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Preparation method and application of hexamethylenetetramine complete antigen

A technology of hexamethylenetetramine and complete antigen, which is applied to the preparation method of peptides, chemical instruments and methods, animal/human proteins, etc., can solve the problem of inability to synthesize hexamethylenetetramine complete antigen and six times of detection Methyltetramine and other problems, to achieve the effect of short detection time, high accuracy, maintenance and supervision

Inactive Publication Date: 2015-08-05
HEBEI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there is no report on the method of synthesizing the complete antigen of hexamethylenetetramine at home and abroad, and the method of conventional synthetic artificial antigen in the prior art is adopted, such as the diazotization method, the carbodiimide method, and the active ester method. , mixed anhydride method, etc., it is impossible to synthesize the ideal hexamethylenetetramine complete antigen
At the same time, there is no relevant report on the detection of hexamethylenetetramine by enzyme-linked immunoassay at home and abroad.

Method used

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  • Preparation method and application of hexamethylenetetramine complete antigen
  • Preparation method and application of hexamethylenetetramine complete antigen
  • Preparation method and application of hexamethylenetetramine complete antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Preparation of Hexamethylenetetramine Complete Antigen

[0040] Prepare 5ml of complex phosphate buffer solution with a pH value of 7.4, an ion concentration of 100 mmol, and 200 mg of bovine serum albumin (carrier protein); weigh 100 mg of hexamethylenetetramine and dissolve it in 400 μl of methanol. Drop into the prepared compound phosphate buffer; then add 3ml of 25% glutaraldehyde aqueous solution by volume, mix well, let stand at room temperature for 1 hour, refrigerate overnight at 4°C, and dialyze continuously with phosphate buffer for 5 day, the medium was changed once a day to obtain the complete antigen of hexamethylenetetramine (complete antigen HMTA-BSA).

Embodiment 2

[0041] Example 2 Identification of Hexamethylenetetramine Complete Antigen

[0042] Precisely prepare standard solutions of hexamethylenetetramine and bovine serum albumin (BSA) with deionized water. Prepare the conjugate HMTA-BSA standard solution with deionized water, measure the protein concentration by Kjeldahl method, and make the protein concentration of the conjugate solution consistent with the BSA protein concentration. Scan the spectrum to judge whether the coupling is successful. See the result figure 1 and figure 2 .

[0043] The results show that: the absorbance value corresponding to the maximum absorption wavelength of the HMT antigen is significantly different from that of HST and BSA, which is determined by figure 1 and figure 2 The comparison shows that after hexamethylenetetramine is coupled with the carrier protein, the ultraviolet spectrum scanning pattern of the conjugate is compared with that of the carrier protein BSA, and the spectral curve chan...

Embodiment 3

[0046] Example 3 Preparation of polyclonal antibody against hexamethylenetetramine

[0047] Immunization of experimental animals: Accurately measure 0.1ml of the complete antigen HMTA-BSA, dissolve it in 0.4ml of phosphate buffered saline (PBS) with a concentration of 0.01 mol / L and a pH value of 7.4, and mix the obtained solution with 0.5ml of Freund’s The complete adjuvant was fully emulsified for the first immunization, and 0.1ml of complete antigen HMTA-BSA was dissolved in 0.9ml of PBS with a concentration of 0.01 mol / L and a pH value of 7.4 for booster immunization, and fully emulsified with 1ml of Freund's incomplete adjuvant Afterwards, immunize two male white rabbits with multiple points (6-9 points) on both sides of the spine under the skin, and boost the immunization for four weeks, 2 weeks, 4 weeks, 8 weeks, and 12 weeks after the initial immunization respectively. The first time, on the 10th day after the fifth immunization, blood was collected from the ear vein o...

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Abstract

The invention discloses a preparation method of a hexamethylenetetramine complete antigen, which includes following steps: (A) preparing a 0.16-0.3 mg / ml hexamethylenetetramine solution; (B) preparing a composite phosphate buffer solution containing bovine serum albumin; (C) adding dropwisely the hexamethylenetetramine solution into the composite phosphate buffer solution and adding a glutaraldehyde water solution, mixing the mixed solution uniformly and allowing the mixed solution to stand for 1-2 h at room temperature; and (D) filling a dialysis bag with the mixed solution in the step (C) after stay overnight at 4 DEG C, dialyzing the mixed solution with a phosphate buffer solution for 3-5 days, wherein the liquid is changed for 1-2 times every day to obtain the hexamethylenetetramine complete antigen. Meanwhile, the invention also provides a quantitative detection method of hexamethylenetetramine with the antigen and an antibody prepared therewith. The antigen and the antibody are strong in specificity and sensitivity. The detection method is low in detection limit, is high in accuracy, is simple in steps, is easy to operate and control, and can be widely used in detection of the hexamethylenetetramine in the field of food safety detection.

Description

technical field [0001] The invention relates to the field of food safety detection, in particular to a method for preparing a complete antigen of hexamethylenetetramine and the application of the complete antigen in the detection of hexamethylenetetramine. Background technique [0002] Hexamethylenetetramine (commonly known as urotropine), molecular formula C 6 h 12 N 4 , is a chemical raw material, and was included in the fifth batch of "List of Non-edible Substances and Food Additives That May Be Added Illegally in Food" by the Ministry of Health. Hexamethylenetetramine is prohibited from being added to food or in the process of food processing. used in . Some offenders mix it into food such as bean curd sticks, vermicelli, and aquatic products, which will have the effects of whitening, keeping fresh, increasing taste, and preserving. Hexamethylenetetramine itself is of low toxicity and can be taken as a drug. However, it can decompose formaldehyde under acidic condit...

Claims

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Application Information

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IPC IPC(8): C07K14/765C07K1/107G01N33/543G01N33/531
CPCC07K14/765C07K19/00G01N33/531G01N33/543G01N2430/00
Inventor 王庭欣贡东军信海红周丽岩孟墨冯智敏吕宝新
Owner HEBEI UNIVERSITY
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