Development and clinical application of a rapid IgM‐IgG combined antibody test for SARS‐CoV‐2 infection diagnosis

Abstract The outbreak of the novel coronavirus disease (COVID‐19) quickly spread all over China and to more than 20 other countries. Although the virus (severe acute respiratory syndrome coronavirus [SARS‐Cov‐2]) nucleic acid real‐time polymerase chain reaction (PCR) test has become the standard method for diagnosis of SARS‐CoV‐2 infection, these real‐time PCR test kits have many limitations. In addition, high false‐negative rates were reported. There is an urgent need for an accurate and rapid test method to quickly identify a large number of infected patients and asymptomatic carriers to prevent virus transmission and assure timely treatment of patients. We have developed a rapid and simple point‐of‐care lateral flow immunoassay that can detect immunoglobulin M (IgM) and IgG antibodies simultaneously against SARS‐CoV‐2 virus in human blood within 15 minutes which can detect patients at different infection stages. With this test kit, we carried out clinical studies to validate its clinical efficacy uses. The clinical detection sensitivity and specificity of this test were measured using blood samples collected from 397 PCR confirmed COVID‐19 patients and 128 negative patients at eight different clinical sites. The overall testing sensitivity was 88.66% and specificity was 90.63%. In addition, we evaluated clinical diagnosis results obtained from different types of venous and fingerstick blood samples. The results indicated great detection consistency among samples from fingerstick blood, serum and plasma of venous blood. The IgM‐IgG combined assay has better utility and sensitivity compared with a single IgM or IgG test. It can be used for the rapid screening of SARS‐CoV‐2 carriers, symptomatic or asymptomatic, in hospitals, clinics, and test laboratories.

into an epidemic that quickly spread all over China and to more than 20 other countries. 6 It has been listed as a public health emergency of international concern. 7 The outbreak of this disease has caused the Chinese government to take drastic measures to contain the outbreak, including the quarantine of millions of residents in Wuhan and other affected cities. Countrywide interventions include delaying the resumption of workplaces, and encouraging citizens to stay and work from home, and so on.
However, these efforts are limited by one hard problem: how to differentiate the COVID-19 cases from the healthy. For confirmed COVID-19 cases, reported common clinical symptoms include fever, cough, myalgia, or fatigue. 8 Yet these symptoms are not unique features of COVID-19 because these symptoms are similar to that of other virus-infected diseases such as influenza. 9 Currently, virus nucleic acid real-time polymerase chain reaction (RT-PCR), CT imaging, and some hematology parameters are the primary tools for clinical diagnosis of the infection. 10  Therefore, there is an urgent need for a rapid, simple to use, sensitive, and accurate test to quickly identify infected patients of SARS-CoV-2 to prevent virus transmission and to assure timely treatment of patients.
Testing of specific antibodies of SARS-CoV-2 in patient blood is a good choice for rapid, simple, highly sensitive diagnosis of COVID-19.
It is widely accepted that immunoglobulin M (IgM) provides the first line of defense during viral infections, Before the generation of adaptive, high-affinity IgG responses that are important for long term immunity and immunological memory. 12 It was reported that after SARS infection, IgM antibody could be detected in patient blood after 3 to 6 days and IgG could be detected after 8 days. 13,14 Since COVID-19 belongs to the same large family of viruses as those that cause the MERS and SARS outbreak, we assume its antibody generation process is similar, and detection of the IgG and IgM antibody against SARS-CoV-2 will be an indication of infection.  Several different designs of antigen were tested and optimized.
Eventually, MK201027 was picked into the testing product. Bovine serum albumin (BSA), and goat anti-human IgG and IgM antibodies, rabbit IgG, and goat anti-rabbit IgG antibodies were obtained from Sigma-Aldrich. Forty-nanometer gold nanoparticle (AuNP) colloids, NC membrane, and plastic pad were obtained from Shanghai KinBio Inc, the glass fiber conjugate pad was obtained from Whatman. The phosphate-buffered saline (PBS) was purchased from Sigma-Aldrich.  | 1519 20 minutes. The supernatant was discarded, and 1 mL of 1 mg/mL BSA in PBS was added to the AuNP conjugate to be resuspended.

| Preparation of AuNP conjugates
The centrifugation and suspension processes were repeated twice, and the final suspension solution was PBS. The AuNP-rabbit IgG conjugates were prepared and purified by the same procedure.

