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ELISA kit for detecting estradiol and detection method thereof

An enzyme-linked immunosorbent reagent, estradiol technology, applied in the direction of biological testing, measuring devices, material inspection products, etc., can solve the problems of restrictions on popularization and application, unsuitability for on-site monitoring and screening of a large number of samples, high cost, etc., to increase the antigen sexual effect

Inactive Publication Date: 2006-05-03
BEIJING WANGER BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Due to the cumbersome sample pretreatment and high cost of the commonly used gas chromatography (GC) analysis method, it is not suitable for on-site monitoring and screening of a large number of samples, and its popularization and application is limited.

Method used

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  • ELISA kit for detecting estradiol and detection method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] The preparation of embodiment 1 kit components

[0067] 1. Antigen Synthesis

[0068] a. Synthesis of hapten Dissolve 100mg of estradiol in 4mL of acetone, add 0.42mM ethyl bromobutyrate, reflux in a water bath at 70°C for 4 hours, drain the solvent, add 4mL of 2N NaOH and continue to reflux for 1.5h, then use 6N Adjust the pH to 2 with HCL, extract twice with petroleum ether, combine the second organic phase, and spin dry to obtain a pale yellow solid powder that is the hapten.

[0069] b. Synthesis of immunogen Weigh 20 mg of the above-prepared hapten and dissolve it in 1 mL N, N'-dimethylformamide (DMF), add 40 mg of hydroxysuccinimide (NHS) dissolved in 1 mL of deionized water, and 40 mg of carbonized Diimine (EDC), 30mgN, N'-dicyclohexylcarbodiimide (DCC) solution, stirred and reacted overnight, the above solution was added dropwise to 30% N, N'-dimethylformamide ( DMF) dissolved in 20 mg hemocyanin (KLH) solution, reacted overnight, obtained after dialysis.

[...

Embodiment 2

[0090] The preparation of embodiment 2 kit components

[0091] 1. Antigen and Antibody Preparation

[0092] (1) Preparation of coating agent: goats were used as immunized animals, and pathogen-free goats were obtained by immunizing rabbit-derived antibodies as immunogens.

[0093](2) The preparation of polyclonal antibody adopts New Zealand white rabbit as immunization animal, uses estradiol and hemocyanin (KLH) conjugate as immunogen, and immunization dose is 1mg / kg, and immunogen is mixed with equal amount during the first immunization Freund's complete adjuvant was mixed to make an emulsifier, which was injected subcutaneously at multiple points on the back of the neck, and the same dose of immunogen plus an equal amount of Freund's incomplete adjuvant was mixed and emulsified at intervals of 4 weeks, and the booster immunization was performed once, a total of 5 immunizations, and the last Without adjuvant. Blood was collected 10 days after the last immunization, serum an...

Embodiment 3

[0098] Embodiment 3 detects the formation of the ELISA kit of estradiol

[0099] Set up the ELISA kit for detecting estradiol to include the following components:

[0100] (1) A microtiter plate coated with a conjugate of estradiol and ovalbumin;

[0101] (2) horseradish peroxidase-labeled goat anti-mouse anti-antibody diluent;

[0102] (3) 6 bottles of estradiol standard solution, the concentrations are 0μg / L, 0.5μg / L, 1.5μg / L, 4.5μg / L, 13.5μg / L, 40.5μg / L;

[0103] (4) Estradiol monoclonal antibody working solution has a concentration of 0.5 μgL;

[0104] (5) Substrate chromogenic solution A liquid is carbamide peroxide, and B liquid is tetramethylbenzidine;

[0105] (6) The concentrated washing solution is a phosphate buffer solution containing 0.01M pH 7.4, 0.8% Tween 20 and 0.5% sodium azide preservative;

[0106] (7) The stop solution is 1mol / L hydrochloric acid;

[0107] (8) The concentrated complex solution is a phosphate buffer solution containing 0.2% N,N'-dimeth...

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Abstract

The invention relates to an enzyme immune agent box for detecting destradiol which comprises: enzyme mark plate which coats antibody or destradiol antigen, enzyme mark material, standard solution, antibody working solution, base material color developing solution, compression cleaning liquid, ending solution and compression twin solution. The invention also discloses a method for applying the detecting method, which comprises: first doing sample front process, then using the agent box to detect, at last analyzing the detected result.

Description

technical field [0001] The invention relates to the technical field of veterinary drug residue detection and analysis, in particular to an ELISA kit and method for detecting estradiol in animal tissues, feed, urine and other samples. Background technique [0002] Estradiol (E2) is a widely used steroidal anabolic hormone, which is widely used to promote the growth of ruminants. Because it can promote animal growth and increase the lean meat ratio of livestock and poultry, it has been used in the poultry breeding industry. However, due to the high residue of estradiol in animal tissues, it endangers human health through the food chain, causing female children to develop ahead of schedule, and male children's mammary gland development is feminized; male reproductive system abnormalities may cause lesions; and lead to female breast cancer and intrauterine The incidence of membranous dysplasia is increasing, and estradiol has obvious carcinogenicity, and developed countries suc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/52G01N33/535G01N33/543
Inventor 沈建忠何方洋冯才伟万宇平吴小平冯才茂汪善良李军赵正苗张照亮史为民张素霞丁双阳罗晓琴孙倩
Owner BEIJING WANGER BIOTECH
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