CN110257343B

CN110257343B - Hybridoma cell strain, monoclonal antibody produced by hybridoma cell strain and application of monoclonal antibody

Info

Publication number: CN110257343B
Application number: CN201910585669.4A
Authority: CN
Inventors: 孙冠如; 安晓红; 张立民
Original assignee: Beijing Haisikai Biological Technology Co ltd
Current assignee: Beijing Haisikai Biological Technology Co ltd
Priority date: 2019-07-01
Filing date: 2019-07-01
Publication date: 2021-08-20
Anticipated expiration: 2039-07-01
Also published as: CN110257343A

Abstract

The invention discloses a hybridoma cell strain, a monoclonal antibody generated by the hybridoma cell strain and application of the monoclonal antibody. The hybridoma cell line secretes a monoclonal antibody that binds to 11dhTxB 2. The monoclonal antibody for resisting the 11dhTxB2 antigen prepared by the invention has high titer, good activity, high sensitivity, strong specificity and good stability. The monoclonal antibody of the invention has high stability, and can maintain high stability and reliability under severe environmental conditions, which is a valuable progress in clinical and laboratory applications.

Description

Hybridoma cell strain, monoclonal antibody produced by hybridoma cell strain and application of monoclonal antibody

Technical Field

The invention relates to the field of biological agents, and particularly relates to a hybridoma cell strain, a monoclonal antibody generated by the hybridoma cell strain and application of the hybridoma cell strain.

Background

Aspirin is an ancient analgesic and antipyretic drug, which was proposed as early as 50 s to prevent arteriosclerosis. It has been demonstrated that people with risk factors for coronary heart disease and myocardial infarction (e.g., hyperlipidemia, hypertension, smoking, diabetes, hormonal imbalance, stress, etc.) often have abnormal platelet function, manifested by increased platelet adhesion and aggregation, which is associated with the formation of microthrombosis in the atherosclerotic lesions. In addition, the increased transient platelet aggregation and release responses in the coronary circulation are a factor in the onset and persistence of coronary artery spasm.

Aspirin is a potent platelet inhibitor, and can inhibit the synthesis of thromboxane A2(TxA2), and has significant effects of inhibiting the adhesion, aggregation and release of platelets, thereby preventing microthrombosis, atherosclerosis and myocardial infarction. Research has proved that aspirin can significantly reduce early mortality and re-infarction rate of myocardial infarction. But recent studies have shown that not all individuals respond consistently to aspirin. About 30% of individuals who take drugs are resistant to aspirin, i.e., do not function as often, and therefore monitoring aspirin efficacy becomes more critical and important.

Direct measurement of patient response to aspirin refers to the determination of circulating levels of TxA2, unfortunately TxA2 has a short life in blood and thus TxA2 is difficult to analyze, but it is enzymatically decomposed into a number of metabolites including 11-dehydrothromboxane B2(11dhTxB2) and 11-dehydro-2, 3-dinor thromboxane B2(11dh2,3DTxB2), which are absorbed by the kidney and excreted in the urine. 11dhTxB2 is very stable in urine, is a biologically inactive substance, is a downstream metabolite of TxA2, has a long half-life, and is not affected by platelet activity and other analytical variables in vivo. Experts point out that the detection of the 11dhTxB2 level in urine can more directly reflect the inhibition effect of aspirin on platelet aggregation, the detection is noninvasive, the operation is simple, the consumption of manpower and material resources is low, and the analysis variation degree is small, so that the method is superior to other functional detection. Studies have now demonstrated that 11dhTxB2 levels in urine are significantly correlated with aspirin hyporesponsiveness and the occurrence of cardiovascular events in patients and can be used to assess the clinical efficacy of aspirin. Therefore, the hybridoma cell strain secreting the 11dhTxB2 monoclonal antibody with high specificity and high sensitivity is an important prerequisite for application of the hybridoma cell strain to 11dhTxB2 immunological detection.

