Abstract
More than forty antigen testing kits have been approved to response the prevalence of SARS-CoV-2 and its variant strains. However, the approved antigen testing kits are not capable of quantitative detection. Here, we successfully developed a lateral flow immunoassay based on colloidal gold nanoparticles (CGNP-based LFIA) for nucleocapsid (N) protein of SARS-CoV-2 quantitative detection. Delta strain (NMDC60042793) of SARS-CoV-2 have been cultured and analyzed by our developed digital PCR and LFIA methods to explore the relationship between N protein amount and N gene level. It indicated that the linear relationship (y = 47 ×) between N protein molecule number and N gene copy number exhibited very well (R2 = 0.995), the virus titers and N protein amount can be roughly estimated according to nucleic acid testing. Additionally, detection limits (LODs) of nine approved antigen testing kits also have been evaluated according to the Guidelines for the registration review of 2019-nCoV antigen testing reagents. Only three antigen testing kits had LODs as stated in the instructions, the LODs of Kits have been converted into the N gene and N protein levels, according to the established relationships among virus titer vers. N gene and antigen. Results demonstrated that the sensitivity of nucleic acid testing is at least 1835 times higher than that of antigen testing. We expect that the relationship investigation and testing kits evaluation have the important directive significance to precise epidemic prevention.
Keywords: SARS-CoV-2, Antigen detection, Kits, Relationship evaluation
Graphical abstract
1. Introduction
The pandemic of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has indescribably blown to the world's society and economy. During the pandemic, an emphasis has been always placed on SARS-CoV-2 testing to screen and diagnose suspicious populations, so that varieties of detection methods have emerged. Reverse transcription quantitative real time polymerase chain reaction (RT-qPCR) has been considered the gold standard approach for SARS-CoV-2 infection because of its high sensitivity and specify [1]. However, nucleic acid testing requires specialized instruments and environment, skilled and time-consuming operations, and high cost [2]. It has been reported that positive viral ribonucleic acid (RNA) of SARS-CoV-2 obtained by RT-qPCR does not indicate whether the subject is still contagious or not [3], but antigen testing could be predictors for the presence of cultivable SARS-CoV-2 [4,5]. In response to the COVID-19 pandemic development trend, the World Health Organization (WHO), Centers for Disease Control (CDC) and other international organizations have continuously updated the operational guidelines for COVID-19 prevention and control. It is noteworthy that many countries, including China, the United States, Japan, etc., have recognized the advantages and values of antigen detection in the early diagnosis of COVID-19 infection. There is an increasing demand for home self-testing and clinical application of antigen detection, because antigen testing based on lateral flow immunoassay (LFIA) can provide detection results on site within minutes [6].
Up to now, more than forty antigen testing kits based on LFIA have been approved by National Medical Products Administration (NMPA) to response the prevalence of SARS-CoV-2 and its variant strains. Whereas, the sensitivity of antigen testing is generally inferior to nucleic acid testing, which can lead to false negative results in early infection or when viral load is low [7,8]. And the approved LFIA-based antigen testing kits are barely capable of quantitative detection, for the same sample testing, the sensitivity relationship between nucleic acid testing and antigen testing is also unclear. Additionally, the performances of the antigen testing kits, especially the detection of limit (LOD), will directly affect the detection results. However, the amount of antigen in the archived samples were unclear or undetermined, which lead to it is challenging to evaluate the sensitivity of antigen testing kits.
Therefore, as illustrated in Scheme 1 , we developed a LFIA method based on colloidal gold nanoparticles (CGNP-LFIA) for quantitative detection of SARS-CoV-2 nucleocapsid (N) protein. And delta strain (NMDC60042793) of SARS-CoV-2 was cultured to establish the relationship between virus and N protein amount. Simultaneously, the N gene level also has been quantified by our developed digital PCR method [9], which is a technology providing absolute copy number quantification for highly accurate and sensitive detection. The relationship between N protein amount and N gene level has been preliminarily investigated. Based on the cultured SARS-CoV-2 delta strain, nine antigen testing kits approved by NMPA have been evaluated, and the LOD was converted from virus titer claimed in the instruction to antigen concentration and N gene copy number. These results are expected to have the important directive significance to precise epidemic prevention.
Scheme 1.
Illustration of the relationship investigation and testing Kits evaluation.
