Rapid, ultrasensitive and highly specific biosensor for the diagnosis of SARS-CoV-2 in clinical blood samples

Lu Zeng ab, Yue Li ab, Jie Liu ab, Lingling Guo ab, Zhongxing Wang ab, Xinxin Xu ab, Shanshan Song ab, Changlong Hao ab, Liqiang Liu ab, Meiguo Xin *c and Chuanlai Xu *ab
aInternational Joint Research Laboratory for Biointerface and Biodetection, and School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, P. R. China. E-mail: xcl@jiangnan.edu.cn
bState Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, P. R. China
cSchool of Food Science and Technology, Foshan University, Foshan, Guangdong 528000, P. R. China. E-mail: meiguo@fosu.edu.cn

Received 3rd May 2020 , Accepted 29th May 2020

First published on 29th May 2020


In this study, a lateral flow combined IgG–IgM immunochromatographic assay is developed for the rapid, simultaneous detection of IgM and IgG antibodies against SARS-CoV-2 in clinical blood samples within 15 min. The clinical detection sensitivity and specificity of the assay strips is investigated in samples of blood from inpatients with COVID-19. The sensitivity and specificity of this assay are 85.29% and 100.00%, respectively. Compared with a single IgG and IgM test, the combined IgG–IgM immunochromatographic strip test has higher sensitivity. Our results demonstrate that the combined IgG–IgM immunochromatographic strip is suitable for the rapid screening of SARS-CoV-2 infection, among confirmed COVID-19 patients, suspect patients and asymptomatic SARS-CoV-2 carriers.


1. Introduction

In December 2019, an outbreak of pulmonary disease caused by an unknown pathogen with clinical presentations greatly resembling viral pneumonia, was first seen in the city of Wuhan, Hubei province, China.1,2 Sequencing of samples from the lower respiratory tract of patients, subsequently identified a novel coronavirus and was first named 2019 novel coronavirus (2019-nCoV) by the World Health Organization.3,4 The new virus has been recently named as SARS-CoV-2 by the coronavirus study group of the International Committee on Taxonomy of Viruses.5 More significantly, a pandemic of coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 has now been spread worldwide. On January 31, 2020, the World Health Organization declared that COVID-19 was listed as a Public Health Emergency of International Concern.6 Most patients with COVID-19 display a broad range of symptoms that may include fatigue, myalgia, diarrhea, fever, headaches, heart palpitations, chest distress, other acute symptoms or may even die of the disease.7,8 However, asymptomatic or mildly symptomatic cases have also been reported.9,10 Yet there are no specific antiviral agents or vaccines available for the treatment or prevention of SARS-CoV-2 infection. The increase in the number of SARS-CoV-2 infection stresses the importance of developing a rapid, sensitive and specific diagnostic tests to confirm infection and mitigate the spread.

Currently, nucleic acid detection-based real-time reverse transcriptase-polymerase chain reaction (RT-PCR) has been widely used as the current standard diagnostic method for identifying SARS-CoV-2 infection.11–14 However, the entire test process is inherently time-consuming in 2 hours to obtain the data. The method requires specialized equipment, certified molecular testing laboratories and well-trained technicians. The results obtained by RT-PCR can also include some false-negatives or failed assays, especially in the diagnosis of suspected cases. Therefore, the development of a rapid, sensitive and specific test as a supplement approach to quickly identify patients with COVID-19 is urgently needed, thereby controlling viral spread and ensuring treatment of affected patients. The specific antibody production of patients with SARS-CoV-2 facilitated the early development of an antibody-based immunoassay. IgM is the first antibody elicited in an immune response following infection, prior to the generation of adaptive, high-affinity IgG responses that play an important role in long-lived immunity and immunological memory.15–17 SARS-CoV-2 is closely related to severe acute respiratory syndrome coronavirus (SARS-CoV) that caused the SARS outbreak. It has been reported that IgM could be detected in the blood of patients after 3–6 days following SARS infection. Whereas, IgG could be detected after 8 days.18 Therefore, a combined IgM and IgG antibody-based immunoassay could be an important supplementary diagnostic method for SARS-CoV-2 infected patients.

