A kind of heterocyclic amine 8-meiqx hapten, antibody and its preparation method and application
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An 8-meiqx, 21mg8-meiqx technology, applied in the field of small molecule compound immunochemistry and trace residue analysis, can solve the problems of complex meat products, not widely used, high instrument dependence, and achieve rapid enzyme-linked immunoassay detection. method, reliable technical support, high affinity effect
Active Publication Date: 2020-07-24
TIANJIN UNIV OF SCI & TECH
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[0003] Heterocyclic amines only exist in trace levels in cooked meat products, and the components of cooked meat products are quite complex. High-efficiency separation, enrichment and sensitive detection methods are often used. However, due to the high polarity of most heterocyclic amines And difficult to volatilize, only a few heterocyclic amines can be detected by GC or GC-MS after derivatization, but the application is not widely
With the development of ultra-performance liquid chromatography (UPLC), a powerful separation technique, ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) has also been applied to the detection of heterocyclic amines, but this type of method High dependence on instruments, high technical requirements for detection, and cumbersome process
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Embodiment 1
[0062] 1. Preparation of Heterocyclic Amine 8-MeIQx Hapten
[0063] Dissolve 20mg of succinic anhydride in 5mL of anhydrous dichloromethane, add 21mg of 8-MeIQx in 15mL of anhydrous dichloromethane solution and reflux at 40°C for 3h, and analyze by thin layer chromatography (Thin Layer Chromatography, TLC) Monitoring (developing agent components and ratios are dichloromethane: anhydrous methanol: ammonia water = 9:1:0.1, v / v), after the reaction is terminated, the solvent is removed by rotary evaporation at 60°C, that is, the heterocyclic amine 8-MeIQx half Antigen (molecular weight: 313.31).
[0064] 2. Preparation of heterocyclic amine 8-MeIQx immunogen:
[0065] Weigh 0.97mg of heterocyclic amine 8-MeIQx hapten in a 4mL brown bottle and dissolve it with 400μLDMF; weigh 1.07mg of NHS and 0.9mg of EDC into the brown bottle, stir at room temperature for 18h to activate; weigh 20mg of KLH protein and dissolve in 4mL of Phosphate buffer at pH 8.0; slowly add the prepared activ...
Embodiment 2
[0075] Use the heterocyclic amine antibody obtained in Example 1 to carry out immunodetection, take 8-MeIQx as the standard target to establish a standard curve, and the method detection results are used as quinoline heterocyclic amines (IQ, MeIQ) and quinoxaline heterocyclic amines in food. The total content of amines (IQx, 8-MeIQx, 4,8-DiMeIQx, 7,8-DiMeIQx, 4,7,8-TriMeIQx) and pyridine heterocyclic amines (PhIP) 8 kinds of polar heterocyclic amines.
[0076] (1) Coating: Dilute the coating antigen appropriately with coating solution, then coat 100 μL per well on a 96-well ELISA plate, and incubate overnight at 4°C or 3h at 37°C.
[0077] (2) Plate washing: add washing solution PBST, add 250 μL to each well, shake on the shaker for 2 minutes, discard PBST, repeat plate washing 3 times;
[0078] (3) Blocking: Add 200 μL of blocking solution to each well, block at 37°C for 1 hour, then discard the blocking solution, and wash the plate three times with PBST;
[0079] (4) Adding...
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Abstract
The invention provides a heterocyclic aromatic amine 8-MeIQx hapten and antibody and a preparing method and application of the antibody, and relates to synthesis of heterocyclic aromatic amine haptens, preparation of artificial complete antigens and antibodies and building of ensyme-linked immunoassay methods of the antibodies. According to the heterocyclic aromatic amine 8-MeIQx hapten and antibody and the preparing method and application of the antibody, aiming at the requirements for high throughput of heterocyclic aromatic amine detection and high sample preparation speed, a sensitive, efficient, quick and accurate immunoassay technology is built and solves the problems that existing physical-chemical analysis methods are cumbersome, complicated, high in cost and low in analysis speed.The method for detecting heterocyclic aromatic amines in food is built with the antibody, has good sensitivity, can simultaneously recognize eight polarity heterocyclic aromatic amines including 8-MeIQx, and has good compatibility, and the detection limit (in terms of 8-MeIQx) is 10.92 microgram / L; meanwhile, the detection method is low in cost, easy to operate and suitable for quick detection ofthe total quantity of the polarity heterocyclic aromatic amines in the food.
Description
technical field [0001] The invention belongs to the technical field of immunochemistry and trace residue analysis of small molecule compounds; relates to organic synthesis, immunochemistry, biochemistry and physical and chemical testing technology, etc.; especially relates to the design and synthesis of small molecule compound heterocyclic amine 8-MeIQx hapten and artificial antigen And the preparation of specific antibody of immunized animals and the establishment of immunoassay method. Background technique [0002] Heterocyclic amines (HAAs) are a class of polycyclic aromatic compounds containing N-heterocycles, which are produced by the high-temperature reaction of free amino acids, sarcosine, creatinine and carbohydrates, and have carcinogenic and mutagenic activities. Heterocyclic amines are widely found in fried foods, coffee drinks, alcohol, cigarette smoke, human body fluids, river water and the atmosphere. So far, more than 25 heterocyclic amines have been isolated...
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