Chemiluminiscence immunoassay kit for detecting zilpaterol

Abstract

The invention provides a chemiluminiscence immunoassay kit for detecting zilpaterol, and belongs to the field of immunological detection. The kit comprises a non-transparent white ELISA plate coated with a zilpaterol-carrier protein conjugate, a zilpaterol standard substance, a zilpaterol-peroxidase labeled antibody working solution, a luminescent substrate solution, a concentrated sample diluent and a concentrated washing liquid. The zilpaterol-carrier protein conjugate is obtained by coupling zilpaterol and a carrier protein through a mixed anhydride method or a carbodiimide method, and the concentrated washing liquid contains 0.05% of Twain-20. Compared with a traditional enzyme-linked immunosorbent assay method, the kit has higher sensitivity degree, is short in detection time and low in detection cost, and can be used for detection of the residual amount of zilpaterol in animal urine, blood samples, tissues, viscera and other samples.

Description

technical field

[0001] The invention relates to a chemiluminescent immunoassay kit for detecting zilpaterol, which is used for detecting the content or residue of zilpaterol in animal-derived food such as animal tissue, viscera, blood, urine, feed, and feed raw materials quantity. It belongs to the field of immunological detection. Background technique

[0002] use beta 2 - Adrenergic receptor agonists can increase the muscle / fat ratio, and can significantly improve the lean meat rate of reared animals. However, if it is used in large quantities or in excess for a long time, and the safe withdrawal period is not observed, it will accumulate in animal tissues, and human consumption of this animal tissue will cause poisoning or even death. Therefore, many countries and international organizations have banned this kind of drug. Compounds are used in food animal feeding. Athletes use such drugs in large doses, which can increase the muscle / fat ratio and improve muscle streng...

Examples

Embodiment 1

[0041] 1. Preparation of each component of the kit

[0042] (1) Preparation of zilpaterol hapten: zilpaterol was acidified, and reacted with sodium nitrite in a dark and low-temperature environment at 4°C to generate an intermediate containing diazonium cations. The diazotized zilpaterol is used as a hapten for subsequent synthesis of immune antigens and coating antigens;

[0043] (2) Preparation of zilpaterol-bovine serum albumin (BSA) immunogen: coupling zilpaterol and bovine serum albumin (BSA) by diazotization method to obtain immune antigen;

[0044] Preparation of zilpaterol-oval serum albumin (OVA) coated antigen: coupling zilpaterol and egg serum albumin (OVA) by diazotization method to obtain coated antigen;

[0045]Preparation of zilpaterol-peroxidase-labeled antibody: For 6-8 week-old female BALB / c mice (body weight 18-20 g), the high-dose immunization scheme is as follows: 160 μg zilpaterol is used for the first immunization -BSA was mixed with an equal amount of...

Embodiment 2

[0080] The chemiluminescent immunoassay kit for detection of zilpaterol includes the following components:

[0081] (1) 96-well opaque white ELISA plate (8 wells x 12 strips) coated with zilpaterol-mouse serum albumin conjugate, vacuum-sealed in an aluminum foil bag;

[0082] (2) 6 bottles of zilpaterol standard solution, the concentrations are respectively:

[0083] 0 ng / mL, 0.01 ng / mL, 0.05 ng / mL, 0.25 ng / mL, 1.25 ng / mL, 6.25 ng / mL

[0084] (3) zilpaterol-horseradish peroxidase labeled antibody solution;

[0085] (4) Luminescence substrate A solution (luminol and enhancer), luminescence substrate B solution (urea hydrogen peroxide);

[0086] (5) 2 times concentrated sample diluent. Before use, please dilute 1:1 (1 part of concentrated diluent + 1 part of deionized water) to become the working sample diluent, and the diluted working sample diluent is 0.05 mol / L, pH 7.4 Tris-HCl buffer;

[0087] (6) 20 times concentrated washing solution. Before use, please dilute at 1:19...

