Double-antibody sandwich ELISA (enzyme-linked immuno sorbent assay) detection kit for detecting new coronavirus

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a detection kit for detecting new coronavirus. According to the invention, a new coronavirus NP protein is taken as an immunogen, monoclonal antibodies aiming at specific epitopes of the new coronavirus NP protein are screened, a specific strain is taken as a capture antibody, the other strain is labeled by horse radish peroxidase and taken as a detection antibody, and a double-antibody sandwich ELISA method for detecting the SARS-CoV-2 NP protein is established. Compared with the prior art, the method has the advantages that the detection time is greatly shortened, the detection can be completed in only 6 hours, and a foundation is laid for rapid detection of an SARS-CoV-2 clinical sample.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a detection kit for detecting a novel coronavirus. BACKGROUND

[0002] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), also known as the novel coronavirus, is an enveloped positive single-stranded RNA virus, and its genome contains nearly 30,000 bases, with four structural proteins, namely spike (S) protein, envelope (E) protein, membrane (M) protein and nucleocapsid (N) protein. Similar to other RNA viruses, SARS-CoV-2 shows the characteristics of rapid mutation, thereby forming different viral lineages, showing higher transmissibility, enhanced virulence and / or the ability to evade immunity (Yang Xiaoyu, Niu Peihua, Tian Xiaoling, et al. Feasibility study on identification of four genotypes of novel coronavirus based on RT-qPCR method [J]. Virus Journal, 2024, 40(02): 266-273.). The 2019 coronavirus disease (COVID-19) caused by SARS-CoV-2 has caused a raging wave in the global range, and with its high infectivity and mortality, it has brought severe challenges to global health care. Patients infected with SARS-CoV-2 may have symptoms such as asymptomatic or fever, cough, dyspnea, and may progress to multiple organ failure. All populations are susceptible to the novel coronavirus, and the mortality rate is higher in the elderly and groups with chronic underlying diseases.

[0003] SARS-CoV-2 is a single-stranded RNA virus with a moderate mutation rate and recombination frequency. The accumulation of mutations between different variants of SARS-CoV-2 circulating in the human population is mainly concentrated in the RBD region of the S protein, which is located in the 330-530 amino acid residues of the S protein and is responsible for the high-affinity binding of Spike to angiotensin converting enzyme 2 (ACE2) (Jackson CB, Farzan M, Chen B, et al. Mechanisms of SARS-CoV-2 entry into cells[J]. Nat Rev Mol Cell Biol, 2022, 23:3-20.). According to the mutation sites of the spike protein, the epidemic variants are successively divided into Alpha, Beta, Gamma, Delta, and Omicron, etc. Omicron has rapidly replaced Delta and become the main strain globally since its emergence, and is the most variable variant of SARS-CoV-2. Omicron contains more than 50 mutations, among which 32 mutations in the S protein, 15 of which are located in the RBD region of the S protein (Liu L, Iketani S, Guo Y, et al. Striking antibody evasion manifested by the Omicron variant of SARS-CoV-2[J]. Nature, 2021, 602(7898):676-681.). This region is the main target of monoclonal therapy and most SARS-CoV-2 vaccines. According to the surveillance data of domestic cases of SARS-CoV-2 variants in China from 2023 to 2025, the dominant circulating strain of SARS-CoV-2 in China has undergone multiple replacements from the initial Omicron sublineage BA.5.2, BF.7, to the current Omicron sublineage XDV, JD.1, etc. Therefore, it is very important to establish a detection method that can quickly detect new variants.

[0004] Currently, there are three main methods for early diagnosis of SARS-CoV-2 infection, including molecular detection (detecting viral RNA), antibody detection (detecting IgG and IgM antibodies), and antigen detection (detecting viral proteins). Nucleic acid detection is currently the 'gold standard' for detecting the novel coronavirus, with high early diagnostic value, high sensitivity and specificity, but it requires supporting equipment, and different variants of SARS-CoV-2 are constantly emerging, with different mutations in the S protein, which may result in detection off-target. Antibody detection is convenient and rapid, but there is a possibility of residual antibody interference. Compared with molecular detection and antibody detection, antigen detection is convenient and rapid, suitable for early rapid diagnosis, and has been widely used. Monoclonal antibodies are specific to a single antigen epitope, with high specificity and sensitivity, and have been widely used in the diagnosis and treatment of diseases. Nucleocapsid protein (NP) is highly expressed during viral infection and has a high content in mature virions, and has good immunogenicity. During viral infection, NP protein enters the host cell with viral RNA, promoting its replication, and participating in virion assembly and release. Serological diagnosis found that the specific antibodies against NP protein in the serum of SARS patients had higher sensitivity and longer duration than other structural proteins of SARS-CoV. The N gene is more conservative and stable, with an amino acid homology of 90% between SARS-CoV-2 and SARS-CoV, and fewer mutations during long-term epidemic (ZHENG M, GAO Y, WANG G, et al. Functional exhaustion of antivirallymphocytes in COVID-19 patients [J]. Cellular&molecular immunology, 2020, 17(5): 533-535.). Therefore, NP protein is often used for serological testing and is used as a diagnostic marker.

