Preparation of VP1s and the S domain of VP1
HuNoV GI.1 and GII.4 ORF2 gene fragments (GenBank Nos. M87661 and KM114291, respectively) were subcloned separately into the pET-28a prokaryotic expression plasmid. HuNoV GI and GII VP1 proteins were expressed from respective recombinant plasmid vectors pET28a-ORF2 GI.1 and GII.4 in Escherichia coli (E. coli) BL21 cells .
HuNoV GII.4 ORF2 (GenBank No. KM114291) was used as a template to amplify the nucleic acid fragment of the S domain. The upstream primer was 5′-GAATTCATGAAGATGGCGTCGAGTG-3′; the downstream primer was 5′- CTCGAGCTCAACTGTGGGTGGCAC-3′. EcoR I and Xho I restriction sites were appended to the 5′ ends of forward and reverse primers, respectively. After amplification and digestion, the nucleic acid fragment of the S domain was inserted into a pSmart vector (Frdbio, China) to generate recombinant pSmart-S. The recombinant S domain of VP1 was induced and expressed, as described in a previous study .
Preparation of anti-S domain of VP1 MAbs
The MAbs against the S domain of VP1 were prepared as described in our previous study . Three MAbs (H9E, B4H, and J5D, further abbreviated as H, B, and J, respectively) were selected for their ability to bind the pSmart-S expression product, but not with E. coli residual proteins. The three MAbs also demonstrated specificity against both GI and GII HuNoV VP1, which was confirmed by Western Blot analysis.
Selection of MAbs recognizing different epitopes of the S domain of VP1 by ELISA assay
Each reaction of the ICA assay utilizes a labeled MAb and an immobilized (“capture”) MAb. Since the conditions for the labeled MAb (40 μg/ml) and the immobilized Mab (2.0 mg/ml) were different, all six pair permutations of the three MAbs (B-H, H-B, B-J, J-B, H-J, and J-H) were tested. First, the three MAbs (B, H, and J) were two-fold serially diluted and tested for their single epitope saturation concentrations using a recombinant S domain of VP1 (100.0 μg/well). A concentration one step lower than the concentration with a significant decrease in OD450 value was defined as a single epitope saturation concentration of the MAbs and named B1 (or H1, J1). The S domain of VP1 (100.0 μg/well) was coated overnight at 4 °C, and 100.0 μl epitope-saturated MAb (e.g., B) was added and incubated at 37 °C for 1 h. After washing, 100.0 μl of saturated MAb (e.g., H) in the test pair was incubated under similar conditions. The OD450 was recorded as BH2. The displacement factor (I) was defined as the ratio of the increased effect of cross-reactivity over the separate effect of the second antibody. If I > 10% , this indicated that the recognition sites of the two MAbs were distinct. I values was calculated using the formula I (e.g., BH) = (BH2 - B1)/H1 × 100%.
Preparation of the colloidal gold-labeled MAbs
Colloidal gold particles with a mean particle diameter of 25.0 nm were produced. One hundred ml of 0.01% (w/v) chloroauric acid (HAuCl4) (Aladdin, Shanghai, China) was thoroughly boiled for 3 min, and 2.0 ml of 1.0% (w/v) sodium citrate (Aladdin, Shanghai, China) quickly added into the solution with magnetic stirring for 30 min. The color gradually changed from yellow to black–blue and finally brilliant red. After stirring for a few minutes at low speed, the colloidal gold suspension could cool down and stored in the dark at room temperature. The total volume was made up to the original volume (100.0 ml) using ultrapure water. To determine the size and distribution of the gold nanoparticles, a transmission electron microscopy (Tecnai G2 spirit Biotwin, USA) was used to scan the colloidal gold solution at 120 KV. The OD value of the colloidal gold solution was measured at 400–680 nm using an ultraviolet spectrophotometer (Tecan Sunrise, Switzerland).
