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ELISA kit for detecting penicillin G and detection method thereof

An enzyme-linked immunosorbent reagent and penicillin technology, which can be used in biological testing, measuring devices, material inspection products, etc., can solve the problems of cumbersome detection operations, high costs, and limitations in promotion and application of detection and analysis methods.

Inactive Publication Date: 2006-05-03
贵州勤邦食品安全科学技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The currently used detection and analysis methods and detection operations are cumbersome and expensive, and are not suitable for on-site monitoring and screening of a large number of samples, which limits the promotion and application

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] The preparation of embodiment 1 kit components

[0070] 1. Antigen preparation

[0071] a. Hapten: penicillin G and 2-aminoacetic acid were synthesized into penicillin G hapten.

[0072] b. Immunogen: The immunogen was obtained by coupling penicillin G hapten and hemocyanin (KLH) by mixed anhydride method (isobutyl chloroformate).

[0073] Dissolve 2g of penicillin G hapten in 30ml, 50% N,N'-dimethylformamide solution, then dissolve 0.5ml of isobutyl chloroformate in 5ml of anhydrous dioxane and add to the hapten solution The reaction was stirred at room temperature for 4 hours, 32 g of hemocyanin was dissolved in 70 ml of pH9.6 carbonate buffer solution, and the hemocyanin was added dropwise to the hapten and stirred overnight at 4°C. The reacted artificial antigen was dialyzed against 0.2M phosphate buffer for 7 days, and the solution was changed 4 times a day. Finally, the antigen is concentrated and freeze-dried for storage.

[0074] c. Coating source: Coupling ...

Embodiment 2

[0094] The preparation of embodiment 2 kit components

[0095] 1. Preparation of coating agent and antibody

[0096] (1) Preparation of coating agent: Goat anti-mouse anti-antibody was obtained by immunizing pathogen-free goat with mouse antibody as immunogen.

[0097] (2) Antibody preparation

[0098] a. Animal immunization The immunogen penicillin G hapten and hemocyanin were injected into Balb / c mice at an immunization dose of 80 μg / mouse to make them produce polyclonal antibodies.

[0099] b. Cell fusion and cloning Spleen cells from immunized Balb / c mice were fused with SP2 / 0 myeloma cells at a ratio of 5:1 to obtain hybridoma cell lines of monoclonal antibodies.

[0100] c. Cell cryopreservation and recovery, the hybridoma cells were made into 1×10 cryopreservation medium 6 cells / mL for long-term storage in liquid nitrogen. When recovering, take out the cryopreservation tube, put it into a 37°C water bath to thaw quickly, remove the cryopreservation solution by centr...

Embodiment 3

[0106] Embodiment 3 detects the formation of the ELISA kit of penicillin G

[0107] An ELISA kit for detecting penicillin G was set up to include the following components:

[0108] (1) A microtiter plate coated with penicillin G hapten and hemocyanin;

[0109] (2) horseradish peroxidase labeled goat anti-mouse anti-antibody;

[0110] (3) Penicillin G standard solution, the concentrations are 0 μg / L, 0.1 μg / L, 0.3 μg / L, 0.9 μg / L, 2.7 μg / L, 8.1 μg / L;

[0111] (4) The substrate chromogenic solution is composed of A liquid and B liquid, the chromogenic liquid A liquid is hydrogen peroxide, and the chromogenic liquid B liquid is o-phenylenediamine;

[0112] (5) The concentrated washing solution is 0.01M, pH 7.4, containing 0.8% Tween 20 and 0.1% sodium azide (NaN 3 ) of phosphate buffered saline.

[0113] (6) The stop solution is 2mol / L sulfuric acid;

[0114] (7) The concentrated complex solution is 0.01M phosphate buffer saline containing 1% gelatin.

[0115] (8) The concen...

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Abstract

The invention relates to an enzyme immune agent box for detecting ampicillin G, which comprises: enzyme mark plate which coats ampicillin G antibody or antigen, enzyme mark material, ampicillin G standard solution, base material color developing solution, compression cleaning liquid, ending solution, compression twin solution and antibody working solution. The invention also discloses a method for applying the detecting method, which comprises: first doing sample front process, then using the agent box to detect, at last analyzing the detected result.

Description

technical field [0001] The invention relates to an ELISA kit and method for detecting penicillin G. Background technique [0002] Penicillin G (Benzyl penicillin, BPG) is a widely used antibiotic, which is used to prevent, treat, diagnose animal diseases or regulate animal physiological functions. Reasonable use can effectively control animal diseases, improve the survival rate of animals, and ensure the safety of the breeding industry. healthy growth. However, excessive intake of penicillin G by animals can lead to high residues in animal foods. When people eat foods containing excessive penicillin G residues, it will cause allergic reactions and drug resistance. Therefore, countries such as Europe and the United States and my country have successively required limited use. [0003] The currently used detection and analysis methods and detection operations are cumbersome and expensive, and are not suitable for on-site monitoring and screening of a large number of samples...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543G01N33/52G01N33/535
Inventor 沈建忠何方洋万宇平冯才伟吴小平冯才茂汪善良李军赵正苗张照亮史为民张素霞丁双阳罗晓琴孙倩
Owner 贵州勤邦食品安全科学技术有限公司
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