Immunoassay method for simultaneous detection of furaltadone metabolites, malachite green and cryptomalachite green

A microporous, artificial antigen technology, applied in chemical instruments and methods, measuring devices, animal/human proteins, etc., to achieve high sensitivity and specificity

Active Publication Date: 2019-04-12
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The technical problem to be solved by the present invention is to overcome the deficiencies of the existing AMOZ, MG and LMG single-residue immunoassay technology, and provide a kind of AMOZ hapten and artificial antigen, LMG hapten and artificial antigen that can be applied to simultaneously detect AMOZ, MG and LMG. Antigen and bispecific antibody that can recognize both AMOZ and LMG

Method used

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  • Immunoassay method for simultaneous detection of furaltadone metabolites, malachite green and cryptomalachite green
  • Immunoassay method for simultaneous detection of furaltadone metabolites, malachite green and cryptomalachite green
  • Immunoassay method for simultaneous detection of furaltadone metabolites, malachite green and cryptomalachite green

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1 Synthesis of furaltadone metabolite hapten and cryptomalachite green hapten

[0058] The furaltadone metabolite hapten AMOZMB was synthesized as follows: 140mg 4-mercaptobenzaldehyde and 225mg AMOZ were dissolved in 20mL anhydrous methanol and 5mL anhydrous methanol respectively, and the methanol solution of AMOZ was slowly added dropwise during stirring into 4-mercaptobenzaldehyde methanol solution and stirred overnight at room temperature. After the reaction was completed, it was filtered, washed twice with distilled water and anhydrous methanol, and dried to obtain the product AMOZMB with the following structural formula. ESI-MS (negative) m / z: 320 (M-H) - , 1 H-NMR (500MHz, d-Chloroform) δ7.90(s,1H),7.50–7.40(m,2H),7.37–7.27(m,2H),5.41(p,J=13.9Hz,1H),4.80 (dd, J=24.8,13.6Hz,1H),4.56(dd,J=24.7,13.7Hz,1H),3.56(t,J=9.4Hz,4H),3.21(s,1H),2.93(dd, J=24.8, 14.0Hz, 1H), 2.81–2.63 (m, 5H).

[0059]

[0060] The cryptomalachite green hapten LMGMB is synthesi...

Embodiment 2

[0062] Example 2 Preparation of furaltadone metabolite artificial antigen and cryptomalachite green artificial antigen

[0063] Both the AMOZ artificial antigen and the LMG artificial antigen are prepared by a bifunctional reagent cross-linking method, and the bifunctional reagent cross-linking method includes the following steps:

[0064] (1) Dissolve 1 / 80mmol of BSA or 1 / 60mmol of OVA in 2mL of 0.01mol / L PBS solution, and record it as solution A;

[0065] (2) Dissolve 1 / 40mmol bifunctional reagent SMCC (succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate) in 0.2mL N,N-dimethyl In formamide, it is recorded as liquid B;

[0066] (3) Under magnetic stirring, add liquid B dropwise to liquid A, react at 4°C overnight, dialyze with 0.01mol / L PBS solution (pH7.4) for 2 days, change the dialysate twice a day, and obtain solution C.

[0067] (4) Dissolve 1 mmol of hapten AMOZMB (or hapten LMGMB) in 0.2 mL of N,N-dimethylformamide, and add this solution dropwise to solution ...

Embodiment 3

[0072] Example 3 Preparation of furaltadone metabolite monoclonal antibody and cryptomalachite green monoclonal antibody

[0073] Female Balb / c mice were immunized with the artificial antigens AMOZMB-BSA and LMGMB-BSA prepared in Example 2, respectively. For the first immunization, take 0.5 μg of AMOZMB-BSA artificial antigen or LMGMB-BSA artificial antigen and emulsify with an equal volume of Freund's complete adjuvant, and adopt subcutaneous multi-point immunization.

[0074] After the first immunization, multiple booster immunizations were carried out. The steps were as follows: take 0.5 μg of the above-mentioned artificial antigen and emulsify it with the same amount of Freund's incomplete adjuvant, and the interval period was 2 weeks. One week after each booster immunization, the tail blood was taken to determine the titer of antiserum. When the titer is stable, the mouse with the best immune effect is selected for a booster immunization, and splenocytes are collected 3 ...

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Abstract

The invention provides an immunodetection method capable of detecting Furaltadone metabolite AMOZ, MG (malachite green) and LMG (leucomalachite green) simultaneously. An AMOZ hapten and an LMG hapten are synthesized scientifically and coupled with carrier proteins respectively, and artificial antigens are prepared. Hybridoma cell strains aiming at the two objects are prepared respectively from the AMOZ artificial antigen and the LMG artificial antigen, a hybrid-hybridoma cell strain capable of secreting an anti-AMOZ and anti-LMG bispecific antibody is prepared with a hybrid-hybridoma technique, a multi-residue immunodetection method is established successfully, simultaneous detection of AMOZ, MG and LMG is realized, the limit of detection of AMOZ is 0.1 ng / mL, and the IC50 is 1.3 ng / mL; the limit of detection of LMG is 4.8 ng / mL, and the IC50 is 45.3 ng / mL. The method has high specificity and higher sensitivity, has broad application prospect in multi-residue detection of AMOZ, MG and LMG in food and is simple and convenient to operate.

Description

technical field [0001] The invention relates to the technical field of food safety detection, and more specifically, relates to an immunological detection method for simultaneous detection of furaltadone metabolites AMOZ, malachite green and cryptomalachite green. Background technique [0002] Furaltadone and malachite green are prohibited drugs frequently detected in aquatic products. The furaltadone metabolite AMOZ (CAS No.: 43056-63-9) is a metabolite of furaltadone in animals, which can stably exist in animal tissues for a long time in the form of protein conjugates, and can be identified by detecting metabolites. Analysis of furaltadone residues. Cryptomalachite Green (LMG, CAS No.: 129-73-7) is the main metabolite of Malachite Green (MG, CAS No.: 2437-29-8) in aquatic animals. It can accumulate in animal tissues and is stable for a long time. exist. [0003] The hazards caused by AMOZ, MG and LMG are mainly manifested in their toxic effects, possible allergic reacti...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07D263/26C07C323/31C07K14/765C07K14/77C07K14/795G01N33/577G01N33/531G01N21/78G01N21/31
CPCC07C323/31C07D263/26C07K14/765C07K14/77C07K14/795C07K19/00G01N21/31G01N21/78G01N33/531G01N33/577
Inventor 肖治理王锋王弘沈玉栋杨金易孙远明王宇戴尽波刘凤银
Owner SOUTH CHINA AGRI UNIV
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