Both physical and chemical crosslinking methods have been used for the conjugation of gold colloids and MAbs . Chemical crosslinking is more stable but the functional groups in MAb are likely to be affected. All the functional domains are retained when MAbs are conjugated by the physical method . In this study, the physical method was used for conjugation . The optimal pH, dose, and BSA concentration during the conjugation of gold colloids and MAbs were evaluated (Fig. S3 and Table S4–6, Additional files 4 and 5). Briefly, 200.0 μl of a MAb (i.e.H9E) was incubated with 0.5 ml of colloidal gold (pH 9.0) for 30 min at room temperature and with gentle stirring. Then, 50.0 μl BSA (Amresco, United States) at different concentrations were added into the colloidal gold as a blocking buffer to stabilize the gold-labeled antibody. After 15 min incubation, centrifugation at 8000×g at 4 °C for 20 min allowed the collection of the colloidal gold-antibody complex as a pellet, and unbound antibodies were retained in the supernatant. The presence of black massive deposits on the wall of the tube should be avoided during the centrifugal process. The conjugated colloidal gold-antibody was re-suspended in 50.0 μl of dissolution buffer (PBS, pH 9.0 containing 10.0% w/v sucrose (Sangon Biotech, Shanghai, China), 0.2% (w/v) PVA-205 (Aladdin, Shanghai, China), 0.2% (v/v) Tween-20 (Aladdin, Shanghai, China), and BSA (3.0, 2.5, 2.0, 1.5, 1.0 and 0.5%, w/v). The conjugation was confirmed by UV-vis spectroscopy using unlabeled gold particles. A 50.0 μl colloidal gold-antibody mixture was evenly dispensed on the 0.5 cm × 2.5 cm conjugated pad and dried for 3 h at room temperature.
Selection of MAbs used on the colloidal gold platform
ICA performance was critically dependent on a combination of the optimal antibody sandwich pair with colloidal gold. ELISA results were considered together with ICA testing. The capture antibody on the Test line (T line) and the control antibody (goat-anti-mouse Ig G, Beyotime Biotech, Shanghai, China) on the Control line (C line) (Fig. 1) measured 2.0 mg/ml, and the labeled antibody exceeded 40 μg/ml. A 0.3 μg/ml of the S domain of VP1 was used. The final selection was based on a combination of the color effect of both C and T lines.
HuNoVs in clinical samples (including suspected HuNoVs)
A total of 122 fecal specimens were collected and tested. Five HuNoVs clinical samples [57,404 (GI.1), 3010 (GI.1), 1704 (GII.4), 1717 (GII.4), 1028 (GII.4)] were provided by Dr. Ningbo Liao (Zhejiang Provincial Center for Disease Control and Prevention (CDC)). In total 117 clinical diarrheal samples were provided by the Affiliated Hospital of Guangzhou Medical University (3), the Affiliated Hospital of Sun Yat-sen University (7), Chinese CDC (39), and Anhui Provincial CDC (68). The samples were tested by both RT-qPCR and the developed ICA. Clinical samples provided by Zhejiang Provincial and Chinese CDC were obtained from patients with acute gastroenteritis in 2018. The samples from Anhui provincial CDC and Affiliated Hospital of Guangzhou Medical University were collected in 2017. The samples from the Affiliated Hospital of Sun Yat-sen University were obtained between 2015 and 2017.
