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Hybridoma cell line and application thereof

A technology of hybridoma cell lines and hybridoma cells, applied in the field of hybridoma cell lines, to achieve good application value, high specificity, and good sensitivity

Inactive Publication Date: 2011-07-20
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to provide a hybridoma cell line capable of producing an IgM subtype antibody that reacts with FB1 in response to the current problems in detecting fumonisin FB1 and its application

Method used

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  • Hybridoma cell line and application thereof
  • Hybridoma cell line and application thereof
  • Hybridoma cell line and application thereof

Examples

Experimental program
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Effect test

preparation example Construction

[0045] 2. Preparation of Hybridoma Cells

[0046] The splenocytes and SP2 / 0 cells of the immunized mice were collected by conventional methods and fused with 50% PEG4000 at a ratio of 10:1. Use HAT culture medium for selective culture, 10-15 days after fusion, take the supernatant and use indirect ELISA method to screen for secretion of anti-FB 1 The positive clones were subcloned by limiting dilution method.

[0047] The operation steps of the indirect ELISA method are as follows: use 2 μg / mL FB 1 -OVA-coated microtiter plate, use immune mouse serum 1:100 as positive control, culture supernatant without clonal growth and normal mouse serum as negative control, add 1:10000 goat anti-mouse IgG-HRP 100 μL to each well, and finally measure 450nm value, where OD 450 If the value is greater than 2 times of the negative control, it can be preliminarily judged as a positive clone.

[0048] 3. Establishment of hybridoma cell lines

[0049] Repeat step 2 for 4 times of cell fusion...

Embodiment 2

[0057] Example 2, Application of 4E10 cell line to prepare anti-FB 1 monoclonal antibody

[0058] 1. Antibody preparation

[0059] Select healthy BALB / C mice, inject 0.5mL pristane intraperitoneally into the mice firstly, and then inject 1×10 hybridoma cells into each mouse intraperitoneally after 7-10 days 6 -2×10 6 After the abdomen of the mouse was significantly enlarged to a certain extent, the ascites was extracted with a No. 9 needle. The collected ascites was centrifuged at 13,000 rpm for 30 min at 4°C, and the supernatant was collected.

[0060] The collected ascites was saturated (NH 4 ) 2 SO 4 The precipitation method was used for purification, and the specific steps were as follows: take the supernatant in the above steps, add an equal volume of PBS buffer, then add an equal volume of acetic acid buffer and 33 μL octanoic acid, and stir magnetically for 30 minutes. Centrifuge at 3000rpm for 30min at 4°C, take the supernatant; add an equal volume of saturated ...

Embodiment 3

[0079] Embodiment 3, using monoclonal antibody for detection kit

[0080] From the above research results, we have developed an immunoassay kit for fumonisin FB1. Since the anti-FB1 monoclonal antibody of the present invention has high sensitivity and stable physical and chemical properties, it can be easily prepared and detected according to standard methods. Toxin kit.

[0081] The following is the specific composition of a detection kit prepared by the present invention utilizing the anti-fumonisin FB1 monoclonal antibody:

[0082] Coating antigen: the conjugate of fumonisin FB1 hapten and hemocyanin KLH, 50 μl / bottle, diluted 3000 times with coating buffer before use;

[0083] Detection antibody: Purified mouse ascites, 100 μl / bottle, diluted 6000 times with PBS buffer and premixed with samples;

[0084] Substrate: Tetramethylbenzidine (TMB), 5mg / bottle;

[0085] HRP-labeled goat anti-mouse IgG: the working concentration is 1:10000, diluted with pH7.4 phosphate buffer for...

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Abstract

The invention relates to a hybridoma cell line and application thereof. The hybridoma cell line can generate anti-fumonisin FB1 monoclonal antibodies, and is used for detecting the pollution condition of fumonisin FB1. The hybridoma cell line is characterized in that: the collection number of the hybridoma cell line is CCTCC NO: C201008. The invention has the advantages that: the cell line can specifically secrete the monoclonal antibodies which aim at the fumonisin FB1, and has high sensitivity and specificity; and the antibodies generated by the cell line can be applied to various ways, andhave high application value in practical production.

Description

technical field [0001] The invention relates to a hybridoma cell strain and application thereof. The hybridoma cell strain can produce a monoclonal antibody against fumonisin FB1 and is used for detecting fumonisin FB1 contamination. Background technique [0002] Fumonisin (FB) is mainly a mycotoxin produced by Fusarium moniliforme. where FB 1 It is the main component of pollutants and the main cause of toxic effects. Facebook 1 Widely present in corn and its products worldwide, it poses a potential threat to human health and animal husbandry development. Research proves FB 1 Can cause leukoencephalomalacia (equineleukoencephalomalacia, ELEM) in horses and pulmonary edema syndrome in pigs (porcinepulmonary edema, PPE). Epidemiological data show that FB in human diet 1 Pollution is associated with a high incidence of esophageal cancer. Therefore, it is urgent to establish a fast, sensitive and simple detection method. [0003] Currently, it is used to analyze FB in fo...

Claims

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Application Information

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IPC IPC(8): C12N5/18C07K16/18G01N33/577C12N15/09C12P21/08
Inventor 祭芳史建荣薛秋燕林凡云徐剑宏
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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