In this study, we developed an immunochromatographic strips using gold nanoparticles (GNPs) labeled antibody against the S protein of SARS-CoV-2 for the rapid, qualitative detection of IgG and IgM antibodies induced by SARS-CoV-2 in the blood of infected patients (within 15 min), respectively. Furthermore, we investigated the detection, sensitivity and specificity of the assay strips from blood samples from inpatients with COVID-19, with diagnoses based on symptoms and radiology and confirmed by PCR. The results indicated that the immunochromatographic strip test has ultrasensitive and highly specificity which can be applied for rapid screening of carriers of SARS-CoV-2 that are confirmed patients with COVID-19, or asymptomatic. The combined IgG–IgM immunochromatographic strip has potential to become a powerful supplementary approach for the diagnosis of COVID-19.

2. Results and discussion

2.1. Principle of the combined IgG–IgM immunochromatographic strip

The combined IgG–IgM immunochromatographic test strip is a qualitative lateral flow immunoassay, based on the reaction of IgG and IgM antibodies in blood samples with the GNPs-labeled SARS-CoV-2 antigen. The antigen/antibody is captured by the anti-human IgM or IgG antibody to form a visual band, indicating the presence of IgG and IgM antibodies against SARS-CoV-2. The assay is performed as follows: the test sample was pretreated with sample dilution buffer, then applied to the sample well of the test cassette. The sample will then move forward to the absorption pad by capillary action. If IgM and IgG antibodies are present, they will be bound by the SARS-CoV-2 antigen. The complex will be captured by the anti-human IgM antibody and anti-human IgG antibody, respectively. Ultimately, red visible bands in both of the detection lines are formed, indicating the result is positive for both IgG and IgM antibodies. If the C line and M line appear, the SARS-CoV-2 IgM has been detected and the result is positive for the presence of IgM antibody. If the C line and G line appear, the SARS-CoV-2 IgG has been detected and the result is positive for the IgG antibody. In the absence of either antibody, the M line and G line are colorless and the result is negative. Any remaining GNPs migrates to the C line zone, and the red C line should be observed, otherwise the strip is invalid. The interpretation of different test results is shown in Fig. 2.
image file: d0qm00294a-f1.tif
Fig. 1 Expression sequence of recombinant RBD antigen.

image file: d0qm00294a-f2.tif
Fig. 2 (A) Schematic representation of the IgM–IgG combined immunochromatographic strip for the diagnosis of SARS-CoV-2. (B) Interpretation of different test results.

2.2. Characterization of the GNPs

GNPs have unique chemical and physical properties and have extensive applications in biomedical science.19–21 The prepared GNPs were characterized by high resolution TEM and the UV-Vis spectrum was recorded. As shown in Fig. 3, the TEM image demonstrated that the GNPs were uniformly dispersed, with an average diameter of 35 nm. In addition, UV-Vis spectrum has a shoulder peak at 530 nm and displayed a broad absorption band. These results indicated that the GNPs was successfully synthesized.
image file: d0qm00294a-f3.tif
Fig. 3 Characterization of the GNPs. (A) The TEM image of GNPs. (B) UV-Vis spectrum.

2.3. Combined IgG–IgM immunochromatographic strip for the detection of SARS-CoV-2 in clinical blood samples

A total of 40 samples from patients with COVID-19 were analyzed using combined IgG–IgM immunochromatographic strip for the detection of SARS-CoV-2 in serum samples. As shown in Fig. 4, the M line or G line, or both lines could be observed. Viral proteins stimulate the immune system to elicit an antibody response, first producing IgM antibody. A red M line indicates that IgM antibody against SARS-CoV-2 was produced. As the disease progresses, IgG antibody gradually produce and is detectable. Although the disease course was not available, IgM antibody and IgG antibody against SARS-CoV-2 infection could be detected using the combined IgM–IgG immunochromatographic assay.
image file: d0qm00294a-f4.tif
Fig. 4 The strip images of COVID-19 infection tested by the IgM–IgG combined immunochromatographic strip.