2. Experiments
2.1. Reagents and equipment
Mouse monoclonal antibodies against SARS-CoV-2 N protein were provided by Wuhan XiZi Biotech Co., Ltd. (Wuhan, China). Goat anti-mouse IgG was purchased from Beijing Yongjia Venture Company (Beijing, China). The Certified Reference Material of SARS-CoV-2 N protein (GBW(E)091097) was provided by the National Institute of Metrology (Beijing, China). Colloidal gold nanoparticle (CGNP) solution was prepared in our lab [10]. PEG20000, bovine serum albumin (BSA), polyvinylpyrrolidone K30, sucrose, ProClin300 and Tween-20 were purchased from Aladdin Reagent Co., Ltd. (Shanghai, China). Sample pad, glass-fiber membrane, PVC pad, and absorbent pad were obtained from Kinbio Tech Co., Ltd. (Shanghai, China). Nitrocellulose (NC) membrane (CN95) was purchased from Sartorius (Gottingen, Germany). All solvents and other chemicals were of analytical reagent grade. Delta strain (NMDC60042793) of SARS-CoV-2 was cultured by Kunming Institute of Zoology, Chinese Academy of Sciences (Kunming, China).
XYZ 3D film spraying instrument, CNC cutting machine (CTS300) and microcomputer automatic cutting machine (ZQ2402) were supplied by Kinbio Tech Co., Ltd. (Shanghai, China). Ultrapure water was purified with Milli-Q system from Millipore Corp. (Bedford, MA, USA).
2.2. CGNP-LFIA test strips fabrication
The CGNP solution was prepared as our previous study [10], and the pH was adjusted to 8.0 with K2CO3 solution (0.2 M). Then, 30 μg of Antibody against N protein dissolved in phosphate buffer was added dropwise into the CGNP solution, after 1 h, 1% of PEG20000 and 10% of BSA solution were added to block the unconjugated sites with 30 min. Finally, the supernatant was discarded after centrifuged at 8000 × g at 4 °C for 30 min, and the precipitate was resuspensed with 1.0 M Tris-HCl containing 0.5% polyvinylpyrrolidone K30, 10% sucrose, 1% BSA, and 0.05% ProClin300. The prepared detection probes solution was sprayed on the conjugated pad at a spray rate of 5 μL/cm by using an XYZ 3D film spraying instrument, and the pad was dried at 37 °C for 2 h with a vacuum dryer.
Goat anti-mouse IgG (0.4 mg/mL) and mouse monoclonal antibody against N protein (0.8 mg/mL) were dispensed onto the NC membrane with 0.8 μL/cm as the control (C) and test (T) lines, respectively, and then dried at 37 °C for 16 h. The CGNP-LFIA test strip was fabricated by attaching the conjugated pad, sample pad, NC membrane, and absorption pad to an adhesive PVC bottom plate, stacking one on top of each other, with an overlap of 2.0 mm, and then cut into 3 mm-wide and 60 mm-long test strips (as illustrated in Scheme 1). All test strips were packaged in a plastic bag containing a desiccant gel and stored at room temperature until use.
2.3. Quantitative curve of CGNP-LFIA construction
Certified Reference Material of SARS-CoV-2 N protein (GBW(E)091097) was diluted to a series of standard solutions at 0.263, 0.525, 1.050, 2.100, 4.202, 8.403, and 16.806 ng/mL with lysis buffer. The dilutions were detected by the CGNP-LFIA test strips, and the qualitative results were obtained by naked eye after 15 min. With the help of homemade portable reader, the signals of T and C lines were recorded, and the ratio of T and C (RT/C) was calculated and used to plotted with the concentration of N protein to construct the quantitative curve for detection.
2.4. Virus titer quantification
Delta strain (NMDC60042793) of SARS-CoV-2 have been cultured with three culture bottles. The virus titer was quantified by an endpoint titration assay. Briefly, serial 10-fold dilutions of cultured SARS-CoV-2 stock were prepared using viral culture media and added into seeded 96-well plates. The 96-well plates were cultured at 37 °C (5% CO2) for 5–6 days and then observed for cytopathic effect. The 50% tissue culture infectious dose (TCID50) was finally calculated by the Reed–Muench method.
2.5. Relationship investigation among virus titers, N protein and N gene levels
The cultured SARS-CoV-2 with the average virus titer at 106.13 TCID50/mL was diluted to 105.13, 104.13, 103.83, 103.53, 103.22, and 103.13 TCID50/mL, the dilutions were simultaneously detected by our developed CGNP-LFIA and digital PCR methods according to the processes in our previous work. The data obtained by the methods were plotted with the software Origin 8.5.
2.6. Antigen testing Kits evaluation
According to the Guidelines for the registration review of 2019-nCoV antigen testing reagents, the cultured delta strain of SARS-CoV-2 with an average titer of 106.13 TCID50/mL was diluted with Mdium DMEM to the claimed LODs of nine approved testing Kits, the dilutions were pretreated with specific buffer of each Kit and tested with twenty repeats. If the results were not consistent with their claimed LODs, their actual LODs were redefined. Briefly, the cultured SARS-CoV-2 were diluted to three different titers and twenty repeated tests were conducted on each dilution titer, the lowest titer that was detected with positive result was defined as the actual LODs of testing Kits.
