CN113493508A

CN113493508A - Double-antibody sandwich ELISA kit for detecting new coronavirus N protein

Info

Publication number: CN113493508A
Application number: CN202110659663.4A
Authority: CN
Inventors: 马晓飞; 武鹏程; 武雷; 孔双泉; 金丽珠; 尹长城
Original assignee: Beijing Protein Innovation Co ltd
Current assignee: Beijing Protein Innovation Co ltd
Priority date: 2021-06-15
Filing date: 2021-06-15
Publication date: 2021-10-12

Abstract

The invention provides a double-antibody sandwich ELISA detection kit of a novel coronavirus (SARS-CoV-2) and a using method thereof, wherein the method is realized based on an antibody, and the antibody pair is obtained by screening according to the following method: preparing immunogen, immunizing animal, screening positive hybridoma and establishing strain, preparing ascites, purifying antibody, and pairing and optimizing 22 strains of antibody obtained by purification to obtain a pair of paired antibodies capable of detecting SARS-CoV-2: coating antibody 64360A #4:27, detection antibody 64360A #5: 42. The ELISA detection method of the invention has simple operation and high sensitivity, can specifically detect the structural protein N protein of the new coronavirus, does not generate cross reaction with the SARS virus N protein, and has the detection sensitivity of 100 pg/mL.

Description

Double-antibody sandwich ELISA kit for detecting new coronavirus N protein

Description

Technical Field

The present invention relates to the field of detection and diagnosis of new coronaviruses. More particularly, the invention relates to a double-antibody sandwich ELISA method for detecting a novel coronavirus, which has important application values in screening, detecting and prognosis evaluation of the novel coronavirus infection.

Background

The novel coronavirus (SARS-CoV-2) belongs to the large family of coronaviruses together with MERS virus and SARS virus causing severe acute respiratory syndrome. SARS-CoV-2 has a positive-sense, single-stranded RNA genome of 30kb in size. Its nucleocapsid protein (N) is an outer membrane composed of membrane protein (M), envelope protein (E) and spike protein (S), and wraps the viral genome. The N protein is a phosphorylated protein consisting of two structural domains, can be combined with viral RNA, participates in RNA synthesis and protein translation, is a structural protein with the most expression copies in the virus replication process, and has the characteristic of stable property, so that the unique advantage of the protein as the virus antigen detection is determined.

At present, 15 new coronavirus detection reagents are totally approved by the national drug administration, wherein 10 nucleic acid detection reagents and 5 antibody detection reagents are adopted, and related detection of virus antigens is not approved. The nucleic acid detection is a gold standard for pathogen detection, the method has high requirements on laboratory conditions, detection personnel and instruments, has the characteristics of strong specificity and weak sensitivity, and has the detection accuracy of only 30-50% on infected cases. The antibody detection reagent is used for detecting IgM or IgG antibodies which are produced by stimulating a human body after viruses enter the human body in serum, wherein the IgM antibodies appear earlier, and the IgG antibodies appear later. For suspected cases in which the new coronavirus nucleic acid test is negative, antibody detection can be used as a supplementary detection index. The antibody detection can also be used cooperatively in the suspected cases of the new coronavirus nucleic acid detection.

Compared with nucleic acid detection, the antigen detection has the advantages of convenience, rapidness, low price and the like, and compared with antibody detection, the antigen detection has no window period and can be used for detecting a patient who just appears infection symptoms in time. For the new coronavirus infection with strong infectivity, the improvement of the detection convenience degree and the sensitivity are particularly important, the antigen detection has the advantage that the existing antibody detection cannot replace the detection, the situation of coronavirus infection and prevention and treatment is expected to be long-term, the high-sensitivity detection method has strong demand, and the high-affinity paired antibody is particularly important in the development of antigen detection products. The length of the N protein of the new coronavirus is 422 amino acids, and the sequence alignment shows that the consistency of the N protein and the N protein sequence (the Genbank ID is AAP51234) with the SARS coronavirus strain number GD01 reaches 90.5%, and the antibody is difficult to distinguish between the two in the commonly prepared antibody, which increases the difficulty of antibody preparation. The double-antibody sandwich ELISA method adopts a traditional double-antibody enzyme-linked immunosorbent assay, an enzymatic chemiluminescence assay or an immune colloidal gold test strip method, and detection is completed by a specific antibody.

Disclosure of Invention

The first purpose of the present invention is to provide a pair of monoclonal antibodies which can be paired and specifically recognize SARS-CoV-2 virus N protein but not SARS virus N protein.

Another objective of the invention is to provide a method for preparing and screening the specific antibody pair.

Another objective of the invention is to provide a double-antibody sandwich ELISA method and a kit for detecting SARS-Cov-2 virus N protein.

In a first aspect of the present invention, there is provided a pair of monoclonal antibodies, each of which is a coating antibody and a detection antibody, and comprises:

(a) light chain variable region

(i) Comprises the amino acid sequence of SEQ ID NO: 01, or a CDR-H1 region of the amino acid sequence shown in seq id No. 01.

