CN114574449A

CN114574449A - Hybridoma cell strain secreting monoclonal antibody against novel coronavirus N protein and application of hybridoma cell strain

Info

Publication number: CN114574449A
Application number: CN202210395330.XA
Authority: CN
Inventors: 杨帆; 刘万建; 杨致亭; 刘洋; 李文; 吴姝青; 王婷; 田永帅
Original assignee: Qingdao Shuojing Biotechnology Co ltd
Current assignee: Qingdao Shuojing Biotechnology Co ltd
Priority date: 2022-04-14
Filing date: 2022-04-14
Publication date: 2022-06-03
Anticipated expiration: 2042-04-14
Also published as: CN114574449B

Abstract

The invention belongs to the technical field of immunology and in vitro diagnosis, and relates to a hybridoma cell strain secreting a monoclonal antibody against a novel coronavirus N protein and application thereof, wherein the hybridoma cell strain is named as 16F3 and is preserved in China center for type culture Collection with the preservation number of CCTCC NO: C202113. the monoclonal antibody secreted by the hybridoma cell strain is an anti-new coronavirus N protein antibody. The hybridoma cell strain 16F3 and the passage cell strain thereof provided by the invention can stably secrete a monoclonal antibody for resisting novel coronavirus N protein, and the secreted antibody has high titer, high affinity, high specificity and high sensitivity; the antibody aims at novel coronavirus N protein, the subtype is IgG1, the subtype is single, and the antibody titer is high; the antibody can be specifically combined with the N protein of the novel coronavirus 2019-nCoV, and has good specificity.

Description

Hybridoma cell strain secreting monoclonal antibody against novel coronavirus N protein and application of hybridoma cell strain

Technical Field

The invention belongs to the technical field of immunology and in vitro diagnosis, and relates to a hybridoma cell strain capable of secreting a monoclonal antibody against a novel coronavirus N protein, a monoclonal antibody secreted by the hybridoma cell strain and application of the monoclonal antibody.

Background

The novel coronavirus causes respiratory symptoms such as fever, cough, shortness of breath and dyspnea, and can cause acute respiratory syndrome and even death in severe cases. At present, the main method for detecting the virus is a PCR technology, mRNA level is detected by PCR, the detection time is long, and 2-3 h is needed; the PCR detection has higher requirements on equipment, environment and operators, and the detection efficiency is lower; PCR detection has requirements on the timeliness of a sample, the detection needs to be finished within a certain time after the sample is collected, otherwise, the detection result is influenced; in addition, the cost of nucleic acid detection is relatively high.

The antibody detection method is characterized in that the detection is carried out on the protein level, the operation is simple and rapid, the detection time can be shortened to about 10min, and the detection efficiency is improved; however, the antibody detection is to collect serum, whole blood or plasma for detection, which is easily interfered by other antibodies in a sample, the sensitivity and accuracy of the product are relatively low, and missed detection and false positive are easily caused in the detection; the antibody exists in the cured patient and the patient with the recovered yang, and under the condition, the antibody detection method cannot distinguish the cured patient from the patient with the recovered yang; generally, the production of antibodies takes about 1 week, and antibody screening is rapid and convenient, but is not suitable for population screening and discovery in the early stage of infection.

Compared with antibody detection and nucleic acid detection methods, the antigen detection method can also use a throat swab sample, can screen in the early stage of virus infection, has low requirements on fields and equipment, can detect at any time and any place, improves the detection efficiency, and can discover and isolate as early as possible. Therefore, the preparation of the antibody with high titer, high sensitivity and specificity and the application of the antibody in detection have important significance.

Disclosure of Invention

The invention aims to provide a hybridoma cell strain secreting an anti-novel coronavirus N protein monoclonal antibody and application thereof.

The technical scheme of the invention is as follows:

the invention provides a hybridoma cell strain 16F3 secreting monoclonal antibody against novel coronavirus N protein, which is preserved in China center for type culture Collection (CCTCC for short) and is addressed to Wuhan, Wuhan university in China; the preservation number is CCTCC NO: C202113.

the invention also provides a preparation method of the hybridoma cell strain, namely, the gene recombinant protein of the novel coronavirus N protein is used for mouse immunization, B lymphocytes of spleen, thymus and limbs of an immunized mouse are taken for cell fusion with mouse myeloma cells, and the hybridoma cell strain is obtained by subcloning and screening and is named as 16F3, and the cell strain can stably secrete the anti-novel coronavirus N protein monoclonal antibody.

