Chromatography column for purifying melamine, kit containing the chromatography column and method for purifying melamine by using the kit

A melamine and chromatographic column technology, applied in chemical instruments and methods, organic chemistry, and other chemical processes, can solve the problems of complex polyclonal antibody preparation methods, high antibody yield and titer, and reduce analysis costs and environment Contamination, simple operation, high selectivity effect

Inactive Publication Date: 2014-01-15
JIANGSU INST OF POULTRY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, although there are reports in the literature on the use of immunoaffinity columns to purify melamine in the matrix, the preparation methods for polyclonal antibodies used in the literature are complicated. This patent uses commercial reagent 4,6-diamino-1,3,5-triazine -2-Acetic acid is used as a hapten to prepare polyclonal antibodies against melamine, the preparation method is simple, and the antibody yield and titer are high

Method used

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  • Chromatography column for purifying melamine, kit containing the chromatography column and method for purifying melamine by using the kit
  • Chromatography column for purifying melamine, kit containing the chromatography column and method for purifying melamine by using the kit
  • Chromatography column for purifying melamine, kit containing the chromatography column and method for purifying melamine by using the kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Preparation of melamine polyclonal antibody

[0029](1) Animal immunization: 4,6-diamino-1,3,5-triazine-2-acetic acid, a structural analog of melamine, was used as the hapten, coupled with the carrier protein BSA by the active ester method as the immunogen, and three Healthy, male New Zealand white rabbits with a weight of about 1.5kg are immunized animals. The immunization dose is 1mg / kg body weight. The same volume of complete Freund's adjuvant is mixed at the first immunization. After 14 days, an equal volume of incomplete Freund's adjuvant is added. The booster immunization was carried out with the same dose of adjuvant, and the booster immunization was carried out 2 weeks, 4 weeks, and 6 weeks after the initial immunization, respectively. The way of immunization is multi-point subcutaneous injection on the back. Seven days after each booster immunization, blood was collected from the rabbit's ear vein for titer detection until the serum titer and specifi...

Embodiment 2

[0038] Example 2 Preparation of Immunoaffinity Chromatography Column (IAC)

[0039] (1) Antibody preparation: The polyclonal antibody purified by saturated ammonium sulfate method was reacted with 0.1 mol / L NaHCO 3 (Containing 0.5 mol / L NaCl, pH=8.3) was dialyzed overnight. After the dialysis was completed, the polyclonal antibody was transferred to a straight tube and stored at -20°C.

[0040] (2) Antibody coupling Accurately weigh 1 g of CNBr-Sepharose 4B dry gel, fully swell in 10 mL, 1 mmol / L, HCl solution, transfer to a sand core funnel and wash with 10 times the volume of coupling buffer to balance . Transfer the washed wet glue to a clean glass bottle, quickly add 2mL of a certain concentration of antibody solution, and slowly stir and react at 4°C for 24h. After the reaction, wash off the uncoupled antibody with more than 10 times the volume of coupling buffer, and collect the washing solution. Set the volume of the collected washing solution to 100mL, measure i...

Embodiment 3

[0045] The determination of embodiment 3 IAC column capacity:

[0046] Take out the IAC column prepared in Example 2, remove the plugs at the upper and lower ends of the column, connect the column to a vacuum pump, adjust the flow rate so that the liquid flows out at a speed not exceeding 1 mL / min, and after the liquid is discharged, use the melamine standard Dilute with PBS to 0.5 μg / mL, load the sample at the same flow rate as above, collect the sample filtrate, measure the content of melamine, calculate the volume of the washing filtrate and the content of melamine in the filtrate. After the liquid is drained, wash the IAC column with 10mL of pure water as eluent, and finally elute with 4mL of 90% methanol water, collect, blow with nitrogen, redissolve in 1mL of methanol, filter through a 0.22μm microporous membrane, and measure by HPLC. Column capacity is calculated by the single-point metering method. Then, the lower end of the column was sealed with a stopper, 3 mL ...

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Abstract

The invention relates to a kit for purifying melamine residues in animal derived food such as milk, milk powder and egg. The kit comprises an immunoaffinity chromatographic column for purification of melamine, an eluent, an eluate, a regeneration liquid and a balance preservation solution. An adsorbent loaded in the immunoaffinity chromatographic column for the purification of melamine is composed of a solid carrier and a melamine polyclonal antibody coupled with the carrier; the melamine polyclonal antibody is obtained by using a conjugate of a melamine hapten with a carrier protein as an immunogen; and the melamine hapten is a melamine analogue 4, 6-diamino-1, 3, 5-triazine-2-acetic acid. The purification method provided by the invention combines with a chromatography method or a chromatography tandem mass spectrometry method for efficient detection of melamine content and has high selectivity. The method has the advantages of simple operation and good purification effect and avoids the interference of impurities on the determination results; and the immunoaffinity chromatographic column can be used repeatedly to reduce analysis cost and environmental pollution.

Description

technical field [0001] The invention relates to a method for preparing and using a melamine immunoaffinity chromatographic column, and belongs to the technical field of immunoaffinity chromatography and melamine detection. Background technique [0002] Melamine (Chemical formula: C3H6N6), commonly known as melamine, protein essence, IUPAC named "1,3,5-triazine-2,4,6-triamino", is a triazine nitrogen-containing heterocycle Organic compounds are used as chemical raw materials. It is white monoclinic crystal, almost odorless, slightly soluble in water (3.1g / L at room temperature), soluble in methanol, formaldehyde, acetic acid, hot glycol, glycerin, pyridine, etc., insoluble in acetone, ethers, harmful to the body Harmful, not used in food processing or food additives. [0003] Melamine is the raw material for the manufacture of melamine formaldehyde. The resin is often used in the manufacture of daily utensils, decorative veneers, fabric finishing agents, etc., and the most...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): B01J20/281C07D251/62
Inventor 施祖灏张小燕高玉时唐修君唐梦君蒲俊华陈大伟葛庆联林伊莱
Owner JIANGSU INST OF POULTRY SCI
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