3. Results and discussion
3.1. Development of CGNP-based LFIA for N protein detection
Based on our previous study [10], a pair of antibody against N protein with higher affinity than before have been screened and applied, the parameters, such as buffer system and amount of antibody, were optimized again for obtaining better performances, and the CGNP-based LFIA coupled with homemade portable reader has been successfully developed for quantitatively detecting N protein of SARS-CoV-2. Certified Reference Material of SARS-CoV-2 N protein (GBW(E)091,097) was diluted to a series of standard solutions (0.263, 0.525, 1.050, 2.100, 4.202, 8.403, and 16.806 ng/mL), which were detected by the developed CGNP-LFIA method with three repeats. As shown in Fig. 1 a, a weak band appeared on the T line when the concentration of N protein was 0.263 ng/mL, and the signal of T line increases with the increasing N protein concentration. Under the assistant of homemade portable reader, the signals of the LFIA were digitized, it is reported that the ratio of signal on T and C lines (RT/C) can effectively offset the effects of the inherent heterogeneity of test strips and the sample matrix [11], thus RT/C was employed as output signal to plot against the concentration of N protein. As displayed in Fig. 1b, there is a good linearity between the RT/C and logarithmic concentration of N protein with correlation coefficient (R2) higher than 0.99, and the equation was y = 0.733 lg(x) + 0.407 (EQ 1). The developed CGNP-LFIA has been used to detect the nasal swab samples from three volunteers, there was a clear distinction between infected and uninfected people (Fig. 1c), and after one week, Fig. 1d indicated that the two infected volunteers recovered but the healthy one was infected with SARS-CoV-2. The results demonstrated the developed LFIA has the capacity of rapid and accuracy diagnosis of infected people.
Fig. 1.
CGNP-LFIA method for N protein detection. (a) Photo of the CGNP-LFIA for the series of N protein detection. (b) Quantitative curve of the CGNP-LFIA, the inset picture is the linear range from 0.263 ng/mL to 16.806 ng/mL. (c) Result of nasal swab samples from three volunteers. (d) Result of the sample from the same volunteers after one week.
3.2. Relationship between N protein amount and N gene level
The relationship between SARS-CoV-2 RNA levels and antigen concentrations is not clear. In this work, delta strain (NMDC60042793) of SARS-CoV-2 have been cultured at an average titer of 106.13 TCID50/mL, which was diluted and analyzed by our developed CGNP-based LFIA and digital PCR [9] methods to simultaneously detect N protein amount and N gene level. The digital PCR method, developed successfully in our previous work [9], can provide absolute copy number quantification with the limit of detection (LOD) at 2.0 copies/μL. As for N protein amount detection, according to the output signal obtained by CGNP-based LFIA and quantitative detection equation EQ.1, the N protein concentration was calculated as (16.00 ± 3.2), (2.40 ± 0.4), (1.25 ± 0.1), (0.56 ± 0.1), (0.52 ± 0.03), (0.44 ± 0.01) ng/mL, when the cultured delta strain of SARS-CoV-2 were diluted to a series of titers (from 103.13 to 105.13 TCID50/mL). And the relationship between N protein amount and virus titer has been plotted in Fig. 2 a, the linear analysis was plotted with forcing the intercept to be 0.0, which displayed that there was a good linearity (R2 = 0.994). The LOD of the developed CGNP-based LFIA evaluated by the culture SARS-CoV-2 was 1092 TCID50/mL, and calculating to N protein concentration was 0.13 ng/mL according to the equation in Fig. 2a. Simultaneously, N gene copy numbers of the cultured delta strain and its dilutions were also quantified by our developed digital PCR method. And the N protein concentration was converted into molecule number via Avogadro constant (6.02 × 1023 mol−1). Interestingly, Fig. 2b indicated that the linear relationship (y = 47 ×) between N protein molecule number and N gene copy number exhibits very well (R2 = 0.995). And it indicates that one copy per mL of total RNA corresponds to forty-seven molecules per mL of N protein. Thus, the N protein level can be calculated according to the N gene copy numbers. These results demonstrated that the virus titers and N protein amount can be roughly estimated according to nucleic acid testing, which has important directive significance to precise epidemic prevention.
Fig. 2.
Relationships among virus titers and N protein amount, N gene level. (a) SARS-CoV-2 titer vers. N protein concentration; (b) N gene copy number vers. N protein molecule number.