[0005] Patent CN 113493508 A provides a pair of monoclonal antibodies suitable for detecting the N protein of the new coronavirus, which are prepared by a double antibody sandwich ELISA kit. Rabbit anti-RVFV NP PcAb polyclonal antibody is used as a capture antibody, N protein of MN908947.3 is used as an immunogen, and the screened monoclonal antibody is labeled with HRP as a detection antibody, with a dilution ratio of 1:10,000. However, the overall detection time of the double antibody sandwich ELISA method established by this method is as long as 20 h, which seriously restricts the detection efficiency of the N protein of the new coronavirus and cannot meet the demand for immediate or rapid detection. Therefore, there is an urgent need for a method that can significantly shorten the detection time and achieve 'completion on the same day' or even within a few hours to meet the urgent needs of clinical rapid diagnosis and emergency response. SUMMARY

[0006] The present application takes the novel coronavirus NP protein as an immunogen, screens monoclonal antibodies against specific epitopes of the novel coronavirus NP protein, takes a specific strain as a capture antibody, and takes another strain labeled with horseradish peroxidase as a detection antibody to establish a double antibody sandwich ELISA method for detecting SARS-CoV-2 NP protein. The method greatly shortens the detection time compared with the prior art, and the detection can be completed in only 6 hours, laying a foundation for rapid detection of SARS-CoV-2 clinical samples. Based on this, the present application is completed.

[0007] In a first aspect, the present application provides a monoclonal antibody combination for detecting novel coronavirus NP protein, the monoclonal antibody combination comprising monoclonal antibody 9E3 and monoclonal antibody 8B1, The heavy chain variable region of the monoclonal antibody 9E3 comprises three complementarity determining regions of CDR1, CDR2 and CDR3, the amino acid sequence of CDR1 is shown as SEQ ID NO. 1, the amino acid sequence of CDR2 is shown as SEQ ID NO. 2, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 3; The light chain variable region of the monoclonal antibody 9E3 comprises three complementarity determining regions of CDR1, CDR2 and CDR3, the amino acid sequence of CDR1 is shown as SEQ ID NO. 4, the amino acid sequence of CDR2 is shown as SEQ ID NO. 5, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 6; The heavy chain variable region of the monoclonal antibody 8B1 comprises three complementarity determining regions of CDR1, CDR2 and CDR3, the amino acid sequence of CDR1 is shown as SEQ ID NO. 7, the amino acid sequence of CDR2 is shown as SEQ ID NO. 8, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 9; The light chain variable region of the monoclonal antibody 8B1 comprises three complementarity determining regions of CDR1, CDR2 and CDR3, the amino acid sequence of CDR1 is shown as SEQ ID NO. 10, the amino acid sequence of CDR2 is shown as SEQ ID NO. 11, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 12.

[0008] In a second aspect, the present application provides the use of the monoclonal antibody combination of the first aspect in preparing a reagent for detecting novel coronavirus NP protein; the monoclonal antibody combination comprising monoclonal antibody 9E3 and monoclonal antibody 8B1, The heavy chain variable region of the monoclonal antibody 9E3 comprises three complementarity determining regions of CDR1, CDR2 and CDR3, the amino acid sequence of CDR1 is shown as SEQ ID NO. 1, the amino acid sequence of CDR2 is shown as SEQ ID NO. 2, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 3; The light chain variable region of the monoclonal antibody 9E3 comprises three complementarity determining regions of CDR1, CDR2 and CDR3, the amino acid sequence of CDR1 is shown as SEQ ID NO. 4, the amino acid sequence of CDR2 is shown as SEQ ID NO. 5, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 6; The heavy chain variable region of the monoclonal antibody 8B1 comprises three complementarity determining regions of CDR1, CDR2 and CDR3, the amino acid sequence of CDR1 is shown as SEQ ID NO. 7, the amino acid sequence of CDR2 is shown as SEQ ID NO. 8, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 9; The light chain variable region of the monoclonal antibody 8B1 comprises three complementarity determining regions of CDR1, CDR2 and CDR3, the amino acid sequence of CDR1 is shown as SEQ ID NO. 10, the amino acid sequence of CDR2 is shown as SEQ ID NO. 11, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 12.

