Preparation of highly specific monoclonal antibodies against SARS‐CoV‐2 nucleocapsid protein and the preliminary development of antigen detection test strips

Abstract The coronavirus disease 2019 (COVID‐19) is outbreaking all over the world. To help fight this disease, it is necessary to establish an effective and rapid detection method. The nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is involved in viral replication, assembly, and immune regulation and plays an important role in the viral life cycle. Moreover, the N protein also could be a diagnostic factor and potential drug target. Therefore, by synthesizing the N gene sequence of SARS‐CoV‐2, constructing the pET‐28a (+)‐N recombinant plasmid, we expressed the N protein in Escherichia coli and obtained 15 monoclonal antibody (mAbs) against SARS‐CoV‐2‐N protein by the hybridomas and ascites, then an immunochromatographic test strip method detecting N antigen was established. In this study, we obtained 14 high‐titer and high‐specificity monoclonal antibodies, and the test strips exclusively react with the SARS‐CoV‐2‐N protein and no cross‐reactivity with other coronavirus and also recognize the recombinant N protein of Delta (B.1.617.2) variant. These mAbs can be used for the early and rapid diagnosis of SARS‐CoV‐2 infection through serological antigen.

steps in the replication cycle and is used as a serological marker of infection. 5 Liu 6 et al. used N protein as an antigen to detect SARS-CoV-2 immunoglobulin M (IgM) and IgG antibodies by enzyme-linked immunosorbent assay (ELISA), and the results were 68.2% and 70.1% positive, respectively, demonstrating that N protein detection of SARS-CoV-2 antibodies is a highly sensitive screening method for the diagnosis of COVID-19. Serum samples are also generally more stable than viral RNA and less variable than nasopharyngeal or oropharyngeal viral RNA samples due to the uniform distribution of proteins in the blood, minimizing false negatives in test results. 7 These research studies suggested that the data about the research of nucleocapsid protein may provide a theoretical basis for the diagnosis and treatment of SARS-CoV-2 infection.
In this study, the BALB/c mice were immunized with recombinant SARS-CoV-2 N protein, anti-SARS-CoV-2 N monoclonal antibodies with high titer and specificity were screened by cell fusion technique. Then, through paired screening of these monoclonal antibodies, a rapid and convenient colloidal gold immunochromatographic strip detection method was prepared, hoping to provide a basis for early diagnosis of clinical novel coronavirus infection.

| The paired screening of monoclonal antibodies
In the preparation of antigen detection immunochromatography test strip by double antibody sandwich method, it was necessary to screen a pair of monoclonal antibodies which could specifically bind to SARS-CoV-2 N antigen and were not affected by the spatial structure. 8

| Determination of mAbs subtypes
According to the titer of monoclonal antibody and the results of paired screening, the mAbs with high titer and pairing effect were selected to determine the subtypes. And the subtype of the mAbs against SARS-CoV-2 N were tested with a Monoclonal Antibody Subtyping Kit (IgG1, IgG2a, IgG2b, and IgG3).

| The preparation of colloidal gold solution
To ensure cleanliness, the sterilized 1 L glass flask was rinsed multiple times with deionized water, then added 500 ml deionized water and heated to boil in a magnetic agitator. Dissolved 0.1125 g sodium citrate in 3 ml boiled water in a 15 ml centrifuge tube, subsequently, 5 ml HAuCl 4 solution (1%) was added with heating and vigorous stirring until the mixture reached a wine red. Then the mixture solution was kept to state for 5 min and stopped, eventually, the solution was naturally cooled to room temperature and store at 4°C.

| The preparation of immunogold labeling
According to the results of paired screening of monoclonal antibodies, the antibodies with better results were labeled with a colloidal gold solution.

| Labeling colloidal gold solution
The colloidal gold-mAb conjugation was then performed using optimum pH and antibody quantity. Briefly, mAb with the best labeling quantity was added into 10 ml of colloidal gold solution (adjusted to optimum pH value), and the mixture was stirred vigorously for 30 min in a magnetic agitator. Then, 1 ml Bovine albumin (BSA) (10%) was added to the solution with vigorously stirring for 30 min, and the solution was centrifuged at 12,000 rpm for 30 min, and the colloidal gold solution was resuspended with 500 μl of resuspended solution (containing 20% sucrose, 5% trehalose, 10% BSA). Finally, the colloidal gold-mAb (SARS-CoV-2 N protein) was obtained.

Preparation of conjugate pad
The labeled colloidal gold-mAb was sprayed on the fiber membrane by a film-gold spraying instrument at the amount of 4 μl/cm, which was used as a conjugate pad. Then we placed them into a 37°C oven and were allowed to dry overnight.

