Synthesis method of highly specific folic acid complete antigen and application of folic acid complete antigen

A complete antigen, high specificity technology, applied in the field of folic acid complete antigen synthesis, to achieve the effect of high specificity and high sensitivity

Active Publication Date: 2014-09-10
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] The purpose of the present invention is to provide a novel method for synthesizing a complete folic acid antigen in view of the deficiencies and defects of the existing folic acid antigen synthesis techn...

Method used

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  • Synthesis method of highly specific folic acid complete antigen and application of folic acid complete antigen
  • Synthesis method of highly specific folic acid complete antigen and application of folic acid complete antigen
  • Synthesis method of highly specific folic acid complete antigen and application of folic acid complete antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Preparation of Modified Carrier Protein

[0034] 50 mg of bovine serum albumin was dissolved in 5 mL of pH9.6 carbonate buffer solution, and then 100 mg of 6-aminocaproic acid and 30 mg of water-soluble carbodiimide were added. Adjust the pH value of the system at 6-8, and react at room temperature for 4 hours. Dialyze with PBS buffer solution for 2 days, change the water 4 times during the period, and obtain bovine serum albumin carrier protein B with modified tether.

Embodiment 2

[0035] Example 2 Preparation of Folic Acid Complete Antigen

[0036] Take 25mg of folic acid, add 2.5mL DMSO to dissolve, adjust the pH value to 7.4, then add 10mg NHS and 23mg DCC respectively, mix well at room temperature in the dark, stir and react for 4h, then add 100mg of carrier protein B, adjust the pH value to 8.5, in The reaction was carried out at room temperature 25° C. and protected from light for 2 h. Dialyze with PBS buffer for 2 days, during which the water was changed 4 times, and the complete antigen of folic acid was obtained.

Embodiment 3

[0037] Example 3 Preparation of Folic Acid Coating Source

[0038] Take 25mg of folic acid, add 2.5mL DMSO to dissolve, and pre-cool at zero temperature for 30 minutes. At zero temperature, add tri-n-butylamine and isobutyl chloroformate (the molar ratio of the hapten folic acid, tri-n-butylamine, and isobutyl chloroformate is 1: 1.2: 1.2), and react at 0°C for 1 hour. Take 100mg ovalbumin / bovine serum albumin, add 10mL 0.1M pH9.6 carbonate buffer, and pre-cool at 0°C for 30 minutes. At 0°C, the activated hapten solution was slowly added dropwise to the protein solution, reacted at 0°C for 1 hour, and then reacted at room temperature for 24 hours. Dialyze with PBS buffer solution for 2 days, change the water 4 times during the period, and obtain the folic acid-coated source.

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Abstract

The invention relates to a synthesis method of a highly specific folic acid complete antigen and an application of the folic acid complete antigen and belongs to the technical field of biochemical industry. The synthesis method disclosed by the invention comprises the steps of adding 6-aminocaproic acid and water-soluble carbodiimide into a vector protein solution and then reacting at a room temperature and carrying out dialysis to obtain a vector protein B modified by a linker arm, dissolving folic acid(N-{4-[(2-amino-1,4-dihydro-4-oxo-6-pteridinyl)methylamino]benzoyl}-L-glutamic acid) in dimethyl sulfoxide, adding dicyclohexyl carbodiimide and hydroxysuccinimide, reacting to obtain a folic acid derivative and then coupling the folic acid derivative with an amino group on the linker arm of the vector protein B to obtain the complete antigen. The results show that the titer of the antiserum obtained by immunizing animals with the antigen disclosed by the invention reaches up to 81000, the detection limit is 0.042ng/mL, and the half-inhibitory concentration is 0.3ng/mL. The produced antibody has high specificity and high sensitivity. The antigen or antibody disclosed by the invention is used for preparing an immunoaffinity column, establishing an enzyme linked immunosorbent assay method and colloidal gold strip rapid detection method and detecting the residue of folic acid.

Description

technical field [0001] The invention relates to a highly specific folic acid complete antigen synthesis method and application thereof, belonging to the technical field of biochemical industry. Background technique [0002] Folic acid (folic acid or folate), also known as dishoyl glutamic acid, one of the B vitamins, is the carrier of single-carbon transfer in the metabolism of amino acids, pyrimidines and purines, and acts as a coenzyme for the synthesis of nucleoproteins; The coenzyme of the internal enzyme reaction; the lack of folic acid will lead to the restriction of the synthesis of dTMP, which will hinder the synthesis of DNA and produce megaloblastic anemia. Current studies have confirmed that folic acid deficiency in women before pregnancy or early pregnancy is one of the important causes of neural tube defects. Folic acid is closely related to the metabolism of homocysteine, and has important biological significance in preventing the occurrence of cardiovascular ...

Claims

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Application Information

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IPC IPC(8): C07K14/765C07K14/77C07K16/44G01N33/53
CPCC07K14/765C07K14/77C07K16/44C07K19/00
Inventor 马伟胥传来孔德昭匡华徐丽广刘丽强宋珊珊吴晓玲
Owner JIANGNAN UNIV
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