[0009] In a third aspect, the present application provides a double antibody sandwich ELISA kit for detecting the NP protein of the new coronavirus, wherein the kit comprises the monoclonal antibody combination of the first aspect, wherein the monoclonal antibody 9E3 is used as a capture antibody, and the monoclonal antibody 8B1 is labeled with HRP and used as a detection antibody (8B1-HRP).

[0010] Further, the kit further comprises a blocking solution, a dilution solution, a washing solution, a color developing solution and a termination solution.

[0011] Further, the mass concentration of the capture antibody is 1-1.50 μg / mL, preferably 1.25 μg / mL.

[0012] Further, the concentration of the detection antibody is 0.01-0.10 μg / mL, preferably 0.0625 μg / mL.

[0013] Further, the coating condition is 4℃ for 2-4 h, preferably 4℃ for 3 h.

[0014] Further, the blocking solution is selected from skimmed milk powder, horse serum, rabbit serum or BSA.

[0015] Preferably, the blocking solution is BSA.

[0016] Further, the blocking condition is 5% BSA blocking for 0.5-2 h, preferably 5% BSA blocking for 2 h.

[0017] Further, the optimal incubation time of the antigen is 30-90 min, preferably 60 min.

[0018] Further, the optimal incubation time of the detection antibody 8B1-HRP is 0.5-2 h, preferably 0.5 h.

[0019] Further, the optimal color development time of the TMB single-component color developing solution is 5-15 min at room temperature, preferably 10 min.

[0020] Advantages The present application successfully prepared specific monoclonal antibodies against SARS-CoV-2 NP protein, and established a double antibody sandwich ELISA antigen detection method based on the paired antibodies obtained by screening. After optimization, the optimal coating antibody concentration of the method is 1.25 μg / mL, and the detection antibody concentration is as low as 0.0625 μg / mL; batch and intra-batch repeatability tests show that the coefficient of variation is less than 9%, with good repeatability; 66 clinical samples were detected, and the results of the method established in the present application were consistent with the results of the RT-qPCR detection method. The specificity test results show that the method has no cross-reactivity to the proteins of EBOV GP / NP, RVFV Gn / NP, LASV GP1 / GP2 and CCHFV NP and other viruses. The method established in the present application can be used for rapid detection, and the detection time is shortened from 20 h to 6 h compared with the prior art; the enzyme-labeled antibody usage is less, and the dilution ratio is increased from 1:10000 to 1:32000 compared with the prior art; the repeatability is high, and the batch and intra-batch variation coefficients are less than 9%; the specificity is strong, and the method has no reactivity to a variety of virus proteins; the standard curve is wide, and the range is expanded from 0.5-5 ng / mL to 7.8-1000 ng / mL compared with the prior art. The method established in the present application has the advantages of rapidity, stability, strong specificity, etc., and provides technical support for the rapid diagnosis and epidemiological monitoring of the novel coronavirus. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 SARS-CoV-2 NP monoclonal cell strain screening and protein purification. Note: A: Comparison of SARS-CoV-2 NP stable expression cell strain yield; B: IFA verification of 3-4D monoclonal cell strain; C: Western Blot (left) and SDS-PAGE (right) detection of SARS-CoV-2 NP purified protein.

[0022] Figure 2 For the preparation of monoclonal antibody and subtype identification. Note: A: reaction identification of monoclonal hybridoma cell supernatant and NP protein; B: antibody subtype identification; C: cross-reactivity identification of antibody.

[0023] Figure 3 For the identification of antibody binding titer and competitive relationship. Note: A: detection of antibody binding titer; B: identification of competitive relationship between 8B1 and 9E3 antibodies.

[0024] Figure 4 For the standard curve of double antibody sandwich ELISA detection method.

[0025] Figure 5 For the specific experimental results. DETAILED DESCRIPTION

[0026] The specific embodiments of the present application will be further described below. It should be noted that the description of these embodiments is used to help understand the present application, but does not constitute a limitation on the present application. In addition, the technical features involved in the following described embodiments can be combined with each other as long as they do not conflict with each other.

[0027] The experimental methods in the following examples are all routine methods unless otherwise specified. The test materials used in the following examples are all commercially available unless otherwise specified.

