3,5-dinitrosalicylic acid hydrazine enzyme-linked immunologic detecting kit and its use method

A technology of dinitrosalicylic acid and enzyme-linked immunoassay, which is applied in the field of immunochemistry, can solve the problems of high instrument cost, time-consuming and laborious processing, and complicated processing, and achieve simplified operation, improved specificity and sensitivity, and simple pre-treatment Effect

Active Publication Date: 2009-04-15
SOUTH CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0003] The currently reported detection methods for 3,5-dinitrosalicylic acid hydrazine residues mainly include liquid-mass chromatography (LC-MS) and high-performance liquid chromatography-mass spectrometry (HPLC-MS). The pretreatment is very complicated, the cost of the instrument is high, and it is time-consuming and laborious
Enzyme-linked immunoassay (ELISA) can make up for these deficiencies, so it can be used for the rapid detection of pesticide and veterinary drug residues, but there is no report on the ELISA of 3,5-dinitrosalicylic acid hydrazine. Using enzyme-linked immunosorbent immunoassay method for fast, sensitive and convenient detection, the key is to find a simple method for pretreatment of samples for the detection of 3,5-dinitrosalicylic acid hydrazine residues, as well as suitable antibodies, coating agents, etc., but at present There is no relevant technical report on the enzyme-linked immunoassay method for 3,5-dinitrosalicylic acid hydrazine

Method used

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  • 3,5-dinitrosalicylic acid hydrazine enzyme-linked immunologic detecting kit and its use method
  • 3,5-dinitrosalicylic acid hydrazine enzyme-linked immunologic detecting kit and its use method
  • 3,5-dinitrosalicylic acid hydrazine enzyme-linked immunologic detecting kit and its use method

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Experimental program
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Effect test

Embodiment 1

[0026] The synthesis of embodiment 1 hapten

[0027] Add 10mmol of m-carboxybenzaldehyde into a 50ml round bottom flask, slowly add methanol until the m-carboxybenzaldehyde is completely dissolved, add 8mmol of 3,5-dinitrosalicylic acid hydrazine while stirring, and stir at room temperature overnight; after the reaction, filter, 20mL The precipitate was washed twice with methanol, dried, and the yellow powdery solid was CPDNSH: APCI-MS (negative) m / z: 374 [M-H] - . 1 HNMR (600MHz, d 6 -DMSO, TMS): δ14.05(s, 1H); 8.82(d, J=3.0Hz, 1H); 8.58(d, J=3.0Hz, 1H); 8.48(s, 1H); 8.35(s, 1H); 7.97(t, J=7.8Hz, 1H); 7.59(t, J=7.7Hz, 1H).

[0028] Its structure is shown in formula (I):

[0029]

Embodiment 2

[0030] Synthesis of Example 2 Antigen

[0031] Dissolve 0.1mmol of the hapten CPDNSH in 2 mL of DMF, stir and add DCC 27.5 mg and NHS 14.4 mg, and react overnight with magnetic stirring at 4°C. After centrifugation, the supernatant is liquid A; weigh 140 mg of BSA and dissolve in 10 mL at a concentration of 0.1 mol / Add 1 mL of DMF to PBS (pH 8.0) in L, stir and dissolve to prepare liquid B; under magnetic stirring, liquid A is gradually dropped into liquid B, and react at 4°C for 12 hours; after centrifugation, take the supernatant and use physiological Saline dialysis was performed for 3 days, and the dialysate was changed 3 times a day to obtain the whole antigen CPDNSH-BSA. Packed at a concentration of 1g / L. Freeze and store in -20°C refrigerator for immunization. The structure of CPDNSH-BSA is shown in formula (II):

[0032]

Embodiment 3

[0033] The synthesis of embodiment 3 coating original

[0034] Dissolve 0.1mmol of the hapten CPDNSH in 2mL of DMF, stir and add 27.5mg of DCC and 14.4mg of NHS, and react overnight with magnetic stirring at 4°C. After centrifugation, the supernatant is A liquid; Add 1 mL of DMF to 0.1mol / L PBS (PH8.0), stir and dissolve to prepare solution B; under magnetic stirring, gradually drop solution A into solution B, and react at 4°C for 12 hours; after centrifugation, take the supernatant for 4 nights. Dialyze with normal saline for 3 days at ℃, change the dialysate 3 times a day, and obtain the coated original CPDNSH-OVA. Packed at a concentration of 1g / L. Store frozen in a -20°C refrigerator for wrapping.

[0035] The coating former CPDNSH-OVA of preparation has structure shown in formula (III):

[0036]

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Abstract

The invention discloses a 3, 5-dinitrosalicylic acid hydrazide enzyme-linked immunodetection test kit which comprises a box body, specific antibody solution, 3, 5-dinitrosalicylic acid hydrazine standard solution, an ELISA plate, sample diluent, washing fluid, second antibody solution, colored solution A, colored solution B and stopping solution; wherein, the specific antibody is a rabbit anti-3,5-dinitrosalicylic acid hydrazide antibody; and half antigens and ovalbumin conjugates are coated inside the hole of the ELISA plate; and the second antibody solution is horseradish peroxidase labeled goat anti-rabbit antibody diluent. After the sample to be tested is hydrolyzed by hydrochloric acid, derived by o-nitrobenzaldehyde, extracted by ethyl acetate, re-dissolved by the sample diluent and degreased by n-hexane, and the like, the test kit is used for detection and analysis. The test kit is strong in specificity, and the minimum detection limit is 74.8ppt, thereby being suitable for rapid detection of 3, 5-dinitrosalicylic acid hydrazine in feed, meat, urine, milk and other samples by the health, quality supervision and customs departments, meat processing plants and livestock farms.

Description

technical field [0001] The invention belongs to the technical field of immunochemistry, and in particular relates to a 3,5-dinitrosalicylic acid hydrazine enzyme-linked immunoassay kit and a use method thereof. Background technique [0002] As a metabolite of nifursol, 3,5-dinitrosalicylate hydrazine can indicate the use of the banned veterinary drug nifursol. 3,5-dinitrosalicylic acid hydrazine residues have been detected many times in my country's chicken products exported to the EU, which seriously restricts the export of animal-derived foods in my country and damages the good image of my country's animal husbandry. Therefore, in order to protect people's health, ensure the quality of imported and exported meat products, and facilitate the vigorous development of my country's animal husbandry, it is necessary to find a fast, sensitive and convenient method to detect 3,5-dinitrohydrin in animal muscle tissue. Hydrazine sylate remains. [0003] Currently reported methods f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543G01N33/53
Inventor 沈玉栋张世伟孙远明雷红涛王弘肖治理杨金易
Owner SOUTH CHINA AGRI UNIV
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