(ii) Comprises the amino acid sequence of SEQ ID NO: 02, or a CDR-H2 region of the amino acid sequence shown in SEQ ID NO.

(iii) Comprises the amino acid sequence of SEQ ID NO: 03, and the CDR-H3 region.

(b) Heavy chain variable region

(i) Comprises the amino acid sequence of SEQ ID NO: 04, or a CDR-L1 region of the amino acid sequence shown in SEQ ID No.

(ii) Comprises the amino acid sequence of SEQ ID NO: 05 of the amino acid sequence shown in SEQ ID NO. 05.

(iii) Comprises the amino acid sequence of SEQ ID NO: 06, or a CDR-L3 region of the amino acid sequence shown in SEQ ID NO.

The detection antibody comprises the following components:

(a) light chain variable region

(i) Comprises the amino acid sequence of SEQ ID NO: 07, or a CDR-H1 region of the amino acid sequence set forth in seq id no.

(ii) Comprises the amino acid sequence of SEQ ID NO: 08, and a CDR-H2 region of the amino acid sequence shown in seq id no.

(iii) Comprises the amino acid sequence of SEQ ID NO: 09, or a CDR-H3 region of the amino acid sequence shown in seq id no.

(b) Heavy chain variable region

(i) Comprises the amino acid sequence of SEQ ID NO: 10, or a CDR-L1 region of the amino acid sequence set forth in seq id No. 10.

(ii) Comprises the amino acid sequence of SEQ ID NO: 11, or a CDR-L2 region of the amino acid sequence set forth in seq id no.

(iii) Comprises the amino acid sequence of SEQ ID NO: 12, or a CDR-L3 region of the amino acid sequence set forth in seq id no.

In a second aspect of the invention, there is provided a pair of monoclonal antibodies which can be paired in a double antibody sandwich ELISA method for detecting a novel coronavirus, wherein the paired antibodies are prepared and screened by the following method:

preparing immunogen, namely performing eukaryotic expression and prokaryotic expression on N protein, purifying, treating at 60 ℃ for 30 minutes, mixing with Freund complete adjuvant and Freund incomplete adjuvant, preparing immunogen, and performing cross immunization on mice;

preparing positive hybridoma, fusing immune mouse spleen cell with SP2/0 myeloma cell, performing positive screening by taking SARS-CoV-2 virus N protein which is expressed by eukaryotic system and is subjected to heat treatment as positive antigen, performing negative screening by indirect ELISA by taking SARS virus N protein which is expressed by eukaryotic system and is subjected to heat treatment as negative antigen, and obtaining positive hybridoma;

the monoclonal antibody is a positive hybridoma cell subclone strain, ascites is prepared and purified to obtain 22 monoclonal antibodies, and the 22 monoclonal antibodies are marked by HRP and biotin;

and (3) pairing the sandwich antibodies, namely pairing the 22 monoclonal antibodies with the enzyme-labeled antibodies to obtain the coated antibodies and the enzyme-labeled antibodies which are suitable for the double-antibody sandwich ELISA method for detecting the new coronavirus.

In a third aspect of the present invention, there is provided a method for establishing a double antibody sandwich ELISA method for detecting a novel coronavirus based on the pair of monoclonal antibodies, comprising:

and determining the optimal working concentration of the paired coated antibody and enzyme-labeled antibody, the optimal sealant under the optimal coating condition, the optimal sealing condition, the components of an enzyme-labeled secondary antibody diluent, the optimal action time of the enzyme-labeled antibody, the action time of a substrate and the critical value of a double-antibody sandwich ELISA method, and selecting the combination of the optimal parameters.

Preferably, the coating antibody is 64360A #4:27, the coating concentration is 5 μ g/mL-1 μ g/mL, and the optimal coating concentration is 1 μ g/mL.

Preferably, the detection antibody 64360A #5:42 is labeled with horseradish peroxidase or biotin at a dilution ratio of 1:5000 to 1:20000, preferably 1:10000, after labeling with horseradish peroxidase (HRP) and at a dilution ratio of 1:5000 to 1:20000, preferably 1:5000, after labeling with biotin.

Preferably, the ELISA kit has a minimum detection limit of 100 pg/mL.

Technical means for solving the technical problems

The invention obtains a pair of hybridoma cell strains secreting anti-SARS-CoV-2-N antibody by large-scale cell fusion and screening, and the preparation method of the hybridoma cell strains comprises the following steps:

1) analyzing according to the published N protein coding sequence of SARS-CoV-2 virus, selecting N protein full length region for recombination expression according to the structure, antigenicity, hydrophilicity and hydrophobicity of constituent amino acids and secondary structure on cell membrane, in order to promote the soluble expression of recombinant protein, utilizing eukaryotic cell to make secretion expression, fusing histidine label on its C end to facilitate purification, after gene synthesis cloning into expression plasmid vector pcDNA3.4, transfecting Expi293 cell to culture, separating and purifying the expression product from culture medium supernatant to use as immunogen and antibody screening.