| Preparation of COVID-19 rapid test of IgG-IgM
The main body of the test strip consists of five parts, including plastic backing, sample pad, conjugate pad, absorbent pad, and NC membrane. Every component of the strip should be given a pretreatment described as follows: the NC membrane was attached to a plastic backing layer for cutting and handling. The anti-human-IgM, antihuman-IgG and anti-rabbit-IgG were immobilized at test M, G, and control line (C line),respectively. Conjugate pad was sprayed with mixture of AuNP-COVID-19 recombinant antigen conjugate and AuNPrabbit-IgG. Sample pad was pretreated with BSA (3%, w/v) and Tween-20 (0.5%, w/v) before use.

| Testing of COVID-19 samples using the LFIA system 2.4.1 | Patient and sample collection
The patients were recruited who conform to the diagnostic criteria of a suspected case of COVID-19 according to the guidelines of diagnosis and treatment of COVID-19 15 including typical epidemiological history and clinical characteristics. These samples were collected from various hospitals and CDC testing laboratories (total eight) at six different provinces of China. The tests were conducted at the sites by clinical staff who followed the test procedure described in the product inserts. The respiratory tract specimen, including pharyngeal swab and sputum, was used to confirm COVID-19 cases, and the blood, including serum and plasma, was used to test the IgM and IgG antibody.

| Sample testing
Before testing, the pouched device was opened immediately before use. When refrigerated blood samples were used for the test, they were warmed to room temperature (15°C-30°C). During testing, 20 μL whole blood sample (or 10 μL of serum/plasma samples) was pipetted into the sample port followed by adding two to three drops (70-100 μL) of dilution buffer (10 mM PBS buffer) to drive capillary action along the strip. The entire test took about 15 minutes to finish.

| Data analysis
The rapid SARS-CoV-2 IgG-IgM combined antibody test kits were tested at eight hospitals and Chinese CDC laboratories in different provinces, with a total of 397 clinical positive and 128 clinical negative patient blood samples. The test data was collected and analyzed. The specificity and sensitivity of the rapid test kits were calculated according to the following formulas: Specificity % 100 true negative true negative false positive , Sensitivity % 100 true positive true positive false negative . is conjugated to colloidal gold nanoparticles and sprayed on conjugation pads. The AuNP-rabbit IgG conjugates were also sprayed on conjugation pads for binding to anti-rabbit IgG antibody which is immobilized on the control line ( Figure 1A).
When testing, 10 to 15 μL specimen is added into the sample port followed by the addition of sample dilution buffer. The mechanism of the assay is based on the hydration and transport of reagents as they interact with the specimen across the strip via chromatographic lateral flow ( Figure 1A).  3.3 | SARS-CoV-2 IgG-IgM combined antibody test in different types of blood samples The above results have verified the sensitivity and specificity of kit detection in un-inactivated vein blood. However, it is more convenient to collect fingerstick blood outside hospitals and clinics.
To achieve a simpler operating process, we tested the performance of SARS-CoV-2 IgG-IgM combined antibody kit with peripheral blood.
Patient fingerstick blood and vein blood and plasma from the same patient were tested. As shown in Table 3 To make the kit suitable for different stages of the disease, we developed an IgG-IgM combined antibody test for COVID-19 infection ( Figure 1). It was also been confirmed that the detection sensibility was higher in IgG-IgM combined antibody test than in individual IgG or IgM antibody test (Table 1). Therefore, we more recommend the no publication on them. We believe the good test products will be used in clinical sites and the information will emerge. We will carry out comparison studies later.
Certainly, this test cannot confirm virus presence, only provide evidence of recent infection, but it provides important immunological evidence for physicians to make the correct diagnosis along with other tests and to start treatment of patients.
In addition, possible cross-reactivity with other coronaviruses and flu viruses were not studied, and the change level of antibody was not compared in the different stages of SARS-CoV-2 infection. We believe the combination of nucleic acid RT-PCR and the IgM-IgG antibody test can provide more accurate SARS-CoV-2 infection diagnosis.

| CONCLUSION
We developed a rapid SARS-CoV-2 IgG-IgM combined antibody test using lateral flow immune assay techniques. It takes less than 15 minutes to generate results and determine whether there is a recent SARS-CoV-2 infection. It is easy to use, and no additional equipment is required. Results from this study demonstrated that this test is sensitive and specific. This rapid test has great potential benefits for the fast screening of SARS-CoV-2 infections, and it has already generated tremendous interest and increased clinical uses after a short time testing in Chinese hospitals.