[0028] Experimental materials pcDNA3.1(+) vector, CHO-K1 cells, SP2 / 0 cells, SARS-CoV-2 inactivated virus, Ebola virus NP and GP proteins, Rift Valley fever virus Gn and NP proteins, Lassa fever virus GP1 and GP2 proteins, and Crimean-Congo hemorrhagic fever virus NP protein were preserved in the laboratory. SPF BALB / c mice were purchased from Beijing Vito Lee Company.

[0029] Mouse anti-His tag antibody and HRP labeled antibody / protein kit were purchased from Wuhan Synergy Biotech Co., Ltd.; DyLight 594 labeled goat anti-mouse IgG was purchased from Abbkine; HRP labeled goat anti-mouse IgG was purchased from Beijing Boaolong Company; High Affinity Ni-NTA Resin was purchased from Jinshirui Biotech Co., Ltd.; protein A / G Agarose was purchased from Shanghai Biyun Tian Biotechnology Co., Ltd.; LTX transfection reagent and skimmed milk powder were purchased from ThermmoFisher Scientific; F12K medium dry powder was purchased from Wuhan Punsai Life Science and Technology Co., Ltd.; CHO MaxH1 was purchased from Shanghai Maibang Biological Technology Co., Ltd.; RPMI1640 medium was purchased from American Gibco Company; viral RNA extraction kit was purchased from Tiangen Biochemical Technology Co., Ltd.; Probe qPCR Mix MultiPlus was purchased from Baori Biotechnology Co., Ltd.; PhantaMax Master Mix and fetal bovine serum were purchased from Nanjing Novozyme Biological Technology Co., Ltd.; 96-well enzyme-labeled plate was purchased from Costar Company; BSA bovine serum albumin was purchased from VWR Company; ELISA developing solution, ELISA termination solution and PBS powder were purchased from Beijing Solabio Technology Co., Ltd.; Tween-20, Freund's (in) complete adjuvant and 10 kDA ultrafiltration centrifuge tube were purchased from Merck Company.

[0030] The amino acid sequence of SARS-CoV2-NP is as shown in SEQ ID NO. 13: MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSRGTSPARMAGNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQKKQQTVTLLPAADLDDFSKQLQQSMSSADSTQA.

[0031] Example 1 Construction of SARS-CoV-2 NP eukaryotic plasmid and protein expression and purification The SARS-CoV-2 NP (Delta strain, 2022) gene was derived from laboratory self-test. The gene was cloned into the linearized pcDNA3.1-SP vector by seamless cloning, with a signal peptide (SEQ ID NO. 14: METDTLLLWVLLLWVPGSTGD) added to the N segment and a His-tag added to the C segment. The plasmid was named pcDNA3.1-SARS-CoV-2-NP-his; a flag-tag was added to the C segment, and the plasmid was named pcDNA3.1-SARS-CoV-2-NP-flag (the upstream primer for constructing the plasmid is shown in SEQ ID NO. 15, and the downstream primer is shown in SEQ ID NO. 17). See Table 1 for details of the cloning primers.

[0032] Table 1 Cloning primer sequences CHO-K1 cells were plated in a well plate, and when the cell density reached 80%, the plasmid was transfected into CHO-K1 cells using LTX transfection reagent, and drug pressure screening was continued in F12K medium containing 10% FBS and adding G418. After flow cytometry sorting, the surviving cell lines were incubated with mouse His-tag antibody, and indirect immunofluorescence (IFA) and Western Blot (WB) tests were performed to compare the expression of NP protein in each cell line. The cell line with the highest expression was selected for expansion culture, and finally the F12K medium was replaced with CHO MaxH1, and the supernatant was continuously collected.

[0033] The collected cell supernatant was purified using the nickel column standard operating procedure. After confirming the accuracy of the purified protein, desalting column desalting and concentration titration were used for storage.

[0034] After G418 drug screening, cells that did not integrate the resistance gene had died. The supernatant of the surviving monoclonal cell lines (4-5H, 1-11D, 2-7H, 3-4D, 3-4G, 3-7G, 4-3B, 4-11B, 6-5C) was subjected to WB test, and mouse His-tag antibody was incubated to compare the expression of SARS-CoV-2 NP protein. The results showed that the size of the SARS-CoV-2 NP protein secreted in the supernatant of the 3-4D monoclonal cell line was consistent with the theoretical value and the expression was the highest Figure 1 A). IFA test was performed on the cell line incubated with mouse His-tag antibody to detect the positive rate of the cells, and the positive cell ratio was > 90% Figure 1B), so this cell line was selected for expansion culture and the supernatant was collected for protein purification. The purified protein was subjected to SDS-PAGE. The results showed that the size of the SARS-CoV-2 NP protein was consistent with the theoretical value and the purity was > 90% (Fig. 2C), which can be used for subsequent experiments. Figure 1

