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Application of endosulfan artificial antigen in enzyme linked immunosorbent assay kit

An enzyme-linked immunosorbent reagent and kit technology, used in biological testing, animal/human proteins, serum albumin, etc., can solve the problems of hazard, mammalian cumulative toxicity, long validity period, etc., and achieve high specificity and detection method. Efficient and accurate results

Active Publication Date: 2020-08-07
BEIJING KWINBON BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The drug has a long validity period, bioaccumulation effect, great harm to fish, shellfish, etc., and cumulative toxicity to mammals

Method used

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  • Application of endosulfan artificial antigen in enzyme linked immunosorbent assay kit
  • Application of endosulfan artificial antigen in enzyme linked immunosorbent assay kit
  • Application of endosulfan artificial antigen in enzyme linked immunosorbent assay kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] The preparation of embodiment 1 kit components

[0025] 1. Preparation of endosulfan hapten

[0026] Take 3.6g of endosulfan, add 100ml of 1,4-dioxane to dissolve, stir well, then add 3.0g of molecular sieve, add 1.02g of succinic semialdehyde, stir well, pass in hydrochloric acid gas, and stir at room temperature for 5h in the dark; stop For reaction, add 0.5mol / L NaOH solution, shake 200ml fully, add 200ml ethyl acetate, shake fully, let stand, separate the water phase, add 80ml water to the organic phase, wash, shake, stand still, separate the water phase, organic phase Dry and evaporate to dryness over anhydrous sodium sulfate, apply to a silica gel column, and elute and purify with dichloromethane / methanol (v / v, 10 / 1) to obtain 2.1 g of succinic acetal endosulfan hapten product with a yield of 45.75%.

[0027] 2. Antigen preparation

[0028] Immunogen preparation—the endosulfan hapten was coupled with bovine serum albumin (BSA) to obtain the immunogen.

[0029] ...

Embodiment 2

[0041] Embodiment 2 detects the formation of the ELISA kit of endosulfan

[0042] An enzyme-linked immunosorbent assay kit for detecting endosulfan was set up to include the following components:

[0043] (1) A microtiter plate coated with an endosulfan-coupled antigen;

[0044] (2) 6 bottles of endosulfan standard solution, the concentrations are 0μg / L, 1μg / L, 3μg / L, 9μg / L, 27μg / L, 81μg / L;

[0045](3) endosulfan antibody labeled with horseradish peroxidase;

[0046] (4) Substrate chromogenic solution is made up of A liquid and B liquid, and A liquid is carbamide peroxide, and B liquid is tetramethylbenzidine;

[0047] (5) The stop solution is 2mol / L sulfuric acid;

[0048] (6) The washing solution has a pH value of 7.4, contains 0.5% to 1.0% Tween-20, 0.01‰ to 0.03‰ sodium azide preservative, and 0.1 to 0.3mol / L phosphate buffer, and the percentage is weight volume percentage;

Embodiment 3

[0049] The detection of endosulfan in embodiment 3 vegetables and fruits

[0050] 1. Detection with kit

[0051] Number the corresponding microwells of the samples and standards in sequence, make 2 parallel wells for each sample and standard, and record the positions of the standard wells and sample wells. Add 50 μl of standard / sample to corresponding microwells, then add 50 μl / well of enzyme conjugate working solution, and react for 30 minutes at 25°C in a dark environment. Shake the liquid in the hole dry, wash 4-5 times, and pat dry. Add 50 μl / well of substrate solution A, then add 50 μl / well of substrate solution B, mix well, and react for 15 minutes at 25°C in a dark environment. Add 50 μl / well of stop solution, mix well, set the microplate reader at 450 nm, and measure the OD value of each well.

[0052] 2. Analysis of test results

[0053] The percent absorbance of a standard or sample is equal to the average of the absorbance values ​​of the standard or sample (dou...

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Abstract

The invention provides an enzyme linked immunosorbent assay kit for detecting endosulfan. The kit comprises an ELISA plate coated with a coating antigen, a endosulfan standard substance solution, an endosulfan antibody, an enzyme conjugate concentrated solution, an enzyme conjugate diluent, a substrate developing solution, a stop solution and a washing solution. The coating antigen is an endosulfan coupling antigen, the enzyme conjugate is an enzyme-labeled endosulfan antibody, and the endosulfan antibody is obtained by immunizing animals with an immunogen. The invention also discloses a method for detecting endosulfan by using the enzyme linked immunosorbent assay kit. The method comprises the following steps of pretreating a sample, detecting by using the kit, and finally analyzing the detection result. The enzyme linked immunosorbent assay kit provided by the invention can be used for detecting the content of endosulfan in vegetable and fruit samples, is simple and convenient to operate, is low in cost and high in sensitivity, can be monitored on a site, and is suitable for screening a large number of samples.

Description

technical field [0001] The invention relates to an application technology of an endosulfan artificial antigen in an ELISA kit, in particular to an endosulfan artificial antigen molecular structure and an ELISA kit for detecting endosulfan, which can qualitatively and quantitatively detect vegetables and Residues of endosulfan drug in fruit. Background technique [0002] Endosulfan is a synthetic organochlorine compound widely used as a pesticide insecticide. Because the drug has the advantages of wide insecticidal range, low price, and low toxicity to bees, it is widely used in various crops. my country began to produce endosulfan in the 1990s. By 2011, there were 47 registered endosulfan products, involving 43 pharmaceutical companies, making it the second largest producer of the drug. The drug has a long period of validity, has bioaccumulation effect, is relatively harmful to fish and shellfish, and has cumulative toxicity to mammals. A large number of literatures have ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/58G01N33/53C07K14/765
CPCG01N33/577G01N33/581G01N33/5308C07K14/765
Inventor 王兆芹马玉华何方洋崔廷婷万六龙陶燕飞冯才伟
Owner BEIJING KWINBON BIOTECH
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