2.4. The sensitivity and specificity of the combined IgG–IgM immunochromatographic strip

A total of 80 samples from 34 SARS-CoV-2 positive patients and 6 suspected cases confirmed by real-time RT-PCR and 40 non-SARS-CoV-2 clinical patients were subjected to the analysis. Single IgG, single IgM and combined IgM–IgG immunochromatographic strips were used to test IgG and IgM antibodies. As shown in Fig. 5, the detection rate of the IgM–IgG immunochromatographic strip was significantly higher than when single IgG or IgM immunochromatographic strips used. Additionally, the IgG positive rate in the confirmed cases was 61.76%, the IgM positive rate in the confirmed cases was 82.35%, and the positive rate was increased to 85.29% combining IgG–IgM (Table 2). Obviously, combining IgG–IgM would increase the sensitivity of the immunochromatographic strip. Furthermore, 40 blood samples from the non-SARS-CoV-2 infected subjects, all tested negative, reaching 100.00% specificity.
image file: d0qm00294a-f5.tif
Fig. 5 The detection rate of single IgG, single IgM and combined IgM–IgG immunochromatographic strip to test non-COVID-19 patients, COVID-19 confirmed patients and suspicious patients.
Table 1 Information of SARS-CoV-2 infection confirmed and suspected patients
Total
Number 71
Gender Male 42 (71)
Female 29 (71)
Age, mean 41 (16–75) 71
Presenting symptoms Dry cough, fever, and fatigue 71
SARS-CoV-2 infection confirmed by PCR 44
Suspected SARS-CoV-2 infection 27


Table 2 Detection results of COVID-19 confirmed cases by the single IgG, single IgM and IgM–IgG combined immunochromatographic strip
IgM IgG Combined IgM–IgG
Confirmed, no. 34 34 34
Positive results, no. 28 21 29
Sensitivity (%) 82.35 61.76 85.29
Specificity (%) 100


2.5. The combined IgG–IgM immunochromatographic strip in nucleic-negative patients

Although the nucleic acid testing is regarded as the “gold standard”, the false-negative cannot be ignored due to certain limitations. 6 suspected cases with the real-time RT-PCR negative were tested by the combined IgG–IgM immunochromatographic strip. The results are shown in Table 3. Of the 6 blood samples from the real-time RT-PCR negative blood samples, 4 (66.67%) tested IgM positive, 3 (50.00%) tested IgM positive, and 4 (66.67%) tested IgM–IgG positive. The combined IgG–IgM immunochromatographic strip showed good detectability in nucleic-negative patients, and could be used as a supplementary approach when the nucleic acid test is negative.
Table 3 Detectability in real-time RT-PCR negative patients using combined IgM–IgG immunochromatographic strip
Real-time RT-PCR IgM IgG Combined IgM–IgG
Patients, no. 6 6 6 6
Negative results, no. 6 2 3 2
Positive results, no. 0 4 3 4
Detection rate (%) 66.67 50.00 66.67


In this study, SARS-CoV-2 was detected in blood samples from patients with confirmed COVID-19 as well as those suspected of having COVID-19. The assays targeted the SARS-CoV-2 induction of IgG and IgM in patient blood. Furthermore, all the blood samples from controls tested negative, indicating the highly specific nature of this assay. The detection rate in confirmed patient was generally higher than seen in suspected patients, demonstrating that IgG and/or IgM detection in patient blood is reliable indicator of COVID-19 infection. It is not surprising that IgM detection is more sensitive that IgG detection in COVID-19 patients, because IgM is generated in the early stage of this disease.

Although this study was conducted in one hospital with a limited number of patients, the data demonstrated the reliability of the GNPs based immunochromatographic assay for SARS-CoV-2 diagnosis. Because more patient clinical information was not available, the data needs further statistical analysis such as correlation with the disease course and the other diagnostic criteria.