Disclosure of Invention

In order to solve the problems, the invention provides a hybridoma cell strain, wherein the hybridoma cell strain has a preservation number of: CGMCC NO.17700, which secretes a monoclonal antibody that binds 11dhTxB 2. The hybridoma cell strain is preserved in China General Microbiological Culture Collection Center (CGMCC), and is classified and named as: 11 dehydrothrombus B2 cell line, the preservation date is: 6, 3.2019, the preservation address is: xilu No.1, Beijing, Chaoyang, Beijing, and institute for microbiology, China academy of sciences.

In one embodiment, the monoclonal antibody is of the IgG2b, kappa type.

In one embodiment, the hybridoma cell line is prepared by:

step 1: preparing 11dhTxB2-KLH or 11dhTxB2-BSA coupled antigen by using KLH protein or BSA protein;

step 2: immunizing animals, mixing the coupled antigen with Freund complete adjuvant or incomplete adjuvant, emulsifying to form water-in-oil state, immunizing for the first time with Freund complete adjuvant, enhancing immunity with incomplete adjuvant, and immunizing animals;

and step 3: cell fusion, namely taking spleen cells of the immune animals, and fusing the spleen cells with myeloma cells SP2/0-Ag14 to prepare fused cells; adding feeder layer cells into the fused cells, and culturing by using HAT complete culture medium; and

and 4, step 4: screening hybridoma cells, detecting an antibody of cell culture supernatant, selecting a cell hole with a high OD value, detecting the inhibition rate of 11dhTxB2 micromolecules, cloning to obtain a stable hybridoma cell strain, and selecting the stable hybridoma cell strain with the preservation number: a hybridoma cell strain of CGMCC NO. 17700.

In one embodiment, PEG, preferably PEG4000, is added to the cell fusion in step 2.

In one embodiment, a monoclonal antibody that binds to 11dhTxB2 produced by a hybridoma cell line having a accession number of CGMCC No.17700 is provided.

In one embodiment, the monoclonal antibody further includes fragments of the monoclonal antibody and derivatives thereof.

In one embodiment, the invention provides the use of a monoclonal antibody as described above in the preparation of a reagent or kit for evaluating aspirin as a platelet inhibitor.

The invention adopts a hybridoma monoclonal antibody technology, a BALB/c mouse is immunized by a synthesized 11dhTxB2 antigen, the mouse with higher antibody titer is selected for cell fusion after serum is screened, 1 hybridoma cell strain capable of stably secreting the antibody is screened by a limiting dilution method after fusion, then a purified antibody is prepared, the titer of the antibody is detected by ELISA, a detection kit is prepared to detect a clinical sample, the detection kit is continuously prepared to detect the clinical sample after repeated freeze thawing and heat acceleration treatment, the prepared monoclonal antibody capable of secreting the 11dhTxB2 is proved to be capable of being specifically combined with a target antigen, and the antibody is a monoclonal antibody with high titer, high sensitivity, high specificity and high stability.

The monoclonal antibody for resisting the 11dhTxB2 antigen prepared by the invention has high titer, good activity, high sensitivity, strong specificity and good stability. The monoclonal antibody of the invention has high stability, and can maintain high stability and reliability under severe environmental conditions, which is a valuable progress in clinical and laboratory applications.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments described in the present application, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.

Fig. 1 is a graph of the 11dhTxB2 small molecule inhibition of the invention.

Detailed Description

In order to make the technical solutions in the present application better understood, the present invention will be further described with reference to the following examples, and it is obvious that the described examples are only a part of the examples of the present application, but not all of the examples. 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 application.

EXAMPLE preparation of hybridoma cell line 16C8

The invention finally obtains the monoclonal antibody hybridoma cell strain with high sensitivity to 11dhTxB2 by immunizing a mouse with 11dhTxB2-KLH (or 11dhTxB2-BSA) antigen, fusing cells, culturing in HAT selective culture medium, and screening cell supernatant by indirect ELISA and indirect competition ELISA.