2) The spleen cells of the immune qualified mice are taken aseptically as antigen-sensitized B cells, the B cells are fused with myeloma cells SP2/0 strain according to a conventional method, and then the B cells are screened by a conventional fused cell HAT screening method, so as to obtain the fused cell growth clone.

3) Antibody preparation and screening for the N protein that specifically recognizes SARS-Cov-2: the SARS virus caused SARS virus in 2003 (commonly called as SARS) has high similarity in nucleic acid sequence and main structure protein sequence composition with the pneumonia caused by new type coronavirus infection in 2019, which increases the difficulty in developing and identifying two virus protein antibody, in the protein sequence alignment result of figure 1, the N protein amino acid sequence deduced from SARS-Cov-2 virus genome sequence is numbered as GenBank as MN908947.3, the length is 422 amino acids, the N protein amino acid sequence deduced from SARS-Cov virus genome sequence is numbered as AY278489.2, the sequence consistency between the two is 90.5%, there are only a few scattered different amino acids, and the length of the continuous different amino acids is within two, according to the general rule that the length of antigen epitope is 4-7 amino acids, there are difficulties in preparing antibodies that can distinguish between the two proteins. Meanwhile, the N protein is a protein rich in phosphorylation modification, blood or a throat swab sample needs to be subjected to heat inactivation before actual detection, and the protein is subjected to structural change and partial group modification change.

The monoclonal antibody is prepared from the hybridoma cell strain, and the preparation method comprises the following two steps:

1) in vitro, the hybridoma cells are cultured in a serum-free culture medium, and the culture supernatant is harvested and separated by immunoaffinity chromatography to purify the required monoclonal antibody.

2) And (3) inoculating hybridoma cells in the abdominal cavity of the animal, harvesting ascites of the animal, and separating and purifying the required monoclonal antibody. The invention relates to an N monoclonal antibody obtained by purifying an antibody prepared by a ascites method through Protein A/G column affinity chromatography.

Advantages and advantageous effects of the invention

The antibody pair for ELISA detection of the invention can specifically recognize SARS-CoV-2 virus N protein, but not the N protein of SARS virus occurring in 2003, has very high specificity, can distinguish two virus proteins, and can also distinguish novel human coronavirus HCoV-HKU1 occurring in 2004-2005 and MERS-CoV coronavirus causing Middle East respiratory syndrome (Middle East respiratory syndrome) in 2012 according to sequence homology comparison. On the other hand, the standard antigen of the ELISA kit is SARS-CoV-2 virus N protein which is subjected to heat inactivation at 60 ℃, and can be used for screening and detecting the new coronary pneumonia caused by SARS-CoV-2.

Description of the drawings:

FIG. 1: comparison of SARS-Cov-2 and SARS-Cov virus N protein sequences

The GenBank accession number is MN908947.3 is the amino acid sequence of the N protein deduced from the genome sequence of SARS-Cov-2 virus, the GenBank accession number is AY278489.2 is the amino acid sequence of the N protein deduced from the genome sequence of SARS-Cov virus, and the sequence identity of the N protein and the N protein is 90.5%.

FIG. 2: the recombinant protein of SARS-Cov-2 virus N cultured and purified in serum-free mode in Expi293 cells is shown by M as molecular weight marker, 1 as the medium supernatant, 2 as the medium supernatant flow through, 3 and 4 as 15mM imidazole elution products, 5-6 as 60mM imidazole elution products, respectively, and the size of the expression product is about 56kDa, and separated by 12% SDS-PAGE gel.

FIG. 3: double antibody sandwich ELISA for detecting SARS-CoV-2 virus N protein

Detailed Description

The present invention is further described with reference to the drawings and the detailed description, so that the technical solutions of the present invention can be more clearly understood by those skilled in the art, and the present invention is not limited thereto.

