Analytical kit of enzyme linked immunosorbent assay for residual carbaryl

A technology of enzyme-linked immunosorbent adsorption and analytical reagents, which is applied in the direction of material analysis, material analysis by optical means, biological testing, etc., can solve the problems of complex operation, low sensitivity, and unstable detection results, and achieve simple pretreatment process, The effect of less time consumption and lower cost of sample testing

Inactive Publication Date: 2005-10-26
LUDUN BIOLOGICAL SCI & TECH DEV XIAN
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Problems solved by technology

[0008] In order to solve the shortcomings of the current chromatographic analysis method for carbaryl pesticide residues such as high cost, complicated operation, poor specificity, low sensitivity and unstable detection results in the rapid detection method of enzyme inhibition, the present invention provides a high specificity, high sensitivity, An enzyme-linked immunosorbent assay kit for carbaryl residues with high accuracy, low cost, simple operation method and rapid detection of large quantities of samples

Method used

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  • Analytical kit of enzyme linked immunosorbent assay for residual carbaryl
  • Analytical kit of enzyme linked immunosorbent assay for residual carbaryl

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] synthetic hapten

[0017] 5.6 g of NaOH (0.139 mol) was dissolved in 56 mL of distilled water; 20 g of 1-naphthol (0.139 mol) was added to the solution. The mixture was stirred at 85 °C for 1 h and cooled to room temperature. Slowly add excess triphosgene (19.3g triphosgene + 100mL toluene; triphosgene is highly toxic, be careful) in a fume hood, and stir the reaction at room temperature for 1h. Anhydrous Na for organic phase 2 SO 4 After drying, the organic solvent was removed under reduced pressure in vacuo to obtain a brown oily substance. It was dissolved in dichloromethane, and the fraction at 100°C was collected by vacuum distillation (1 mmHg) to give 13.7 g (48%) of an intermediate product (naphthyl chloroformate) as a light yellow oil.

[0018] 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 the intermediate product (30.3mmol) in 11mL of 1,4-dioxane, cool to 4°C, then mix with 7.5mL of cold 4mol / L ...

Embodiment 2

[0020] Preparation of immunogen and enzyme-labeled hapten

[0021] (1) Immunogen

[0022] Dissolve about 12.2 μmol of the hapten in 200 μL of anhydrous N,N'-dimethylformamide (DMF), then add 2.9 μL (about 12.2 μmol) of tri-n-butylamine, 1.6 μL (about 12.3 μmol) of chlorine Isobutyl formate was stirred at room temperature for 1 h. The reaction solution was diluted with 1.8 mL of anhydrous DMF.

[0023] Dissolve 30mg of BSA in 2mL of carbonate buffer solution (0.05mol / L, pH 9.6), add 100μL of the first step reaction solution dropwise to the BSA solution under stirring, add it in about 20min, and stir at room temperature for reaction 3h. Then dialyzed, centrifuged, freeze-dried, and stored at 4°C.

[0024] (2) Enzyme-labeled hapten

[0025] The method used was the same as that of immunogen synthesis, replacing BSA with horseradish peroxidase (HRP). Finally, an equal volume of glycerol was added to the conjugate and stored at -20°C.

Embodiment 3

[0027] Preparation of carbaryl monoclonal antibody

[0028] (1) To immunize Balb / c mice, the dose of immunogen is 100 μg / mouse each time, and the immunogen is diluted to a certain concentration before immunization. Freund's complete adjuvant was used to emulsify with the immunogen for the first immunization, and Freund's incomplete adjuvant was used for booster immunization, once every 2 weeks for a total of 5 immunizations, and the injection site was subcutaneous on the back of the mouse. For the last immunization, mice were injected intraperitoneally without any adjuvant.

[0029] (2) Prepare feeder cells, take splenocytes from immunized mice and fuse them with Sp2 / 0 myeloma, the ratio of splenocytes to myeloma cells is 10:1-3, and the fusion agent is PEG2000;

[0030] (3) On the 8th day after fusion, the antibody level in the culture supernatant of fusion cells was detected by indirect non-competitive ELISA method, and the wells with OD value greater than 1.0 were regarded...

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Abstract

This inventon shows a reagent box , which is used for enzyme linked immunosorbent assay of remained carbaryl. It belongs to enzyme linked immunosorbent assay technology. The common methods of carbaryl rationalize analyze are too complex, slow and its cost is high. This invention overcomes these defaults. It can quickly detect the remained carbaryl in water, soil and food samples. In the process, the antigen on the enzyme target rivals carbaryl . It reacts with the antigens in the solution. And the result is showed by enzyme catalysis color reaction. You can estimate the consistency of carbaryl by detecting the solution, whose consistency of carbaryl is known and drawing calibration vurve. The retention period of the box is more than 6 months.

Description

1. Technical field [0001] The invention relates to an enzyme-linked immunosorbent assay (ELISA) kit for carbaryl residues, which is mainly suitable for the rapid determination of carbaryl residues in large quantities of vegetables, water, soil and other samples. Enzyme Immunoassay [0002] technology field. 2. Background technology [0003] Carbamate insecticides are the main types of insecticides produced and applied in my country, and carbaryl is one of them, and its trade name is carbaryl. In my country, carbaryl has a large production volume and is widely used. It is used to control various crop pests such as rice planthoppers, rice leafhoppers, cotton pink bollworms, and soybean borers. It is also used to control livestock and poultry parasites and household hygiene. Insecticide, its main toxic metabolite is 1-naphthol. Carbaryl is considered to be a safe insecticide, but studies by many scholars have shown that carbaryl has toxic side effects on humans and animals: s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543
Inventor 许艇潘峰李季井德明
Owner LUDUN BIOLOGICAL SCI & TECH DEV XIAN
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