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Butachlor polarized fluorescence immunity detection method

An immunodetection method and polarization fluorescence technology, applied in the field of biological immunoassay analysis, can solve problems such as polarization fluorescence immunoassay of pesticides that have not yet been seen

Inactive Publication Date: 2012-05-30
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there is no relevant report on the polarization fluorescence immunoassay of pesticides in China

Method used

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  • Butachlor polarized fluorescence immunity detection method
  • Butachlor polarized fluorescence immunity detection method
  • Butachlor polarized fluorescence immunity detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Preparation method of fluorescently labeled hapten

[0034] In the following, the preparation method of hexamethylene diamine fluorescein isothiocyanate (abbreviated as HDF, n=6) labeled hapten is taken as an example to illustrate, other methods of labeling haptens with fluorescein isothiocyanate and aminofluorescein are the same as this similar.

[0035] Dissolve 15.2 mg of butachlor hapten (BMPA), 16.0 mg of N,N'-dicyclohexylcarbodiimide and 9.7 mg of N-hydroxysuccinimide (NHS) in N,N-dimethylformamide (DMF), stirred and reacted overnight, added 4.5mg HDF to the resulting supernatant, and stirred and reacted overnight in the dark at room temperature, and the product was separated with chloroform+methanol (4:04, v / v ) as developing agent. Products with different Rf were extracted with methanol, and the resulting extract (named BMPA-HDF) was used for activity identification.

Embodiment 2

[0036] Example 2 Identification of fluorescently labeled hapten activity

[0037] The polarized fluorescence value of the fluorescently labeled hapten combined with 100-fold diluted antiserum was measured on a PE Victor-3 instrument, and the activity of the fluorescently labeled hapten was identified by comparing the change in mp value after the fluorescently labeled hapten was combined with the specific antibody. As shown in Table 1, products with different Rf values ​​have different polarization values ​​after reacting with antibodies, and products with Rf=0.9 have the largest signal difference before and after reaction, which can specifically bind to antibodies, indicating that the product is active, so Rf=0.9 The product was used as a fluorescently labeled hapten (MBPA-HDF) for subsequent studies.

[0038] Table 1

[0039]

Embodiment 3

[0040] Example 3 standard curve drawing

[0041] Borate buffer (BB solution) was used as diluent for all sample dilutions. Add 100 μL of fluorescently labeled haptens and 20 μL of butachlor standards of different concentrations to the wells of the fluorescent microplate successively, and then add 100 μL of 400-fold diluted butachlor antibody. After 15 minutes, the FP value is passed by Wallac VICTOR 3 1420 multi-label analyzer for determination. Take the mP value as the ordinate, and the logarithm value of the standard concentration as the abscissa, and apply the four-parameter logarithmic equation of originPro7.5 software for curve fitting to establish a competitive inhibition curve, see the attached figure 1 . attached figure 1 The standard curve of butachlor polarized fluorescence immunoassay for the butachlor polarized fluorescence immunoassay established for utilizing hexamethylenediamine fluorescein isothiocyanate (n=6) to label the hapten, which is consistent with t...

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Abstract

The invention discloses a butachlor polarization fluorescence immunoassay method, mainly comprising the following steps: mixing the sample to be analyzed with the fluorescent-labeled hapten, adding the butachlor antibodies, directly measuring the polarization fluorescence signals of the reaction system, and calculating the butachlor concentration in the sample to be analyzed according to the standard curve set up by the butachlor standard sample. The method can be operated at room temperature, and the entire reaction time is very short; the method can simultaneously detect a plurality of samples in one step, without washing; after being equipped with a mechanical hand, the invention can realize sample adding and automated reading, which is a high-flux automated immunoassay method.

Description

technical field [0001] The invention belongs to the technical field of biological immune detection and analysis, and in particular relates to a rapid homogeneous fluorescence immunological detection method for herbicides. Background technique [0002] In recent years, incidents of mass poisoning caused by irrational use of pesticides and excessive pesticide residues in agricultural products have occurred frequently, and pesticide residue standards have also become the main content of technical barriers to trade in developed countries. The problem of pesticide residues not only seriously endangers the health of the people, but also seriously affects the international competitiveness of my country's agricultural products. [0003] Butachlor is a highly efficient pre-emergence selective chloroacetanilide herbicide, which is mainly used to remove weeds in rice fields in my country. It is one of the most produced and used herbicide varieties, but butachlor is It has strong stabil...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/53G01N21/76G01N33/577
Inventor 雷红涛吴青薛钢沈玉栋孙远明王弘徐小艳李美英肖治理柳春红
Owner SOUTH CHINA AGRI UNIV
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