The invention also provides an anti-novel coronavirus N protein monoclonal antibody secreted and generated by the hybridoma cell strain 16F 3. The monoclonal antibody secreted by the hybridoma cell line 16F3 was designated monoclonal antibody 16F 3.

Furthermore, the monoclonal antibody is IgG1 subtype, and has specificity to the N protein of the novel coronavirus.

The invention also protects the application of the monoclonal antibody in detecting the novel coronavirus N protein.

Furthermore, the monoclonal antibody is used for preparing a detection reagent for detecting the novel coronavirus N protein.

The invention has the beneficial effects that:

the hybridoma cell strain 16F3 and the passage cell strain thereof provided by the invention can stably secrete a monoclonal antibody for resisting novel coronavirus N protein, and the secreted antibody has high titer, high affinity, high specificity and high sensitivity; the antibody aims at novel coronavirus N protein, the subtype is IgG1, the subtype is single, and the antibody titer is high; the antibody can be specifically combined with the N protein of the novel coronavirus 2019-nCoV, and has good specificity.

The biological material deposit information is as follows:

the hybridoma cell strain 16F3 is preserved in China center for type culture Collection (CCTCC for short), and the preservation unit address is the preservation center of Wuhan university, eight paths of Wuchang district in Wuhan City, Hubei province, China; the preservation number is CCTCC NO: c202113; the preservation time is 2020, 12 and 30 days.

Drawings

FIG. 1 shows the results of the measurement of titer and affinity of monoclonal antibodies against the N protein of the novel coronavirus;

FIG. 2 shows the result of detecting the titer consistency of the hybridoma cell strain 16F3 at different passage times;

FIG. 3 shows the results of the double antibody sandwich method;

FIG. 4 shows the results of detection by the colloidal gold method of an antibody.

Detailed Description

For further understanding of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments and the accompanying drawings, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The experimental procedures in the following examples, unless otherwise specified, were carried out in a conventional manner according to the conventional techniques in the art or according to the product specifications. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1

Preparation of hybridoma cell strain capable of secreting anti-novel coronavirus N protein monoclonal antibody

1. Immunization of mice

Female balb/c mice, 6 weeks old, were immunized with recombinant N protein of the novel coronavirus. The protein is mixed with equal volume of Freund's complete adjuvant according to the dosage of 100 mug/mouse, the emulsification is complete, and the mice are immunized subcutaneously in the back. Three weeks later, the immunization was boosted, and the protein was mixed with an equal volume of Freund's incomplete adjuvant at a dose of 50. mu.g/mouse, emulsified completely, and the mice were immunized subcutaneously at multiple sites on the back. After 1 week, the mice were subjected to tail-off blood collection to detect serum titer. Serum titer reaches over 5 ten thousand, and fusion is prepared. 3 days before fusion, mice were immunized by intraperitoneal injection without adjuvant, and the immunization dose was 100 μ g/mouse.

2. Cell fusion

In the process of preparing the hybridoma, the quality of fetal calf serum is adjusted, the fetal calf serum with good quality and stable batch is selected, the concentration of the fetal calf serum is optimized, high-quality myeloma cells and mouse immune lymphocytes are selected, and the cell fusion efficiency is enhanced.

During fusion, taking spleen, thymus and limb lymph nodes of an immunized mouse in a sterile environment, grinding dispersed cells, cracking erythrocytes by using an erythrocyte lysate, collecting mouse B lymphocytes and mouse myeloma cells sp2/0 after centrifugation, mixing according to the proportion of 5-10: 1, centrifuging, cleaning cells by using a serum-free DMEM medium to ensure that no fetal calf serum exists in the mixed cells, performing cell fusion by using 0.8ml of PEG in a water bath at 37 ℃, dropwise adding the PEG within 1min while gently mixing, and standing for 30s to ensure that the PEG fully acts.