1. Antigen preparation

Weighing 3mg of KLH protein (or BSA protein), and preparing 20mg/mL of KLH protein solution by using 0.1M potassium phosphate (pH7.4); weighing 1mg of 11dhTxB2 micromolecules, and adding 500 mu L DMSO for dissolving; adding 11dhTxB2 micromolecules into KLH protein (or BSA protein) solution, and uniformly mixing by using an oscillator; weighing EDC5mg, directly adding into KLH protein (or BSA protein) solution, placing in a shaker overnight for 12h in total; dialyzing with 0.01M PBS for 3 times in ice bath, each time for 3 h; NANODROP2000 measures 11dhTxB 2-KLH-conjugated antigen concentration.

2. Animal immunization

11dhTxB2-KLH (or 11dhTxB2-BSA) coupled antigen is used for immunizing 10 SPF-grade BALB/c female mice with the age of 6 weeks, the antigen is mixed and emulsified with Freund's complete adjuvant (first immunization) or incomplete adjuvant (booster immunization) according to the proportion of 1:1 to form a water-in-oil state, the mice are immunized through subcutaneous multiple points at the back of the neck of the mice for 2 times of booster immunization, each time of immunization is separated by 2 weeks, serum is collected 7 days after the last booster immunization, and the titer and the inhibition rate are detected.

3. Cell fusion

3.1. Abdominal feeder layer cell preparation

Injecting 5ml of incomplete culture solution into the abdominal cavity of the mouse one day before fusion, slightly shaking, extracting the abdominal cavity solution, centrifuging at 1000rpm, discarding the supernatant, suspending the precipitate with a small amount of culture solution, and counting cells for later use;

3.2. spleen cell suspension preparation

Dissecting a mouse, taking a spleen, dispersing splenocytes by a mechanical method, filtering by a filter screen to obtain splenocyte suspension, washing with incomplete culture solution, and counting;

3.3. cell fusion

Mixing splenocytes and myeloma cells SP2/0-Ag14 in a 50mL centrifuge tube according to the ratio of 10:1-5:1, adding incomplete culture solution to 30mL, and mixing thoroughly; centrifuging at 1000rpm for 5min, and discarding the supernatant; lightly vibrating to make the precipitated cells loose and uniform into paste; dropwise adding 1mL of 50% PEG4000 preheated to 37 ℃ into the mixture along the tube wall by using a 1mL pipette within about 1min, and standing for 30 s; sucking the incomplete culture medium by using a 1mL pipette, dropwise adding the incomplete culture medium, and slowly stopping the action of PEG 4000; centrifuging at 800rpm for 5min, and discarding the supernatant; adding 25ml feeder layer cells into the fused cells, paving a 96-well plate with a HAT complete culture medium for 20 plates, changing HAT half amount of liquid after 4 days, observing the cells after 6-7 days, and detecting cell supernatant;

3.4. hybridoma cell selection

And (3) detecting cell supernatant: detecting the antibody of the cell culture supernatant by adopting an enzyme-linked immunosorbent assay (ELISA), selecting a cell hole with a higher OD value, and detecting the inhibition rate of 11dhTxB2 micromolecules; cloning: observing positive cells under a microscope, and counting; calculating the volume number of cell suspension liquid required by 100 cells paved on each plate, adding the cell suspension liquid into a plate of 20mL HT complete culture medium, uniformly mixing, paving the plate, adding 200 mu L/hole of cell suspension liquid with the same volume, and culturing at 37 ℃ under the condition of 5% CO 2; observing the cells after 7-10 days, repeating the steps, and carrying out next cloning; after 3 times cloning, the cell cloning density was adjusted to 0.3/well cell cloning 4 plate, 3/well cell cloning 2 plate, 30/well cell cloning 1 plate. After 3-4 times of subcloning, ELISA detection achieves full positive and stable inhibition rate, and the cell is fixed, frozen, preserved and expanded for culture. As a result, 9 hybridoma cell lines were obtained altogether, and the supernatant was subjected to hybridoma cell supernatant inhibition assay shown in Table 1, wherein the inhibition assay was performed using standards containing 0ng/ml 11dhTxB2 and 1ng/ml 11dhTxB2, respectively, and the results showed that 16C8 showed the strongest competitive inhibition effect, and therefore 1 hybridoma cell line (16C8) was selected as the cell line prepared using the monoclonal antibody, and the secreted monoclonal antibody was determined to be of IgG2b, kappa type by using the antibody typing reagent.