[0035] Example 2 Preparation and purification of SARS-CoV-2 NP monoclonal antibody SARS-CoV-2 NP protein (1 mg / mL) was emulsified with an equal volume of Freund's (incomplete) adjuvant, and 6-8 week-old BALB / c mice were injected subcutaneously with 200 μL per mouse. The mice were boosted at 2 weeks and 4 weeks after the initial immunization. After three immunizations, the mice were subjected to submandibular venous blood collection and serum separation. The serum antibody titer produced by the mice was detected by indirect ELISA. Mice with serum titers meeting the fusion requirements were selected for intraperitoneal booster immunization with SARS-CoV-2 NP protein without adjuvant. Three days after the booster immunization, the mouse spleen cells were fused with SP2 / 0 cells. Ten days after the fusion, the supernatant of the hybridoma cells was detected by indirect ELISA, and positive hybridoma cells were selected. Flow cytometry was then used to sort the positive hybridoma cells. After the positive monoclonal cell strains were detected again by indirect ELISA, the positive monoclonal cell strains were selected for expansion culture. The expanded monoclonal cell strains were injected into the peritoneal cavity of previously sensitized BALB / c mice, and the ascites were collected 7 days later to obtain the corresponding monoclonal antibodies.

[0036] The ascites was purified according to the Protein A / G filler operation manual. The purified antibody was subjected to SDS-PAGE detection, and after confirmation, desalting column desalting and concentration titration were performed, and the sample was aliquoted and frozen.

[0037] The spleen cells of mice with qualified serum titers after three immunizations were hybridoma cell fusion tested with SP2 / 0 cells. Six SARS-CoV-2 NP monoclonal hybridoma cell strains were screened by indirect ELISA and flow cytometry, and were named 7B5, 7B6, 8B1, 8B2, 9A9 and 9E3, respectively (Fig. 3A). Figure 2 A).

[0038] The antibody subtype detection results showed that the six monoclonal antibodies were all IgG1 type, and the light chains were all κ type (Fig. 3B). Figure 2 B).

[0039] The cross-reactivity detection results showed that the six monoclonal antibodies could all react with SARS-CoV-2 NP-flag expressed by intracellular transfection (Fig. 3C). Figure 2 ​C). After immunization with SARS-CoV-2 NP-His, the selected antibodies may recognize either the SARS-CoV-2 NP protein or the His tag. However, after replacing the His tag with the Flag tag, the fusion protein contains both the SARS-CoV-2 NP protein and the Flag tag. If the antibody can react with the fusion protein, then the recognition epitope can only be the SARS-CoV-2 NP protein, thus excluding specific reactions with the His-tag.

[0040] Example 3: Identification of SARS-CoV-2 NP monoclonal antibody binding activity and competition relationship The ELISA plate was coated with SARS-CoV-2 NP protein. The monoclonal antibodies screened in Example 2 were initially diluted 10,000 ng / mL to 0.001 ng / mL in a 10-fold serial dilution. Each concentration was used as the primary antibody for incubation. The binding activity of the antibodies to SARS-CoV-2 NP protein was detected by indirect ELISA.

[0041] Monoclonal antibodies were HRP-labeled using an HRP-labeled antibody / protein kit. Unlabeled antibodies were used as capture antibodies to coat ELISA plates, SARS-CoV-2 NP protein was used as the detection sample, and HRP-labeled antibodies were used as the detection antibody as the secondary antibody. A competitive ELISA was employed to detect antibody competition.

[0042] The results showed that the antibody binding titers of strains 8B1 and 9E3 were superior to those of other strains. Figure 3 A), EC 50 The concentrations were 38 ng / mL and 61 ng / mL, respectively, and the competition test results showed that 8B1 and 9E3 had no competitive relationship. Figure 3 Therefore, 8B1 and 9E3 were selected to establish a SARS-CoV-2 NP double antibody sandwich ELISA antigen detection method.

[0043] Example 4: Determination of SARS-CoV-2 NP monoclonal antibody sequence The hybridoma cells were obtained by cDNA rapid amplification of cDNA ends (RACE) to obtain cDNA. The cDNA was used as a template to configure a PCR reaction system using Phanta Max Master Mix, and each hybridoma cell contained two reactions corresponding to heavy chain and light chain, respectively. Each reaction contained 2 X Mix 5 μL, 0.5 μL of upstream and downstream primers, 1 μL of template, 3 μL of deionized water, and the primer sequences are shown in Table 2. The reaction conditions were as follows: 95℃-2 min; 95℃-15 s, 60℃-15 s, 72℃-30 s, 35 cycles; 72℃-5 min. The PCR products were separated by 1.5% agarose gel electrophoresis, and the target band was around 750 bp. After recovery, it was sent to a biological company for sequencing, and the sequencing results were analyzed using the Kingsbury antibody variable region sequence analysis tool.

