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Clenbuterol ELISA method and reagent kit and method for making same

An enzyme-linked immunoassay and kit technology, which is applied in the detection field, can solve the problems of inhomogeneity, low specificity, and no detection of enzyme-labeled antibody batches, and achieve good coating effect, high fermentation density, and reduced The effect of testing costs

Active Publication Date: 2008-10-22
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Polyclonal antibodies are easy to obtain but have low specificity. Some researchers have reported that anti-CBL polyclonal antibodies have different IgG subtypes, which show different binding abilities to CBL and the carrier protein BSA.
Monoclonal antibodies do not have this problem, but it is worth noting that the use of hybridoma cell culture for monoclonal antibody (McAb) has a long cycle and low yield (the average yield is only 10-100mg / L)
In addition, regardless of whether it is a polyclonal antibody or a monoclonal antibody, the in vitro labeling process of the enzyme must be carried out when the enzyme-linked immunoassay is used, and the following disadvantages will inevitably exist: 1) The chemical covalent cross-linking process is harmful to the enzyme and antibody. Cause certain denaturation, thus affecting the activity; 2) The inhomogeneity of enzyme-labeled antibody batch products requires correction before use; 3) The products need to be purified before and after the enzyme-antibody coupling, and the steps are cumbersome
[0009] In the patent application with application number 200610036447.X, the applicant disclosed a detection clenbuterol enzyme-linked immunosorbent kit and its detection method and a sample preparation method of animal tissue before detection, but did not realize the use of its double function Genetic engineering antibody immunization for residual detection, and no related reports have been seen in the prior art

Method used

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  • Clenbuterol ELISA method and reagent kit and method for making same
  • Clenbuterol ELISA method and reagent kit and method for making same
  • Clenbuterol ELISA method and reagent kit and method for making same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1 Preparation of clenbuterol bifunctional genetically engineered antibody

[0067] Design degenerate primers:

[0068] V H (Back): 5'-TTACTCGCGGCCCAGCCGCCATGGCCCAGGTSMARCTGCAGSAGTCWGG-3'

[0069] V H (For): 5'-GCCAGAGCCACCTCCGCCTGAACCGTCCACCTGAGGAGACGGTGAC CGTGGTGCC-3'

[0070] V L (Back): 5'-TCAGGCGGAGGTGGCTCTGGCGGTGGGGATCGGACATTGAG CTC ACC CAG TCTCCA-3'

[0071] V L (For): 5'-GAGTCATTCTGCGGCCGCCCGTTTTATTTTCCAGCTTGGTCCC-3'

[0072] R 1 : 5'-CCATGATTACGCCAAGCTTTGGAGCC-3'

[0073] R 2 : 5'-CGATCTAAAGTTTTGTCGTCTTTTCC-3'

[0074] where primer V H (Back) Contains Sfi I restriction site, V H (For) containing (Gly4Ser) 3 Linker peptide partial sequence; V L (For) Contains Not I restriction site, V L (Back) contains (Gly 4 Ser) 3 Partial sequence of connecting peptide, 21 bases overlap with partial sequence of VH(For). R 1 , R 2 These are vector-specific primers for PCR identification of inserts.

[0075]Animals were immunized with synthetic CBL ar...

Embodiment 2

[0077] The preparation of embodiment 2 ELISA kit components

[0078] (1) Preparation of concentrated washing buffer: phosphate buffer containing 0.5 to 1.5% Tween 20, the pH of the buffer is 7.4, and the concentration is 0.1mol / L, which is 15 to 25 times the concentration used in the existing conventional method .

[0079] (2) Preparation of blocking solution: 1.0-5.0 g of skimmed milk powder was dissolved in 100 mL of distilled water.

[0080] (3) Preparation of substrate buffer: 30 μL of 30% hydrogen peroxide was dissolved in 19 mL of pH 5.0 phosphate-citric acid buffer, and stored at 4° C. Phosphate-citrate buffer uses 0.2M Na 2 HPO 4 25.7mL, 24.3mL of 0.1M citric acid, and 50mL of distilled water.

[0081] (4) Preparation of substrate solution: When the enzyme is horseradish peroxidase (HRP), the substrate solution is to dissolve 80 mg of 3,3,5,5-tetramethylbenzidine (TMB) in 10 mL of pH 5.0 Store in phosphate-citrate buffer at 4°C. Described phosphoric acid-citric a...

Embodiment 3

[0086] Example 3 The formation of the ELISA kit for detection of clenbuterol

[0087] The structure of the ELISA kit for detection of clenbuterol is as attached figure 2 ~ attached Figure 7 As shown, in addition to the kit box, it contains the following components:

[0088] (1) ELISA plate coated with clenbuterol antigen, 96 wells;

[0089] (2) Clenbuterol bifunctional genetic engineering antibody working solution, 7mL / bottle;

[0090] (3) 6 bottles of clenbuterol standard solution, the concentrations are 0μg / L, 0.1μg / L, 0.3μg / L, 0.9μg / L, 2.7μg / L, 8.1μg / L, 1mL / bottle;

[0091] (4) Substrate solution, 7mL / bottle;

[0092] (5) Substrate buffer, 7mL / bottle;

[0093] (6) Stop solution, 7mL / bottle;

[0094] (7) Concentrated washing solution, 50mL / bottle;

[0095] (8) Instruction manual, 1 copy;

[0096] (9) Cover film, 2 sheets;

[0097] (10) Ziplock bag (with desiccant), 1 piece.

[0098] attached figure 2 Middle 1: clenbuterol standard solution; 2: clenbuterol bifun...

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Abstract

The invention discloses a clenbuterol enzyme-linked immunity detection method, a reagent kit and a method for preparing the same. The detection method comprises the following steps that: a clenbuterol antigen is coated on a solid-phase vector; a guide sample or a sample to be detected is added and then a clenbuterol bifunctional genetic engineering antibody is added; after reaction, substrate solution is added for color development; the percent absorbency is measured; the concentration of clenbuterol in the sample to be detected is calculated according to a standard curve of the clenbuterol and the percent absorbency value of the sample to be detected; etc. The invention also discloses the reagent kit for realizing the detection method and the method for preparing the same. The clenbuterol enzyme-linked immunity detection method, the reagent kit for realizing the detection method and the method for preparing the same adopt the clenbuterol bifunctional genetic engineering antibody and the direct competition enzyme linked immunosorbent assay technology, have high sensibility and good stability, greatly simplify the operation steps and the reaction time, reduce the cost, are very suitable for screening a large quantity of samples, and have important practical significance.

Description

technical field [0001] The invention belongs to the technical field of detection, and in particular relates to an immunological detection method of a bifunctional genetic engineering antibody for clenbuterol residue in animal-derived food, a kit for realizing the detection method and a preparation method of the kit. Background technique [0002] Clenbuterol (CBL) is a kind of β2-adrenergic agonist (BAA), which belongs to catecholamines (epinephrine and norepinephrine) substances. As early as the early 1970s, the ability of catecholamines and their analogs to improve the nutritional allocation and carcass composition of the body was recognized by the research department of the American cyanamide company. Then it is used on a large scale in livestock and poultry production to increase lean meat percentage, reduce fat deposition, and increase milk production. Among them, clenbuterol and ractopamine are the most widely used in production because of their high oral efficiency. ...

Claims

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Application Information

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IPC IPC(8): G01N33/53G01N21/25G01N33/543
Inventor 孙远明杨金易王弘雷红涛沈玉栋潘科肖治理柳春红梁燕刘细霞
Owner SOUTH CHINA AGRI UNIV