CN116751286A

CN116751286A - Monoclonal antibody pair for resisting novel coronavirus N protein and application thereof

Info

Publication number: CN116751286A
Application number: CN202310307286.7A
Authority: CN
Inventors: 张涛; 邹柳; 洪明凤; 盛布恩; 张松; 周国栋
Original assignee: Xiamen Tongrenxin Biology Technology Co ltd
Current assignee: Xiamen Tongrenxin Biology Technology Co ltd
Priority date: 2023-03-27
Filing date: 2023-03-27
Publication date: 2023-09-15

Abstract

The application relates to the technical field of biomedicine, and particularly discloses a monoclonal antibody pair for resisting novel coronavirus N protein and application thereof. The monoclonal antibody pair comprises two monoclonal antibodies; the monoclonal antibody comprises a heavy chain variable region and a light chain variable region; one monoclonal antibody in the pair was designated as the 7E8C9 antibody and the other monoclonal antibody was designated as the 10H1C7 antibody. The application also discloses a nucleic acid molecule for encoding the monoclonal antibody and a hybridoma cell strain for secreting the monoclonal antibody. In addition, the application also discloses a detection kit for detecting the novel coronavirus, which comprises the monoclonal antibody pair, wherein the 7E8C9 antibody is used as a capture antibody, and the 10H1C7 antibody is used as a labeled antibody. The monoclonal antibody for resisting the novel coronavirus N protein provided by the application has better specificity for the novel coronavirus N protein.

Description

Monoclonal antibody pair for resisting novel coronavirus N protein and application thereof

Technical Field

The application relates to the technical field of biomedicine, in particular to a monoclonal antibody pair for resisting novel coronavirus N protein and application thereof.

Background

Currently, there are two main methods for novel coronavirus detection: nucleic acid PCR detection (detection of viral RNA) and antigen rapid detection (detection of viral proteins). The nucleic acid PCR detection sensitivity is high, and the method is a gold detection standard. However, nucleic acid detection takes a long time (4-6 hours) and requires a specialized operator to perform and expensive PCR equipment. The novel coronavirus antigen chromatographic detection strip has the advantages of short time consumption (10-30 min), no need of professional operation and no need of special equipment, and can be used for basic medical institutions and home self-test markets.

The immunochromatography detection technology is an emerging immunoassay technology in the 90 th century, and is characterized in that an antigen-antibody immunological reaction and a chromatographic reaction are applied, and a dry-sheet test paper is used for achieving the purpose of rapidly and accurately developing colors to detect an object to be detected. The novel coronavirus antigen chromatographic detection strip is based on the principle that an antibody is coated and immobilized on a detection line (T line) of the test strip in advance, and a probe with an immune mark is combined with the detection line. The general probes are microparticles with colorimetric or fluorescent signals, such as colloidal gold, latex particles or fluorescent microspheres. The sample to be tested is added to the sample pad, and when the sample flows to the binding pad, the sample is combined with the probe through antigen-antibody interaction to form a sample-probe complex. When flowing to the T-line, the complex will be captured by the antibody on the T-line, forming a labeled sandwich complex, which develops the T-line.

The novel coronavirus nucleocapsid protein (Nucleocapsid protein, N protein) is one of the structural proteins mainly expressed in infection, the sequence is relatively conserved, and the novel coronavirus nucleocapsid protein is an ideal target for early diagnosis and detection of virus infection.

Disclosure of Invention

In order to improve the specificity detection of novel coronavirus N protein, the application provides a monoclonal antibody pair for resisting novel coronavirus N protein and application thereof.

In a first aspect, the present application provides a monoclonal antibody pair against a novel coronavirus N protein, which adopts the following technical scheme:

a pair of monoclonal antibodies against a novel coronavirus N protein, the pair of monoclonal antibodies comprising two monoclonal antibodies; the monoclonal antibody comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3; the light chain variable region includes a light chain CDR1, a light chain CDR2, and a light chain CDR3.

One monoclonal antibody in the monoclonal antibody pair is named 7E8C9 antibody, and the sequence information is as follows:

the heavy chain CDR1 comprises the amino acid sequence as set forth in SEQ ID NO:1 or an amino acid sequence as set forth in SEQ ID NO:1 or an amino acid sequence comprising the above sequence compared to an amino acid sequence having 1 or 2 conservative amino acid substitutions;

the heavy chain CDR2 comprises the amino acid sequence as set forth in SEQ ID NO:2, or an amino acid sequence as set forth in SEQ ID NO:2, or an amino acid sequence comprising the above sequence, with 1 or 2 conservative amino acid substitutions compared to the sequence shown in seq id no;

the heavy chain CDR3 comprises the amino acid sequence as set forth in SEQ ID NO:3, or an amino acid sequence as set forth in SEQ ID NO:3, or an amino acid sequence comprising the above sequence, with 1 or 2 conservative amino acid substitutions compared to the sequence shown in figure 3;

the light chain CDR1 comprises the amino acid sequence as set forth in SEQ ID NO:4, or an amino acid sequence as set forth in SEQ ID NO:4, or an amino acid sequence comprising the above sequence, with 1 or 2 conservative amino acid substitutions compared to the sequence shown in figure 4;