3. Conclusion

In this study, we developed a lateral flow combined IgM–IgG immunochromatographic assay for the rapid detection (within 15 min) of IgM and/or IgG antibodies against SARS-CoV-2. No specialized equipment or certified molecular testing laboratories are required. Therefore, this combined IgM–IgG immunochromatographic strip test was a highly sensitive and specific assay for rapid screening of symptomatic and asymptomatic SARS-CoV-2-infected patients.

4. Experimental section

4.1. Materials

Chloroauric acid, trisodium citrate, bovine serum albumin (BSA), anti-human IgM and IgG, goat anti-rabbit IgG antibody, and other antibodies were purchased from Sigma-Aldrich (Shanghai, China). Polyvinylchloride (PVC) backing card, conjugate pad, absorption pad and sample pad were purchased from Gold bio Tech Co., Ltd (Shanghai, China). Nitrocellulose (NC) high-flow-plus membranes (Pura-bind RP) were supplied by Whatman-Xinhua Filter Paper Co. (Hangzhou, China).

SARS-CoV-2 specific antigen preparation: subunit S1 protein of coronavirus spike glycoprotein harbors a receptor binding domain (RBD), which is responsible for identifying and promoting enter into cells and is the main target of antibodies. Besides, nucleocapsid protein (NP) is the most abundant protein in SARS-CoV-2 and has strong immunogenicity. Therefore, one week after SARS infection, anti-NP and anti-RBD antibody (IgM and IgG) could be detected in serum.22 An increase was discovered in IgG or IgM antibody levels against NP or RBD at 10 days or later after symptoms onset.23 However, earlier seropositivity was appeared for anti-RBD than anti-NP for both IgG and IgM. Furthermore, rates of seropositivity for anti-RBD IgM (94%) and IgG (100%) were higher than anti-NP IgM (88%) and IgG (94%).23 Thus, RBD was used as SARS-CoV-2 specific antigen in this study. The recombinant RBD (YP_009724390.1, Arg319-Phe541, Fig. 1) was transiently expressed in HEK293F cells and purified by nickel affinity column in manual. Detailed expression and purification experiments were assisted by Wuxi Determine Bio-Tech Co., Ltd (Wuxi, China).

4.2. GNPs synthesis

Chloroauric acid was reduced by the trisodium citrate method to prepare GNPs with an average diameter of 35 nm.24 In brief, 100 mL of 0.01% (w/v) chloroauric acid solution was heated and after boiling, 1 mL of 1% trisodium citrate solution was quickly added into chloroauric acid solution, with continuous stirring. Once the color of the reaction turned wine red, the solution was boiled for 15 min. Then, the c solution was cooled to room temperature and stored at 4 °C for further use. The fabricated GNPs were characterized by transmission electron microscopy (TEM).

4.3. GNPs conjugate preparation

The GNPs–SARS-CoV-2 antigen conjugation and the GNPs–rabbit IgG antibody conjugation were prepared as described previously, with minor modifications.25–28 Briefly, the pH value of the GNPs solution (10 mL) was adjusted to 8.2 by the addition of 0.1 M K2CO3. After this, the RBD recombinant protein was added to the solution. After incubation for 1 h at room temperature, BSA (0.5% w/v, 1 mL) was added to the mixture to block the surface of the GNPs, to avoid nonspecific adsorption. After incubation for 2 h at room temperature, the solution was then centrifuged for 30 min at 7000 × g, the supernatant was discarded, and 5 mL of 0.02 M PBS (pH 7.4) containing 5% sucrose, 1% BSA, 0.5% polyethylene glycol 6000 was added and the precipitate was washed three times. Finally, the precipitate was resuspended in 1 mL of 0.02 M PBS containing 0.02% NaN3. The GNPs-labeled rabbit IgG antibody was prepared as described above and the conjugations were stored at 4 °C until further use.