[0044] Table 2 Antibody sequencing primer sequences The sequencing results were labeled using the IMGT method. Specifically, as shown in Table 3.

[0045] Table 3 Antibody amino acid sequence Example 5 Establishment of SARS-CoV-2 NP double antibody sandwich ELISA antigen detection method and optimization of reaction conditions The two antibodies with the highest binding activity and no competitive relationship were selected for the establishment of a double antibody sandwich ELISA detection method. To determine the order of the two antibodies, the unlabelled two antibodies were used as capture antibodies to coat the enzyme-labeled plate at 4℃ overnight, and then washed with PBST. 5% skim milk was used for incubation at 37℃ for 1 h, and then washed with PBST. SARS-CoV-2 NP was added as an antigen and incubated at 37℃ for 1 h, and then washed with PBST. The HRP-labeled complementary antibody was added and incubated at 37℃ for 1 h, and then washed with PBST. TMB double-component color developing liquid was added and incubated at room temperature for 10 min, then 50 μL of stop solution was added, and the OD 450 nm value of each well was determined on a microplate reader. The best antibody combination was selected based on the maximum P / N value.

[0046] After the capture antibody and detection antibody were determined, the best coating amount of the capture antibody, the best dilution of the enzyme-labeled antibody, the best blocking liquid and blocking time, the best incubation time of the antigen and enzyme-labeled antibody, and the best color developing time were determined by using the chessboard method to determine the best reaction conditions of the double antibody sandwich ELISA. The OD 450 nm value of the positive well was around 1.0, and the OD 450nm value <0.1, and the minimum amount of antibody used is the determination standard. The capture antibody (10 μg / mL) and enzyme-labeled antibody (500 ng / mL) are diluted by PBS at a ratio of 2:1 to determine the optimal use concentration of the capture antibody and enzyme-labeled antibody. On this basis, the capture antibody incubation time is set to 4°C for 3 h, 4°C overnight, 4°C overnight followed by 37°C for 2 h, and 37°C for 2 h; the blocking solution is 5% BSA, 5% skim milk powder, 1% horse serum, and 1% rabbit serum; the blocking time is 0.5 h, 1 h, 1.5 h, and 2 h; the sample incubation time is 0.5 h, 1 h, 1.5 h, and 2 h; the enzyme-labeled antibody incubation time is 0.5 h, 1 h, 1.5 h, and 2 h; and the color development time is 5 min, 10 min, and 15 min for ELISA reaction condition optimization.

[0047] After screening, it is determined that 9E3 is used as the capture antibody and 8B1-HRP is used as the detection antibody, which has a significant effect. Figure 3 B), the optimal coating concentration of 9E3 is 1.25 μg / mL; the optimal concentration of 8B1-HRP is 0.0625 μg / mL; the optimal coating condition is 4°C for 3 h; the blocking condition is 37°C for 2 h with 5% BSA; the optimal incubation time of the antigen is 37°C for 60 min; the optimal incubation time of the detection antibody 8B1-HRP is 37°C for 30 min; and the optimal color development time of TMB single-component color developing solution is 10 min at room temperature. (The specific results are shown in Tables 4-9).

[0048] Table 4 Optimal coating concentration and detection concentration Table 5 Optimal coating time Table 6 Optimal blocking agent Table 7 Optimal blocking time Table 8 Optimal sample incubation time Table 9 Optimal secondary antibody incubation time and color development time Example 6 Standard curve On the basis of the optimization results in Example 5, the SARS CoV-2 NP recombinant protein (1000 ng / mL) is diluted by a ratio of 2:1, and then detected by the established double antibody sandwich ELISA method. PBS is used as a blank control, and the OD 450nm values, a standard curve was drawn according to the results, and EC was calculated 50 .

[0049] It was determined that the standard curve of the detection method was y=0.11+2.61 / (1+10^(1.98-X)*1.35), which showed a typical S-shaped curve (four-parameter fitting) in the range of 7.8125-1000 ng / mL. The OD value slowly rose with the increase of concentration in the low concentration section (0-10 ng / mL), steeply rose in the medium concentration section (10-250 ng / mL), and gently rose in the high concentration section (250-1000 ng / mL). The EC 50 value was 94.66 ng / mL ( Figure 4 ).