EXAMPLE 1 preparation of recombinant SARS-Cov-2 Virus N protein immunogen

The complete open reading frame region was synthesized by referring to the nucleotide sequence encoding the N protein in the novel coronavirus genome sequence numbered MN908947.3 in GenBank and cloned into a plasmid vector pUC57 (Nanjing Kingsler Biotech Co., Ltd.) to design the upstream primer N-Cov-NF: 5' -GGATCGAATTCGATGTCTGATAATG-3 ' and a downstream primer n-Cov-NR: 5'-ACGCTCGAGTTAGTGGTGGTGGTGGTGGTGGGCCTGAGTTGAG TCAGC-3', an EcoRI cleavage site is introduced through an upstream primer, a histidine purification tag and an XhoI cleavage site are introduced through a downstream primer, and the primers are synthesized by Beijing Liuhua Dagenescience and technology Co. According to the standard nucleic acid amplification process, a target gene is amplified from a plasmid vector, and is subjected to EcoRI/XhoI double enzyme digestion and recovery, then the target gene is cloned into a eukaryotic expression vector pcDNA3.4 (purchased from Invitrogen Inc) linearized fragment subjected to the same double enzyme digestion, and after sequencing identification, plasmid preparation is carried out, wherein the reference is Expi293^TMExpi293 cells were transfected and cultured in Expression System Kit (Seimer Feissuer science and technology (China) Co., Ltd., product No. A14635) instructions to maintain cell viability at 70% or more, and purified after 120 hours of culture. Since the recombinant N protein carries a histidine tag, affinity purification was performed using a nickel column. After elution with imidazole solutions of different concentrations, the fractions and flow-through were separately subjected to SDS-PAGE for separation and detection, and FIG. 2 shows the expression and purification results of recombinant N protein fused with histidine tag. The purity of the recombinant N protein is more than 90 percent, the concentration is about 1-1.5mg/mL, and the requirements of immune animals and antibody screening and identification can be met.

Example 2 establishment of hybridoma cell lines and antibody screening

Animal immunization

Six female Balb/C mice (purchased from Beijing Wintolite laboratory animals technologies, Inc.) of 4-6 weeks old, numbered in order 64360# A, 64360# B, 64360# C, 64360# D, 64360# E, and 64360# F were immunized with the recombinant N protein of example 1 after inactivation at 60 ℃ and emulsified with Freund's complete adjuvant (purchased from Sigma). The dose per mouse was 60. mu.g/mouse by abdominal subcutaneous injection. The booster was administered every 14 days, and the antigen was emulsified with Freund's incomplete adjuvant (Sigma Co.) at a dose of 30. mu.g/mouse. 7 days after 3 times of booster immunization, the multi-antibody titer of the anti-immunogen in the serum of the mice is detected by indirect ELISA (wavelength of 450nm), the mice with the highest titer are injected by tail vein for impact immunization, the antigen is uniformly heated by physiological saline, and the dosage is 50 mu g/mouse.

Second, cell fusion

Aseptically preparing mouse spleen fine powder with up-to-standard immunityThe cell suspension was mixed with mouse myeloma cells sp2/0(ATCC) at a ratio of 5:1, centrifuged at 1500rpm for 5min, the supernatant was discarded and the tube was placed in a 37 ℃ water bath, 1mL of PEG1500(Roche Co.) was added slowly over 1 minute, and the cells were agitated. After standing in warm water for 1min, 10mL of serum-free IMDM (Sigma Co.) was added, and the mixture was mixed, centrifuged at 1000rpm, and after 5min the supernatant was discarded, 10mL of serum (PAA Co.) was added to gently blow up the cells, and 5mL of thymocytes mixed with 10 XHAT (Sigma Co.) were added and mixed. Then, 25mL of a semi-solid medium containing 2.1% nitrocellulose (Sigma) was added thereto, mixed well, and then poured into 20 cell culture dishes uniformly. Placing the cell culture dish into a wet box, and adding 5% CO at 37 deg.C₂Culturing in an incubator.

Screening and preserving positive hybridoma

The cell mass is moderate in size and density 7 days after fusion, round, solid and large cell masses are sucked under a dissecting mirror and are put into a 96-hole culture plate with a prepared culture medium in advance, the culture plate is put into a 37 ℃ incubator for culture, and CO is added₂The concentration was 5%.

Fourth, ELISA screening positive hybridoma cell

After 3 days of hybridoma culture, the amount of cells accounted for approximately floor area 2/3, and 200. mu.l of complete medium containing feeder cells and 1% HT (Sigma) was added. And performing second ELISA screening after two days to obtain hybridoma with OD value higher than 0.5 detected by indirect ELISA of immunogen as positive clone for subsequent culture. These positive clones were transferred to 24-well plate cultures prepared in advance with medium (containing feeder cells and HT). Five days later 100. mu.l of the supernatant was used for a third ELISA screening. The screening process is as follows, 100 mul supernatant is taken and is used for carrying out the specificity screening of the ELISA method by taking the eukaryotic expression SARS-Cov-2 virus N protein (Beijing Hua big protein research and development center, Inc., the product number PT-00117) which is inactivated by heat at 60 ℃ and the prokaryotic expression SARS virus N protein (Beijing Hua big protein research and development center, Inc., the product number PT-20304) as envelope antigen. For screening, the antigen protein was diluted to 2. mu.g/ml with PBS buffer. Add 100. mu.l antigen per well, coat for 12 hours at 4 ℃, discard solution, wash plate 3 times with wash solution (PBS buffer containing 0.05% Tween) and drain, add 300. mu.L 2% (v/w) cattle per wellTaking serum protein as a sealing solution, incubating for 3h at 37 ℃, repeatedly washing the plate for 3 times, adding cell culture supernatant to be identified, after incubation and screening, sealing, finishing adding a sample to be detected in the hole of the ELISA plate, incubating for 1h at 37 ℃, washing the plate for 3 times, adding 1: mu.l HRP-labeled goat anti-mouse antibody (purchased from Takara Shuzo) diluted 5000 times, incubated at 37 ℃ for 1H, washed for 3 times, drained, added 100. mu.L of TMB color development buffer per well, incubated at room temperature for 8min, and added 50. mu.l of H per well₂SO₄(2M) the reaction was stopped and the absorbance at 450nm was read on a microplate reader.

