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Methyl paraoxon enzyme-linked immunosorbent assay method

An enzyme-linked immunosorbent assay and methyl paraoxon technology, applied in the field of immunoassay, can solve the problem of lack of detection and establishment of ELISA method, and achieve the effects of high selectivity and sensitivity, low cost, good stability and repeatability

Inactive Publication Date: 2008-12-31
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is still a lack of establishment of an ELISA method for the detection of methyl paraoxon residues in actual samples

Method used

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  • Methyl paraoxon enzyme-linked immunosorbent assay method
  • Methyl paraoxon enzyme-linked immunosorbent assay method
  • Methyl paraoxon enzyme-linked immunosorbent assay method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1, the steps of indirect competition ELISA experimental method are as follows:

[0053]The methyl paraoxon standard was prepared in advance into a 1000 μg / mL methanol solution as a working mother solution, and stored at 4°C until use. Prepare PBST solution (0.01mol / L, pH7.4, 0.15mol / L NaCl, 0.5% Tween-20), and prepare a series of reaction solutions based on this to dilute competitor standard solution and antiserum.

[0054] a. Coating: Coat the enzyme-linked reaction plate with the coating agent at a set concentration, 100 μL / well, overnight at 4°C.

[0055] b. Washing: Wash the reaction plate three times with PBST, 3 min each time, 200 μL / well, and then spin dry the reaction plate.

[0056] c. Blocking: CBS containing 0.1% gelatin, 200 μL / well. Blocking at 37° C. for 2 hours.

[0057] d. Washing: Same as b

[0058] e. Competition: Dilute the methyl paraoxon mother solution into 0.01μg / mL, 0.1μg / mL, 0.5μg / mL, 1μg / mL, 5μg / mL, 10μg / mL, 20μg / mL series with st...

Embodiment 2

[0065] Embodiment 2, actual sample detection

[0066] Take the negative sample (cotton) as a representative sample, cut it to about 5mm×5mm, and mix well. Weigh 1.0g samples respectively and place them in 100mL conical flasks with stoppers, then add a certain concentration of methyl paraoxon standard acetone solution to it, so that the final concentration of methyl paraoxon in cotton is 0.1 μg / g, 1μg / g, 10μg / g, stand at room temperature for 15min. Add 20mL ethyl acetate to the Erlenmeyer flask, extract in an ultrasonic generator for 20min, and filter the extract. The residue was then ultrasonically extracted with 10 mL of ethyl acetate for 5 min, and the two filtrates were combined. Concentrate the filtrate to near dryness in a water bath rotary evaporator at 40°C, dissolve it with acetone and adjust the volume to 1 mL, then dry it with nitrogen, dissolve it in PBS containing 10%-20% methanol, and use it for ELISA detection; Oxonphosphorus has a certain degree of hydrophobi...

Embodiment 3

[0067] Embodiment 3, determination of recovery rate and sample extraction method

[0068] Take the negative sample (cotton) as a representative sample, cut it to about 5mm×5mm, and mix well. Weigh 1.0g samples respectively and place them in 100mL conical flasks with stoppers, then add a certain concentration of methyl paraoxon standard acetone solution to it, so that the final concentration of methyl paraoxon in cotton is 0.1 μg / g, 1μg / g, 10μg / g, stand at room temperature for 15min.

[0069] Add 20mL ethyl acetate to the Erlenmeyer flask, extract in an ultrasonic generator for 20min, and filter the extract. The residue was then ultrasonically extracted with 10 mL of ethyl acetate for 5 min, and the two filtrates were combined.

[0070] Concentrate the filtrate to near dryness in a 40°C water-bath rotary evaporator, dissolve it with acetone and make the volume to 1 mL, then dry it with nitrogen, and dissolve it in PBS containing 10-20% methanol.

[0071] Pipette 50 μL of the...

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Abstract

The invention discloses an enzyme-linked immunosorbent assay method of paraoxon-methyl, belonging to the technical field of immunoassay. The enzyme-linked immunosorbent assay method utilizes a synthetic paraoxon-methyl immunogen for immunization, thereby obtaining a polyclonal antibody; the paraoxon-methyl is taken as a standard product, and a conjugate of the paraoxon-methyl hapten and OVA is taken as a coating antigen, thereby establishing the indirect competitive enzyme-linked immunosorbent assay method of the paraoxon-methyl in textiles (cotton). The enzyme-linked immunosorbent assay method establishes the indirect ELISA method of the paraoxon-methyl pesticide metabolite in the textiles, thereby providing the rapid and high-efficient detection means for the residual detection of the paraoxon-methyl in the textiles; as the enzyme-linked immunosorbent assay method adopts the polyclonal antibody, the cost is lower, the stability and the repeatability are better. The sensitivity is 0.01ppm and the linear range is 0.1 to 20ppm. The high specificity and the high affinity of the immune reaction allow the ELISA to have very high selectivity and sensitivity.

Description

technical field [0001] An enzyme-linked immunosorbent detection method for methyl paraoxon, relates to a method for detecting pesticide metabolite residues, more specifically, an enzyme-linked immunosorbent assay (ELISA) is used to detect the detection of methyl paraoxon in textile raw material cotton The residue belongs to the technical field of immunoassay. Background technique [0002] Studies since the 1960s have further found that in addition to the pesticide itself, its metabolites also have residual toxicity problems, and the toxicity is stronger than that of the parent pesticide. This discovery not only promoted the strengthening of the research on the degradation of pesticide residues, but also triggered the research on the toxicity of metabolites in the process of pesticide degradation. Methyl parathion (M 1605) is a common organophosphorus pesticide, and methyl paraoxon (M 1600) is its metabolite. The toxicity of methyl paraoxon is more toxic than the original p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543G01N21/78
Inventor 胥传来殷祥刚尹丽梅袁媛徐一平马伟彭池方
Owner JIANGNAN UNIV
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