4.4. GNPs-based immunochromatographic strip preparation

The assembly of the immunochromatographic strip is illustrated in Fig. 2A. The prepared immunochromatographic strip contained five components: the PVC backing card, NC membrane, sample pad, conjugate pad and absorption pad. Sample pad was treated with 0.01 M PBS containing 0.5% BSA and dried overnight at 37 °C. The GNPs-labeled rabbit IgG antibody and GNPs-labeled RBD were diluted with the suspension buffer (0.02 M Tris–HCl containing 5% sucrose, 0.1% PEG, 0.1% Tween-20, 5% trehalose, 0.2% BSA, and 5% Brij-30), and sprayed onto the conjugate pad followed by drying at 37 °C for 1 h. In this study, the GNPs-labeled rabbit IgG antibody (8, 4, 2 and 1 μg mL−1) and GNPs-labeled RBD (8, 4, 2 and 1 μg mL−1) were optimized. Finally, the GNPs-labeled rabbit IgG antibody and GNPs-labeled RBD were sprayed on the conjugate pad at concentrations of 2 μg mL−1. One quality control line (C line) and two detection lines (G and M lines) were fixed onto the NC membrane with pore size of 140 micrometer. Anti-rabbit IgG antibody was immobilized onto the C line. The M and G lines were separately fixed with anti-human IgM antibody and anti-human IgG antibody for the detection of the IgM and IgG antibodies against SARS-CoV-2. Then, the sample pad, conjugate pad, NC membrane and absorption pad were laminated and pasted onto the PVC backing card.

4.5. Immunochromatographic strip for the detection of SARS-CoV-2 in clinical blood samples

As shown in Table 1, we collected 80 blood samples from 71 patients (42 males vs. 29 females), with a mean age of 41 years (16–75 years) at the Fourth Hospital of Foshan City, China, from January 24, 2020 to March 10, 2020. Nine of patients were collected twice. Most of the patients showed apparent symptoms of COVID-19, such as dry cough, fever, and fatigue. Among the blood samples, 44 of samples was confirmed as SARS-CoV-2 infection by real-time RT-PCR, 27 of samples was suspected cases. In addition, 40 blood samples from non-COVID-19 subjects collected from hospital volunteers from Wuhan city and treated as the control group.

Considering that the non-specific IgM or IgG antibodies in serum can bind with fixed anti-human IgM or IgG, we have taken some measures to avoid non-specific adsorption. First, SARS-CoV-2 antigen RBD was fused with a His-tag, that can avoid the binding of non-specific antibodies to GNPs-labeled RBD. Second, the amounts of RBD labeling as well as anti-human IgM and anti-human IgG spraying were increased to ensure that specific antibodies can richly bind to GNPs-labeled RBD and then enough immune-complex GNPs–RBD–IgM or GNPs–RBD–IgG can be captured by M line or G line. In addition, endogenous substances may interfere with the test results. The clinical serum sample was diluted before testing, resulting in a content reduction of non-specific endogenous substances. Specific IgM or IgG antibody could be detected due to its high affinity.

Serum samples (30 μL) were pretreated with 5-fold dilution with 0.15 mM NaCl containing 0.1% Tween-20, 100 μL of serum samples were vertically loaded into the sample well. The tests were conducted by trained personnel after hospital admission. Each sample was repeated six times. And result was recorded after 15 min. The sensitivity and specificity of the assay strips were investigated using blood samples from inpatients diagnosed with COVID-19, based on symptoms and radiology, and confirmed by PCR. This study was approved by the ethics commission of the Fourth Hospital of Foshan City, with a permission of the testing results.

Authors contribution

Chuanlai Xu participated in the design of experiments. Lu Zeng, Yue Li, Jie Liu, Shanshan Song, Changlong Hao performed the experiments. Lingling Guo, Zhongxing Wang, Xinxin Xu ran the data analysis. Shanshan Song contributed analysis tools. Liqiang Liu, Meiguo Xin, Chuanlai Xu drafted the manuscript, and all authors read and approved the manuscript prior to submission.

Conflicts of interest

The authors declare no competing financial interest.

Acknowledgements

This work is financially supported by National Key R&D Program (2018YFC1602604).

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