Six mice are preliminarily screened to obtain 260 hybridoma cells capable of recognizing the N protein, screening results of 60 hybridomas of 64360# D are shown in table 1, antibodies secreted by most hybridoma cells can simultaneously recognize two different antigens, and 22 strains obtained by screening can only recognize the SARS-Cov-2 virus N protein but not be combined with the SARS virus N protein and can be used as candidate antibodies of a subsequent double-antibody sandwich ELISA method.

TABLE 1 hybridoma specificity screening for preparation of mouse # 64360D

EXAMPLE 3 preparation of monoclonal antibody by ascites Induction method

Cells in logarithmic growth phase were washed with serum-free medium and suspended, and counted at about 5X 10⁵1ml of suspended cells were injected intraperitoneally into mice previously sensitized with paraffin oil. Ascites collection was started 7 days later. The ascites fluid taken out was centrifuged at 4000rpm at 4 ℃ and the middle ascites fluid was carefully aspirated for 10min and collected in a centrifuge tube for purification. The antibody was purified according to the instructions of HiTrap rProtein A FF (available from GE, cat. No. 17-5079-02).

Example 4 monoclonal antibody subclass identification and affinity determination

Subclass identification, goat anti-mouse IgG (Beijing China fir Biotechnology Co., Ltd.) coated with 100mM PBS (pH7.4) was diluted to 0.5. mu.g/m 1, 100. mu.l was added to each well, the liquid was emptied overnight at 4 ℃, washed 3 times with PBS (PBS-T) containing 0.05% Tween, and 200. mu.l of blocking solution was added to each well(PBS containing 2% BSA and 3% sucrose), incubated at 37 ℃ for 1 h. The liquid was decanted and washed 3 times with PBS-T. Mu.l of hybridoma culture supernatant or purified antibody was added to each well, incubated at 37 ℃ for 1h, the liquid was emptied, and washed 3 times with PBS-T. HRP-labeled goat anti-mouse (kappa, lambda) antibody was diluted with a blocking solution 1:1000 or HRP-labeled goat anti-mouse (IgM, IgG1, IgG2a, IgG2b, IgG3, IgA) antibody (Southern Biotech Co.) was added to each well of the antibody sample to be tested in a volume of 100. mu.l per well, incubated at 37 ℃ for 1 hour, the solution was emptied, and washed 3 times with PBS-T. 50 μ l of 0.15% ABTS (Southern Biotech Co., cat. 0202-1) and 0.03% H was added to each well₂O₂The citric acid buffer (pH4.0) was developed, and the absorbance at a wavelength of 405nm was measured within 10 min.

Second, determination of affinity constant

Recombinant N protein was coated at a concentration of 2. mu.g/m 1, 100. mu.1/well, coated overnight at 4 ℃ and washed 3 times with PBS-T. Add 200. mu.l of blocking solution to each well and block at 37 ℃ and wash 3 times with PBS-T. The monoclonal antibody purified in example 4 was prepared from 1:200 began a 2-fold gradient dilution, and finally 1 well was blank, incubated at 37 ℃ for 1h, and washed 3 times with PBS-T. HRP-labeled goat anti-mouse secondary antibody was diluted 1:20000 per well, incubated at 37 ℃ for 1h, and washed 3 times with PBS-T. Mu.l of a buffer containing 0.1% TMB (Sigma) and 0.03% H was added to each well₂O₂The reaction solution was developed in the citric acid phosphate buffer for 10min, and 50. mu.l of a 0.5M sulfuric acid solution was added to terminate the reaction. Measuring absorbance at 450nm with microplate reader, and plotting antibody dilution concentration and OD using GraphPad Prism v8 as data analysis plotting software₄₅₀The affinity was calculated corresponding to the curve.

Example 5 establishment of double antibody Sandwich ELISA method

5.1 pairing of monoclonal antibodies with enzyme-labeled monoclonal antibodies

The specific operation is as follows:

coating the purified antibody according to the concentration of 1 mu g/mL and 100 mu L/hole, and incubating overnight at 4 ℃;

blocking, namely blocking 1% BSA at 200 mu L/hole for 2h at 37 ℃;

adding SARS-CoV2-N standard protein (protein is subjected to heat treatment at 60 ℃) with the concentration of 5ng/mL, 0.5ng/mL and 0.05ng/mL in sequence according to 100 mu L/hole, incubating for 2h at 37 ℃, washing PBST for several times, adding enzyme-labeled antibody with the concentration of 100 mu L/hole, performing water bath at 37 ℃ for 1h, washing PBST for three times, and 5min for each time;

adding TMB color developing solution into the solution at a rate of 100 μ L/hole, and allowing the solution to act at room temperature for 15 min;

the reaction was stopped by adding 2M sulfuric acid at 50. mu.L/well, and the OD450 readings are shown in Table 2, which shows that the mAbs 64360A #4:27 and 64360A #5:42 were most effective in pairing.

