis was observed with an EVOSTM M7000 imaging system.

Preparation of colloidal gold-recombinant antibodies

Colloidal gold particles were prepared by the Frans method (Wang et al., 2014). In 192 ml of ultrapure water with high-speed magnetic stirring, 8 ml of 1% chloroauric acid (HAuCl4) solution was added and boiled for 5 min, then 12.8 ml of 1% trisodium citrate solution was added. After boiling for 3 min, the colloidal gold pellet solution was cooled gradually. Bhosphate buffer (pH7.4) was added with an appropriate amount of monoclonal antibodies (SV01-13 and SV06-21) to a final concentration of 20 μg/mL, shaken and mixed well, added with 100 μL of prepared colloidal gold pellet solution, and shaken at 50–60 rpm at room temperature for 1 h. Thereafter, we added 20 μL of bovine serum albumin solution to block the reaction for 2 h, followed by centrifugation for 15 min at 500 × g. The supernatant was discarded, and the precipitate was resuspended by adding compound solution containing 1% BSA, 0.02% NaN3,5% trehalose and 20% sucrose.

Real-time reverse transcription PCR

We extracted RNA of SFTSV according to the TRIzol operating instructions (Invitrogen, Waltham, MA, USA). Total RNA was extracted using TRIzol and processed with RQ1 RNase-free DNase I (Promega, Madison, WI, USA) to eliminate residual DNA. DNA fragments were generated with the specific primers F (5′-AGCATGAATTCTCACGGAGC′) and R (5′-CGCTCTTCAAGGTTCTGCTT-3′) of the target gene (SFTSV-NP). According to the manufacturer’s instructions, the reaction was performed in a 25-μL reaction solution containing 17 μL of reaction solution A, 3 μL of reaction solution B and 5 μL of RNA elution. The RT-qPCR thermal cycling conditions were as follows: reverse transcription at 50 °C for 15 min and an initial denaturation step at 95 °C for 15 min, followed by 45 cycles of 94 °C for 15 s and 55 °C for 45 s. The amplification assay was performed on a 7500 Real-time PCR Detection System (Applied Biosystems, Waltham, MA, USA).

Evaluation of different strains of SFTSV

We used the developed ICA test to detect SFTSV strains preserved by the Zhejiang Provincial Center for Disease Control and Prevention, including SFTSV genotypes A, B, and D. The results of the ICA were interpreted with reference to the color card (Fig. 1C). Besides, we assessed the detection limits of different SFTSV strains, including dilutions and Ct values.The measurement sequence of all samples is randomly numbered.

Cross-reactivity of SFTSV detection

We collected nine clinical samples of dengue virus preserved by the Zhejiang Provincial Center for Disease Control and Prevention for testing, plus, Chikungunya virus and Zika virus, and evaluated the cross-reactivity capability of the test strip against various pathogens.

Detection of clinical samples

We utilized clinically suspected SFTSV infected serum samples preserved by the Zhejiang Provincial Center for Disease Control and Prevention. We dropped 75 μL of serum onto the ICA test strip to assess its detection capabilities for clinical specimens. Besides, considering that SFTSV mainly occurs in rural areas, in order to simplify the pre-processing procedures for blood samples, we tested whole blood samples from six healthy individuals and one positive case to evaluate the detection capability of the ICA test strip for whole blood samples.

Statistical methods

GraphPad Prism 8.0 software was used for graphical and statistical analyses.

Preparation of recombinant immunogens

The recombinant pET-30a(+) plasmid expressing his-tagged SFTSV-NP was transformed into the Escherichia coli expression strain BL21 (DE3). After induction, the protein was purified using an Ni2+-NTA resin column. We separated the proteins using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) experiments and stained them with Coomassie Brilliant Blue R-250, which showed a single band corresponding to his-tagged SFTSV-NP (Fig. 2A). Western blotting was performed using an anti-his tag antibody (Fig. 2B), which indicated successful expression of the recombinant SFTSV-NP.

Immunization of mice with different adjuvants

In this study, we employed three different adjuvant immunization schemes (SupraAjuvant, QuickAntibody-Mouse and Freund’s adjuvant). The comparative analysis revealed that the SupraAjuvant yielded the most effective immune titers, followed by QuickAntibody-Mouse, and lastly Freund’s adjuvant. Thus, it was evident that the peptide hydrogel adjuvant could indeed stimulate the host to generate a stronger immune response (Fig. 3A).

Preparation and validation of anti-SFTSV-NP monoclonal antibodies

After obtaining multiple positive clones by the hybridoma technique, two monoclonal antibodies, SV01-13 and SV06-21, were obtained by screening two monoclonal antibodies with high affinity for SFTSV. We performed performance validation on both of the obtained monoclonal antibodies, SV01-13 and SV06-21. SV01-13 and SV06-21 possessed good potency, and the working concentration could reach 15.625 ng/ml, as detected using indirect ELISA (Fig. 3B). Vero cells were then cultured and infected with the SFTSV virus. After the appearance of a cytopathic effect, the cells were collected. Western blotting experiments were conducted using SV01-13 and SV06-21 as the primary antibodies at a dilution of 1:12,000, with anti-mouse IgG as the secondary antibody (Fig. 2B). Similarly, Vero cells were infected with the SFTSV virus and fixed after the appearance of the cytopathic effect. Indirect immunofluorescence assays were performed using SV01-13 and SV06-21 as the primary antibodies at a dilution of 1:10,000, and fluorescence-labeled anti-mouse IgG as the secondary antibody (Fig. 2C). The experimental results showed that both monoclonal antibodies could specifically bind to the NP protein of the SFTSV virus strain, demonstrating excellent sensitivity.

Evaluation of different strains of SFTSV

Through testing different strains of SFTSV, the results indicated a 100% detection rate for the immunochromatographic strip (ICA) test for the various SFTSV strains. In comparison with the qRT-PCR results, it was evident that the average detection limit of this assay kit was a Ct value of 30.142, demonstrating excellent detection capabilities (Fig. 4A and Table 1).

Cross-reactivity of SFTSV detection

We collected nine clinical serum samples of Dengue virus preserved by the Zhejiang Provincial Center for Disease Control and Prevention, and single samples of Zika Virus and Chikungunya Virus, to evaluate the cross-detection capability of the test strip against various pathogens (Fig. 4A and Table 2). None of the 11 samples tested positive.

Detection of clinical samples

We analyzed 36 clinical serum samples and compared the results with those of qRT-PCR detection (Fig. 4B). We observed that among the 36 clinical serum samples, the positive detection rate of qRT-PCR-positive samples (30 cases) with Ct values in the range of 15–35 was 96.67% (29 cases), and the negative detection rate of qRT-PCR-negative samples (seven cases) with Ct values ≥ 35 and was 85.71% (six cases) (Fig. 4B). The only serum sample with a false-negative ICA test result had a Ct value of 34.86, which is in the error interval of the ICA test (Fig. 3C and Table 3). Additionally, the colloidal gold kit ICA test was 100% correct for whole blood samples, correctly detecting six negative samples and one positive sample (Fig. 4C).