[0050] Example 7 Specificity experiment Ebolavirus GP protein (EBOV-GP), ebolavirus NP protein (EBOV-NP), lassa virus GP1 protein (LASV-GP1), lassa virus GP2 protein (LASV-GP2), rift valley fever virus NP protein (RVFV-NP), rift valley fever virus Gn protein (RVFV Gn) and Crimean-Congo hemorrhagic fever virus NP protein (CCHFV-NP) were used as detection samples, SARS-CoV-2 NP protein was used as a positive control, and PBS was used as a negative control. The double antibody sandwich ELISA method established in the application was used for specificity detection. 9E3 was coated at a concentration of 1.25 μg / mL at 4°C for 3 h, followed by 5% BSA blocking at 37°C for 2 h, antigen incubation at 37°C for 60 min, detection antibody 8B1-HRP incubation at a concentration of 0.0625 μg / mL at 37°C for 30 min, and finally TMB single-component color developing liquid color development at room temperature for 10 min and value reading. The OD 450 nm values of each sample and the negative control were compared. When the sample value was greater than the OD 50 nm of the negative control + 3s, it was determined to be positive.

[0051] It was determined that when the sample OD 450 nm value was greater than 0.208, it was determined to be positive, and the OD 450When the nm value is less than 0.208, it is determined to be negative, and the results of the samples of Ebola virus GP protein (EBOV-GP), Ebola virus NP protein (EBOV-NP), Lassa virus GP1 protein (LASV-GP1), Lassa virus GP2 protein (LASV-GP2), Rift Valley fever virus Gn protein (RVFV Gn), Rift Valley fever virus NP protein (RVFV-NP), Crimean-Congo hemorrhagic fever virus NP protein (CCHFV-NP) are determined to be negative, and the result of the SARS-CoV-2 NP protein sample is determined to be positive. Figure 5

[0052] Example 8 Reproducibility experiment Five plates coated with the same batch were selected, and six holes were taken at the same position of each plate. Three positive samples and three negative samples were each 3 copies, and five repeats were made for each sample. The OD 450 nm value was measured. 450 The nm value was calculated according to the statistical method. The coefficient of variation (CV) of the sample was calculated, and the reproducibility of the method was evaluated. Plates coated with different batches were selected, and batch reproducibility experiments were performed according to the above method.

[0053] Three positive samples (P1, P2, P3) and three negative samples (N1, N2, N3) were selected for reproducibility test, and the final result showed that the CV was less than 9%, indicating that the reproducibility and stability of the established double antibody sandwich ELISA method could be used for clinical detection of SARS-CoV-2 (Table 10).

[0054] Table 10 Reproducibility results Example 9 Clinical sample detection 66 clinical samples from dogs were detected simultaneously by two methods: one was detected by reverse transcription-real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) method, and the detection result of this method was used as the gold standard for evaluating the accuracy of the target method in this study. The specific steps are as follows: the sample is extracted according to the standard operation manual of the virus RNA extraction kit, and the extracted RNA is aliquoted and frozen; the qPCR reaction system is configured, each reaction contains 2 X Mix 5 μL, 0.5 μL of upstream and downstream primers and probe, 1 μL of template, 2.5 μL of deionized water, and the primer sequences are shown in Table 3; the qPCR reaction conditions are 95℃-2min pre-denaturation, 95℃-10s, 60℃-30s, 40 cycles. The second is to use the detection method optimized in the previous stage for detection, and the results are compared with the RT-qPCR detection results, aiming to evaluate the accuracy of the method.

[0055] ​Table 11 RT-qPCR primer sequences 66 clinical samples from dogs were detected by RT-qPCR method and double antibody sandwich ELISA method respectively, and the results of RT-qPCR method and double antibody sandwich ELISA method showed that the 66 samples were all negative. The detection results of the method established in this study and RT-qPCR method were consistent.