the light chain CDR2 comprises the amino acid sequence as set forth in SEQ ID NO:5, or an amino acid sequence as set forth in SEQ ID NO:5, or an amino acid sequence comprising the above sequence, with 1 or 2 conservative amino acid substitutions compared to the sequence shown in seq id no;

the light chain CDR3 comprises the amino acid sequence as set forth in SEQ ID NO:6, or an amino acid sequence as set forth in SEQ ID NO:6, or an amino acid sequence comprising the above sequence, with 1 or 2 conservative amino acid substitutions compared to the sequence shown in seq id no;

the other monoclonal antibody in the monoclonal antibody pair was designated as 10H1C7 antibody, and the sequence information was as follows:

the heavy chain CDR1 comprises the amino acid sequence as set forth in SEQ ID NO:7, or an amino acid sequence as set forth in SEQ ID NO:7, or an amino acid sequence comprising the above sequence, with 1 or 2 conservative amino acid substitutions compared to the sequence shown in seq id no;

the heavy chain CDR2 comprises the amino acid sequence as set forth in SEQ ID NO:8, or an amino acid sequence as set forth in SEQ ID NO:8, or an amino acid sequence comprising the above sequence, with 1 or 2 conservative amino acid substitutions compared to the sequence shown in seq id no;

the heavy chain CDR3 comprises the amino acid sequence as set forth in SEQ ID NO:9, or an amino acid sequence as set forth in SEQ ID NO:9, or an amino acid sequence comprising the above sequence, with 1 or 2 conservative amino acid substitutions compared to the sequence shown in seq id no;

the light chain CDR1 comprises the amino acid sequence as set forth in SEQ ID NO:10, or an amino acid sequence as set forth in SEQ ID NO:10, or an amino acid sequence comprising the above sequence, with 1 or 2 conservative amino acid substitutions compared to the sequence shown in seq id no;

the light chain CDR2 comprises the amino acid sequence as set forth in SEQ ID NO:11, or an amino acid sequence as set forth in SEQ ID NO:11, or an amino acid sequence comprising the above sequence, with 1 or 2 conservative amino acid substitutions compared to the sequence shown in seq id no;

the light chain CDR3 comprises the amino acid sequence as set forth in SEQ ID NO:12, or an amino acid sequence as set forth in SEQ ID NO:12, or an amino acid sequence comprising the above sequence, with 1 or 2 conservative amino acid substitutions.

In the 7E8C9 antibody, the heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:13, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:14, and a polypeptide having the amino acid sequence shown in seq id no.

In the 10H1C7 antibody, the heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:15, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:16, and a polypeptide having the amino acid sequence shown in seq id no.

In a second aspect, the present application provides a nucleic acid molecule, which adopts the following technical scheme:

a nucleic acid molecule encoding the amino acid sequence of the monoclonal antibody described above.

The sequence of the nucleic acid molecule encoding the heavy chain variable region of the 7E8C9 antibody comprises the sequence as shown in SEQ ID NO:17, and the sequence of the nucleic acid molecule encoding the monoclonal antibody light chain variable region comprises the nucleotide sequence set forth in SEQ ID NO:18, and a nucleotide sequence shown in seq id no.

The sequence of the nucleic acid molecule encoding the heavy chain variable region of the 10H1C7 antibody comprises the sequence as shown in SEQ ID NO:19, and the sequence of the nucleic acid molecule encoding the monoclonal antibody light chain variable region comprises the nucleotide sequence set forth in SEQ ID NO:20, and a nucleotide sequence shown in seq id no.

In a third aspect, the application provides a monoclonal antibody hybridoma cell strain resisting novel coronavirus N protein, which adopts the following technical scheme:

a hybridoma cell line of a monoclonal antibody against novel coronavirus N protein, which hybridoma cell line secretes said monoclonal antibody.

In a fourth aspect, the present application provides a detection kit for detecting a novel coronavirus, which adopts the following technical scheme:

a detection kit for detecting a novel coronavirus, the detection kit comprising the above monoclonal antibody pair against a novel coronavirus N protein.

Preferably, in the detection kit, the 7E8C9 antibody is used as a capture antibody and the 10H1C7 antibody is used as a labeling antibody.

In a fifth aspect, the present application provides the use of the monoclonal antibody pair, the nucleic acid molecule or the detection kit for detecting novel coronaviruses in the preparation of a reagent for detecting novel coronavirus N protein antigens.

In summary, the application has the following beneficial effects:

the application provides a monoclonal antibody pair for resisting novel coronavirus N protein, which is respectively named as a 7E8C9 antibody and a 10H1C7 antibody. Wherein, the 7E8C9 antibody is used as a capture antibody, and the 10H1C7 antibody is used as a labeled antibody, thereby having good specificity and sensitivity to novel coronavirus N protein.

Drawings

FIG. 1 is a schematic diagram of a gold-labeled strip.

FIG. 2 shows the detection of the Omikovia strains at different dilution factors by the colloidal gold chromatographic strip prepared by the application.

Fig. 3 shows the detection of delta strains at different dilution factors by the colloidal gold chromatographic strip prepared by the application.

Detailed Description

The application is described in further detail below with reference to the drawings and examples.

Examples

Example 1

The present example provides a method for preparing hybridoma cells and a preliminary screening process for positive hybridoma cells, including animal immunization, cellular immunization, screening of positive hybridoma cells, and cloning of positive hybridoma cells. Through screening, 12 monoclonal antibody hybridoma cell strains are obtained in total in the embodiment.

