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Monoclonal antibody for detecting dibutyl phthalate and cresyl butyl phthalate and application thereof

A technology of tolylbutyl phthalate and dibutyl phthalate, applied in the field of veterinary drug residue analysis and immunology, can solve the problem of poor sensitivity and specificity, single type of phthalate, and low antibody sensitivity and other problems, to achieve the effect of less physical harm, good market value, and high method accuracy

Inactive Publication Date: 2018-08-03
HUAZHONG AGRI UNIV
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AI Technical Summary

Problems solved by technology

[0004] CN 103257115 A uses 4-amino phthalate as hapten to prepare a monoclonal antibody that can recognize DBP; CN 104004719 A uses 4-amino phthalate as hapten to prepare a monoclonal antibody that can recognize DBP Monoclonal antibodies to DBP, BBP, DiBP, DNP, DMP, CN 103869070 A The compound obtained by reacting phthalic anhydride with n-butanol and 4-aminobutyric acid is used as a hapten to prepare a monoclonal antibody capable of recognizing DBP and DiBP Cloned antibody; CN 103204925 A A phthalic acid was prepared by esterifying phthalic acid with 6-(fluorenylmethoxycarbonyl-amino)-1-hexanol, and then removing the protecting group of fluorenylmethoxycarbonyl Dicarboxylate general hapten, the prepared antibody has low sensitivity to DBP, DEHP and DINP
It can be seen that the current enzyme-linked immunoassay methods for phthalates mainly focus on the research of haptens, and the monoclonal antibodies prepared by using existing haptens either have poor sensitivity and specificity, or can recognize phthalates. The species is single, and there is no monoclonal antibody that can recognize both DBP and BBP in the prior art

Method used

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  • Monoclonal antibody for detecting dibutyl phthalate and cresyl butyl phthalate and application thereof
  • Monoclonal antibody for detecting dibutyl phthalate and cresyl butyl phthalate and application thereof
  • Monoclonal antibody for detecting dibutyl phthalate and cresyl butyl phthalate and application thereof

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Experimental program
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Effect test

Embodiment 1

[0027] The preparation of embodiment 1 immunogen and coating former

[0028] 1.1 Preparation of immunogen 4-DBHP-CDI-HSA

[0029] Weigh 3 g of 4-hydroxyphthalic acid and dissolve it in 4 mL of n-butanol, add concentrated H 2 SO 4 0.3mL catalyst, stirred and refluxed at 100°C for 4h, then rotary evaporated, and the product was washed with 10% Na 2 CO 3 After washing until the water layer is colorless (pH value is 8), the organic layer is rotary evaporated, that is, the hapten 4-hydroxydibutylphthalate (4-DBHP). Weigh 28mg 4-DBHP and dissolve it in 200μL 1,4-dioxane, add 50mg CDI (N,N-carbonyldiimidazole) and 60mg DMAP (4-dimethylaminopyridine) at the same time, activate the mixture overnight at room temperature, and activate the The reaction solution was added dropwise to 40 mg of HSA, reacted overnight at 4°C, centrifuged to remove the supernatant, dialyzed for 3 days, and changed the dialysate twice a day. The dialysate was pH7.4 phosphate buffer. Coated with original 4...

Embodiment 2

[0034] The preparation of embodiment 2 monoclonal antibody

[0035]Preparation of hybridoma cells: referring to "Animal Immunology" by Yang Hanchun, Balb / C mice (purchased from Hubei Provincial Center for Disease Control and Prevention) were immunized with the immunogen 4-DBHP-CDI-HSA conjugate prepared in Example 1. The immunization procedure is as follows: for basic immunization, the protein emulsion containing 50 μg of immunogen emulsified with an equal volume of Freund's complete adjuvant is injected subcutaneously on the back of the mouse's neck, and then emulsified with Freund's incomplete adjuvant every 15 days. Protein emulsion containing 100 μg immunogen was used for booster immunization. from three immunizations. Tail blood was collected on the 8th day after each immunization, the serum was separated, and the serum antibody titer was detected by indirect ELISA. Immunization qualified mice (high titer, good sensitivity) stop immunization in preparation for fusion. ...

Embodiment 3

[0040] The establishment of embodiment 3 indirect competition ELISA detection method

[0041] 3.1 Preparation of reagents (the reagents used in this example were prepared by the following methods unless otherwise specified)

[0042] Phosphate buffer: NaCl 8.0g, KH 2 PO 4 0.2g, Na 2 HPO 4 12H 2 O 2.9g, KCl 0.2g, add double distilled water to 1000mL, adjust pH to 7.4;

[0043] Coating solution: Take Na 2 CO 3 1.59g, NaHCO 3 2.93g, add triple distilled water to 1000mL, adjust the pH value to 9.6;

[0044] Washing liquid: NaCl 8.0g, KH 2 PO 4 0.2g, Na 2 HPO 4 12H 2 O 2.9g, KCl 0.2g, Tween 200.5mL, add double distilled water to 1000mL, adjust pH to 7.4;

[0045] Blocking solution: Ovalbumin 1g dissolved in 100mL phosphate buffer;

[0046] Substrate solution A: 3,3',5',5-tetramethylbenzidinediamine (TMB) 200mg, absolute ethanol 100mL, add double distilled water to 1000mL;

[0047] Substrate B: Na 2 HPO 4 14.6g, citric acid 9.3g, 0.75% urea hydrogen peroxide 6.4...

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Abstract

The invention discloses a specific monoclonal antibody capable of recognizing dibutyl phthalate and cresyl butyl phthalate, and the invention also discloses a method for detecting dibutyl phthalate and phthalate Enzyme-linked immunosorbent method and kit for cresyl butyl phthalate. The monoclonal antibody of the present invention is secreted by the hybridoma cell line DBP3B2 with the preservation number CCTCC NO: C201497. Compared with the prior art, the monoclonal antibody prepared by the present invention can simultaneously recognize dibutyl phthalate and cresyl butyl phthalate, and the lowest detection limit (IC50) of DBP is only 44.33 μg / L, The lowest detection limit for BBP is only 62.59 μg / L, and it has a high cross-reactivity rate for BBP, so it has high detection sensitivity and specificity for DBP and BBP, two phthalate plasticizers .

Description

technical field [0001] The invention belongs to the technical field of veterinary drug residue analysis and immunology, and specifically relates to a monoclonal antibody capable of recognizing dibutyl phthalate and cresyl butyl phthalate and an application thereof. Background technique [0002] Phthalate esters (phthalate esters, PAEs), commonly known as plasticizers or phthalates, are derivatives of phthalic acid. The basic structure of phthalates is composed of a benzene ring and two side chains. The two side chain groups can be the same or different. There are more than 20 types depending on the side chains. The common ones are phthalic acid di Methyl phthalate (DMP), diethyl phthalate (DEP), dibutyl phthalate (DBP), dipropyl phthalate (DPrP), Bis(2-ethylhexyl)phthalate (di(2-ethylhexyl)phthalate, DEHP), etc. Phthalates are often used as plasticizers in industrial production. These substances are added to plastic products to improve the flexibility of plastics. Because ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/44C12N5/20G01N33/577G01N33/543
Inventor 袁宗辉王玉莲赵兰彭大鹏潘源虎陈冬梅陶燕飞刘振利
Owner HUAZHONG AGRI UNIV
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