Labeling the NC membrane
The diluted goat anti-mouse IgG (1 mg/ml) and the monoclonal antibody (1 mg/ml) were sprayed in the control (C) line and testing (T) line located in nitrocellulose membranes, respectively.
Then we also placed them into a 37°C oven and were allowed to dry overnight.
The assembly of the immunochromatography test (ICT) strip According to the structure of the colloidal gold immunochromatography test strip, 9 the trips were assembled using the labeled colloidal gold-mAb conjugate pad, NC membrane with test line and control line, PVC base plate, and absorbent pad.

| The evaluation of the test trips
To detect the sensitivity of the immunochromatographic strips, the recombinant pET-28a ((+)-N protein was diluted to 1:2, 1:4,

| Preparation of SARS-CoV-2-N recombinant protein
The designed primers for the SARS-CoV-2 N gene amplified an amost1200 bp band by polymerase chain reaction (PCR), which was observed in agarose gel electrophoresis (1%). ( Figure 1A). The recombinant pET-28a (+)-N plasmid was transformed into E. coli BL21 and the protein expression was induced by IPTG, then the supernatant was purified using Ni-NTA affinity chromatography after centrifugation. After elution with different concentrations of imidazole, the protein was identified by SDS-PAGE electrophoresis. As shown in Figure 1B, the band of pET-28a (+)-N protein was about 50 kDa.

| Preparation and purification of monoclonal antibodies against SARS-CoV-2 N protein
There were 15 monoclonal antibodies against SARS-CoV-2 N protein obtained after immunization, cell fusion, and screening, and they were

| Determination the titer of monoclonal antibodies
The recombinant SARS-CoV-2 N protein was coated for a 96-well plate at 4°C (5 μg/well) overnight, and 14 diluted mAbs were added successively to detect the antibody titer by indirect ELISA method. As shown in Figure 3

| DISCUSSION
The SARS-CoV-2 can cause acute respiratory disease, and the common signs of infection include respiratory symptoms, fever, cough, shortness of breath, and dyspnea. In more severe cases, the infection can lead to pneumonia, severe acute respiratory syndrome, kidney failure, and death. 10 The SARS-CoV-2 mainly spread through the respiratory, so the global epidemic is still spreading and potentially long-lasting. Early diagnosis and prevention will be a crucial step without specific treatment for novel coronavirus infections.
F I G U R E 6 Sensitivity detection of SARS-CoV-2-N antigen dipstick. (A) Schematic representation of the dipstick; (B) N10 gold conjugated, N3 test line, the detection sensitivity is 960 ng/ml; (C) N15 gold conjugated, N3 test line, the detection sensitivity is 960 ng/ml; (D) N3 gold conjugated, N10 test line, the detection sensitivity is 960 ng/ml; (E) N15 gold conjugated, N10 test line, the detection sensitivity is 480 ng/ml; (F) N3 gold conjugated, N15 test line, the detection sensitivity is 960 ng/ml; (G) N10 gold conjugated, N15 test line, the detection sensitivity is 240 ng/ml. SARS-CoV-2, severe acute respiratory syndrome coronavirus 2 The current common method diagnostic of SARS-CoV-2 is based on the detection of genomic RNA by molecular assays, such as realtime fluorescent reverse-transcription polymerase chain reaction (RT-PCR), which possesses high sensitivity and specificity. However, the sample pretreatments before the experiment were extensive, such as total RNA extraction and high-cost RT-PCR reagents, as well as the need for PCR instruments. 11 These challenges add to some extent to the workload and the risk of infection for healthcare workers. Furthermore, among pathogenic specimens, blood specimens are easier to obtain and more stable than nasal swabs, stool samples, and so on. 12 According to the recent findings, the SARS-CoV-2 N protein is a critical component of the virus replication, involved in the viral particle assembly and the release of viral particles, and a major diagnostic marker of infection and immune protection. 13 And the antibodies to the N protein are the most sensitive diagnostic marker in the serological diagnosis of SARS infection. 14 These results suggest that the protein in the serum of SARS-CoV-2-infected patients persistently stimulates the organism for a longer period of time compared with other structures of the virus. Moreover, in the diagnosis of SARS-COV-2, rapid antigen testing may present a potential diagnostic advantage because RNA and virion test results maybe not necessarily consistent in the detection. 15 Therefore, in this study, we expressed and purified SARS-CoV-2 N protein, then 15 stable strains of hybridoma cells anti-N-protein monoclonal antibodies were obtained, and one mAb with lower concentration was removed in the subsequent experiments, and we detected the titer, paired screening of the remaining 14 mAbs, and developed a SARS-CoV-2 antigen test trip based on colloidal gold immunochromatographic strip technique, as shown in Figure 6, the sensitivity could reach 200 ng/ml when detecting recombinant SARS-CoV-2 N antigen. Thus, this study can theoretically be used to detect SARS-CoV-2 N antigen and to make an early diagnosis of SARS-CoV-2 infection.
In this study, several immunological methods such as ELISA based on antibodies to SARS-CoV-2 N protein, 16 immunofluorescence assays, 17 and test strip methods 18 have been established and used for serological testing. However, these antibody-based assays just reflect SARS-CoV-2 infection to some extent, serological antibody diagnosis is mainly based on the detection of IgM or IgG antibodies in the serum.
Unfortunately, there is a "window period" for antibody diagnosis and it limits the use of antibodies in the early detection of disease because the period that antibodies emerge after the release of antigen from the virus into the host blood system may take at least 10-28 days. 19  Recently, a novel variant B.1.617.2 has firstly been found in India which increased rapidly in other countries. 22 And according to a recent study, the Delta variant has an enhanced transmission capacity and increased virulence, in addition, this variant may promote the fusion of the spike protein with cells or inhibit antibodies from binding to it. 23