After the fusion was completed, the fusion was terminated with 20ml of serum-free DMEM, and the addition was completed within 10min from slow to fast. After termination, the cells were incubated in a carbon dioxide incubator for 15min, centrifuged at 800rpm/min for 10min, the fused cells were resuspended in HAT medium, placed in a 96-well cell culture plate, and placed in a carbon dioxide incubator for culture.

3. Hybridoma cell screening and subcloning

On the fifth day after the fusion, the HAT medium was replaced with the whole medium, and on the seventh day, the culture supernatant was examined by indirect ELISA. The specific detection method comprises the following steps:

(1) coating: the recombinant N protein of the novel coronavirus was diluted to 0.5. mu.g/ml with CBS buffer, coated to an ELISA plate at 100. mu.g/well, and coated overnight at 4 ℃. The plate was washed 5 times with PBST buffer and the plate was patted dry.

(2) And (3) sealing: the microplate was blocked with 2% glycine in PBST buffer, 200. mu.g/well and incubated at 37 ℃ for 2 h. The plate was washed 5 times with PBST buffer and the plate was patted dry.

(3) And (3) detection: adding cell supernatant to be detected into an enzyme label plate at 100 mu g/hole, taking mouse immune serum as a positive control, taking HAT culture medium as a blank control, and incubating for 1h at 37 ℃. The plate was washed 5 times with PBST buffer and the plate was patted dry.

(4) Adding an enzyme-labeled secondary antibody: the goat anti-mouse antibody marked by the HRP is diluted by 2 ten thousand times by PBST buffer solution, added into an enzyme label plate, 100 mu g/hole and incubated for 1h at 37 ℃. The plate was washed 5 times with PBST buffer and the plate was patted dry.

(5) Color development: developing with TMB developing solution in dark, 100 μ g/well, incubating at 37 deg.C for 15 min; the termination was performed with 2M sulfuric acid stop solution, 50. mu.g/well, and detection was performed at a wavelength of 450 nm.

After the first screening is finished, the HAT culture medium is used again for replacing all the liquid, indirect ELISA screening is carried out every other day, and the screening results of two times are selected to be strong positive (OD)₄₅₀Stronger than positive control) cells were subcloned, the remaining positive wells (OD)₄₅₀>1) And (5) performing cell cryopreservation. And subcloning by adopting a multiple dilution method, taking 200-500 cells, transferring the cells to an A1 hole of a new 96-hole cell culture plate, and performing subcloning by respectively diluting the cells by 2 times in the longitudinal direction and the transverse direction. And detecting the culture supernatant by using an indirect ELISA method after seven days, selecting a positive hole with a single cell mass, and continuously subcloning until the positive rate of a 96-well plate reaches 100%. And continuously repeating the subcloning for 2 times, wherein the positive rate is 100%, and the subcloning is finished, so that the stability of the hybridoma cell strain is ensured.

4. Hybridoma cell line preservation

Selecting a hybridoma cell strain with stable growth, carrying out amplification culture, collecting cells when the cell density reaches 80%, using a cell cryopreservation solution to cryopreserve the cells, and storing the cells in a cell bank of the company; meanwhile, the culture medium is preserved, and the preservation number is CCTCC NO: C202113.

example 2

Preparation of monoclonal antibody against novel coronavirus N protein

1. Preparation of ascites

Selecting 12-week old balb/c female mice, injecting 500 μ l liquid paraffin into abdominal cavity of each female mouse, and injecting hybridoma cells into abdominal cavity after one week at dosage of 10⁶One/only. After 5 days, the mice were continuously observed, and after the abdominal distension was evident, ascites were collected, centrifuged at 12000rpm/min to remove impurities, and stored in a refrigerator at-80 ℃.

2. Purification of ascites

The ascites fluid was removed from the refrigerator, thawed, centrifuged and filtered through a 0.22 μm filter. Purification was performed with reference to the Bogelong protein-A column instructions. Collecting purified antibody, dialyzing with 20mM PB, collecting antibody after 48h, filtering with 0.22 μm filter membrane, subpackaging, and storing in-80 deg.C refrigerator for subsequent detection and verification.

The specific purification method is as follows:

(1) balancing: the Protein-A column was taken and equilibrated with 10 volumes of loading buffer.

(2) Loading: adding the processed ascites into a chromatographic column, and collecting the flow-through liquid by using a centrifugal tube. After all the sample passes through the column, the flow-through liquid is loaded again, and meanwhile, the flow-through liquid is collected and marked (in the loading process, the flow rate is controlled to be slow, so that the sample and the purification column are fully combined).