The method specifically comprises the following steps:

1. immunization of animals

The immunized subjects were 8-week-old BALB/C females.

The antigen used for immunization is a novel coronavirus N protein expressed by escherichia coli recombination, which comprises a sequence as shown in SEQ ID NO:37, and a nucleotide sequence shown in seq id no.

The immunization mode is specifically as follows:

(1) Preparation of the immunogen:

the novel coronavirus N protein expressed by escherichia coli is mixed with Freund's Complete adjuvant (F5881, sigma company) in equal volume to prepare a first immunogen;

the novel coronavirus N protein expressed by escherichia coli is mixed with Freund's Incomplete adjuvant (Freund's Adj μvant, incomplex, product number is F5506, sigma company) in an equal volume to prepare a second immunogen;

and mixing the novel coronavirus N protein recombinantly expressed by the escherichia coli with normal saline in an equal volume to prepare the third immunogen.

(2) Immunization process:

primary immunization: 50 μg of the first immunogen was injected at 3-4 points subcutaneously on the back of BALB/C female mice;

two weeks later, a second immunization was performed and 50 μg of the second immunogen was injected subcutaneously at 3-4 points on the back of BALB/C females;

two weeks later, a third immunization was performed and 50. Mu.g of the second immunogen was injected subcutaneously at 3-4 points on the back of BALB/C female mice;

two weeks later, a fourth immunization-impact immunization was performed, and 50 μg of the third immunogen was intraperitoneally injected into BALB/C female mice;

a fifth immunization was performed 24h later and 50 μg of the third immunogen was intravenously injected into the BALB/C female rat tail.

2. Cellular immunity

Cell immunization was initiated the third day after the fifth immunization of BALB/C female mice.

The cellular immune process specifically comprises the following steps:

(1) Preparation of spleen cell suspension: on the third day after the fifth immunization of the BALB/C female mice, the eyeballs of the BALB/C female mice are removed for blood sampling, and the serum of the BALB/C female mice is separated as a positive control in antibody detection; meanwhile, the cervical dislocation kills the BALB/c female mice, and spleen is taken to prepare spleen cell suspension;

preparation of myeloma cell suspension: recovering myeloma cells (ATCC, product number BNCC 100908) two weeks in advance (ensuring that myeloma cells are in logarithmic growth phase when in use) to obtain myeloma cell suspension;

preparation of feeder cells: the day before cell fusion, blank BALB/c female mouse peritoneal macrophages and spleen cells were taken and cultured in 96-well plates to obtain feeder layer cell-containing plates (cell concentration 1X 10) ⁴ Well), feeder cells are prepared;

(2) Cell fusion process:

using PEG (polyethylene glycol) to mediate cell fusion, taking spleen cell suspension and myeloma cell suspension, and the ratio of the cell number is 5:1 in a serum-free 1640 medium (cat No. C3010-0500, vivacell), centrifuging at 1200rpm for 5min, and removing supernatant;

flick the bottom of the centrifuge tube with finger to mix the two kinds of cells loosely, put in a beaker filled with 37 ℃ water for heat preservation, add 1ml 50% peg1500 (pH 8.0, cat No. 10783641001, rogowski) to fuse cells in 1min, shake while adding, and stand for 30s after adding; adding serum-free 1640 culture medium (product number C3010-0500, vivacell), centrifuging at 800rpm for 5min, suspending the precipitate with HAT culture medium, packaging into cell plate containing feeder cells to obtain cell plate containing fusion cell-feeder cells, and placing at 37deg.C and 5% CO ₂ Is cultured in a cell culture box;

wherein, the preparation of 500ml HAT medium requires the following reagents: 100ml of fetal bovine serum FBS (product No. 11011-8611, ilicis Purpureae), 5ml of Penicillin-streptomycin double antibody (Penicillin-streptomycin, 100×, product No. E607011-0100, shanghai, inc.), 10ml of HAT medium additive (HAT Media S μ sample, 50×, product No. H0262, sigma Co.), 385ml of 1640 medium (product No. C3010-0500, vivacell).

3. Screening of positive hybridoma cells

And culturing the cell plate containing the fused cell-feeder layer cells until the time of 4 days for half liquid exchange, continuously culturing until the time of 7 days for full liquid exchange, and screening positive holes by using a conventional indirect ELISA method when the fused cells cover 10-50% of the bottoms of the holes.

The indirect ELISA method specifically comprises the following steps:

(1) And (3) wrapping the plate: novel coronavirus N protein expressed by recombinant E.coli is used as coating antigen, and 0.05mol/L CB buffer (Na ₂ CO ₃ 31.8g，NaHCO ₃ 58.8g, 2L of ultrapure water) was added to the mixture to 2. Mu.g/ml, an ELISA plate was added at 100. Mu.L/well, the mixture was dried by shaking after coating at 4℃overnight, the mixture was blocked with 1% by mass/volume of gelatin-PBS buffer, and the mixture was blocked at 300. Mu.L/well, and the mixture was dried by shaking after blocking overnight at 4℃for further use.

