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Hybridoma cell line 4f4 and its antibody

A hybridoma cell line, monoclonal antibody technology, applied in the direction of antifungal/algae/lichen immunoglobulin, instruments, biochemical equipment and methods, etc., to achieve the effect of good specificity and high antibody titer

Active Publication Date: 2019-05-24
SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the preparation of TA monoclonal antibody and its product development are in a blank stage, and there is no report

Method used

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  • Hybridoma cell line 4f4 and its antibody
  • Hybridoma cell line 4f4 and its antibody
  • Hybridoma cell line 4f4 and its antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1, animal immunization and detection of mouse serum titer after immunization

[0023] Five female BALB / c mice aged 6-8 weeks were selected and immunized with the TAO-KLH immunogen of the hapten TAO-coupled hemocyanin (KLH) and Freund's adjuvant, subcutaneously injected 100 μg, 2- Booster immunization once every 3 weeks (the first immunization immunogen is mixed with Freund's complete adjuvant, and then the immunogen is mixed with Freund's incomplete adjuvant). Among them, the hapten TAO is obtained by derivatizing Tenuaconic acid (TA) with carboxymethylhydroxylamine hemihydrochloride and then introducing the linker.

[0024] After the fourth immunization, the blood was collected for detection, and the titer of the antiserum against the TAO-BSA detection source was determined by the indirect ELISA method. The detection conditions were as follows:

[0025] Coating antigen: TAO-BSA detection source, Alternaria tennonic acid (competition antigen) produced by Alt...

Embodiment 2

[0035] Embodiment 2, cell fusion

[0036] 1. Preparation of myeloma cells

[0037] One week before fusion, SP2 / 0 cells were expanded in DMEM medium containing 10% FBS. By the time of confluence, the cells have covered about 6 bottles of T25 cell culture flasks. On the day of confluence, collect SP2 / 0 cells into a 50mL centrifuge tube and centrifuge at 1000rpm for 5min. Discard the supernatant, then add 20mL DMEM basal medium, blow off the cells and count.

[0038] 2. Splenocyte preparation

[0039] Mice whose serum ELISA titer was above 1:10000 after four immunizations were finally immunized 3 days before fusion, and 100 μg of antigen was injected intraperitoneally. Euthanize the mouse to be fused by cervical dislocation on the day of fusion. Soak in 75% alcohol for 5 min. Aseptically take the spleen, and put the spleen into a petri dish with 10 mL of DMEM basic culture inside. Remove the mesh into another dish, transfer the spleen to the mesh, and grind the spleen viscer...

Embodiment 3

[0058] Embodiment 3, fusion screening and subcloning

[0059] 1. Fusion screening

[0060] On the day before the test, coat 5 μg / mL antigen on the ELISA plate with PBS, overnight, absorb the cell supernatant 100 μL / well for ELISA test the next day. According to the ELISA results, judge the positive well (OD value of sample well / OD value of negative well ≥ 2.1 is judged as a positive well. Use a single-channel pipette to pick the positive wells detected on the entire plate, perform a second confirmation test, further confirm the positive wells, and subclone the cells in the confirmed positive wells.

[0061] 2. Subcloning

[0062] Pipette the cells in the positive wells, count them, add N / 4mL DMEM medium to the centrifuge tube, take 100μl of cell suspension into the centrifuge tube, leave 1mL after blowing, add DMEM to 4mL, blow evenly, leave 100μl (about 2 drop) at the bottom of the tube. Add DMEM to 5mL in the centrifuge tube, mix well and add dropwise to the first three r...

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Abstract

The invention relates to a hybridoma cell strain 4F4 and an antibody thereof. The hybridoma cell strain 4F4 is preserved in China Center for Type Culture Collection on Dec. 27, 2016 with the preservation number of CCTCC NO:C201702. The hybridoma cell strain 4F4 generates an anti-tenuazonic acid monoclonal antibody, specifically detects tenuazonic acid, and is of great significance to detect the tenuazonic acid.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a hybridoma cell line 4F4, and also relates to an antibody produced by the hybridoma cell line 4F4. Background technique [0002] Alternariol tenuazonic acid (Tenuazonic acid, TA) is one of the toxic metabolites produced by Alternaria, Magnaporthe oryzae, Pointer sorghum, etc. under specific conditions. ), Alternariol monomethyl ether (Alternariol monomethyl ether, AME), an Alternaria mycotoxin discovered. TA has acute toxicity, subacute toxicity, potential carcinogenicity, and cytotoxicity, which can cause dizziness, salivation, and vomiting in mammals, followed by tachycardia, massive esophageal and gastrointestinal bleeding, circulatory failure, and death. Because TA has a variety of biological characteristics and is the most toxic of Alternaria mycotoxins, it has been reported that TA can synergistically enhance other mycotoxins, which has huge potential harm, and has...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C07K16/14G01N33/577G01N33/543
CPCC07K16/14
Inventor 马良张宇昊钟红
Owner SOUTHWEST UNIV
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