Claims

1. A combination of monoclonal antibodies for detecting the novel coronavirus NP protein, characterized in that, The monoclonal antibody combination comprises monoclonal antibody 9E3 and monoclonal antibody 8B1, The heavy chain variable region of the monoclonal antibody 9E3 comprises three complementarity determining regions of CDR1, CDR2 and CDR3, the amino acid sequence of CDR1 is shown as SEQ ID NO. 1, the amino acid sequence of CDR2 is shown as SEQ ID NO. 2, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 3; The light chain variable region of the monoclonal antibody 9E3 comprises three complementarity determining regions of CDR1, CDR2 and CDR3, the amino acid sequence of CDR1 is shown as SEQ ID NO. 4, the amino acid sequence of CDR2 is shown as SEQ ID NO. 5, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 6; The heavy chain variable region of the monoclonal antibody 8B1 comprises three complementarity determining regions of CDR1, CDR2 and CDR3, the amino acid sequence of CDR1 is shown as SEQ ID NO. 7, the amino acid sequence of CDR2 is shown as SEQ ID NO. 8, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 9; The light chain variable region of the monoclonal antibody 8B1 comprises three complementarity determining regions of CDR1, CDR2 and CDR3, the amino acid sequence of CDR1 is shown as SEQ ID NO. 10, the amino acid sequence of CDR2 is shown as SEQ ID NO. 11, and the amino acid sequence of CDR3 is shown as SEQ ID NO.

12.

2. The use of the monoclonal antibody combination of claim 1 in the preparation of a reagent for detecting the NP protein of the novel coronavirus; the monoclonal antibody combination comprises monoclonal antibody 9E3 and monoclonal antibody 8B1, The heavy chain variable region of the monoclonal antibody 9E3 comprises three complementarity determining regions of CDR1, CDR2 and CDR3, the amino acid sequence of CDR1 is shown as SEQ ID NO. 1, the amino acid sequence of CDR2 is shown as SEQ ID NO. 2, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 3; The light chain variable region of the monoclonal antibody 9E3 comprises three complementarity determining regions of CDR1, CDR2 and CDR3, the amino acid sequence of CDR1 is shown as SEQ ID NO. 4, the amino acid sequence of CDR2 is shown as SEQ ID NO. 5, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 6; The heavy chain variable region of the monoclonal antibody 8B1 comprises three complementarity determining regions of CDR1, CDR2 and CDR3, the amino acid sequence of CDR1 is shown as SEQ ID NO. 7, the amino acid sequence of CDR2 is shown as SEQ ID NO. 8, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 9; The light chain variable region of the monoclonal antibody 8B1 comprises three complementarity determining regions of CDR1, CDR2 and CDR3, the amino acid sequence of CDR1 is shown as SEQ ID NO. 10, the amino acid sequence of CDR2 is shown as SEQ ID NO. 11, and the amino acid sequence of CDR3 is shown as SEQ ID NO.

12. The light chain variable region of the monoclonal antibody 8B1 comprises three complementarity determining regions of CDR1, CDR2 and CDR3, the amino acid sequence of CDR1 is shown as SEQ ID NO. 10, the amino acid sequence of CDR2 is shown as SEQ ID NO. 11, and the amino acid sequence of CDR3 is shown as SEQ ID NO.

12.

3. A double antibody sandwich ELISA kit for detecting the novel coronavirus NP protein, characterized in that, The kit comprises the monoclonal antibody combination of claim 1, wherein the monoclonal antibody 9E3 is used as a capture antibody, and the monoclonal antibody 8B1 is labeled with HRP as a detection antibody (8B1-HRP).

4. The kit of claim 3, wherein The kit further comprises a blocking solution, a dilution solution, a washing solution, a color developing solution and a termination solution.

5. The kit of claim 3, wherein The mass concentration of the capture antibody is 1-1.50 μg / mL; and the concentration of the detection antibody is 0.01-0.10 μg / mL.

6. The kit of claim 3, wherein The mass concentration of the capture antibody is 1.25 μg / mL; and the concentration of the detection antibody is 0.0625 μg / mL.

7. The kit of claim 3, wherein The coating condition is 4℃ for 2-4h, preferably 4℃ for 3h.

8. The kit of claim 3, wherein The blocking solution is selected from 5% skimmed milk powder, 1% horse serum, 1% rabbit serum or 5% BSA, preferably 5% BSA.

9. The kit of claim 3, wherein The blocking condition is 5% BSA blocking for 0.5-2h; the optimal incubation time of the antigen is 30-90 min; the optimal incubation time of the detection antibody 8B1-HRP is 0.5-2h; and the optimal color developing time of the TMB single-component color developing solution is 5-15 min at room temperature.

10. The kit of claim 3, wherein The blocking condition is 5% BSA blocking for 2h; the optimal incubation time of the antigen is 60 min; the optimal incubation time of the detection antibody 8B1-HRP is 0.5h; and the optimal color developing time of the TMB single-component color developing solution is 10 min at room temperature. The blocking condition is 5% BSA blocking for 2h; the optimal incubation time of the antigen is 60 min; the optimal incubation time of the detection antibody 8B1-HRP is 0.5h; and the optimal color developing time of the TMB single-component color developing solution is 10 min at room temperature.

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