Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting beta-receptor stimulant

An enzyme-linked immunosorbent reagent and receptor stimulant technology, which is applied in the field of veterinary drug residue analysis and immunology, can solve the problems of unfavorable standardization, insufficient sensitivity, and limited types of drugs, and achieve high precision, good sensitivity, and high sensitivity.

Inactive Publication Date: 2012-11-07
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The sensitivity of polyclonal antibodies in the above studies is high, but it is not conducive to standardization; although monoclonal antibodies are conducive to standardized production, their sensitivity is not enough, and the types of drugs detected are limited

Method used

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  • Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting beta-receptor stimulant
  • Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting beta-receptor stimulant
  • Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting beta-receptor stimulant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Preparation of Immunogen and Coating Original

[0029] 1.1 Synthesis of albuterol immune hapten (SAL-SA)

[0030] Salbutamol immune hapten (SAL-SA) was synthesized according to the following route: Weigh 80 mg (0.33 mmol) of salbutamol base, dissolve it in 10 mL of methanol, and evaporate it into a yellow oil by rotary evaporation, then add 16 mL of absolute ethanol to dissolve it, keep stirring, and then dropwise Add 36 mg of succinic anhydride (dissolved in pyridine solution, 0.36 mmol). After white turbidity appeared, the structure of succinic anhydride was detected by thin-layer chromatography. After reacting at room temperature for 3 h, stop stirring, centrifuge at 2750 r / min for 15 min, discard the supernatant, wash the precipitate 3 times with absolute ethanol, and evaporate the solvent to dryness to obtain a white solid that is the hapten SAL-SA.

[0031]

[0032] 1.2 Preparation of salbutamol immunogen (SAL-SA-BSA)

[0033] Dissolve 40 mg of SAL...

Embodiment 2

[0039] Example 2 Preparation of Monoclonal Antibody

[0040] 2.1 Immunization of mice

[0041] Refer to the method in Xue Qingshan's "Principles and Techniques of In Vitro Culture" Science Press, 2001 edition: use the SAL-SA-BSA conjugate prepared in Example 1 as the immunogen, and immunize Balb / C female mice. The immunization program adopts one basic immunization and several booster immunizations. For the first immunization, a protein emulsion containing 100 μg of immunogen emulsified with an equal volume of Freund’s complete adjuvant was injected subcutaneously on the back of the neck of the mouse for basic immunization, and then every 15 days with protein emulsion containing 100 μg of immunogen emulsified with Freund’s incomplete adjuvant Protein emulsion of immunogen for booster immunization. From the third immunization, the tail blood was collected on the 8th day after each immunization, the serum was separated, and the serum antibody titer was detected by indirect ELIS...

Embodiment 3

[0046] Example 3 Establishment of Indirect Competitive ELISA Detection Method

[0047] 3.1 Reagent preparation

[0048] Carbonate buffer (pH9.6): Accurately weigh Na 2 CO 3 1.59g, NaHCO 3 2.93g, dissolved in a small amount of ultrapure water, and adjusted to 1000mL.

[0049] Washing solution (pH7.4): Accurately weigh 8.00g of NaCl, KH 2 PO 4 0.20g, Na 2 HPO 4 12H 2 O 2.90g, KCl 0.20g, dissolved in a small amount of ultrapure water, Tween 20 0.50mL was added, and the volume was adjusted to 1000mL.

[0050] Phosphate buffer (pH7.4): Accurately weigh 8.00g of NaCl, KH 2 PO 4 0.20g, Na 2 HPO 4 12H 2 Dissolve 2.90g of O, 0.20g of KCl in a small amount of ultrapure water, and dilute to 1000mL.

[0051] Blocking solution: Accurately weigh 10.00 g of ovalbumin, add 1000 mL of phosphate buffer, stir and mix until the protein is completely dissolved.

[0052] Substrate mixture: Accurately absorb 10mL of substrate B solution (purchased from Wuhan Feiyuan Technology Co....

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Abstract

The invention discloses a monoclonal antibody capable of identifying a beta-receptor stimulant. The monoclonal antibody is secreted by a hybridoma cell line Sal, which is preserved in China Center for Type Culture Collection, and has a preservation number of CCTCC NO:C201187. The invention also discloses an enzyme-linked immunosorbent assay method and a kit for detecting the beta-receptor stimulant, and application thereof to detection of the beta-receptor stimulant. Compared with prior art, the monoclonal antibody prepared by the invention can identify 8 beta-receptor stimulant drugs, has a wide range of identification; and the monoclonal antibody, the ELISA kit and the ELISA method provided by the invention have high sensitivity, and can detect beta-receptor stimulant residues with lower concentration.

Description

technical field [0001] The invention belongs to the technical field of veterinary drug residue analysis and immunology, and specifically relates to a monoclonal antibody capable of recognizing eight kinds of β-receptor stimulants, an enzyme-linked immunosorbent method and a kit. Background technique [0002] β-receptor agonists are a group of phenylethanolamine derivatives similar in structure and physiological function to adrenaline and norepinephrine, which can bind to β-adrenergic receptors on most tissue cells in animals and activate β-adrenergic receptors. - Adrenergic receptors, thereby accelerating the decomposition of intracellular fat and the oxidation of free fatty acids. Therefore, the main effect of β-receptor stimulants is to promote the growth of animal muscle tissue, reduce the content of carcass fat, and increase the lean meat rate. At the same time, the growth rate and feed utilization rate can be improved to varying degrees. The European Union and some ot...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577
Inventor 袁宗辉王玉莲章玲燕彭大鹏潘源虎陶燕飞刘振利
Owner HUAZHONG AGRI UNIV
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