Detection of HuNoVs by RT-qPCR and calculation of viral genomic copies
Real-time RT-PCR was performed using a commercial one-step RT-qPCR kit (Sangon Biotech, China), consisting of 12.5 μl 2 × one-step RT-qPCR Master Mix (with SYBR Green), 0.65 μl RT enzyme Mix, and 0.4 μl of each primer (0.16 μmol/l) (See Table S7, Additional file 6) . The RNA template (2.0 μl) and RNase free ddH2O were added to make a total volume of 25.0 μl. The amplification reactions were as follows: reverse transcription at 50 °C for 30 min; heat-denaturation at 95 °C for 3 min; 40 cycles with denaturation at 95 °C for 10 s, annealing and extension at 60 °C for 30 s. Fluorescence signals were collected at the end of each extension step. The highest dilution from real-time RT-PCR was used to generate a positive cycle threshold (Ct) signal, which was one real-time RT-PCR unit (RT-qPCRU) . Linear standard curves of Ct values versus log10 viral genomic copies were generated from a continuous 10-fold dilution (See Fig. S4, Additional file 7). Amplified DNA fragments were sequenced on an ABI 3730XL (Personalbio, China). Automated genotypes were analyzed using a Norovirus Typing Tool Version 2.0 (www.rivm.nl/mpf/norovirus/typingtool).
Exposure of the S domain of VP1 from viral capsid in clinical samples
Pretreatment included physical or chemical methods. Heat-denaturation was used for physical treatment. Briefly, 10.0% (w/v) fecal sample was diluted with PBS, placed in a water bath at a specific temperature (60 °C, 70 °C, 80 °C, 90 °C, 100 °C) for 1 to 5 min at an interval of 1 min. A reducing agent (DTT) (Thermo Fisher, China) and alkaline conditions were used for chemical treatment. The pH in PBS was adjusted from 6.0 to 10.0. The fecal samples were dissolved to 10.0% with PBS buffers (pH 6.0, 7.0, 8.0, 9.0 or 10.0) at room temperature (25 °C) for 5, 10, 15, 20, 25 and 30 min. DTT was added to the homogenized solution of the clinical sample at a final concentration of 1.0%. Sandwich ELISA was used to detect HuNoVs, as previously described .
Performing an ICA test
The procedure used to prepare ICA strips is described in Additional file 8. The sample preparation process was as follows: 10.0% (w/v) homogenized solution of stool slurries was prepared with PBS (pH 9.0) (10,000×g, 10 min). DTT was added to a final concentration of 1.0% and incubated at 25 °C for 10 min. 50 μl of the mixtures were added to the sample pad. In the presence of adequate amounts of HuNoVs antigens, the binding of the gold-labeled antigen complex occurred at both the T and C lines. The presence of C line confirmed that the test was valid.
Limit of detection (LOD) of ICA for the purified S domain of VP1 in clinical samples
The purified S domain of the VP1 was used at concentrations of 22.4 ng/ml, 11.2 ng/ml, 5.6 ng/ml, 2.8 ng/ml, 1.4 ng/ml and 0.7 ng/ml. PBS was used as blank control. Two HuNoVs positive samples (57,404 GI.1 and 1717 GII.4) were two-fold diluted with 1.6 × 105 to 5 × 106 gc/g (GI) and 1.1 × 105 to 3.5 × 106 gc/g (GII). All experiments were performed in triplicate to confirm the reproducibility of the results.
Evaluating ICA specificity
To determine the specificity of antibodies in the colloidal gold test, 4 Rotavirus, 3 Sapovirus, 2 Astrovirus and 4 Adenovirus (stool sample provided by Zhejiang CDC, the affiliated Hospital of Guangzhou Medical University and the affiliated Hospital of Sun Yat-sen University) and 3 Salmonella (ATCC 14028, CMCC 50115 and CICC 21482) were tested. The copies of Rotavirus, Sapovirus, Astrovirus, and Adenovirus RNA were more than 107 gc/g feces.
Thermal acceleration tests were used to determine the stability of the ICA strips, at 60 °C . The activity of the antibodies on each assay was determined using the lowest detectable S domain of the VP1 concentration (1.4 ng/ml). The identical strips were tested at appropriate intervals (1, 2, 3, 7, 14, 21, and 28 days). The shelf-life was estimated to be at room temperature.
One-way ANOVA was used for data analysis. SPSSAU, an online data analysis tool, was used to perform all the statistical analyses (www.spssau.com).