(3) Balancing: and after the sample loading is finished, balancing the column by using a sample loading buffer solution with 10 times of the column volume, and simultaneously detecting the outlet liquid by using a Coomassie brilliant blue reagent, wherein when the concentration of the liquid protein is lower than 1mg/ml, the balance is complete.

(4) And (3) elution: mu.l of neutralization buffer was added to 600. mu.l of EP tube in advance, and elution was carried out with 10 column volumes of elution buffer. During elution, 4-5 drops of eluent are collected in each tube and are mixed uniformly in time. The concentration of antibody in the eluate was measured with Coomassie Brilliant blue reagent (note the node where the start and end of the collection was collected) and labeled.

(5) Balancing: and (4) balancing by using 10 times of column volume of loading buffer, detecting the pH of the outlet liquid by using a pH test paper, and finishing the balancing when the pH of the outlet liquid is consistent with the pH of the loading buffer. The column was washed with 20% ethanol, stored in 20% ethanol, and stored at 4 ℃.

Example 3

Monoclonal antibody identification

1. Antibody titer and affinity assay

The titer and the affinity of the antibody are detected by adopting an indirect ELISA method, and the specific detection method comprises the following steps:

(1) coating: the recombinant N protein of the novel coronavirus was diluted to 1. mu.g/ml with CBS buffer, coated to an ELISA plate at 100. mu.l/well, and coated overnight at 4 ℃. The plate was washed 5 times with PBST buffer and the plate was patted dry.

(2) And (3) sealing: the microplate was blocked with 2% glycine in PBST buffer, 200. mu.l/well and incubated at 37 ℃ for 2 h. The plate was washed 5 times with PBST buffer and the plate was patted dry.

(3) And (3) detection: the antibody to be detected was diluted to 1mg/ml, from 1: starting at 1000, 12 gradients were diluted 2-fold and added to the microplate, 100. mu.l/well and incubated for 1h at 37 ℃. The plate was washed 5 times with PBST buffer and the plate was patted dry.

(4) Adding an enzyme-labeled secondary antibody: the goat anti-mouse antibody marked by HRP is diluted 2 ten thousand times by PBST buffer solution, added into an enzyme label plate, 100 mu l/hole and incubated for 1h at 37 ℃. The plate was washed 5 times with PBST buffer and the plate was patted dry.

(5) Color development: developing with TMB developing solution in dark, 100 μ l/well, incubating at 37 deg.C for 15 min; the termination was performed with 2M sulfuric acid stop solution, 50. mu.l/well, and detection was performed at a wavelength of 450 nm.

The detection results of the titer and affinity of the anti-novel coronavirus N protein monoclonal antibody are shown in figure 1, and the titer of the antibody reaches 10⁶。

2. Antibody subtype detection

The detection was carried out using an SBA subtype detection kit from Southern Biotech according to the instructions, and the specific detection method was as follows:

(1) coating: the coated antigen in the kit is diluted to 5 mu g/ml by CBS buffer solution, coated into an enzyme label plate at 100 mu l/hole and coated overnight at 4 ℃. The plate was washed 5 times with PBST buffer and the plate was patted dry.

(3) And (3) detection: the antibodies to be detected were added to the microplate (8 per antibody detected), 100. mu.l/well and incubated for 1h at 37 ℃. The plate was washed 5 times with PBST buffer and the plate was patted dry.

(4) Adding an enzyme-labeled secondary antibody: HRP-labeled goat anti-mouse antibodies (containing IgA-HRP, IgM-HRP, IgG1-HRP, IgG2a-HRP, IgG2b-HRP, IgG3-HRP, kappa-HRP, lambda-HRP) were diluted 500-fold with PBST buffer, added to an ELISA plate, 100. mu.l/well, and incubated at 37 ℃ for 1 h. The plate was washed 5 times with PBST buffer and the plate was patted dry.