TABLE 2 partial antibody pairing

5.2 determination of optimal coating concentration of monoclonal antibody and optimal working concentration of enzyme-labeled monoclonal antibody

And determining the optimal coating concentration of the monoclonal antibody and the optimal dilution of the enzyme-labeled secondary antibody by adopting a chessboard titration method. The coated antibody 64360A #4:27 was diluted to 5, 2, and 1. mu.g/mL with a coating solution (pH9.6 carbonate buffer), and then the mixture was applied to an ELISA plate in the longitudinal direction at 100. mu.L/well and coated overnight at 4 ℃. PBST is washed, then is blocked for 2h at 37 ℃ by 1% BSA, after PBST is washed, 100 mu L of standard N protein (the highest concentration is 5ng/mL, 3 times of gradient dilution, total 7 dilution gradients) which is subjected to heat treatment at 60 ℃ for 30min is added, incubation is carried out for 2h at 37 ℃, and PBST is washed and dried; serial dilutions of enzyme-labeled detection antibody 64360a #5:42(2mg/m1) were added at 1:5000, 1:10000, 1:20000, 3 replicates per dilution, 100 μ L/well, incubation for 1h at 37 ℃. After washing, 100. mu.L of TMB substrate solution was added to each well, incubated at 37 ℃ for 30min, and 2M H was added at 50. mu.L/well₂After SO4, the microplate reader reads the values. And fitting a four-parameter curve according to the concentration of the standard substance and the light absorption value of OD450nm, and selecting the monoclonal antibody coating concentration with the optimal curve parameters (including EC50 value, curve highest value and curve lowest value) and the enzyme-labeled polyclonal antibody dilution as the optimal working concentration. Finally, the optimal coating concentration of 64360A #4:27 was determined to be 1. mu.g/mL, and the optimal dilution concentration of enzyme-labeled antibody 64360A #5:42 was 1:10000.

Example 6ELISA kit Assembly

In the assembly of the double-antibody sandwich kit, the 64360A #4:27 monoclonal antibody is usedThe antibody is coated with coating buffer (Na) of pH9.4₂CO₃ 1.5g，NaHCO_3.2.9g, 1000mL of double distilled water) to 1-10. mu.g/mL, typically 1. mu.g/mL, 100. mu.L of coated antibody per well in a 96-well microplate, coating for 12 hours at 4 ℃ and discarding the coated antibody. After washing each well 3 times with 200. mu.L of PBS (pH7.4), 300. mu.L of 1% bovine serum albumin was added to each well as a blocking solution, incubated at 37 ℃ for 3 hours, washed 3 times with PBS, and then dried in a 25 ℃ drying oven for 12 hours. And (3) putting the dried ELISA plate into a sealable plastic bag, vacuumizing, adding a drying agent, and performing hot-pressing sealing for later use. The enzyme-labeled detection antibody in the kit is 64360A #5:42, the antibody is labeled by horse radish peroxidase or biotin, when labeling, 6mg HRP (horse radish peroxidase purchased from Sigma) is weighed and dissolved in 1mL of triple distilled water, 0.30mL of newly-prepared 0.1M NaIO is slowly added dropwise into each 1mL of solution₄The solution was stirred at 4 ℃ in the dark for 35min to activate HRP, changing the color from brown to green. The solution was filled into a dialysis bag and dialyzed overnight at 4 ℃ against 0.01M sodium acetate buffer pH4.4, and the color changed to brown-green. Centrifuging at 4 deg.C and 10000rpm for 10min to remove trace precipitate, adding 0.16M ethylene glycol (0.1 mL per mg enzyme) into dialyzed HRP, stirring at 4 deg.C in dark for 1h, adding purified polyclonal antibody, mixing, and dialyzing with 0.05M carbonate buffer (pH9.5) at 4 deg.C overnight to obtain HRP-antibody mixture. 0.1mL of 5mg/mL NaBH was added to the HRP-antibody mixture₄Solution (0.1-0.2 mg NaB per mg enzyme added)₄Stirring at 4 ℃ in dark for 3 h. Adding equal volume of saturated ammonium sulfate dropwise, stirring at 4 deg.C in dark for 2h, at 4 deg.C 10000rpm, centrifuging for 10min, and discarding the supernatant. The precipitate was dissolved in PBS to give a horseradish peroxidase-labeled enzyme-labeled antibody 64360A #5:42, 1/5000-1/20000 diluted, preferably at 1/10000.