Table 1: inhibition rate of hybridoma cell supernatant

Hybridoma cell strain	OD(0ng/mL)	OD(1ng/mL)
			3G7	0.6	0.376
9C7	0.6	0.466
			8G8	1.828	1.549
6G5	0.88	0.465
			9D4	0.444	0.277
4C9	0.932	0.644
			27B5	0.629	0.392
14C6	0.579	0.36
			16C8	0.961	0.279

4. Antibody purification and Performance testing

The mouse was intraperitoneally injected with liquid paraffin, the hybridoma cell line 16C8 was inoculated into the abdominal cavity of the mouse one week later, the abdominal enlargement of the mouse was observed after about 7 to 8 days, the abdominal ascites was carefully extracted using a 10mL syringe, and the Protein A-Sepharose affinity column was equilibrated with 10mL of a starting buffer (20mM phosphate buffer pH 7.0). And (3) taking 10-25 mL of ascites to be purified to pass through the column, washing with 7-8mL of initial buffer solution, 6-7mL of elution buffer solution and 5mL of initial buffer solution in sequence, and collecting 1mL of eluent in each tube.

5. Antibody titer determination

The titer of the anti-11 dhTxB2 monoclonal antibody was determined by ELISA and the results are shown in table 2. The titer of the anti-11 dhTxB2 monoclonal antibody was determined by ELISA to determine the optimal antibody concentration for inhibition experiments, usually with OD values less than 2, and the optimal final concentration of antibody for ELISA was 31.25ng/mL as shown in the test results in Table 2.

Table 2: antibody titer determination

Antibody concentration	OD value
		4000ng/mL	2.575
2000ng/mL	2.552
		1000ng/mL	2.539
500ng/mL	2.511
		250ng/mL	2.413
125ng/mL	2.294
		62.5ng/mL	2.021
31.25ng/mL	1.781
		0ng/mL	0.086

6. Determination of antibody inhibition Rate

The result of measuring the antibody inhibition rate of the anti-11 dhTxB2 monoclonal antibody by using an indirect competition ELISA method is shown in Table 3 and figure 1, and the result shows that the detection sensitivity to 11dhTxB2 is very good and can reach 78.125 pg/mL.

Table 3: determination of 11dhTxB2 small molecule inhibition rate

11dhTxB2 Small molecule concentration	OD value
		0pg/mL	1.647
78.125pg/mL	1.322
		156pg/mL	1.099
312.5pg/mL	0.858
		625pg/mL	0.616
1250pg/mL	0.419
		2500pg/mL	0.284
5000pg/mL	0.191

It is to be understood that the invention disclosed is not limited to the particular methodology, protocols, and materials described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

1. A hybridoma cell strain is characterized by having a preservation number of: CGMCC NO.17700, which secretes a monoclonal antibody that binds 11dhTxB 2.

2. The hybridoma cell line of claim 1, wherein the monoclonal antibody is of the IgG2b, kappa type.

3. The monoclonal antibody combined with 11dhTxB2 is produced by a hybridoma cell line with the preservation number of CGMCC NO. 17700.

4. The monoclonal antibody of claim 3, further comprising an antigen-binding fragment of the monoclonal antibody.

5. Use of a monoclonal antibody according to any one of claims 3-4 in the manufacture of a reagent or kit for evaluating aspirin as a platelet inhibitor.