Embodiment 3

[0089] The chemiluminescent immunoassay kit for detection of zilpaterol includes the following components:

[0090] (1) 96-well opaque white ELISA plate (8 wells x 12 strips) coated with zilpaterol-egg albumin conjugate, vacuum-sealed in an aluminum foil bag;

[0091] (2) 6 bottles of zilpaterol standard solution, the concentrations are respectively:

[0092] 0 ng / mL, 0.05 ng / mL, 0.2 ng / mL, 0.8 ng / mL, 4.0 ng / mL, 16.0 ng / mL

[0093] (3) zilpaterol-horseradish peroxidase labeled antibody solution;

[0094] (4) Luminescence substrate A solution (luminol and enhancer), luminescence substrate B solution (urea hydrogen peroxide);

[0095] (5) 2 times concentrated sample diluent. Before use, please dilute 1:1 (1 part of concentrated diluent + 1 part of deionized water) to become the working sample diluent, and the diluted working sample diluent is 0.05 mol / L, pH 7.4 glycine-HCl buffer;

[0096] (6) 20 times concentrated washing solution. Before use, please dilute at 1:19 (1 part...

technical field

[0001] The invention relates to a chemiluminescent immunoassay kit for detecting zilpaterol, which is used for detecting the content or residue of zilpaterol in animal-derived food such as animal tissue, viscera, blood, urine, feed, and feed raw materials quantity. It belongs to the field of immunological detection. Background technique

[0002] use beta 2 - Adrenergic receptor agonists can increase the muscle / fat ratio, and can significantly improve the lean meat rate of reared animals. However, if it is used in large quantities or in excess for a long time, and the safe withdrawal period is not observed, it will accumulate in animal tissues, and human consumption of this animal tissue will cause poisoning or even death. Therefore, many countries and international organizations have banned this kind of drug. Compounds are used in food animal feeding. Athletes use such drugs in large doses, which can increase the muscle / fat ratio and improve muscle streng...

Examples

Embodiment 1

[0041] 1. Preparation of each component of the kit

[0042] (1) Preparation of zilpaterol hapten: zilpaterol was acidified, and reacted with sodium nitrite in a dark and low-temperature environment at 4°C to generate an intermediate containing diazonium cations. The diazotized zilpaterol is used as a hapten for subsequent synthesis of immune antigens and coating antigens;

[0043] (2) Preparation of zilpaterol-bovine serum albumin (BSA) immunogen: coupling zilpaterol and bovine serum albumin (BSA) by diazotization method to obtain immune antigen;

[0044] Preparation of zilpaterol-oval serum albumin (OVA) coated antigen: coupling zilpaterol and egg serum albumin (OVA) by diazotization method to obtain coated antigen;

[0045]Preparation of zilpaterol-peroxidase-labeled antibody: For 6-8 week-old female BALB / c mice (body weight 18-20 g), the high-dose immunization scheme is as follows: 160 μg zilpaterol is used for the first immunization -BSA was mixed with an equal amount of...

Embodiment 2

[0080] The chemiluminescent immunoassay kit for detection of zilpaterol includes the following components:

[0081] (1) 96-well opaque white ELISA plate (8 wells x 12 strips) coated with zilpaterol-mouse serum albumin conjugate, vacuum-sealed in an aluminum foil bag;

[0082] (2) 6 bottles of zilpaterol standard solution, the concentrations are respectively:

[0083] 0 ng / mL, 0.01 ng / mL, 0.05 ng / mL, 0.25 ng / mL, 1.25 ng / mL, 6.25 ng / mL

[0084] (3) zilpaterol-horseradish peroxidase labeled antibody solution;

[0085] (4) Luminescence substrate A solution (luminol and enhancer), luminescence substrate B solution (urea hydrogen peroxide);

[0086] (5) 2 times concentrated sample diluent. Before use, please dilute 1:1 (1 part of concentrated diluent + 1 part of deionized water) to become the working sample diluent, and the diluted working sample diluent is 0.05 mol / L, pH 7.4 Tris-HCl buffer;

[0087] (6) 20 times concentrated washing solution. Before use, please dilute at 1:19...

Embodiment 3

[0089] The chemiluminescent immunoassay kit for detection of zilpaterol includes the following components:

[0090] (1) 96-well opaque white ELISA plate (8 wells x 12 strips) coated with zilpaterol-egg albumin conjugate, vacuum-sealed in an aluminum foil bag;

[0091] (2) 6 bottles of zilpaterol standard solution, the concentrations are respectively:

[0092] 0 ng / mL, 0.05 ng / mL, 0.2 ng / mL, 0.8 ng / mL, 4.0 ng / mL, 16.0 ng / mL

[0093] (3) zilpaterol-horseradish peroxidase labeled antibody solution;

[0094] (4) Luminescence substrate A solution (luminol and enhancer), luminescence substrate B solution (urea hydrogen peroxide);

[0095] (5) 2 times concentrated sample diluent. Before use, please dilute 1:1 (1 part of concentrated diluent + 1 part of deionized water) to become the working sample diluent, and the diluted working sample diluent is 0.05 mol / L, pH 7.4 glycine-HCl buffer;

[0096] (6) 20 times concentrated washing solution. Before use, please dilute at 1:19 (1 part...

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