(2) And (3) detection: cell culture supernatant in cell plates containing fused cell-feeder cells was added to an ELISA plate at 100. Mu.L/well, incubated at 37℃for 60min, washed with 0.01mol/L PBST buffer containing Tween-20 and then patted dry, 100. Mu.L/well of HRP-labeled goat anti-mouse secondary antibody (TJ-211229 CN, xiamen Taijing) was added, incubated at 37℃for 60min, washed and patted dry, 100. Mu.L/well of TMB chromogenic solution was added, incubated at 37℃for 10min in the absence of light, and 50. Mu.L/well of 1mol/LHCl was added to terminate the reaction.

Meanwhile, BALB/c female mouse serum obtained by eyeball blood sampling and separation in 'two-cell immunity' is used as a positive control, and fusion cells with higher antibody titer are screened out, namely positive hybridoma cells.

4. Cloning of positive hybridoma cells

Positive hybridoma cells obtained by screening from the cell plate containing the fusion cells-feeder cells are derived from more than two hybridoma cells, so that antibodies secreted by the hybridoma cells obtained by screening are of different qualities. In order to obtain fully homogeneous monoclonal antibodies, positive hybridoma cells need to be cloned.

The day before cloning, preparing feeder cells according to the method of step (1) in the step (II, cell immunity) and plating to obtain a cell plate containing the feeder cells; suspending positive hybridoma cells obtained by screening by using HT culture medium, blowing and mixing by using a liquid transfer device, inoculating to cell plates containing feeder cells, diluting cells in cell plate holes to 1 cell in each hole by using HT culture medium, placing at 37deg.C, 5% CO ₂ The antibody can be detected by wet-culturing for 7-10 days to obtain macroscopic cloned cells.

Wherein, the following reagents are required for preparing 500ml HT medium: 100ml of fetal bovine serum FBS (product No. 11011-8611, ilicis Purpureae), 5ml of Penicillin-streptomycin double antibody (Penicillin-streptomycin, 100X, product No. E607011-0100, shanghai, inc.), 10ml of HT medium additive (HT Media S. Mu. Pplens, product No. H0137, sigma Co.), 385ml of 1640 medium (product No. C3010-0500, vivacell).

Observing under an inverted microscope, marking out a hole in which only a single clone grows, finally primarily screening to obtain 12 monoclonal antibody hybridoma cell strains, and taking the supernatant for the next functional screening. The 12 monoclonal antibody hybridoma cell lines were designated and are specifically shown in table 1.

Table 112 monoclonal antibody hybridoma cell strain and antibody

Example 2

The present example provides a method for preparing monoclonal antibodies. Specifically comprises the preparation of ascites by utilizing hybridoma cells and the purification of monoclonal antibodies.

In this example, 12 monoclonal antibody hybridoma cell lines selected in example 1 were used to prepare monoclonal antibodies, and finally 12 monoclonal antibodies with excellent specificity and sensitivity were prepared. Specifically, the results are shown in Table 1.

The preparation method specifically comprises the following steps:

(1) Preparation of ascites

Abdominal ascites adjuvant (Beijing bolaolone, cat. No. KX 0210048) was injected into 8-10 week old Balb/c mice, and the monoclonal antibody hybridoma cell lines (1X 10) shown in Table 1 were obtained at 10d after injection ⁶ Individual cells/individual) were injected into the abdominal cavity of Balb/c mice, and the ascites of Balb/c mice was collected by a medical syringe over 12 d.

(2) Purification of monoclonal antibodies

Pouring the Balb/c mouse ascites collected in the step (1) into a centrifuge tube, centrifuging at 12500rpm for 20min, collecting supernatant, filtering with a 0.22 μm filter membrane, and purifying by affinity chromatography on a Protein A column (Protein ADiamond, product number AA301307, boge Long Shengwu).

The specific steps of affinity chromatography purification using Protein A column are as follows:

and (3) column loading: adding 5ml Protein A Resin medium into chromatographic column, standing, and washing the chromatographic column with 10 column volumes of ultrapure water;

balance: the chromatography column was equilibrated with 10 column volumes of pre-chilled Protein A column equilibration solution (50 mM Tris-HCl,100mM NaCl, water as solvent, pH 8.0);

loading: loading the sample filtered by the 0.22 mu m filter membrane, wherein the flow rate is 5ml/min;

washing: washing the chromatographic column with 10 column volumes of pre-chilled Protein a column equilibration solution;

eluting: eluting the antibody with an eluent (100mM Glycine,150mM NaCl, water as solvent, pH 3.0) to obtain an elution buffer comprising the antibody; immediately after elution, adding a neutralization buffer (2M Tris-HCl, water as solvent, pH 9.0) into the elution buffer until the solution is neutral pH;

and (3) dialysis: the eluted antibody was dialyzed three times in 1000 times the elution volume of PBS pH7.4 to obtain a purified antibody, and the concentration of the antibody was measured with an ultraviolet-visible spectrophotometer (Mv-5500 PC, shanghai meta-analysis instrument).

Example 3

The present example provides a method for preparing HRP-antibody conjugated complex proteins.

In this example, 12 monoclonal antibodies prepared in example 2 were labeled with HRP, respectively, to finally prepare 12 HRP-antibody-conjugated complex proteins. Specifically, the results are shown in Table 1.

