Preparation method of monoclonal antibody and kit thereof

A monoclonal antibody and kit technology, applied in the field of monoclonal antibody preparation, can solve the problems of time-consuming, poor quality, false positive sensitivity and specificity, and achieve the effect of simple operation, strong purpose and small workload

Inactive Publication Date: 2015-12-30
SINOCARE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, many NT-BNP protein monoclonal antibodies for in vitro diagnostics prepared by many companies and research and development units are mostly formed through cell fusion to antibody formation. Obtaining a monoclonal antibody that can be used for clinical diagnosis requires a large number of screening processes. The technical scheme used in cell screening is to conduct a large number of screening through enzyme-linked immunosorbent assay, and then obtain positive cell lines. This method of screening antibodies has no clear purpose, and there are a large number of false positives or sensitivity in these cell lines. And in the case of poor specificity, then a large amount of purification and step-by-step detection and testing are necessary
As a result, a large amount of human and material resources and scientific research funds are lost, and it takes a long time, resulting in the loss of high-quality antibody cell lines, and may not be able to screen high-quality antibodies.
In view of the cumbersome preparation process of NT-BNP antibody, the relatively long time-consuming, the low titer of the monoclonal antibody produced, and the inability to meet the experimental needs, there is an urgent need to provide a rapid preparation of NT-BNP protein monoclonal antibody and other monoclonal antibodies. exact preparation method

Method used

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  • Preparation method of monoclonal antibody and kit thereof
  • Preparation method of monoclonal antibody and kit thereof
  • Preparation method of monoclonal antibody and kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] The preparation of embodiment 1NT-BNP protein monoclonal antibody

[0052] (1) Animal immunity

[0053] The NT-BNP antigen was diluted to 1 mg / mL, mixed with an equal volume of Freund's complete adjuvant, and fully emulsified, and 5 6-week-old Balb / c mice were subcutaneously inoculated at multiple points, and the inoculation dose of antigen was 50 μg / mouse. After 3 weeks, the same dose of antigen treated with Freund's incomplete adjuvant was injected, and then the antigen was injected every 2 weeks, repeated 3 times, and the dose of each immunization was 25 μg per mouse. Three days before cell fusion, NT-BNP protein without adjuvant was injected intraperitoneally for a booster immunization, and then cell fusion was performed.

[0054] (2) Preparation of splenocytes

[0055] Three days later, blood was collected from the eyeballs of the mice, and the blood was placed at 37°C for 0.5h, at 4°C for 1h, centrifuged at 3000rpm for 5min, and the separated serum was used as p...

Embodiment 2

[0116] The preparation of embodiment 2NT-BNP protein monoclonal antibody

[0117] Repeat the test once according to the monoclonal antibody preparation method in Example 1,

[0118] Positive clones were screened by the indirect ELISA method of this experiment, and transferred to 24 wells for detection. The screened positive clones are as follows:

[0119] Positive clonal cell lines screened out in table 6

[0120] mother clone number

OD 450

mother clone number

OD 450

1

2.824

9

2.877

2

2.589

10

2.379

3

2.318

11

2.914

4

2.754

12

2.644

5

2.631

13

2.241

6

2.482

14

2.684

[0121] 7

2.863

15

0.07

8

2.375

Negative

3.097

[0122] 2. Establishment of NT-BNP protein competition ELISA method

[0123] Carry out competition ELISA test according to the method of Example 1, add 15C4-HRP to each ho...

Embodiment 3

[0144] The preparation of embodiment 3PCT monoclonal antibody

[0145] (1) Animal immunity

[0146] Dilute the PCT (procalcitonin) antigen to 1 mg / mL, mix it with Freund's complete adjuvant in equal volume, and fully emulsify it, and inoculate 5 6-week-old Balb / c mice subcutaneously at multiple points, and the inoculated antigen dose is 50 μg / Only. After 3 weeks, the same dose of antigen treated with Freund's incomplete adjuvant was injected, and then the antigen was injected every 2 weeks, repeated 3 times, and the dose of each immunization was 25 μg per mouse. Three days before cell fusion, PCT protein without adjuvant was injected intraperitoneally for a booster immunization, and then cell fusion was performed.

[0147] (2) Preparation of splenocytes

[0148] Three days later, blood was collected from the eyeballs of the mice, and the blood was placed at 37°C for 0.5h, at 4°C for 1h, centrifuged at 3000rpm for 5min, and the separated serum was used as positive serum. T...

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Abstract

The invention relates to the technical field of preparation of monoclonal antibodies, and in particular relates to a preparation method of a monoclonal antibody and a kit thereof. The preparation method comprises the following steps: performing immunization on an animal by adopting an antigen, and taking immune spleen cells; fusing the immune spleen cells with myeloma cells to obtain fusion cells; detecting the fusion cells by adopting an indirect ELISA (enzyme-linked immunosorbent assay) method to obtain positive hybridoma cells; detecting the positive hybridoma cells by adopting a competitive inhibition ELISA method to obtain hybridoma cells with competitive reactions; sub-cloning the hybridoma cells with competitive reactions to obtain monoclonal antibody hybridoma cells; and excreting the monoclonal antibody hybridoma cells to obtain the monoclonal antibody. According to the preparation method provided by the invention, the monoclonal antibody is screened by adopting a method combining the indirect ELISA method and the competitive inhibition ELISA method, the operation is simple and convenient, the purposiveness is strong, the workload is small, and a high-quality hybridoma cell strain can be locked in a short time.

Description

technical field [0001] The invention relates to the technical field of monoclonal antibody preparation, in particular to a monoclonal antibody preparation method and a kit thereof. Background technique [0002] Heart failure, referred to as heart failure, refers to the failure of the venous return blood to be fully discharged from the heart due to the dysfunction of the systolic function and / or diastolic function of the heart, resulting in blood stasis in the venous system and insufficient blood perfusion in the arterial system, thereby causing cardiac circulation syndrome. The symptoms of this disorder are manifested as pulmonary congestion and vena cava congestion. Clinically, according to the patient's medical history of coronary heart disease, hypertension and other basic cardiovascular diseases, the clinical symptoms of dyspnea, fatigue, lower extremity edema during rest or exercise, tachycardia, shortness of breath, pulmonary rales, Signs of pleural effusion, increase...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/26C12N15/06G01N33/543
Inventor 刘伟
Owner SINOCARE
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