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Preparation and Application of Clenbuterol Monoclonal Antibody

A monoclonal antibody and antibody technology, used in enzyme-linked immunosorbent assay kits and colloidal gold test paper cards, the detection of clenbuterol drug residues in samples, and the preparation of clenbuterol monoclonal antibodies, which can solve the problem of kits. Low sensitivity, poor stability of detection products, cumbersome and time-consuming processing methods, etc.

Active Publication Date: 2016-12-14
BEIJING KWINBON BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the current enzyme-linked immunoassay kit for detecting clenbuterol is not ideal for detecting clenbuterol specificity, the sensitivity of the kit is not high, the stability is not good, the sample pretreatment method is tedious and time-consuming, and the Most of the reagents used are organic reagents, which are harmful to the human body and the environment; the clenbuterol colloidal gold test paper card has the disadvantages of poor product stability and inaccurate test results

Method used

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  • Preparation and Application of Clenbuterol Monoclonal Antibody
  • Preparation and Application of Clenbuterol Monoclonal Antibody
  • Preparation and Application of Clenbuterol Monoclonal Antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] Example 1 Preparation of clenbuterol monoclonal antibody

[0090] 1. Preparation of clenbuterol antigen

[0091] 1. Synthesis of clenbuterol hapten (see Figures 1a-1d )

[0092](1) Add 1.0-10.0g clenbuterol hydrochloride to 20-100ml dimethylformamide (DMF), add 5-25ml triethylamine, 10-20% molar equivalent of 4-dimethylaminopyridine (DMAP ), add dropwise a solution of 1.1-1.5 molar equivalents of tert-butyldimethylsilyl chloride in 10-50ml DMF at 0°C, after the dropwise addition, continue to react at room temperature for 1-10 hours, evaporate most of the solvent, add water , extracted with ethyl acetate, washed with water, dried, and recrystallized in cyclohexane-ethyl acetate after removing the solvent to obtain a white solid, which is O-silylated clenbuterol; (the synthetic route is as follows Figure 1a )

[0093] (2) 11.0-5.0g of the product obtained in step (1), add 20-50ml DMF to dissolve, add 0.5-1.5g NaOH, add dropwise 1.1-1.5 molar equivalent of ethyl bromo...

Embodiment 2

[0130] Example 2 Detection of Clenbuterol ELISA Kit Preparation and Application

[0131] 1. Preparation of clenbuterol ELISA kit components

[0132] The present invention detects the ELISA kit of clenbuterol, which comprises:

[0133] (1) 96-well enzyme plate coated with clenbuterol antigen;

[0134] (2) Enzyme-labeled secondary antibody;

[0135] (3) Clenbuterol antibody working solution;

[0136] (4) clenbuterol standard solution;

[0137] (5) clenbuterol high concentration standard solution;

[0138] (6) Substrate solution A liquid;

[0139] (7) Substrate solution B solution;

[0140] (8) Termination solution;

[0141] (9) Concentrate the washing solution.

[0142] 1. Preparation of ELISA plate

[0143] Use 0.1 mol / L carbonate buffer solution with a pH of 9.6 to dilute the clenbuterol coating source in Example 1 to 0.1-0.5 μg / mL, and add 100 μl of the coating source dilution to the microwells of the microtiter plate. solution, incubated at 37°C for 2 hours, poured...

Embodiment 3

[0219] Example 3 Preparation and Application of Detecting Clenbuterol Colloidal Gold Test Paper Card

[0220] 1. Preparation of test strips

[0221] 1. Preparation of clenbuterol monoclonal antibody-colloidal gold marker

[0222] (1) Preparation of colloidal gold

[0223] Take 100ml of 0.01% chloroauric acid aqueous solution and place it in a conical flask, heat it to boiling with a constant temperature electromagnetic stirrer, add 2ml of 1% trisodium citrate aqueous solution under the condition of continuous stirring at high temperature, continue to stir and heat at a constant speed for 15min, the solution is Translucent red. Cool at room temperature, restore to the original volume with deionized water, and store at 4°C.

[0224] (2) Preparation of gold-labeled antibody

[0225] Adjust the colloidal gold solution to pH 7.2 with 0.1mol / L potassium carbonate, add the clenbuterol monoclonal antibody in Example 2 to the colloidal gold solution according to the standard of add...

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Abstract

The invention provides a clenbuterol monoclonal antibody and application thereof. The invention discloses a preparation method of a clenbuterol monoclonal antibody. The clenbuterol antigen is obtained by synthesizing the clenbuterol hapten and immunizing the clenbuterol hapten with a carrier protein, and immunizing animals with the clenbuterol antigen with high specificity. Sexual monoclonal antibodies. The invention also provides a method of applying the clenbuterol monoclonal antibody to the clenbuterol ELISA kit to detect clenbuterol. The invention also provides a method for applying the clenbuterol monoclonal antibody to the clenbuterol colloidal gold test paper card to detect clenbuterol. The clenbuterol monoclonal antibody prepared by the present invention has high specificity and low cost, and the clenbuterol ELISA kit and colloidal gold test paper card prepared by the clenbuterol monoclonal antibody can detect clenbuterol drug It has the characteristics of convenient operation, low detection cost, high specificity, high sensitivity, high accuracy, high precision, and rapid detection.

Description

technical field [0001] The invention belongs to the technical field of biochemical analysis, and specifically relates to a preparation method and application of a clenbuterol monoclonal antibody, an enzyme-linked immunoassay kit and a colloidal gold test paper card for detecting clenbuterol, which are especially suitable for Detection of clenbuterol drug residues in livestock tissue, urine samples, feed, dairy products and other samples. technical background [0002] Clenbuterol (Clenbuterol, CBL), the scientific name is Clenbuterol hydrochloride, also known as dicyandiamide, chlorpheniramine, and clenbuterol. Clenbuterol is a β-stimulant. In the early 1980s, a company in the United States began to add it to feed to increase lean meat percentage. After eating pigs, it can increase the growth rate, promote protein synthesis in the metabolic process, accelerate the transformation and decomposition of fat, and increase the lean meat percentage of pork, so it is called lean mea...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/44C07C215/68C07C213/00G01N33/577
Inventor 何方洋冯才伟付军权冯才茂聂雯莹杨秀贤刘福林段盈盈
Owner BEIJING KWINBON BIOTECH
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