The preparation method specifically comprises the following steps:

diluting the purified antibody to 2mg/mL with 0.05mol/L CB buffer with the pH value of 9.6; the PE glove is worn for operation, the dialysis membrane is selected according to the actual requirement and dialysis volume of the target protein molecular weight interception, and the proper length is cut; the dialysis membrane is soaked in 0.05mol/LCB buffer solution with the pH value of 9.6 in advance, and the dialysis membrane is soaked and washed once by replacing 0.05mol/L CB buffer solution with the pH value of 9.6. 1mL of the antibody protein solution was transferred to a dialysis membrane. The solution was dialyzed with stirring at 4℃in 0.05mol/L CB buffer at pH 9.6, once every 1 hour, and 5 times total dialysis.

Activation of HRP (hydroformylation): dissolving HRP (horseradish peroxidase, product number RS20220118, rasi reagent) in ultrapure water to obtain HRP solution with concentration of 20mg/mL, adding NaIO ₄ Dissolving in ultrapure water to prepare NaIO with the concentration of 20mg/mL ₄ A solution; after vortex is fully dissolved, HRP solution and NaIO are added ₄ The solutions were mixed at a volume of 1:1, i.e. NaIO ₄ The solution was slowly added to the HRP solution, immediately wrapped in tin foil, and the HRP was activated at 4 ℃ for 30min in the absence of light.

Terminating HRP activation: the ethylene glycol was slowly added dropwise to the HRP-activated centrifuge tube while gently shaking (1. Mu.L of ethylene glycol per 1mg of HRP) and continued to be protected from light at 4℃for 30min.

Adding the activated HRP solution to the antibody dialysis membrane (1 mg antibody to 1mg HRP and 1mg NaIO) ₄ ) The coupling was carried out overnight at 4℃in 0.05mol/LCB buffer at pH 9.6.

The next day, after a further change of 0.05mol/LCB buffer with a pH value of 9.6, the dialysis was continued for 2 hours. And transferring the coupled dialysate to a centrifuge tube after dialysis to obtain the antibody-HRP coupling solution.

Preparation of 20mg/mL NaBH with pure water ₄ The solution was added to the antibody-HRP conjugate solution in the above step in an amount of 2. Mu.L NaBH per 1mg of HRP ₄ The solution is reacted for 2 hours at the temperature of 4 ℃, and the solution is mixed evenly upside down every 0.5 hour to obtain the coupled composite protein.

The conjugated complex protein was precipitated with 50% ammonium sulfate (i.e., an equal volume of saturated concentration of ammonium sulfate was 1:1 mixed with the resulting conjugated complex protein solution), precipitated at 4℃for 15min, centrifuged at 10000rpm for 10min, and the resulting HRP-antibody conjugated complex protein was precipitated on one side of a centrifuge tube, and the supernatant was carefully aspirated with a pipette.

And (3) preserving: the HRP-antibody conjugated complex protein is blown or vortex dissolved and precipitated by a preservation solution and preserved at-20 ℃. The stock solution was 50% glycerol, 10% calf serum, 20mM PBS pH7.4.

Example 4

In this example, the 12 monoclonal antibodies prepared in example 2 and the 12 HRP-antibody conjugated complex protein prepared in example 3 were paired in pairs to screen antibodies capable of recognizing different epitopes of novel coronavirus N protein (including the amino acid sequence shown in SEQ ID NO: 37).

In this example, a double-antibody sandwich ELISA method was used, in which a monoclonal antibody was used as a capture antibody and HRP-antibody-conjugated complex protein was used as a labeled antibody. The method specifically comprises the following steps:

monoclonal antibodies (which are not subjected to HRP labeling) are used as capture antibodies and are respectively coated on the ELISA plates; then adding novel coronavirus N protein into the hole of the ELISA plate, and washing away unbound novel coronavirus N protein after incubation; adding HRP-antibody conjugated complex protein (subjected to HRP labeling) as a labeled antibody, and washing off unbound HRP-antibody conjugated complex protein after incubation; finally, adding a color development liquid for color development, and measuring the result at the wavelength of 280nm by using a spectrophotometer, wherein the detection result is shown in table 2.

Judging a detection result: if color development is possible, it indicates that the labeled antibody and the capture antibody can recognize different epitopes of the novel coronavirus N protein, indicating that the set of capture antibody and labeled antibody is a pair of paired antibodies. If color development is not possible, the labeled antibody cannot bind to the novel coronavirus N protein, and is eluted, and the group of capture antibodies and the labeled antibody are not paired antibodies.

TABLE 2 detection results of pairwise pairing of 12 monoclonal antibodies and 12 HRP-antibody-coupled composite proteins

As can be seen from Table 2, through the above-described test, a pair of monoclonal antibodies having high titers, including a 7E8C9 antibody and a 10H1C7 antibody, were finally selected. Wherein the 7E8C9 antibody is a capture antibody and the 10H1C7 antibody is a labeled antibody. The hybridoma cell producing the 7E8C9 antibody was designated as hybridoma cell 7E8C9, and the hybridoma cell producing the 10H1C7 antibody was designated as hybridoma cell 10H1C7.

Example 5

The present example provides the results of cell sequencing of hybridoma cell 7E8C9 and hybridoma cell 10H1C7.

Amplifying and culturing hybridoma cell 7E8C9 and hybridoma cell 10H1C7, respectively, and collecting 5×10 ⁶ The individual cells/ml were placed in a centrifuge tube, the supernatant was blotted dry, frozen and dry ice was sent to Nanjde bioengineering Co., ltd for hybridoma cell sequencing.

