Indirect chemiluminescent enzyme linked immunosorbent assay (ELISA) kit for detecting residual ractopamine by using monoclonal antibody

A technology of ractopamine and chemiluminescent enzymes, which is applied in chemiluminescence/bioluminescence, analysis through chemical reactions of materials, and measurement devices, etc., can solve the problems of inability to operate on-site, long cycle, expensive needs, etc., and achieve high sensitivity , Sensitivity improvement, high sensitivity and specificity effects

Inactive Publication Date: 2011-01-19
NANKAI UNIV
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Problems solved by technology

Although the measurement results of these methods are very accurate, they cannot meet the needs of actual testing work due to defects such as the need for expensive instruments and equipment, skilled professionals, cumbersome time-consuming, long cycle, high cost, and inability to operate on-site.
ELISA enzyme-linked immunosorbent technology has the characteristics of sensitivity, specificity, rapidity, and simplicity. It has been applied to the detection of ractopamine residues in recent years. Haasnootet et al. established a blocking ELISA method, Shelver et al. established an indirect competitive ELISA method, and domestic Yu Hongxia et al. The ractopamine polyclonal antibody, Qu Qing et al. have studied the ractopamine monoclonal antibody, but the establishment of the chemiluminescent enzyme-linked immunosorbent immunoassay (CL-ELISA) detection method has not been reported yet.

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  • Indirect chemiluminescent enzyme linked immunosorbent assay (ELISA) kit for detecting residual ractopamine by using monoclonal antibody
  • Indirect chemiluminescent enzyme linked immunosorbent assay (ELISA) kit for detecting residual ractopamine by using monoclonal antibody
  • Indirect chemiluminescent enzyme linked immunosorbent assay (ELISA) kit for detecting residual ractopamine by using monoclonal antibody

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1, detect the component of ractopamine kit and preparation process

[0039] 1. The composition of the chemiluminescent ELISA kit for detecting ractopamine

[0040] A, coated with a solid phase carrier (enzyme plate) coated with antigen (conjugate of ractopamine and carrier protein);

[0041] B. Ractopamine standard solution: 0.01ng / mL, 0.05ng / mL, 0.1ng / mL, 1ng / mL, 5ng / mL, 10ng / mL.

[0042] C. Enzyme-labeled goat anti-mouse antibody solution: Enzyme-labeled goat anti-mouse antibody is horseradish peroxidase-goat anti-rabbit IgG stock solution, loaded, and prepared with washing solution to a working concentration of 1:2000 when used.

[0043] D. Ractopamine antibody solution: the monoclonal antibody prepared by immunizing animals with an artificial immune antigen, and the obtained ractopamine antibody was diluted with a washing solution to a working concentration of 1:8000.

[0044] E, luminescent solution: use 0.0001M tris(hydroxymethyl)aminomethane solution...

Embodiment 2

[0059] Embodiment 2, establishment of indirect CL-ELISA detection method

[0060] (1) Optimization of antibody and coated antigen concentration (square matrix)

[0061] Plating plate with coated antigen dissolved in coating buffer: longitudinal serial dilution from the first row dilution 1 / 10, 100 μL / well, incubate at 37°C for 2 hours; wash the plate five times; block, 300 μL / well, 0-4°C, Overnight; Antibody plating: horizontal gradient dilution from the first column 1 / 100 (see Table 1), 100 μL / well, incubate at 37°C for 2 hours; enzyme label dilution is 1 / 2000, 100 μL / well plated at 37°C, 50 minutes; Add luminescent substrate solution: add 100 μL of luminescent substrate solution to each well, and detect after 3 to 5 minutes. Select the best ratio of coating and antibody, and then use this coating concentration to plate the plate, dilute the antibody horizontally, and dilute the enzyme label vertically, and select the best ratio of antibody and enzyme label in the same way. ...

Embodiment 3

[0071] Example 3, Application of Chemiluminescent ELISA Kit for Detection of Ractopamine

[0072] (1) Preparation of reagents

[0073] A. Sample diluent: Dilute the concentrated phosphate buffer solution provided in the kit 10 times with distilled water before use.

[0074] B. Washing solution: Dilute the concentrated washing solution provided in the kit 10 times with distilled water before use.

[0075] C. Luminescence solution: 0.01M luminol+0.001M p-cresol tris(hydroxymethyl)aminomethane solution (pH8.8)+3 / 10000(volume ratio)H 2 o 2 .

[0076] (2) Sample pretreatment

[0077] A. Urine The collected pig urine samples must be clear. If there are impurities, centrifuge at 4°C for 15 minutes and take the supernatant.

[0078] B. Mix animal tissue samples with 4mL 0.1M hydrochloric acid, put them together and use ultrasonic wave to extract for 20min, then centrifuge at 10000g for 15min, take the supernatant and adjust the pH to 9.5±0.5 with 10M NaOH, vortex for 5min, and th...

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Abstract

The invention relates to an indirect chemiluminescent enzyme linked immunosorbent assay (ELISA) kit for detecting residual ractopamine by using a monoclonal antibody, which comprises an ELISA plate with each pore coated with a coating antigen prepared by coupling ractopamine and bovine serum albumin, a ractopamine series standard solution, an ELISA goat-anti-mouse antibody solution, a ractopamine antibody solution, a luminescent solution, a washing solution, a coating solution and a sealing solution. The chemiluminescent ELISA kit carries out chemiluminescent ELISA by using the monoclonal antibody, wherein IC50=0.6 ng/ml. The lowest limit of detection in a chicken sample is 0.10 ng/ml, the coefficient of variation among batches and in a batch is smaller than 13%, and the recovery rate is 88-114%. In the invention, a new system for detecting the residual ractopamine is created by applying the monoclonal antibody and combining chemiluminescence with indirect ELISA, has higher sensitivity and specificity compared with the common ELISA and can play important role in the detection of the residual ractopamine in animal foods (such as milk, animal tissues and urine sample).

Description

【Technical field】 [0001] The invention relates to an ELISA kit, in particular to a ractopamine chemiluminescent ELISA detection kit. 【Background technique】 [0002] Ractopamine (RAC) is a phenylethanolamine beta 2 - Adrenoceptor agonist, which can selectively stimulate the beta of smooth muscle 2 Receptors are mainly used clinically to treat bronchial asthma, congestive heart failure and muscular atrophy, etc. The structural formula of ractopamine is as follows: [0003] [0004] Studies have shown that when the amount of ractopamine added to animal diets is 5-10 times the amount of clinical treatment, the nutrients in the animal body are transferred from fat to muscle, showing a nutrient redistribution effect, and then regulating the nutrient metabolism pathway of the animal body. Enhance fat catabolism, promote protein synthesis, significantly increase carcass lean meat rate, and improve feed return rate, especially for pigs. [0005] Since ractopamine remains in ani...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/535G01N21/76
Inventor 郗日沫王亚宾孟萌徐静张元阳张太昌薛虎寅
Owner NANKAI UNIV
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