Expression and purification of SARS-CoV-2 nucleoprotein
The pET28a(+)-nCoV-N-his plasmid was constructed, and the SARS-CoV-2-N-His recombinant protein was produced using E. coli BL21. The plasmid was transformed into BL21 E. coli, and a single clone was inoculated into 5 mL of 2YT medium and cultured at 37°C and 220 rpm for 14 h. Subsequently, 2 mL of the bacterial solution was added into 1 L of 2YT medium and cultured at 37°C and 150 rpm for 2.5 h. The bacterial broth was incubated with 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 30°C and 150 rpm for 6 h. The bacterial solution was centrifuged, the culture medium discarded, and the bacteria fully resuspended in 50 mL of phosphate-buffered saline (PBS). This solution was sonicated with a cell ultrasonic disruptor, according to the following protocol: 250 W ultrasound for 10 s, pause for 10 s, for 30 cycles, at which time the bacterial solution became transparent. After sonication, the bacterial solution was centrifuged at 15,000 rpm for 20 min, and the supernatant was filtered through a 0.22 μm filter membrane, followed by affinity purification using a His Trap FF crude 5 mL nickel column. The purity of nucleoprotein was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and quantified using ImageJ. The concentration of nucleoprotein was measured by BCA protein assay kit.
Preparation of selenium nanoparticles and nucleoprotein complex
The selenium nanoparticle solution (SeNps) was prepared using ascorbic acid to reduce selenite. The selenium nanoparticle preparation was used to label nucleoproteins at different pH levels, and the pH of SeNps was adiusted with K2CO3. Then, nucleoprotein was added in drops to 100 mL SeNps with gentle stirring at room temperature. The mixture was then softly agitated for 30 min to allow conjugation of nucleoproteins to selenium nanoparticle surfaces by physical adsorption. Subsequently, 1 g bovine serum albumin (BSA) was added to the mixture for 30 min to block the non-coated selenium nanoparticle surfaces, and then the mixture was centrifuged at 10,000 rpm for 10 min. Selenium nanoparticle-conjugated nucleoprotein in a soft-pellet form was collected. Finally, the labeled proteins were resuspended in a working solution (pH 7.4 10 mM PBS containing 0.05% Tween 20, 1% BSA, 5% sucrose, and 5% trehalose) and then stored at 4°C before use. The optimal pH was selected according to color development. At the selected optimal pH, 1.5 mg/mL, 2 mg/mL, and 4 mg/mL were selected as antibody coating concentration at the test line. 5 μg/mL, 10 μg/mL, and 20 μg/mL were the alternative conjugating concentrations of the nucleoproteins. 1 mg/mL was used as the coating concentration of the anti-His antibody at the control line to prepare the kit. The criteria for determining the optimal conditions were the number of false positive results and the degree of color development.
The internal structure of the kit is shown in Fig. 1a and includes a backing, a sample pad, a conjugate pad, a reaction pad, and an absorbent pad. The liquid matrix added to the sample pad is chromatographed to the conjugate pad to re-dissolve the selenium-labeled protein complex fixed to the conjugate pad, and, due to the capillary action, the selenium-labeled nucleoprotein is chromatographed with the liquid matrix toward the absorbent pad in the nitrocellulose membrane. The process for IgM detection is provided as an example in Fig. 1b. When the sample contains anti-SARS-CoV-2 IgM, the IgM conjugates to the selenium nanoparticle-labeled nucleoprotein. When this protein complex conjugate is chromatographed to the position of the test line, the IgM will bind to the anti-human IgM antibody, resulting in the accumulation of color at the position of the test line. When the excess complex conjugate is chromatographed to the control line area, the anti-His antibody coated in this region captures the selenium nanoparticle-labeled His-tag nucleoprotein, resulting in color accumulation. Therefore, when both the control line and the test lines develop color, it is determined that the sample contains anti-nucleoprotein IgM, and the test result is considered positive. When a negative sample is applied to the test, there is no immune binding reaction in the detection area. Thus, no color will be accumulated at the test line, but the control line will develop color normally. Therefore, when the control line develops color and the test line does not develop color, it is determined that the sample does not contain anti-nucleoprotein IgM, and the test result is considered negative. If the control line does not develop color, the condition of color development at the test line is an invalid result. Interpretation of IgG test results works in the same way as for IgM. For the anti-SARS-CoV-2 IgG test, the anti-human IgG antibody was coated at the position of the test strip, and other the conditions remained unchanged.
Preparation of the SARS-CoV-2 antibody detection kit
The anti-human IgM antibody or IgG antibody was coated on the test line, and the anti-His antibody was coated on the control line. After the coating was completed, the reaction pad was dried at 37°C for 4 h. The sample pad was immersed in the sample pad treatment solution (10 mM PBS, pH 7.4, containing 0.05% Tween 20, and 5% serum) for 30 min and dried at 37°C for 4 h. The conjugate pad contained immobilized selenium nanoparticle-SARS-CoV-2 nucleoprotein complex. The test strips were prepared prior to performing the assay, assembled, cut, and ready for use.
Clinical sample validation
Fetal bovine serum (FBS) was chosen as the negative sample for the selection of kit conditions in the early stage. The serum of two healthy persons was used to verify the above conditions. SARS-CoV-2 positive specimen and negative specimen were used to validate the test strips. Among them, 19 cases were diagnosed as positive in Henan Provincial People's Hospital, the COVID-19 designated hospital in Henan Province; 41 cases were confirmed negatives by clinical detection from the First Affiliated Hospital of Henan University. For each kit, 16 μL of plasma and 64 μL of sample processing solution were added. The sample processing solution was 10 mM PBS containing 0.05% Tween-20 and 1% BSA, pH 7.4, and the results were observed after 5 min. The observation results are shown in Figure 1. When both the test line and the control line develop color, the result is positive; when only the control line develops the color, the result is negative; when the control line does not develop a color, the result is invalid. We confirm that the detection results of COVID-19 patients in this study have not been reported in any other submission by us or anyone else.
The sensitivity, specificity, positive predictive value, and negative predictive value of the kit were calculated according to the following formulas :
Sensitivity = [True Positive/(True Positive + False Negative)] × 100%
Specificity = [True negative / (True Negative + False Positive)] × 100%
Positive predictive value = [True Positive / (True Positive + False Positive)] × 100%
Negative predictive value = [True Negative / (False Negative + True Negative)] × 100%