ELISA (enzyme-linked immuno sorbent assay) detection kit for analyzing residual carbaryl and application thereof

A carnacarb and kit technology, which is applied in the field of ELISA detection kits for analyzing carnacarb residues, can solve the problems of complex pretreatment, low sensitivity and high cost of the analysis method, and achieves a simple pretreatment process and less time-consuming. Effect

Active Publication Date: 2017-05-17
CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The purpose of the present invention is to provide a method with high specificity, high sensitivity, high accuracy, and high precision for the shortcomings of the current pesticide residue instrument analysis method, such as high cost, complicated pretreatment, poor specificity, low sensitivity, and difficulty in experimental field detection. , The operation method is simple, and can be used for rapid detection of large batches of samples, ELISA detection kit for analyzing carbaryl residues

Method used

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  • ELISA (enzyme-linked immuno sorbent assay) detection kit for analyzing residual carbaryl and application thereof
  • ELISA (enzyme-linked immuno sorbent assay) detection kit for analyzing residual carbaryl and application thereof
  • ELISA (enzyme-linked immuno sorbent assay) detection kit for analyzing residual carbaryl and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Synthesis of the hapten N-(1-naphthyloxycarbonyl)-6-aminocaproic acid

[0022] 5.6g of NaOH (0.139mol) was dissolved in 56mL of distilled water, and 20g of α-naphthol (0.139mol) was added. The mixture was stirred at 85 °C for 1 h and cooled to room temperature. An excess of triphosgene (19.3 g of triphosgene and 100 mL of toluene) was slowly added in a fume hood, and the reaction was stirred at room temperature for 1 h. Anhydrous Na for organic phase 2 SO 4 After drying, the organic solvent was removed under reduced pressure to give a brown oil. It was dissolved in dichloromethane, vacuum distilled (1 mm Hg), and the fraction at 100°C was collected to obtain 13.7 g of the product naphthyl chloroformate as a pale yellow oil.

[0023] Dissolve 7.37g of 6-aminocaproic acid (56.1mmol) in 7.5mL of 4mol / L NaOH and cool to 4°C. Dissolve 6.25g of naphthyl chloroformate (30.3mmol) in 11mL of 1,4-dioxane, cool to 4°C, then mix with 7.5mL of 4mol / L cold NaOH, divide...

Embodiment 2

[0024] Example 2 Preparation of Carbaryl Coated Antigen

[0025] A conjugated complex was prepared with the hapten N-(1-naphthyloxycarbonyl)-6-aminocaproic acid and bovine serum albumin as the coating antigen. The preparation method is as follows:

[0026] (1) Dissolve 30.2mg of N-(1-naphthyloxycarbonyl)-6-aminocaproic acid, 13.9mg of N-hydroxysuccinimide and 24.3mg of N,N'-dicyclohexylcarbodiimide in 1mL of anhydrous In dimethylformamide, the reaction was stirred overnight at room temperature. The reaction solution was centrifuged (5000 rpm, 10 min), the precipitate was discarded, and the supernatant was collected, which contained active ester. (2) Dissolve 20mg of bovine serum albumin in 2mL of 0.05M, pH 9.6 carbonate buffer solution, add dropwise to the supernatant under stirring, and slowly add in about 20min. Then the stirring reaction was continued at room temperature for 4 h. (3) After the reaction, the reaction solution was put into a dialysis bag and dialyzed with...

Embodiment 3

[0027] Example 3 Construction of Carbaryl Phage Display Nanobody Library

[0028] The active ester method is used to couple the hapten of Example 1 to keyhole limpet hemocyanin, and the specific method is as follows:

[0029]Dissolve equimolar amounts of N-(1-naphthyloxycarbonyl)-6-aminocaproic acid, NHS and DCC in DMF, and stir overnight at room temperature for reaction. The reaction solution was centrifuged to discard the precipitate, and the supernatant was the active ester. The supernatant was added into the keyhole limpet hemocyanin solution under stirring, and the stirring reaction was continued for 4 h at room temperature. The reaction solution was put into a dialysis bag and dialyzed with PBS. Centrifuge, collect the supernatant, and freeze-dry to obtain the conjugate of N-(1-naphthyloxycarbonyl)-6-aminocaproic acid and keyhole limpet hemocyanin.

[0030] Dissolve 1mg of the conjugate in 1mL of normal saline, mix with 1mL of complete Freund's adjuvant, fully emulsif...

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Abstract

The invention provides an ELISA (enzyme-linked immuno sorbent assay) detection kit for analyzing residual carbaryl, which comprises a kit body, a dismountable ELISA plate arranged in the kit body and a reagent arranged in the kit body, wherein every hole of the ELISA plate is coated with carbaryl coating antigen, the reagent comprises an anti-carbaryl nano antibody, a carbaryl standard solution, a second antibody of an enzyme marker, a buffer solution PBS, a scrubbing solution PBST, substrate liquid, a developing solution, a reaction stop solution and the like. In the detection process, the coating antigen adsorbed on a hole wall of the ELISA plate and the carbaryl to be detected compete with each other to react with the antibody, and the competitive result is shown through chromogenic reaction. By detecting the carbaryl with known concentration, and drawing a standard curve, the concentration of carbaryl to be detected can be calculated. The ELISA detection kit provided by the invention has the advantages that the residual carbaryl in water, soil and vegetables can be accurately and sensitively detected, the pretreatment process of a sample is simple and consumes short time, a large quantity of samples can be detected simultaneously, and the sample detection cost is far lower than that of a traditional instrument detection method.

Description

technical field [0001] The invention relates to a pesticide residue analysis technology based on an ELISA method, in particular to an ELISA detection kit for analyzing carbaryl residues and an application thereof. Background technique [0002] Carbaryl is a carbamate pesticide. The routine detection of this kind of pesticide residues is mainly through the application of instrumental analysis methods, including gas chromatography (GC) and high performance liquid chromatography (HPLC). The instruments required for these analytical methods are expensive, the pretreatments such as sample separation, extraction, purification, and derivation are complicated, the analysis speed is slow, and the detection sensitivity is low, which makes it difficult to meet the needs of on-site rapid detection. Many physical and chemical analysis techniques have their own limitations, such as the poor thermal stability of carbaryl, which is difficult to analyze by gas chromatography, and liquid chro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543
CPCG01N33/543
Inventor 许艇王楷刘志平姬培林优优
Owner CHINA AGRI UNIV
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