EXAMPLE 7 method for measuring SARS-CoV-2-N protein content

1. Coating buffer solution is added into a 96-well enzyme label plate according to the concentration of 1 mu g/mL, 100 mu L of 64360A #4:27 monoclonal antibody of the invention is taken as a capture antibody in each well, the plate is washed 3 times by washing solution (PBS-T) after being coated for 12h at 4 ℃, 300 mu L of 2% (v/w) bovine serum albumin is added into each well as blocking solution, 37 DEG CIncubating for 3H for sealing, pouring back to remove liquid after sealing, washing the plate for 3 times by a washing solution (PBS-T), adding a sample to be detected and recombinant N proteins with the concentrations of 5ng/mL, 1.667ng/mL, 0.556ng/mL, 0.185ng/mL, 0.062ng/mL, 0.021ng/mL, 0.007ng/mL and 0ng/mL in sequence as standard substances into each hole, incubating for 1H at 37 ℃, pouring back to remove liquid, washing for 3 times, adding a pre-prepared 64360A #5:42 labeled by horseradish peroxidase as a detection antibody into each hole at 100 mu L/hole, incubating for 1H at 37 ℃, washing the plate for 6 times, adding 100 mu L of developer TMB solution into each hole, incubating for 8min at room temperature, and adding 2M H at 50 mu L/hole₂SO₄The reaction was stopped and the OD read₄₅₀The N protein concentration in the sample to be detected is calculated according to the standard curve, and the result of the standard curve in the attached figure 3 shows that the detection concentration of the ELISA detection kit prepared by the monoclonal antibody is 100 pg/mL.

Example 8 determination of variable region sequences of antibodies

Culturing fresh hybridoma cells, taking the supernatant, verifying the antigen binding properties of the cell line used for cloning, confirming that the cell line can indeed secrete the required antibody, and collecting 10 by centrifugation⁶The hybridoma cells described above. Total RNA from hybridoma cells was extracted by Trizol method, and 9. mu.l of total RNA was added to 2.5. mu.l of oligo (dT) 12-18 primer (10mM) and 5. mu.l of dNTPs, mixed well, incubated at 70 ℃ for 5 minutes and then placed on ice for 5 minutes, or denatured according to the reverse transcriptase used. Then, 5. mu.L of RT buffer (5X), 2.5. mu.L of DTT (0.1M) and 1. mu.L of reverse transcriptase were added and reacted at 42 ℃ for 1 hour. The reaction was terminated by incubation at 70 ℃ for 15 minutes, and the obtained cDNA was stored at-20 ℃. The obtained first strand cDNA was subjected to PCR amplification, and 25pmol each of primers was added to a 50. mu.L reaction system, and the sequences of the primers for amplification of the heavy chain variable region and the light chain variable region are shown in Table 3

TABLE 3 antibody variable region amplification primers

The rest dNTPs and buffer are added according to the conventional method, and finally 1 mu L of cDNA template and 1U of hot start Taq DNA polymerase are added. Setting PCR amplification program as 94 deg.c for 40 sec, 52 deg.c for 40 sec, 72 deg.c for 40 sec, 25 cycles of amplification, final extension at 72 deg.c for 3 min, and setting the product at 4 deg.c for use or direct electrophoresis. Taking 20 muL PCR product to carry out electrophoretic analysis, separating and cutting gel on 1.5% agarose gel for recovery, cloning the obtained heavy chain variable region and light chain variable region to pMD18T plasmid vector (TaKaRa) respectively, sequencing, analyzing the framework region, antigenic determinant (CDR) sequence and deduced amino acid sequence of the obtained DNA sequence of the heavy chain variable region of the antibody by using an on-line analysis tool, adding the constant regions of the heavy chain and the light chain into the sequence of the variable region of the antibody respectively at the downstream by using a gene cloning mode, obtaining an expression vector capable of recombining and expressing the antibody fragment by using a genetic recombination mode to express a recombined complete antibody or fragment in eukaryotic cells, and connecting the heavy chain variable region and the light chain variable region by using a coding sequence of a segment of interchain connecting peptide (such as GGSGGGGSGGGGS) to construct a single chain antibody to express the antibody fragment in proper host cells.