3.3. Evaluation of the approved antigen testing kits
The LODs of nine antigen testing Kits approved by NMPA have been evaluated with our cultured delta strain (NMDC60042793) of SARS-CoV-2 and its dilutions. As shown in Table 1 , except for Kit I, the claimed LODs have been reported ranged from 64 to 850 TCID50/mL SARS-CoV-2 according to the user guide of each Kit. The cultured SARS-CoV-2 delta strain was diluted to the claimed LODs and tested with twenty repeats by each corresponding Kit. However, the LODs of only three kits (A, B, C) were proved to be consistent with their claims, which are 200, 850, 850 TCID50/mL, respectively. The other Kits exhibited false negative when the detected virus titers were at their claimed LODs. In order to evaluate the actual LODs of the other six testing Kits, the cultured SARS-CoV-2 delta strain has been diluted to three different titers and twenty repeated tests were conducted on each dilution titer. The LODs of Kit D, E, F, G, H, I have been defined as 911, 273, 364, 109, 182, 729 TCID50/mL (Table 1). Consequently, the detection performance is ranked as G > H > A > E > F > I > B=C > D according to the redefined LODs. Those experiments were conducted according to the Guidelines for the registration review of 2019-nCoV antigen testing reagents.
Table 1.
Analytical sensitivity comparison of antigen Kits approved by NMPA.
| Kits approved by NMPA | The claimed detection limit (TCID50/mL) | The evaluated detection limit (TCID50/mL) | Copy number（copies/μL) N gene |
N protein concentration (ng/mL) |
|---|---|---|---|---|
| Aa | 200 | 200 | 6716 | 0.024 |
| Ba | 850 | 850 | 28,541 | 0.102 |
| Ca | 850 | 850 | 28,541 | 0.102 |
| D | 137 | 911 | 30,581 | 0.109 |
| E | 160 | 273 | 9174 | 0.033 |
| F | 78 | 364 | 12,232 | 0.044 |
| G | 64 | 109 | 3670 | 0.013 |
| H | 100 | 182 | 6116 | 0.022 |
| I | NR | 729 | 24,465 | 0.087 |
NR: No reported.
The claimed detection limited has been confirmed.
Additionally, the LODs of antigen testing Kits are expressed as virus titer, but nucleic acid and antigen testings are the general approaches for SARS-CoV-2 infection screening. Therefore, the LODs of Kits have been converted into the N gene and N protein levels, according to the established relationships among virus titer vers. N gene and antigen. As shown in Table 1, It indicated that the differences in LOD among the approved antigen detection kits are very significant (95% Confidence Interval), the lowest LOD of the nine Kits is 3670 copies/μL. But the LOD of our developed digital PCR method is 2.0 copies/μL [9], thus, the sensitivity of nucleic acid testing is at least 1835 times higher than that of antigen testing kits. Consequently, the sensitivity of antigen testing is generally inferior to nucleic acid testing [12], resulting in false negative results in the early period of infection or with low viral load, which could explain that some samples are SARS-CoV-2 RNA positive but antigen negative. Recently, there is no reference method for SARS-CoV-2 antigen detection, thus the N protein quantification results were not confirmed, and this is the most limitation of the work.
4. Conclusions
In this work, a CGNP-based LFIA method has been successfully developed for quantitative detection of SARS-CoV-2 N protein. Our cultured delta strain (NMDC60042793) of SARS-CoV-2 and its dilutions have been analyzed by our developed digital PCR and LFIA methods to explore the relationship between N protein amount and N gene level. And nine approved antigen testing kits have also been evaluated, which indicated that only three antigen testing kits had LODs as stated in the instructions and the other six kits had higher LODs than their claimed. And the sensitivity of nucleic acid testing is at least 1835 times higher than that of antigen testing. These results were expected to have the important directive significance for precise epidemic prevention.
Credit author statement
Conceptualization and methodology, Xinhua Dai, Xiang Fang, Lianhua Dong and Minghua Li; Methodology, Tao Peng, Lianhua Dong, Chunyan Niu, Xia Wang, Zhanwei Liang; Software and formal analysis, Lianhua Dong and Tao Peng; Investigation and data curation, Xiaoli Feng, Yi Yang, Wang Qu, and Qingcui Zou; Resources, Xinhua Dai, Lianhua Dong and Minghua Li; Writing—original draft preparation, Tao Peng and Lianhua Dong; Writing—review and editing, Lianhua Dong and Tao Peng; Supervision, Xinhua Dai, Xiang Fang and Lianhua Dong. All authors have read and agreed to the final version of the manuscript.
Consent to participate
As the study was retrospective it was exempt from informed consent.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This work was supported by Basic scientific research project of National institute of metrology (AAKYZZ2126, AKYYJ2009/ZYZJ2001) and the Major Science and Technique Programs in Yunnan Province (202102AA310055). And the authors appreciate the cooperation of other faculty members in our groups.
Data availability
Data will be made available on request.
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Associated Data
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Data Availability Statement
Data will be made available on request.