The subtype was IgG1, as shown by the results of the assay. The results are shown in table 1 below:

TABLE 1 subtype test results

3. Antibody specificity detection

According to the indirect ELISA method in the 1, N protein fragments of the novel coronavirus and other viruses are respectively coated, the specificity of the antibody is good, and the antibody does not react with other viruses. The results are shown in table 2:

TABLE 2 results of specific detection

Viral strains	The result of the detection
		SARS-COV-2NP	2.253
SARS-COVNP	0.109
		OC43-COVNP	0.062
HKU1-COVNP	0.076
		His-tag	0.069

Example 4

Hybridoma cell secreted antibody stability detection

Subculturing the hybridoma 16F3, culturing for 30 generations, collecting culture supernatants at 1, 3, 6, 9, 12, 15, 18, 21, 24, 27 and 30 generations, detecting the quality control product of the novel coronavirus by ELISA method, and analyzing the stability of antibody secreted by the cells. The specific detection method comprises the following steps:

(3) And (3) detection: the cell supernatant to be detected was added to an ELISA plate at 100. mu.l/well and incubated at 37 ℃ for 1 h. The plate was washed 5 times with PBST buffer and the plate was patted dry.

(4) Adding an enzyme-labeled secondary antibody: the HRP-labeled goat anti-mouse antibody was diluted 2 ten thousand times with PBST buffer, added to an ELISA plate at 100. mu.l/well, and incubated at 37 ℃ for 1 h. The plate was washed 5 times with PBST buffer and the plate was patted dry.

As shown in table 3 and fig. 2, it was found that the cells after 30 passages had high consistency before each passage.

TABLE 316F 3 Titers results for different passage times

Example 5

Application of monoclonal antibody 16F3

1. Enzyme immunoassay

The antibody paired with the monoclonal antibody 16F3 is screened by a double antibody sandwich method, and the sensitivity of the paired antibody is detected. The specific detection method comprises the following steps:

(1) coating: monoclonal antibody 16F3 was diluted to 5. mu.g/ml with CBS buffer, coated 100. mu.l/well onto microtiter plates and coated overnight at 4 ℃. The plate was washed 5 times with PBST buffer and the plate was patted dry.

(3) Adding a quality control product: the recombinant N protein of the novel coronavirus is diluted to 5 mu g/ml, 1 mu g/ml, 0.2 mu g/ml, 0.04 mu g/ml and 100 mu l/well and added into an enzyme label plate, and the plate is incubated for 1h at 37 ℃. The plate was washed 5 times with PBST buffer and the plate was patted dry.

(4) Adding an enzyme-labeled antibody: the HRP-labeled antibody was diluted to 5. mu.g/ml with PBST buffer, added to the microplate, 100. mu.l/well, and incubated at 37 ℃ for 1 h. The plate was washed 5 times with PBST buffer and the plate was patted dry.

(5) Color development: developing with TMB developer in dark place, incubating at 37 deg.C for 15min at a concentration of 100 μ l/well; the termination was performed with 2M sulfuric acid stop solution, 50. mu.l/well, and detection was performed at a wavelength of 450 nm.

The detection results are shown in FIG. 3, and the linearity of antibody pairing detection is good.

2. Colloidal gold method

The monoclonal antibody 16F3 was labeled on gold particles and fixed on a specific conjugate pad, the other antibody (as a detection line) and the goat anti-mouse antibody (as a quality control line) were fixed on an NC membrane, and the conjugate pad, the sample pad, the NC membrane, and the absorbent paper were smoothly combined on a PVC plate in a certain order. The recombinant N protein of the novel coronavirus and the sample diluent are detected respectively.

The test results are shown in FIG. 4, the positive test results are good, and the test diluent has no false positive.

Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments described above, or equivalents may be substituted for elements thereof. Any modification, equivalent replacement, or modification made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A hybridoma cell strain 16F3 secreting monoclonal antibodies against novel coronavirus N proteins is preserved in China center for type culture Collection with the preservation number of CCTCC NO: C202113.

2. a monoclonal antibody against a novel coronavirus N protein, which is secreted from the hybridoma cell line 16F3 of claim 1.

3. The monoclonal antibody of claim 2, wherein the monoclonal antibody is of the IgG1 subtype.

4. Use of the monoclonal antibody of claim 2 for the detection of a novel coronavirus N protein.

5. The use according to claim 4, wherein the monoclonal antibody is used for the preparation of a detection reagent for the detection of the N protein of a novel coronavirus.