Sequence listing

<110> Beijing Huada protein research & development center, Ltd

<120> double-antibody sandwich ELISA kit for detecting new coronavirus N protein

<130> 1

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 10

<212> PRT

<213> Mus musculus

<400> 1

Gln Ser Val Ser Thr Ser Asn Tyr Asn Phe

1 5 10

<210> 2

<211> 3

<212> PRT

<213> Mus musculus

<400> 2

Tyr Ala Ser

1

<210> 3

<211> 9

<212> PRT

<213> Mus musculus

<400> 3

Gln His Asn Trp Glu Val Pro Arg Thr

1 5

<210> 4

<211> 8

<212> PRT

<213> Mus musculus

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Gly Phe Ala Phe Asn Tyr Tyr Leu

1 5

<210> 5

<211> 8

<212> PRT

<213> Mus musculus

<400> 5

Ile Asn Pro Gly Ser Gly Ser Thr

1 5

<210> 6

<211> 10

<212> PRT

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His Tyr Tyr Val Ser Ser Gly Met Asp Tyr

1 5 10

<210> 7

<211> 10

<212> PRT

<213> Mus musculus

<400> 7

Gln Ser Val Asp Tyr Asp Gly Asp Ser Tyr

1 5 10

<210> 8

<211> 3

<212> PRT

<213> Mus musculus

<400> 8

Thr Ala Ser

1

<210> 9

<211> 9

<212> PRT

<213> Mus musculus

<400> 9

Gln Gln Ser Asn Glu Asp Pro Leu Thr

1 5

<210> 10

<211> 8

<212> PRT

<213> Mus musculus

<400> 10

Gly Tyr Thr Phe Thr Asp Tyr Ser

1 5

<210> 11

<211> 8

<212> PRT

<213> Mus musculus

<400> 11

Ile Asn Thr Glu Thr Gly Glu Pro

1 5

<210> 12

<211> 8

<212> PRT

<213> Mus musculus

<400> 12

Thr Lys Ser Thr Ala Thr Ala Tyr

1 5

Claims

1. A pair of monoclonal antibodies prepared by an ELISA kit suitable for detecting the N protein of the novel coronavirus is characterized by being obtained by the following method:

preparing immunogen: purifying the SARS-CoV-2 virus N protein of prokaryotic expression and eukaryotic expression, and mixing with Freund's complete adjuvant and Freund's incomplete adjuvant after heat treatment at 60 ℃ for 30 minutes to prepare immunogen for cross immunization of mice;

the monoclonal antibody is a positive hybridoma cell subclone establishing strain, ascites is prepared and purified to obtain 22 monoclonal antibodies, and the 22 monoclonal antibodies are labeled by biotin;

and (3) pairing and screening the 22 monoclonal antibodies by pairing the coating antibody and the enzyme-labeled antibody to obtain the coating antibody 64360A #4:27 and the enzyme-labeled antibody 64360A #5:42 which are suitable for the double-antibody sandwich ELISA method for detecting the new coronavirus.

2. The coated antibody 64360A #4:27 of claim 1, consisting of:

(a) light chain variable region

(i) Comprises the amino acid sequence of SEQ ID NO: 01, or a CDR1 region of the amino acid sequence shown in seq id no.

(ii) Comprises the amino acid sequence of SEQ ID NO: 02, or a CDR2 region of the amino acid sequence shown in SEQ ID NO.

(iii) Comprises the amino acid sequence of SEQ ID NO: 03, and a CDR3 region.

(b) Heavy chain variable region

(i) Comprises the amino acid sequence of SEQ ID NO: 04, or a CDR1 region of the amino acid sequence shown in SEQ ID No.

(ii) Comprises the amino acid sequence of SEQ ID NO: 05 of the amino acid sequence shown in seq id No. 2.

(iii) Comprises the amino acid sequence of SEQ ID NO: 06, or a CDR3 region of the amino acid sequence shown in SEQ ID NO.

3. The detection antibody 64360A #5:42 of claim 1, consisting of:

(a) light chain variable region

(i) Comprises the amino acid sequence of SEQ ID NO: 07, or a CDR1 region of the amino acid sequence set forth in seq id no.

(ii) Comprises the amino acid sequence of SEQ ID NO: 08, or a CDR2 region of the amino acid sequence shown in seq id no.

(iii) Comprises the amino acid sequence of SEQ ID NO: 09, or a CDR3 region of the amino acid sequence shown in seq id no.

(b) Heavy chain variable region

(i) Comprises the amino acid sequence of SEQ ID NO: 10, CDR1 region.

(ii) Comprises the amino acid sequence of SEQ ID NO: 11, CDR2 region.

(iii) Comprises the amino acid sequence of SEQ ID NO: 12, or a CDR3 region of the amino acid sequence set forth in seq id no.

4. The ELISA detection kit method of claim 1 wherein the coating antibody is 64360A #4:27, the coating concentration is 5 μ g/mL-1 μ g/mL, and the optimal coating concentration is 1 μ g/mL.

5. The ELISA detection kit method of claim 1 wherein the detection antibody 64360A #5:42 is labeled with horseradish peroxidase or biotin at a dilution ratio of 1:5000 to 1:20000, preferably 1:10000, and the detection antibody is labeled with biotin at a dilution ratio of 1:5000 to 1:20000, preferably 1: 5000.

6. The ELISA test kit of claim 1 wherein the minimum limit of detection is 100 pg/mL.

7. The ELISA test kit of claim 6 can be used for the detection of N protein of the novel coronavirus SARS-CoV-2 without cross-reaction with SARS virus.