Sequencing results were as follows:

hybridoma cell 7E8C9

(1) Heavy chain sequence information

Leader sequence (base): comprising the amino acid sequence as shown in SEQ ID NO:18, and a nucleotide sequence shown in seq id no.

Leader sequence (amino acid): comprising the amino acid sequence as shown in SEQ ID NO:17, and a sequence of amino acids shown in seq id no.

Heavy chain full length base sequence: comprising the amino acid sequence as shown in SEQ ID NO:16, and a nucleotide sequence shown in seq id no.

Heavy chain full-length amino acid sequence: comprising the amino acid sequence as shown in SEQ ID NO:15, and a polypeptide having the amino acid sequence shown in seq id no.

Wherein the heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:13, and a nucleotide sequence shown in seq id no. The heavy chain variable region includes a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3. Heavy chain CDR1 includes the amino acid sequence as set forth in SEQ ID NO:1, and a polypeptide having the amino acid sequence shown in 1. Heavy chain CDR2 includes the amino acid sequence as set forth in SEQ ID NO:2, and a polypeptide having the amino acid sequence shown in 2. Heavy chain CDR3 includes the amino acid sequence set forth in SEQ ID NO:3, and a polypeptide having the amino acid sequence shown in 3.

Wherein the heavy chain constant region comprises the amino acid sequence as set forth in SEQ ID NO:14, and a polypeptide having the amino acid sequence shown in seq id no.

(2) Light chain sequence information

Leader sequence (base): comprising the amino acid sequence as shown in SEQ ID NO:24, and a nucleotide sequence shown in seq id no.

Leader sequence (amino acid): comprising the amino acid sequence as shown in SEQ ID NO: 23.

Full length base sequence of light chain: comprising the amino acid sequence as shown in SEQ ID NO:22, and a nucleotide sequence shown in seq id no.

Light chain full-length amino acid sequence: comprising the amino acid sequence as shown in SEQ ID NO:21, and a polypeptide comprising the amino acid sequence shown in seq id no.

Wherein the light chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:19, and a polypeptide comprising the amino acid sequence shown in seq id no. The light chain variable region includes a light chain CDR1, a light chain CDR2, and a light chain CDR3. Light chain CDR1 includes the amino acid sequence as set forth in SEQ ID NO:4, and a polypeptide having the amino acid sequence shown in (a) and (b). Light chain CDR2 includes the amino acid sequence as set forth in SEQ ID NO: 5. Light chain CDR3 includes the amino acid sequence set forth in SEQ ID NO:6, and a polypeptide having the amino acid sequence shown in FIG. 6.

Wherein the light chain constant region comprises the amino acid sequence as set forth in SEQ ID NO:20, and a polypeptide having the amino acid sequence shown in seq id no.

(II) hybridoma cell 10H1C7

(1) Heavy chain sequence information

Leader sequence (base): comprising the amino acid sequence as shown in SEQ ID NO:30, and a nucleotide sequence shown in seq id no.

Leader sequence (amino acid): comprising the amino acid sequence as shown in SEQ ID NO: 29.

Heavy chain full length base sequence: comprising the amino acid sequence as shown in SEQ ID NO:28, and a nucleotide sequence shown in seq id no.

Heavy chain full-length amino acid sequence: comprising the amino acid sequence as shown in SEQ ID NO: 27.

Wherein the heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:25, and a polypeptide comprising the amino acid sequence shown in seq id no. The heavy chain variable region includes a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3. Heavy chain CDR1 includes the amino acid sequence as set forth in SEQ ID NO: 7. Heavy chain CDR2 includes the amino acid sequence as set forth in SEQ ID NO:8, and a polypeptide having the amino acid sequence shown in FIG. 8. Heavy chain CDR3 includes the amino acid sequence set forth in SEQ ID NO: 9.

Wherein the heavy chain constant region comprises the amino acid sequence as set forth in SEQ ID NO:26, and a polypeptide comprising the amino acid sequence shown in seq id no.

(2) Light chain sequence information

Leader sequence (base): comprising the amino acid sequence as shown in SEQ ID NO:36, and a nucleotide sequence shown in seq id no.

Leader sequence (amino acid): comprising the amino acid sequence as shown in SEQ ID NO:35, and a nucleotide sequence shown in seq id no.

Full length base sequence of light chain: comprising the amino acid sequence as shown in SEQ ID NO:34, and a nucleotide sequence shown in seq id no.

Light chain full-length amino acid sequence: comprising the amino acid sequence as shown in SEQ ID NO:33, and a nucleotide sequence shown in seq id no.

Wherein the light chain variable region comprises the amino acid sequence as set forth in SEQ ID NO: 31. The light chain variable region includes a light chain CDR1, a light chain CDR2, and a light chain CDR3. Light chain CDR1 includes the amino acid sequence as set forth in SEQ ID NO:10, and a polypeptide having the amino acid sequence shown in FIG. 10. Light chain CDR2 includes the amino acid sequence as set forth in SEQ ID NO:11, and a polypeptide comprising the amino acid sequence shown in seq id no. Light chain CDR3 includes the amino acid sequence set forth in SEQ ID NO:12, and a polypeptide having the amino acid sequence shown in FIG. 12.

