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Isoniazide hapten derivative, isoniazide artificial antigen, hybridoma cell strain, isoniazide monoclonal antibody and application

A monoclonal antibody and artificial antigen technology, applied in microorganisms, biological testing, animal/human peptides, etc., which can solve the problems of antibody cross-reaction, destruction of characteristic differential groups, and difficulty in determination.

Active Publication Date: 2022-07-29
BEIJING DIAGREAT BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Chinese patent CN 110672576 A discloses a method for determining isoniazid by quantum dot fluorescence quenching method, but this method can only test isoniazid in a simple buffer, such as the content of isoniazid in medicines
For the determination of isoniazid in clinical samples, it is difficult to measure due to the complex matrix composition
[0004] Chinese patent CN 111848507 A discloses a hapten structure derived from the hydrazide group of isoniazid, but the derivation site destroys the main metabolites of isoniazid and isoniazid (isoniacin and acetyliso Niacinazine, etc.) characteristic difference group, but exposes the common structure, which will lead to cross-reactivity of the prepared antibody to metabolites
Literature (Kam, S. Tse, et al. "Antibodies specific to isoniazid and isonicotinicacid." The Journal of Immunology 116.4 (1976): 965-969.) discloses isoniazid prepared by derivatizing the hydrazide group of INH Hydrazine antibodies, however, also show significant cross-reactivity to isonicotinic acid in the detection of INH

Method used

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  • Isoniazide hapten derivative, isoniazide artificial antigen, hybridoma cell strain, isoniazide monoclonal antibody and application
  • Isoniazide hapten derivative, isoniazide artificial antigen, hybridoma cell strain, isoniazide monoclonal antibody and application
  • Isoniazide hapten derivative, isoniazide artificial antigen, hybridoma cell strain, isoniazide monoclonal antibody and application

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preparation example Construction

[0039] The present invention provides the preparation method of the isoniazid hapten derivative described in the above technical scheme, comprising the following steps:

[0040] Isoniazid and acetone are subjected to condensation reaction under alkaline conditions to obtain intermediate 1;

[0041] Mix the intermediate 1, monomethyl succinate, persulfate, trifluoroacetic acid, metal salt catalyst and water, and carry out oxidative decarboxylation reaction to obtain intermediate 2;

[0042] The intermediate 2 is acidified after being subjected to an alkaline hydrolysis reaction to obtain the isoniazid hapten derivative;

[0043] .

[0044] In the present invention, unless otherwise specified, the raw materials used are all commercially available products well known to those skilled in the art.

[0045] The isoniazid and acetone of the present invention undergo a condensation reaction under alkaline conditions to obtain intermediate 1.

[0046] In the present invention, the...

Embodiment 1

[0125] Preparation of isoniazid hapten derivative with structure shown in formula I

[0126]

[0127]

[0128]

[0129] 13.7 g of isoniazid and 40 mL of acetone were added to the NaOH aqueous solution (NaOH mass 5 g, 1 mol / L) under stirring conditions, and the reaction was stirred at 50 °C for 2 h. At the end of the reaction, the excess acetone was evaporated under reduced pressure, 20 mL of water was added, the temperature was kept at 12 °C for crystallization for 2 h, filtered, and the obtained solid powder was vacuum-dried at 45 °C to obtain Intermediate 1.

[0130] To 10.0 g of Intermediate 1 was added 300 mL of water, 64.0 g of ammonium persulfate, 6.5 g of TFA and 20 g of AgNO 3 , react at room temperature (25°C) for 2 h, add 30 mL of ethyl acetate and let stand for layers, take the organic layer and concentrate under reduced pressure to evaporate the ethyl acetate to obtain intermediate 2.

[0131] Add 50 mL of water to intermediate 2, then dropwise add NaOH (...

Embodiment 2

[0134] Preparation of isoniazid artificial antigen

[0135] Take 10 mg of the isoniazid hapten derivative prepared in Example 1, add 1 mL of DMSO to dissolve completely, and obtain the isoniazid hapten derivative solution;

[0136] Weigh 10 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, dissolve it in 100 μL of water, add it to the isoniazid hapten derivative solution, and react at room temperature for 1 h. A cross-linked isoniazid hapten derivative solution is obtained;

[0137] Weigh 20 mg of bovine thyroglobulin and dissolve it in 5 mL of PBS to obtain a bovine thyroglobulin solution;

[0138] Mix the cross-linked isoniazid hapten derivative solution and bovine thyroglobulin solution, stir at room temperature for 2 hours to obtain a composite reaction solution, and dialyze the composite reaction solution with PBS buffer (pH=7.4) (dialysis bag 7000Da) 4 times to obtain The isoniazid artificial antigen having the structure shown in formula II, the isonia...

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Abstract

The invention provides an isoniazide hapten derivative, an isoniazide artificial antigen, a hybridoma cell strain, an isoniazide monoclonal antibody and application, and relates to the technical field of drug monitoring. The isoniazide hapten derivative provided by the invention can realize specific recognition of isoniazide, and the isoniazide antigen obtained by coupling protein by taking-CH2-CH2-CH2-COOH as a coupling arm can accurately obtain an antibody with specific binding capacity to isoniazide. The hybridoma cell strain provided by the invention can generate a high-sensitivity isoniazide monoclonal antibody for equivalent identification of isoniazide, and the antibody has no obvious cross reaction with main metabolites (acetyl isoniazide, isonicotinic acid, nicotinamide and aminosalicylic acid) of isoniazide, and can realize high-sensitivity equivalent identification of isoniazide. Therefore, the compound can be applied to preparation of detection reagents (an isoniazide homogeneous enzyme immunoassay reagent and an isoniazide magnetic particle chemiluminescence detection reagent) for detecting isoniazide, and the clinical detection requirements of isoniazide are met.

Description

technical field [0001] The invention belongs to the technical field of drug monitoring, and particularly relates to isoniazid hapten derivatives, isoniazid artificial antigens, hybridoma cell lines, isoniazid monoclonal antibodies and applications. Background technique [0002] Isoniazid (INH, chemical name 4-pyridinecarboxylhydrazide) has antibacterial and bactericidal effects and is a first-line drug for anti-tuberculosis. After INH is absorbed, it needs to reach a certain concentration in the lesion site of the patient in order to inhibit or kill Mycobacterium tuberculosis. However, the effective treatment window of INH is narrow. Too low concentration of INH may lead to treatment failure or drug resistance. When the concentration of INH is too high, it is easy to cause many adverse drug reactions in the liver, kidney and nerve due to high dose and long-term use. . Therefore, in the process of INH medication, it is necessary to monitor the blood drug concentration in or...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D213/86C07K14/47C07K14/765C07K14/77C07K16/44G01N21/76G01N33/53G01N33/543G01N33/577C12N5/20
CPCC07D213/86C07K14/765C07K14/77C07K14/47C07K16/44G01N33/5308G01N33/543G01N33/577G01N33/54326G01N21/76
Inventor 周建平周裕军许秀丽常缘荣
Owner BEIJING DIAGREAT BIOTECH CO LTD
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