Anti-melamine monoclonal antibody and kit for detecting melamine

A monoclonal antibody, anti-melamine technology, applied in anti-animal/human immunoglobulins, measuring devices, biochemical equipment and methods, etc. and high personnel operation problems, to achieve the effect of satisfying large-scale sample detection and low detection cost

Active Publication Date: 2011-08-31
ANIMAL AND PLANT & FOOD DETECTION CENTER JIANGSU ENTRY EXIT INSPECTION AND QUARANTINE BUREAU +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The current method for detecting melamine is high-performance liquid chromatography (HPLC), which has high requirements for equipment and personnel operation, high detection cost, and is difficult to meet the needs of large-scale sample detection

Method used

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  • Anti-melamine monoclonal antibody and kit for detecting melamine
  • Anti-melamine monoclonal antibody and kit for detecting melamine
  • Anti-melamine monoclonal antibody and kit for detecting melamine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] The preparation of embodiment 1 anti-melamine monoclonal antibody

[0035] Preparation of 1.6 carbon melamine-carrier protein conjugates

[0036] (1) Synthesis of 6-carbon melamine

[0037]

[0038] a. Dissolve 5-10 mg of melamine in DMSO, add 6-chloroacetic acid, and react at 90°C for 3 hours;

[0039] b. After cooling the reacted mixture, a precipitate was precipitated in ice water.

[0040] c. Dissolve the precipitate with 1N sodium hydroxide, centrifuge, and take the supernatant.

[0041] d. Adjust the pH to 7.0 with 1N hydrochloric acid, and the precipitated substance is 6-carbon melamine.

[0042] (2) Preparation of 6-carbon melamine-carrier protein conjugate (immunogen)

[0043] a. Dissolve 1-5 mg of 6-carbon melamine in 0.5 ml of anhydrous DMF (dimethylformamide), then add 6 mg of NHS (N-hydroxysuccinimide) and 10 mg of EDC (1-ethyl -3-(3-Dimethylpropylamino)carbodiimide), stirred overnight at room temperature.

[0044] b. Dissolving 20 mg of carrier pr...

Embodiment 2

[0123] Embodiment 2 detects the test kit of melamine

[0124] 1. ELISA plate preparation: Dilute the detection antigen 6-carbon melamine-OVA coupling product with 0.05M carbonate buffer (pH9.6) to 1-0.5μg / ml, add it to the microwell, 100μl / well , overnight at 2-8°C, and the next day, block with 150 μl / well of blocking solution at room temperature for 2 hours, discard the liquid, dry, and package.

[0125] 2. Preparation of HRP-labeled anti-melamine monoclonal antibody solution

[0126] Preparation of HRP-labeled anti-melamine monoclonal antibody: Weigh 5 mg of HRP (horseradish peroxidase) and dissolve it in 1 ml of distilled water, add 200 μl of newly prepared NaIO with a concentration of 0.1 M 4 , stirred at room temperature for 20 minutes in the dark, put the mixture into a dialysis bag, dialyzed with 1 mM acetate buffer (pH 4.4), overnight at 4°C. Get the solution in the dialysis bag then, add 20 μ l concentration and be the carbonate buffer solution (pH 9.5) of 0.2M to m...

Embodiment 3

[0135] The usage method of embodiment 3 kit

[0136] Reagent preparation: Mix the 20-fold concentrated lotion and distilled water (or deionized water) at a ratio of 1:1 to obtain the lotion, which is prepared before use.

[0137] Numbering: Number the microwells corresponding to the melamine standard solution and the sample to be tested in sequence. It is recommended to make duplicate holes for each standard solution and sample to be tested.

[0138] Adding samples: Add 50 μL of melamine standard solution or sample to each well, and then add 50 μL of HRP-labeled anti-melamine monoclonal antibody to each well.

[0139] Incubation: Gently oscillate to mix, seal the plate with a sealing film and place it at room temperature for 30 minutes.

[0140] Washing: Carefully peel off the sealing film, dry the liquid in the well, add 250-300 μL / well of washing solution, fully wash 5 times with an interval of less than 5 seconds, and pat dry with absorbent paper (if it is not removed afte...

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Abstract

The invention provides a preparation method for an anti-melamine monoclonal antibody and a kit for detecting melamine. The preparation method comprises the steps of injecting specific lymphocyte hybridoma cell strains of the melamine into an abdominal cavity of a laboratory mouse; taking ascites of the mouse; depositing the ascites through saturated ammonium sulfate and dialyzing after depositionis dissolved; purifying via affinity chromatography of Protein G; and collecting absorption peak to finally obtain the anti-melamine monoclonal antibody. The anti-melamine monoclonal antibody prepared by the method can be used for producing competition enzyme linked immunity detection reagent kits and colloidal gold test strips to rapidly detect residues of the melamine in food, thereby meeting ademand for rapidly detection of a large quantity of samples.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an anti-melamine monoclonal antibody and an application thereof. Background technique [0002] Melamine (Melamine) referred to as triamine, also known as 2,4,6-triamino-1,3,5-triazine, 1,3,5-triazine-2,4,6-triamine, 2,4, 6- Trisaminocarbamide, melamine, melamine or cyanuronamide, molecular formula C 3 N 6 h 6 or C 3 N 3 (NH 2 ) 3 . Melamine is an important chemical raw material, which is widely used in wood, plastic, paint, paper, textile, leather, electrical, pharmaceutical and other industries. [0003] [0004] Melamine is banned as a food and animal feed additive. Cases of illegally adding melamine not only occurred in China, but fishmeal and pet food imported from Malaysia, Thailand, Peru, Australia and other countries were found to be positive for melamine. The US FDA also found melamine in feed produced in the US. After infant milk powder and other dai...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/18C07K16/18G01N33/577
Inventor 薛峰蒋原吴艳涛高海岗张睿李旭辉陶宏锦张小荣刘秀梵赵红霞
Owner ANIMAL AND PLANT & FOOD DETECTION CENTER JIANGSU ENTRY EXIT INSPECTION AND QUARANTINE BUREAU
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