Wherein the light chain constant region comprises the amino acid sequence as set forth in SEQ ID NO:32, and a polypeptide having the amino acid sequence shown in seq id no.

Example 6

This example provides a colloidal gold chromatographic strip prepared using the 7E8C9 antibody and the 10H1C7 antibody.

The preparation method of the colloidal gold chromatographic strip specifically comprises the following steps:

1. preparation of detection pad

(1) Preparing a coating buffer solution: 2.901g NA was weighed out ₂ HPO ₄ ·12H ₂ O、0.2914g NAH ₂ PO ₄ ·2H ₂ O, 8.5g NaCl and 25g sucrose are dissolved in 1000ml ultra-pure water, the pH is 7.4, and the mixture is preserved at 4 ℃ for standby.

(2) And (3) preparing a detection line: 1mg/ml of 7E8C9 antibody was taken, a detection line was prepared on a nitrocellulose membrane, the spray amount was 1.5. Mu.l/cm, and the spray length was 30cm. The width is 1mm.

(3) Preparing a quality control line: a quality control line was prepared on a nitrocellulose membrane by taking 1mg/ml of goat anti-mouse IgG polyclonal antibody (Hangzhou long biotechnology Co., ltd.) at a spray rate of 1.5. Mu.l/cm and a spray length of 30cm. The distance between the quality control line and the detection line is 5mm.

(4) And (3) taking the nitrocellulose membrane coated with the detection line and the quality control line as a detection pad, attaching the detection pad to a PVC lining plate, and drying the PVC lining plate in a drying oven at 50 ℃ for 24+/-2 hours.

2. Labeling process of gold-labeled conjugate

(1) Preparing related solutions:

0.2M K ₂ CO ₃ preparing a solution: 2.7642g of anhydrous potassium carbonate was weighed out and dissolved in 100ml of pure water.

Preparation of 20% BSA solution: 20g BSA (bovine serum albumin, cat# V900933, sigma) and 100 μl proclin300 were weighed out in 100ml ultra pure water and stored at 4deg.C for further use.

Preparing a compound solution: 0.36g Tris, 0.3g sodium caseinate, 0.2g PEG20000, 100. Mu.l TW-20 (Tween-20), 2g sucrose, 100. Mu.l proclin300 were weighed out in 100ml ultra pure water, pH8.0, and kept at 4℃for further use.

(2) Marking process

1) 100ml of 0.01% colloidal gold solution (model number C805628, michelin) was placed in a clean container, and 100. Mu.l-300. Mu.l of 0.2M K was added ₂ CO ₃ Stirring the solution uniformly;

2) 150-500 μg of 10H1C7 antibody is added and stirred for reaction for 1-2H;

3) Adding 500 μl of 20% BSA solution, stirring, and sealing for 30min;

4) And (3) centrifuging: centrifuging at 10000r/min for 15min, and discarding supernatant;

5) And (3) re-dissolving: concentrating and redissolving the centrifuged precipitate by 40 times of a redissolution to obtain a labeled detection label conjugate, and storing the labeled detection label conjugate at 4 ℃ for later use.

3. Preparation of bond pads

(1) Preparing a gold-labeled conjugate solution: 0.362g Tris, 0.2g sodium caseinate, 0.2g PEG20000, 100. Mu.l TW-20, 3g sucrose, 2g trehalose, 100. Mu.l proclin300 were dissolved in 100ml ultra pure water, pH8.0 and kept at 4℃for further use.

(2) Adding the labeled detection label conjugate into the gold label conjugate solution according to the concentration of 7-15%, uniformly mixing, uniformly coating on a glass fiber membrane (35 ml/sheet), and drying in an oven at 50 ℃ for 24+/-2 hours.

4. Preparation of sample pad

(1) Preparing sample pad treatment liquid: 0.242g Tris, 0.5g sodium caseinate, 0.1g Pvp-40, 500. Mu.l TW-20, 100. Mu.l proclin300 were dissolved in 100ml ultra pure water, pH8.0 and kept at 4℃for further use.

(2) And uniformly coating the sample pad treatment liquid of 32ml per plate on a glass fiber membrane, drying in an oven at 50 ℃ for 24+/-2 hours, and sealing and preserving at room temperature for standby after detection and identification are qualified.

5. Cutting of absorbent paper

Cutting the absorbent paper into pieces of 25-35cm long and 1.7+ -0.05 cm wide by a paper cutter, and placing in a drying room (drying room: daytime temperature is controlled at 30-35deg.C, night is controlled at 35-40deg.C, and humidity is controlled at 10-30%) for use.

6. Assembly of gold-labeled paper (workshop temperature is controlled at 18-28 ℃ and humidity is 10-30%)

FIG. 1 is a schematic diagram of a gold-labeled strip.

According to the structure shown in fig. 1, a sample pad and a bonding pad are sequentially stuck to one end of a PVC board, which is close to a detection line, and the sample pad is partially overlapped on the bonding pad which is partially overlapped on the detection pad; and (3) adhering the water absorbing paper to one end of the PVC plate far away from the detection line, and partially overlapping the water absorbing paper on the detection pad. After the large plate is formed, the large plate is cut into thin strips according to the width of 3 mm.

