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PBDE monoclonal antibody hybridoma cell strain and application thereof

A technology of hybridoma cell lines and polybrominated diphenyl ethers, applied in biochemical equipment and methods, instruments, microorganisms, etc., can solve the problems of high cost, inability to realize rapid detection of a large number of samples, cumbersome operation, etc., and achieve good detection sensitivity and specificity Effect

Inactive Publication Date: 2015-11-25
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] At present, the methods for detecting PBDEs mainly include gas chromatography / electron capture detection (GC / ECD), gas chromatography-negative chemical ionization source mass spectrometry (GC-NCI-MS), liquid chromatography (HPLC), gas chromatography ( GC / MS) and other methods, but these methods have the disadvantages of cumbersome operation, time-consuming, expensive, etc., and cannot realize the rapid detection of a large number of samples. Therefore, it is of great significance to establish a fast and simple method for the detection of PBDEs

Method used

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  • PBDE monoclonal antibody hybridoma cell strain and application thereof
  • PBDE monoclonal antibody hybridoma cell strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] The preparation of embodiment 1 hybridoma cell line 1A1

[0023] (1) Synthesis of complete antigen

[0024] Dissolve 2,4-dibromophenol (1.04g, 4.13mmol) in 8mL of DMF solution, add NaH (0.2g, 8.33mmol), stir in ice bath for 30min, then let stand to room temperature; after keeping room temperature for 15min, add 3- Bromo-4-fluoronitrobenzene (1.01g, 4.13mmol), heated to reflux at 90°C for 2h; add 10mL of water, wash with CH 2 Cl 2 Extract 4 times with 20 mL each time, extract the organic phase with 25 mL of water, then dry the organic phase with anhydrous magnesium sulfate, and remove the solvent with a rotary evaporator; use silica gel column chromatography to purify, and mix with ether:n-hexane volume ratio of 50:50 Solvent elution gave orange liquid 4'-nitro-2,2',4-tribromodiphenyl ether.

[0025] Using Fe / H + Nitro reduction method, adding 5.0mmol of reducing agent iron powder and 5.0mmol of 4'-nitro-2,2',4-tribromodiphenyl ether into a mixed solvent of ethanol 1...

Embodiment 2

[0034] Example 2 Preparation and identification of polybrominated diphenyl ether monoclonal antibody

[0035] Take 8-10 week-old BALB / c mice, and inject 1 mL of paraffin oil into each mouse; 7 days later, each mouse is injected with 1×10 6 Hybridoma cell line 1A1, ascitic fluid was collected from the seventh day, the ascites fluid was purified by octanoic acid-ammonium sulfate method, and the monoclonal antibody obtained was stored at -20°C.

[0036] Determination of the IC of monoclonal antibodies to polybrominated diphenyl ethers using an indirect competition ELISA 50 It is: 20μg / L, indicating that it has good sensitivity to PBDEs and can be used for the detection of PBDEs in immunoassays.

Embodiment 3

[0037] Application of embodiment 3 polybrominated diphenyl ether monoclonal antibody

[0038] The monoclonal antibody prepared by the hybridoma cell line 1A1 through ascites in vivo was applied to the rapid detection of polybrominated diphenyl ethers, and the specific steps were as follows:

[0039] (1) Coating: Dilute the coated PBDE-OVA with 0.05M pH9.6 carbonate buffer starting from 2μg / mL, 100μL / well, and react at 37°C for 2h.

[0040] (2) Washing: Pour off the solution in the plate, spin dry, and wash 3 times with PBST washing solution, 3 minutes each time.

[0041] (3) Blocking: after pat dry, use CBS blocking solution containing 0.01% gelatin, add 200 μL / well blocking solution, and react at 37°C for 2 hours. Wash and tumble dry for later use.

[0042] (4) Adding samples: Dilute the antiserum starting from 1:1000, add PBS containing 0.01% gelatin to the coated wells of each dilution, 100 μL / well, react at 37°C for 1 hour; after fully washing, add 1:3000 dilution of HR...

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Abstract

The invention relates to a PBDE monoclonal antibody hybridoma cell strain and application thereof, and belongs to the field of food safety immunology detection. PBDE complete antigens and an equal amount of a Freund's adjuvant are mixed to be emulsified, and the mixture is subcutaneously injected to the backs of BALB / c mice for immunization. The complete Freund's adjuvant is used in the first immunization, and the incomplete Freund's adjuvant is used in the following immunization. Splenocytes, with high potency and low IC50, of the mice are fused with myeloma cells of the mice according to a PEG method, and the hybridoma cell strain 1A1 is obtained through indirect competition ELISA screening and three times of subcloning. Monoclonal antibodies excreted from the cell strain 1B2 have better specificity and detection sensitivity (the IC50 value is 20 [mu]g / L) on PBDE, and can be used for residue detection of PBDE in food safety.

Description

technical field [0001] The invention relates to a polybrominated diphenyl ether monoclonal antibody hybridoma cell line and an application thereof, belonging to the field of food safety immunology detection. Background technique [0002] Polybrominated diphenyl ethers (PBDEs), as a large class of flame retardant substances of brominated flame retardants, have been more and more widely used in various consumer products due to their excellent flame retardant properties. At the same time, polybrominated diphenyl ethers (PBDEs) are also a class of global organic pollutants, which have attracted much attention due to their persistence, toxicity and potential bioaccumulation. Photochemical degradation is one of the important fates of PBDEs in the environment. The detection of PBDEs has been continuously reported in environmental samples, and the environmental problems caused by these compounds have attracted more and more attention, especially in environmental science. [0003] A...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/44G01N33/577C12R1/91
Inventor 匡华孙成胥传来徐丽广马伟刘丽强宋珊珊胡拥明
Owner JIANGNAN UNIV
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