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Anti-melamine monoclonal antibody and application thereof

A monoclonal antibody and melamine technology, which is applied to instruments, peptides, immunoglobulins, etc., can solve the problems of high cost and time-consuming detection, and achieve the effects of saving time, simple operation, and strong specificity

Inactive Publication Date: 2011-10-05
南京沐赛生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existing detection methods are mainly high-performance liquid chromatography, gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry / mass spectrometry, these detection methods involve the use of large precision instruments, require professional operation, and take a long time. Testing is expensive

Method used

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  • Anti-melamine monoclonal antibody and application thereof
  • Anti-melamine monoclonal antibody and application thereof
  • Anti-melamine monoclonal antibody and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1, the preparation of melamine structural analogue (SM)

[0035] The molecular formula of SM is as follows:

[0036]

[0037] 1. Preparation and characterization of compound 3

[0038] 1. Preparation of Compound 3

[0039]

[0040] 2,3-dihydropyran (compound 1) (0.56g, 6.63mmol), 6-bromo-1-hexanol (1g, 5.52mmol) and pyridine p-toluenesulfonate (0.28g, 1.1mmol ) was dissolved in 15 mL of dichloromethane (DCM), and the reaction solution was stirred at room temperature for 23 hours. with saturated NaHCO 3 solution and saturated brine to wash the reaction solution, anhydrous Na 2 SO 4 dry. The desiccant was removed by filtration, and the solvent was distilled off under reduced pressure to obtain a colorless oily liquid compound 3 (1.4 g, yield 96%).

[0041] 2. Characterization of Compound 3

[0042] 1 HNMR (CDCl 3 , 400MHz) δ4.57(m, 1H), 3.86(m, 1H), 3.74(m, 1H), 3.50(m, 1H), 3.36-3.43(m, 3H), 1.82-1.91(m, 3H) , 1.71 (m, 1H), 1.38-1.64 (m, 10H). ...

Embodiment 2

[0087] Embodiment 2, the preparation of antigen

[0088] For the schematic diagram of antigen: SM-BSA (SM-KLH) coupling reaction, see figure 1 .

[0089] 1. Preparation of SM-BSA (coating antigen)

[0090] Weigh 5mg of SM and 20mg of BSA, fully dissolve in 5ml of hydrochloric acid acidified deionized water (pH4.7), add 46mg of EDC to the mixed solution, and react overnight under stirring at 4°C; It is 0.01M PBS buffer solution (pH7.0); respectively take BSA and SM-BSA with the same concentration (2mg / mL) for mass spectrometry analysis to identify the coupling results.

[0091] For mass spectrometry results, see figure 2 . The molecular weight of BSA is 66.32KD. The average molecular weight of SM-BSA is 70.57KD, an increase of 4.25KD compared with BSA. The molecular weight of SM is 221D. The formation of SM-BSA is through the dehydration of the carboxyl group on the SM side chain and the free amino group on the lysine on the surface of BSA through EDC to form an amide bo...

Embodiment 3

[0094] Embodiment 3, the preparation of monoclonal antibody

[0095] 1. Immunization of mice

[0096] 1. Five BALB / c mice aged 6-8 weeks were taken, and each mouse was subcutaneously injected with 20 μg of SM-KLH emulsified in complete Freund's adjuvant (primary immunization).

[0097] 2. Subcutaneous injection of the same dose of SM-KLH emulsified in Freund's incomplete adjuvant every 15 days (boosting immunization).

[0098] 3. Orbital blood was collected 10 days after the fourth immunization, the titer of antiserum was detected by indirect ELISA, and the specificity of the antibody relative to melamine was detected by indirect competitive ELISA.

[0099] The antiserum titers of 5 mice were all 1:72000. 1 # ,2 # 、3 # The antiserum of the mouse can recognize melamine more specifically than the antiserum of the other 2 mice; when adding the same dose of melamine, 3 # B / B of mouse antiserum 0 The smaller the value, the more obvious the suppression phenomenon.

[0100] 4....

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Abstract

The invention discloses an anti-melamine monoclonal antibody and application thereof. The invention provides a hybridoma cell strain 3B11 with a preservation number of CGMCC NO. 3237. The monoclonal antibody provided by the invention is secreted and generated by the hybridoma cell strain 3B11 with the preservation number of CGMCC NO. 3237. By applying the monoclonal antibody provided by the invention and an indirect competitive ELISA method, melamine in dairy products can be rapidly detected. The monoclonal antibody has the advantages of strong specificity, high sensitivity, simple operation, capability of detecting a large number of samples, and the like. In addition, compared with detection methods of the same type, the method does not need to pretreat liquid samples detected, and only need to use phosphate buffer solution to dissolve solid samples in a proper concentration and then can load and detect the solid samples, so the method is simple to operate and saves time. The monoclonal antibody not only can be applied to melamine detection, but also can be applied to the preparation of melamine-detecting kits, thereby having great value.

Description

technical field [0001] The invention relates to an anti-melamine monoclonal antibody and its application. Background technique [0002] Melamine (English name: Melamine) is a triazine-type nitrogen-containing heterocyclic organic compound, and its properties are pure white monoclinic prism crystals, odorless. Triamine for short, commonly known as melamine, protein essence, also known as 2,4,6-triamino-1,3,5-triazine, 1,3,5-triazine-2,4,6-triamine, 2 , 4,6-trisaminocarbamide, cyanuric acid amide, cyanuric triamide, molecular formula sees formula (I); [0003] [0004] Melamine is a basic organic chemical intermediate product with a wide range of uses, and its main use is as a raw material for the production of melamine-formaldehyde resin (MF). Melamine can also be used as flame retardant, water reducer, formaldehyde cleaner, etc. However, it is prohibited from being used in food and pet feed. Long-term intake of melamine can cause reproductive and urinary system damage,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C07K16/44G01N33/577
Inventor 智刚石晓娟
Owner 南京沐赛生物技术有限公司