Example 7

In this example, the colloidal gold chromatographic strip prepared in example 6 was used for virus detection, and the sensitivity of the colloidal gold chromatographic strip was detected. The subjects were inactivated omikun strain virus broth (Beijing card Mei De Biotechnology Co., ltd.) and inactivated delta strain virus broth (Nanj Bai Biotechnology Co., ltd.).

(1) Sensitivity test of Omikou strain

The titer of the inactivated Omikovia strain virus culture solution is 1.4X10 ⁵ TCID ₅₀ Per mL, using dilutions (composition: 10mM PBS,2% BSA,0.1% Tween 20) in the order 1: 500. 1: 1000. 1: 2000. 1: 4000. 1: after 10000 dilutions, the colloidal gold chromatographic strip prepared in example 6 was used to detect the results.

The detection method comprises the following steps: and (3) dripping 100 mu L of diluted solution on the sample pad, and observing the color development condition of the detection line and the quality control line on the detection pad after 5-10 min. The color development results at the above-mentioned different dilution factors are shown in FIG. 2.

As can be seen from fig. 2, in the armikovia strain 1: the detection bands are also clearly observed after 4000 dilutions, in the amikacin strain 1: weak detection bands were also observed after 10000 dilutions. The lowest detection of the Omikovia strain of the colloidal gold test strip is shown as 14 xTCID ₅₀ /mL。

(2) Delta strain sensitivity test

Titer of inactivated delta strain virus culture medium was 1.0X10 ⁶ TCID ₅₀ Per mL, using dilutions (composition: 10mM PBS,2% BSA,0.1% Tween 20) in the order 1: 5000. 1: 10000. 1: 20000. 1: 40000. 1: after 100000 dilution, the colloid prepared in example 6 was usedAnd (5) detecting a result by using a gold chromatographic strip.

The detection method comprises the following steps: and (3) dripping 100 mu L of diluted solution on the sample pad, and observing the color development condition of the detection line and the quality control line on the detection pad after 5-10 min. The color development results at the above-mentioned different dilution factors are shown in FIG. 3.

As can be seen from fig. 3, in delta strain 1: the detection bands were also evident after 40000 dilution, at the armstrong strain 1: weak detection bands were also observed after 100000 dilution. The titer of the lowest detection delta strain of the colloidal gold test strip is 10 multiplied by TCID ₅₀ /mL。

Example 8

In this example, the colloidal gold chromatographic strip prepared in example 6 was used for virus detection, and the specificity of the colloidal gold chromatographic strip was detected. The detection object is common inactivated virus culture solution. The test results are shown in Table 3.

TABLE 3 specificity detection results of the colloidal gold chromatography strips of the application

As shown in the detection results of Table 3, the colloidal gold chromatographic strip provided by the application is negative to the detection results of common viruses in Table 3, and the combination of the detection results of example 7 shows that the colloidal gold chromatographic strip has better specificity to novel coronavirus N protein.

The present embodiment is only for explanation of the present application and is not to be construed as limiting the present application, and modifications to the present embodiment, which may not creatively contribute to the present application as required by those skilled in the art after reading the present specification, are all protected by patent laws within the scope of claims of the present application.

Claims

1. A monoclonal antibody pair for resisting novel coronavirus N protein is characterized in that,

the monoclonal antibody pair comprises two monoclonal antibodies; the monoclonal antibody comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3; the light chain variable region comprises a light chain CDR1, a light chain CDR2, and a light chain CDR3;

2. The pair of monoclonal antibodies against novel coronavirus N protein of claim 1, wherein in the 7E8C9 antibody, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:13, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:14, an amino acid sequence shown in seq id no; in the 10H1C7 antibody, the heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:15, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:16, and a polypeptide having the amino acid sequence shown in seq id no.

3. A nucleic acid molecule encoding the amino acid sequence of the monoclonal antibody of claim 1 or 2.

4. The nucleic acid molecule of claim 3, wherein the sequence encoding the heavy chain variable region of the 7E8C9 antibody comprises the sequence set forth in SEQ ID NO:17, and the sequence of the nucleic acid molecule encoding the monoclonal antibody light chain variable region comprises the nucleotide sequence set forth in SEQ ID NO:18, a nucleotide sequence shown in seq id no; the sequence of the nucleic acid molecule encoding the heavy chain variable region of the 10H1C7 antibody comprises the sequence as shown in SEQ ID NO:19, and the sequence of the nucleic acid molecule encoding the monoclonal antibody light chain variable region comprises the nucleotide sequence set forth in SEQ ID NO:20, and a nucleotide sequence shown in seq id no.

5. A monoclonal antibody hybridoma cell line against a novel coronavirus N protein, wherein said hybridoma cell line secretes the monoclonal antibody of claim 1 or 2.

6. A test kit for detecting a novel coronavirus, comprising the pair of monoclonal antibodies against the novel coronavirus N protein of claim 1 or 2.

7. The kit for detecting a novel coronavirus according to claim 6, wherein the 7E8C9 antibody is used as a capture antibody and the 10H1C7 antibody is used as a labeled antibody.

8. Use of the monoclonal antibody pair of claim 1 or 2, the nucleic acid molecule of claim 3 or 4 or the detection kit for detecting novel coronavirus of claim 6 or 7 for preparing a reagent for detecting novel coronavirus N protein antigen.