Immune antibody for detecting pesticide organic phosphorus residus and its application

A technology of organophosphorus pesticides and immune antibodies, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., and can solve the problems of limiting the application of immune analysis methods

Inactive Publication Date: 2005-03-23
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is: for the antibodies that exist in the current pesticide residue immunoassay usually have strong specificity to specific substances, most of the established immunoassay methods are only sui...

Method used

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  • Immune antibody for detecting pesticide organic phosphorus residus and its application
  • Immune antibody for detecting pesticide organic phosphorus residus and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Example 1. Preparation of Immunogen and Coated Antigen by Activated Ester Method

[0016] Take equimolar amount (0.1mmol) of diethylphosphonic acid acetic acid (0.0196g) and NHS (0.0109g), 0.11mmol DCC (0.0226g), add to the glass tube, dissolve the mixture with dioxane, room temperature React overnight in the dark, centrifuge at 15,000g for 10min the next day, remove the precipitate, take the supernatant and vacuum-dry it at 35°C, suspend the residue in 3ml dissolved in 20mg bovine serum albumin (BSA) (for the synthesis of artificial immunogen) or ovalbumin (OVA) (used in the synthesis of coating antigen) in sodium borate buffer. The mixture was magnetically stirred at room temperature for 1 hour, and dialyzed against 1 LPBS at 4° C. overnight (4 times of medium change). The coating antigen is a conjugate of hapten and ovalbumin, and the preparation method is the same as that of immunogen. Dialyzed samples were scanned with BACKMAN DU640 UV scanner for full-wavelength...

Embodiment 2

[0018] Example 2. Preparation of immunogen and coated antigen by water-soluble carbodiimide method

[0019] Weigh 0.0392 of diethylphosphonic acid acetic acid and dissolve it in 2ml of dimethylformamide (DMF), and stir at low temperature. Then weigh 8.0 mg of water-soluble carbodiimide (EDC), dissolve it in 1 ml of DMF, and slowly add it to the DMF solution of diethylphosphonic acid-acetic acid. Another 20 mg of BSA (for the synthesis of artificial immunogen) or OVA (for the synthesis of artificial coating antigen) was dissolved in 2 ml of DMF, stirred at 4°C, and slowly added to the above mixture. Continue to stir the reaction at 4°C for 8 hours, then react overnight at 14°C, centrifuge at 2000g for 10 minutes the next day, take the precipitate, dialyze the sample against PBS solution with pH 7.4 for 3 days, and use BACKMAN DU640 UV scanner to scan for full wavelength to identify the coupling results.

[0020] figure 2 a is a conjugate (coating source) of OVA-hapten prepar...

Embodiment 3

[0021] Example 3. Preparation of polyclonal antibody.

[0022] Three New Zealand white rabbits were immunized with the two immunogens prepared according to the method of Example 1 or Example 2. The specific immunization scheme is shown in Table 1.

[0023] Immunization times

[0024]After completing the immunization procedure in Table 1, the two antisera were tested with two coated antigens by square matrix titration to determine the optimal antigen-antibody combination and its optimal working concentration. The results are shown in Table 2. And use the best working conditions of antigen and antibody to analyze the inhibitory effect of different kinds of organophosphorus pesticides on the antigen-antibody binding reaction. Taking diethylphosphonic acid acetic acid as an object, a regression equation was established to calculate its I 10 and I 50 Select 13 pesticides including ethyl parathion, methyl parathion, dimethoate, omethoate, methamidophos, phoxim, chlorpyri...

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Abstract

The invention discloses a rudimental immune antibody which is used to detect organic phosphorus pesticide residue and its application, the character of the invention is using diethyl phosphonic acid acetic acid as hapten, produce artificial immunogen through coupling with bovine serum albumin(BSA), and produce several clone antibody through immune Zelanian giant blanc or produce mono clone antibody through chmice immune, cell amalgamation, filtration, clone planting and other steps, the several and single antibody can be used to indirect emulative ELISA, direct ELISA, or immune test paper to detect the rudimental phosphorus pesticide. The excellences of the invention including: the selected hapten has several organic phosphorus pesticides general structure, the structure reforming is not necessary, it can immune animal after coupling with protein, it can also be used to detect organic phosphorus pesticide in food and water source, this show the rapid and convenient of the immune detecting tehchnology.

Description

Technical field: [0001] The invention relates to an immune antibody for immunological detection of pesticide residues and its application, in particular to an immune antibody for detecting organophosphorus pesticide residues and its application. Background technique: [0002] Pesticides are indispensable substances for the prevention and control of diseases, insects, and weeds in current agricultural production. They play an extremely important role in promoting agricultural production, but at the same time they bring serious environmental problems: ecological imbalance, decline in the quality of agricultural products, and human health. Health is at risk. At present, organophosphorus and pyrethroid pesticides have highly effective insecticidal effects and are the main force of phytochemical protection. With the growing call for environmental protection in recent years, the environmental problems caused by the use of organophosphorus pesticides have attracted increasing atte...

Claims

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Application Information

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IPC IPC(8): G01N33/531G01N33/532G01N33/577
Inventor 刘贤金颜春荣余向阳张存政王冬兰
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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