Preparation of VP1s and S proteins
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 protein was induced and expressed, as described in the previous report .
Preparation of anti-S protein McAbs
The S domain McAbs were prepared as we previously described . Three McAbs (H9E, B4H, and J5D, further abbreviated as H, B, and J, respectively) were selected for their ability to bind pSmart-S expression product, but not with E. coli residual proteins. These three McAbs also demonstrated specificity against both GI and GII HuNoV VP1s via Western Blot.
Selection of McAbs that recognize different epitopes of S protein by ELISA assay
Each reaction of the ICA assay utilizes a labeled McAb and another immobilized (“capture”) McAb. All six pair permutations of the three McAbs (B-H, H-B, B-J, J-B, H-J, and J-H) were tested. First, the three McAbs (B, H, and J) were two-fold serially diluted and tested for their single epitope saturation concentrations using recombinant S protein (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 and named as B1 (or H1, J1). Then S protein (100.0 μg/well) was coated overnight at 4 °C, and a 100.0 μl epitope-saturated McAb (e.g., B) was incubated at 37 °C for 1 h. After washing, 100.0 μl second saturated McAb (e.g., H) in the pair tested was incubated under the same conditions. OD450 was recorded as BH2. Displacement factor (I) was defined as the ratio of the increased effect of cross-reactivity over the separate effect of the second added antibody. If I > 10 % , it indicates that the recognition sites of the two McAbs are distinct. I values were calculated according to the formula I (e.g., BH) = (BH2 - B1)/H1×100 %.
Selection of McAbs used on the colloidal gold platform
The performance of ICA depended critically upon the combination of the optimal antibody sandwich pair with colloidal gold. ELISA results needed to be considered in conjunction 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. 3) was both 2.0 mg/ml, and the labeled antibody was exceeded (40 μg/ml). The concentration of S protein used in the test was 0.3 μg/ml. The final selection was based on the combination of the color effect of both C and T lines.
Source of HuNoVs clinical samples (including suspected HuNoVs)
A total of 122 fecal specimens were collected and tested. Five HuNoVs clinical samples [57404 (GI.1), 3010 (GI.1), 1704 (GII.4), 1717 (GII.4), 1028 (GII.4)] were kindly provided by Dr. Ningbo Liao (Zhejiang Provincial Center for Disease Control and Prevention (CDC)). Total 117 clinical diarrheal samples which kindly provided by the Affiliated Hospital of Guangzhou Medical University (N=3), the Affiliated Hospital of Sun Yat-sen University (N=7), Chinese CDC (N=39) and Anhui Provincial CDC (N=68) were also tested by both RT-qPCR and ICA. Clinical samples provided by Zhejiang Provincial and Chinese CDC were from patients with acute gastroenteritis in 2018. The samples of Anhui provincial CDC and Affiliated Hospital of Guangzhou Medical University were all in 2017. The samples of the Affiliated Hospital of Sun Yat-sen University were from 2015 to 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, 0.4 μl for each primer (0.16 µmol/l) (See Table S4, Additional file 4) . 2.0 μl RNA template and RNase free ddH2O to a total volume of 25.0 μl. Amplification reactions were as follows: reverse transcription at 50 °C for 30 min; heat-denatured 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 of real-time RT-PCR to generate a positive cycle threshold (Ct) signal was one real-time RT-PCR unit (RT-qPCRU) . Ct values versus log10 viral genomic copies linear standard curves were generated from a continuous 10-fold dilution (See Figure S3, Additional file 5). Amplified DNA fragments were sequenced on ABI 3730XL at Personalbio (Shanghai, China). Automated genotypes were analyzed with Norovirus Typing Tool Version 2.0 (www.rivm.nl/mpf/norovirus/typingtool).
Conditions for exposing S protein from viral capsid in clinical samples
Pretreatment comprises of physical or chemical methods. Heat-denaturation was used for the physical treatment. Briefly, ten percent (w/v) fecal sample diluted with ddH2O was put in a water bath at a set temperature (60 °C, 70 °C, 80 °C, 90 °C, 100 °C) for 1 to 5 min with an interval of 1 min. The reducing agent (DTT) (Thermo Fisher, China) and alkaline were used for chemical treatment. The pH in PBS was adjusted to 6.0 to 10.0. 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 minutes. DTT was added to the homogenized solution of clinical sample at a final concentration of 1.0 %. A sandwich ELISA was undertaken to detect HuNoVs, as described previously .
Performing an ICA test
The procedure of preparing ICA strips was described in the Supplementary Material (Figure S4, Table S5-7, See Additional file 6). The sample preparation process was as follows: 10 % (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 (1.0 %). After 10 minutes at 25 °C, 50 μl mixtures were added to the sample pad. If enough HuNoV antigens were present, 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 S protein and clinical samples
Purified S protein was used at a concentration 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 buffer was used as blank control. Two HuNoV positive samples (57404 GI.1 and 1717 GII.4) were two-fold diluted with a range of 1.6×105 to 5×106 gc/g (GI) and 1.1×105 to 3.5×106 gc/g (GII). All analyses were conducted in triplicate to determine the repeatability of the results.
Evaluation of the specificity of ICA
To further evaluate the specificity of antibodies in the colloidal gold test, four Rotavirus, three Sapovirus, two Astrovirus and four Adenovirus (stool sample provided by Zhejiang CDC, the affiliated Hospital of Guangzhou Medical University and the affiliated Hospital of Sun Yat-sen University) and three 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.
To determine the stability of ICA strips, they were examined by thermal acceleration tests at 60 °C. The activity of the antibodies on each assay was tested by using the lowest detectable S protein concentration (1.4 ng/ml). The identical strips were tested at appropriate intervals (1, 2, 3, 7, 14, 21, and 28 days). By comparing the result of Sylvia’s report , the shelf-life could be estimated at room temperature.
One-way ANOVA was used for data analysis. SPSSAU, an online data analysis tool, was used for statistical analysis (www.spssau.com).