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Hybridoma cell line secreting methotrexate monoclonal antibody, preparation method and applications thereof

A hybridoma cell line and hybridoma cell technology, applied in the field of hybridoma cell line secreting methotrexate monoclonal antibody and its preparation, can solve the problems of poor detection effect, poor specificity, wrong measurement results, etc., and achieve high affinity, Large capacity, the effect of increasing the amount of fusion clones

Inactive Publication Date: 2017-10-03
THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when the existing antibodies are used to determine the presence or amount of methotrexate in the test sample for immunoassay, the detection effect is poor due to poor specificity, and erroneous measurement results often occur.

Method used

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  • Hybridoma cell line secreting methotrexate monoclonal antibody, preparation method and applications thereof
  • Hybridoma cell line secreting methotrexate monoclonal antibody, preparation method and applications thereof
  • Hybridoma cell line secreting methotrexate monoclonal antibody, preparation method and applications thereof

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preparation example Construction

[0030] The preparation method of the hybridoma cell line that secretes methotrexate monoclonal antibody according to the embodiment of the present invention, the method comprises the following steps:

[0031] Balb / c mice were immunized with antigen MTX-BSA (bovine serum albumin);

[0032] Spleen cells from immunized mice were collected and fused with Sp2 / 0 myeloma cells, and the fused hybridoma cells were selectively cultured with HAT medium;

[0033] The antibody content in the culture supernatant of the hybridoma cells was detected by an indirect competition ELISA method, and the hybridoma cell lines stably secreting the monoclonal antibody were screened.

[0034] The application of the hybridoma cell line secreting methotrexate monoclonal antibody according to the embodiment of the present invention, the hybridoma cell line is used to prepare anti-methotrexate monoclonal antibody, and the monoclonal antibody can be used to specifically recognize methotrexate whisper.

[0...

Embodiment 1

[0038] Example 1 of the present invention is a hybridoma cell line secreting methotrexate monoclonal antibody, and the biological deposit number of the hybridoma cell line is CCTCC NO: C201726.

[0039] Embodiment 1 of the present invention is a method for preparing a hybridoma cell line secreting methotrexate monoclonal antibody, the method comprising the following steps:

[0040] 1. Animal immunity

[0041] Balb / c mice, adult females about 8 weeks old, mixed and emulsified the antigen with equal volume of complete adjuvant (first immunization) and incomplete adjuvant (boost immunization), mixed fully to the state of water-in-oil for subcutaneous multi-point immunization, 2-3 times of booster immunization, each immunization interval is 2 weeks, orbital blood is collected after 3 times of immunization to detect serum antibody titer, and the serum antibody titer is tested by indirect ELISA at intervals of 2 weeks. On the 5th day before cell fusion, the same method was used for...

Embodiment 2

[0062] Use the hybridoma cell that embodiment 1 obtains to prepare anti-methotrexate monoclonal antibody, comprise the steps:

[0063] The hybridoma cells obtained in Example 1 were injected into BALB / c mice pre-treated with Freund's incomplete adjuvant, and the ascites of the mice was collected. After the collected ascites was pretreated, Protein G-agarose affinity was selected. Chromatographic column purification, the specific steps are as follows:

[0064] 1) Prepare buffer: the initial buffer is pH7.0, 20mmol / L phosphate buffer; the elution buffer is pH2.70.1mmol / L glycine hydrochloride.

[0065] 2) Prepare collection tubes: take 1.5ml centrifuge tubes, add 70 μl pH9.0, concentration 1mol / L Tris-HCl to each centrifuge tube.

[0066] 3) Sample preparation: the sample obtained by precipitation with 50% SAS was dialyzed overnight in the initial buffer solution, and filtered through a 0.22m microporous membrane.

[0067] 4) Purification process: Equilibrate the Protein G-Sep...

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Abstract

The invention provides a hybridoma cell line secreting methotrexate monoclonal antibody, its preparation method and application, and the biological deposit number of the hybridoma cell line is CCTCC NO: C201726. The preparation method of the hybridoma cell line secreting methotrexate monoclonal antibody comprises the steps of: immunizing Balb / c mice with the antigen MTX-BSA; collecting the splenocytes of the immunized mice, and carrying out Fusion, fused hybridoma cells are selectively cultured with HAT medium; the antibody content in the hybridoma cell culture supernatant is detected by indirect competitive ELISA method, and hybridoma cell lines that stably secrete monoclonal antibodies are screened. The hybridoma cell line can produce anti-methotrexate monoclonal antibody, and the monoclonal antibody has high specificity and can be used to specifically recognize methotrexate.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a hybridoma cell line secreting methotrexate monoclonal antibody, a preparation method and an application thereof. Background technique [0002] Methotrexate (MTX) is an anticancer drug, an antirheumatic drug, and belongs to folic acid metabolic antagonist. As an anticancer agent, it is used in the treatment of leukemia and trophoblastic diseases. However, it has serious side effects of inhibiting bone marrow function and causing liver and kidney dysfunction, so it is necessary to conduct multiple clinical examinations during the medication process and carefully observe the patient's state. Usually, calcium tetrahydrofolate (CF) is applied after MTX to relieve the adverse reactions of MTX, but there are still different degrees of toxic reactions, and even the risk of death. How to ensure that large doses of tumors are treated clinically without serious toxic side effect...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/44C12R1/91
CPCC07K16/44C07K2317/33C07K2317/92C12N5/163C12N2510/02
Inventor 方勇飞牟方祥吴红陈思闫彩霞陈欣
Owner THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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