ELISA kit for rapidly testing melamine content and method thereof

A technology of melamine and detection method is applied in the preparation of ELISA kits, the preparation of antibodies and the establishment of detection methods, and the field of antigens, which can solve the problems of long process, low sensitivity, and few kits.

Inactive Publication Date: 2009-10-28
CUSABIO TECH LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Melamine is a small molecular compound. Like many other small molecular compounds, the content of melamine is generally determined by GC, MS, LC-UV, high-performance liquid chromatography, etc., and the methods established by relevant testing institutions are rarely reported.
However, the above methods have disadvantages such as lengthy process, need for derivatization, large amount of reagents and low sensitivity.
[0008] At present, the immunological detection method of melamine has also been reported, but there are not many kits on the market that can effectively detect melamine

Method used

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  • ELISA kit for rapidly testing melamine content and method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Preparation and purification of embodiment 1 antibody

[0021] 1. Antigen preparation: According to the properties and structural characteristics of melamine, design and synthesize melamine derivatives, couple them with carriers, and prepare coated antigen melamine-BSA and immune antigen melamine-OVA.

[0022] 2. Preparation of antibodies by immunizing animals

[0023] The melamine-OVA conjugate was used as the immunogen to inject the experimental Japanese white rabbits, 0.5 mg of the immunogen was injected each time, and an equal volume of adjuvant was added. Freund's complete adjuvant was used for the first immunization, followed by incomplete Freund's adjuvant. A total of four times of immunization, carotid artery blood or heart blood to obtain antiserum.

[0024] 3. Preparation of melamine affinity chromatography column

[0025] Use CNBr-activated+Sepharose+4B as column gel and add melamine to make affinity chromatography column.

[0026] 4. Antibody Purificatio...

Embodiment 2 3

[0033] The preparation of embodiment 2 melamine ELISA kit

[0034] 1. Microwell reaction plate coated with melamine-BSA conjugate, 1 piece (96T or 48T).

[0035] 2. Melamine standard (500ng / ml 300μl, 2 bottles or 1 bottle).

[0036] 3. HRP enzyme-labeled melamine antibody 1×60 μl / bottle or 1×30 μl / bottle (1:100).

[0037] 4. Concentrated washing liquid 1×20ml / bottle or 1×10ml / bottle, each bottle is diluted 25 times with distilled water when used.

[0038] 5. Substrate 1×10ml / bottle or 1×5ml / bottle.

[0039] 6. Reaction termination solution 1×10ml / bottle or 1×5ml / bottle (2N H 2 SO4).

[0040] 7. Sample diluent 1×20ml / bottle (2 bottles or 1 bottle)

Embodiment 3

[0041] The content determination of melamine in embodiment 3 sample

[0042] 1. Sample Processing

[0043] The liquid sample is directly diluted 1000 times with the sample diluent, and the solid sample treatment method: take 1g sample, add 10ml methanol and mix thoroughly. Centrifuge at 5000g or more for 10 minutes. Take 1ml of the supernatant in a test tube and blow dry. Add 2.5ml of PBS solution containing 10% methanol to fully dissolve, and take 150μl for analysis.

[0044] 2. Detection

[0045] (1) Adding samples: set blank wells, standard wells, and sample wells to be tested respectively. Add 100 μl of sample diluent to the blank well, add 50 μl of standard substance or sample to be tested respectively in the remaining wells, pay attention not to have air bubbles, add the sample to the bottom of the microplate well, and then add 50 μl of the diluted solution to the standard well and the well of the sample to be tested Enzyme working solution (not added to blank wells...

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Abstract

The invention relates to the preparation of an antigen and an antibody of a small-molecule addictive melamine or analogues thereof related to food security, and a method for semi-quantifying or quantifying samples potentially containing the melamine or the analogues thereof. Based on the principle of specific reaction of the antigen and the antibody, the invention utilizes a specific antibody of the melamine to test the concentration of the melamine or the analogues thereof in the samples with a blocking-ELISA method. The antigen is an artificially prepared immunogen and a coatingen of the melamine with reactionogenicity and immunogenicity, which are prepared by using immunogen immunoassay animals, and the blocking-ELISA theory is utilized to establish the melamine testing method. The melamine antigen provided by the invention owns good immunogenicity and the coatingen obtained owns good activity. The testing method established is capable of testing the melamine content in milk, dairy products and feedstuff without non-specific reaction with other components in milk, dairy products and feedstuff.

Description

technical field [0001] The invention relates to food safety detection, in particular to the preparation of small molecule antigens and antibodies and the establishment of detection methods. The invention specifically relates to a method for detecting melamine content in a sample by blocking ELISA and preparation of an ELISA kit based on the method. Background technique [0002] Small molecule compounds, known histochemically as haptens, are only immunoreactive with corresponding antigens or sensitized lymphocytes, but not immunogenic. Gas chromatography, high pressure liquid chromatography, and mass spectrometry are generally used for the detection of haptens, but immunological detection methods gradually show their irreplaceable advantages in the detection of haptens. Immunological detection method does not require complex and expensive equipment like traditional chromatography and mass spectrometry methods. In addition, it is easy to operate, simple in the pretreatment pr...

Claims

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Application Information

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IPC IPC(8): C07K14/765C07K14/77C07K16/44G01N33/566G01N33/543G01N33/545G01N33/548G01N33/577
Inventor 华权高
Owner CUSABIO TECH LLC
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