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Melamine antigen, related antibody and preparation method thereof

A technology of melamine and antigen, which is applied in the fields of antigen, related antibody and its preparation, melamine antigen containing polyaldehyde dextran-linked carrier protein, and melamine antigen. Melamine has no nutritional value and other issues

Inactive Publication Date: 2012-10-03
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0011] (2) Illegal use
In addition, the cost of melamine, especially melamine waste, is very low, which is driven by great interests for adulterers and counterfeiters, so this irrelevant chemical raw material is added to milk powder and other foods, resulting in contamination of pet food in the United States such as in 2007 Serious food safety incidents such as the tainted milk powder incident in China in 2008
[0014] 3. The toxicity of melamine
[0015] Melamine itself has almost no nutritional value and cannot really replace protein. After adding it, it can only cause the illusion of increased protein content in the test, and the nutrient content in milk powder will inevitably be much lower than the normal value.
[0046] However, through literature search, etc., so far, there have been no reports on the use of linking reagents or bridge reagents to simultaneously connect melamine and carrier proteins to prepare new melamine antigens, related antibodies and preparation methods thereof.

Method used

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  • Melamine antigen, related antibody and preparation method thereof
  • Melamine antigen, related antibody and preparation method thereof
  • Melamine antigen, related antibody and preparation method thereof

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preparation example Construction

[0159] The preparation method of the melamine antigen monoclonal antibody of the present invention specifically comprises:

[0160] (1) Immunization of mice

[0161] Mice were immunized by conventional methods, specifically:

[0162] Use the melamine-PAD-carrier protein conjugate product as an immunogen to immunize male BALB / C mice for 6-8 weeks. 2 to 3 mice. The melamine-PAD-carrier protein was mixed with an equal volume of Freund's complete adjuvant, fully emulsified, and intraperitoneally injected into each mouse. Booster immunizations were carried out every two weeks, and a total of 5 booster immunizations were carried out. During the first 4 booster immunizations, melamine-PAD-carrier protein was fully emulsified with an equal volume of Freund's incomplete adjuvant, and each mouse was intraperitoneally injected with 0.1 mg of melamine-PAD-carrier protein antigen. The last immunization was done by tail vein injection without adding adjuvant. After 3 days, the splenocyt...

Embodiment 1

[0239] Embodiment 1, the preparation of melamine antigen

[0240] (1) Prepare the following solutions:

[0241] a. 0.25g PAD (made from 2W molecular weight dextran) was dissolved in 5ml 0.1M PB (pH=7.2) buffer

[0242]b. Dissolve 0.25g OVA in 2.55ml 0.1M PB (pH=7.2) buffer

[0243] c. 50mg melamine dissolved in 1ml 20% ethanol solution

[0244] d. Dissolve 50mg of sodium trihydroborocyanide in 0.5ml of 0.1M PB (pH=7.2) buffer solution, the solution is ready for immediate use

[0245] (2) Mix solution a with solution b, add solution d at 40°C, stir and react for 4 hours, dialyze with 0.1M PB buffer, test the dialysate with 2,4-dinitrophenylhydrazine color test, and dialyze When complete, set aside.

[0246] (3) Mix solution c with the solution obtained after complete dialysis, add solution d at 40°C, stir and react for 4 hours, dialyze with 0.1M PB buffer solution, and test the dialysate without free melamine small molecules to obtain melamine - PAD-OVA antigen.

[0247] ...

Embodiment 2

[0248] Embodiment 2, the preparation of melamine antigen

[0249] (1) Prepare the following solutions:

[0250] a. 0.25g PAD (made from 4W molecular weight dextran) was dissolved in 5ml 0.1M PB (pH=7.2) buffer

[0251] b. Dissolve 0.25g OVA in 2.55ml 0.1M PB (pH=7.2) buffer

[0252] c. 50mg melamine dissolved in 1ml 20% ethanol solution

[0253] d. Dissolve 50mg of sodium trihydroborocyanide in 0.5ml of 0.1M PB (pH=7.2) buffer solution, the solution is ready for immediate use

[0254] (2) Mix solution a with solution b, add solution d at 40°C, stir and react for 4 hours, dialyze with 0.1M PB buffer, test the dialysate with 2,4-dinitrophenylhydrazine color test, and dialyze When complete, set aside.

[0255] (3) Mix solution c with the solution obtained after complete dialysis, add solution d at 40°C, stir and react for 4 hours, dialyze with 0.1M PB buffer solution, and test the dialysate without free melamine small molecules to obtain melamine - PAD-OVA antigen.

[0256]...

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Abstract

The invention relates to a melamine antigen, a related antibody and a preparation method thereof. Melamine and carrier protein are coupled by using PAD (polyaldehyde dextran) as bridge reagent; coupling products is used to immunize animals to prepare monoclonal antibodies for detecting melamine. Artificially-prepared melamine antigen and related antibody provided by the invention can be used for researching and developing melamine immunity detection reagents to meet detection demand on low cost, rapidity and convenient operations.

Description

technical field [0001] The present invention relates to technical fields such as medicine and reagents, in particular to an antigen, a related antibody and a preparation method thereof, more specifically to a melamine antigen, a related antibody and a preparation method thereof, and more specifically to a A melamine antigen containing polyaldehyde dextran-linked carrier protein, related antibodies and a preparation method thereof. Background technique [0002] (1) Overview of melamine [0003] 1. Physical and chemical properties [0004] Melamine (melamine) referred to as triamine, protein essence, scientific name triamine triazine, alias melamine, cyanuramide, trimeramide, is a triazine nitrogen-containing heterocyclic organic compound, named by IUPAC as "1,3, 5-Triazine-2,4,6-triamino", CAS: 08-78-1, molecular weight 126.12, molecular formula on the left: [0005] Melamine is a low-toxic, odorless pure white monoclinic prism crystal, similar to protein powder, and its...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/765C07K14/77C07K14/795C07K16/44G01N33/53
Inventor 唐古生丁自更康蔡俊陈英豪秦阳华赵卫国
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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