CN102219852A

CN102219852A - Synthesis method of artificial antigen of heavy metal copper

Info

Publication number: CN102219852A
Application number: CN201010208285XA
Authority: CN
Inventors: 胥传来; 陈莲君; 邓小芳; 马文蔚; 严文静; 勇倩倩
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2010-06-18
Filing date: 2010-06-18
Publication date: 2011-10-19

Abstract

The invention relates to a synthesis method of an artificial antigen of heavy metal copper and belongs to the technical field of biochemical engineering. In the method, based on diethylenetriamine pentaacetic acid (DTPA) serving as a chelant, a copper ion is coupled with a carrier protein bovine serum albumin (BSA) so as to successfully synthesize the artificial antigen of heavy metal copper. The prepared artificial antigen is used for immunoassay method research on heavy metals. The method has brief and effective steps, can be fully used for immunoassay so as to provide a convenient way for research of people in the future, thereby meeting the requirement on heavy metal immunoassay method research in China.

Description

The synthetic method of a kind of heavy metal copper artificial antigen

Technical field

The synthetic method of a kind of heavy metal copper artificial antigen belongs to technical field of biochemical industry.

Background technology

Along with the fast development heavy metal contamination of economy is more and more serious in China.Though, with the phytoremediation heavy-metal contaminated soil is the research focus of current domestic and international academia, but the technology that can effectively reduce heavy metal content in the environment also rests on experimental stage, enter human body in order to reduce heavy metal from food chain, convenient, simply, the technology of heavy metal content is crucial in the testing environment reliably.Heavy metal detection technique commonly used now, as atomic absorption spectrometry, technology such as inductively coupled plasma emission spectrography and dynamic phosphorimetry all need expensive detecting instrument and complicated pre-treatment, as high temperature, high pressure, strong acid digestion etc.And because the Instrument measuring condition changes and detected result is closely related, need the very analytical test personnel operation of specialty, therefore, have only the centralab of research institution just to possess test condition, the sense cycle of sample is longer relatively, the testing process complexity, it is higher to detect cost, quick and easy needs in the tainted food practice of incompatibility China.Current, immunoassay technology has been widely used in the environmental pollution of toxin, sterilant, explosive etc. and the detection of hazardous material.Compare with other detection systems, immunologic detection method has fast, cheap, easy, sensitive and special advantage, can portablely carry out the scene and detect.In recent years, carried out the research of counterweight metalloimmunoassay (MIA) method both at home and abroad, but,, be necessary to prepare the heavy metal copper artificial antigen in order to remedy this blank both at home and abroad still not at the report of cuprase connection immunodetection.

Summary of the invention

The synthetic method that the purpose of this invention is to provide a kind of heavy metal copper artificial antigen.Prepared product is used for the immune analysis method research of heavy metal.For people's research from now on provides essential artificial antigen.

Technical scheme of the present invention: the synthetic method of a kind of heavy metal copper artificial antigen is a sequestrant with diethylene triamine pentacetic acid (DTPA) DTPA, with cupric ion and carrier proteins bovine serum albumin BSA coupling.

Processing step is:

(1) the heavy metal copper artificial antigen is synthetic:

The cupric chloride that takes by weighing 0.147g is dissolved in the 1mL concentrated nitric acid and is prepared into copper ion solution.

Take by weighing in DTPA to the 1.5mL centrifuge tube of 6mg, add the KOH of the 10mol/L of 10 μ L earlier, add the 0.1mol/L of 990 μ L again, the pH value is 9.7 hydroxyethyl piperazine second thiosulfonic acid HEPES solution, promptly is prepared into the DTPA chelating agent solution after the dissolving.

Taking by weighing 0.25g BSA, to be dissolved in 5mL, 0.1mol/L, pH value be the BSA solution that is prepared into 50mg/mL among 7.4 the HEPES.

The first DTPA chelating agent solution of adding 520 μ L in the reactor adds the copper ion solution of 12 μ L again.At room temperature, reacting more than the 1h formation haptens reaction solution on the magnetic stirring apparatus.

The 0.1mol/L, the pH value that almost add the BSA solution of 500 μ L and 200 μ L in the haptens reaction solution simultaneously are 9.7 HEPES, regulate pH value to 9.0.After reaction is spent the night, protein complex is carried out separation and purification with the Cenricon-30 ultra-filtration centrifuge tube.Promptly get the heavy metal copper artificial antigen.

(2) evaluation of heavy metal copper artificial antigen: the heavy metal copper artificial antigen adopts gel electrophoresis to identify its coupling result, utilizes standard protein to obtain typical curve, calculates its coupling ratio.

The conjugate determination of protein concentration: compound concentration is 0,40,60,80,100,120,160,200 μ gmL ^-1Bovine serum albumin solution 1.5mL, add 5mL coomassie brilliant blue staining liquid, mixing immediately, warm 5 minutes of 30 ℃ of water-baths, each concentration is made parallel sample. survey light absorption value, the relation curve of drafting bovine serum albumin concentration and light absorption value at 595nm place.Antigenic solution is diluted by a certain percentage, measure the light absorption value of antigenic solution at the 595nm place, obtain the corresponding proteins concentration of antigenic solution from curve.

Beneficial effect of the present invention: the present invention successfully synthesizes the heavy metal copper artificial antigen, and synthesis step is succinct, effectively, can be used for immunoassay fully, for people's research later on provides approach easily, can satisfy domestic needs to heavy metal research.

Description of drawings

UV scanning figure before and after the preparation of Fig. 1 heavy metal copper artificial antigen.

Embodiment

Embodiment 1

(1) the heavy metal copper artificial antigen is synthetic: the cupric chloride that takes by weighing 0.147g is dissolved in the 1mL concentrated nitric acid and is prepared into copper ion solution.Take by weighing in DTPA to the 1.5mL centrifuge tube of 6mg, add the KOH solution of the 10mol/L of 10 μ L earlier, 0.1mol/L, the pH value that adds 990 μ L again is 9.7 hydroxyethyl piperazine second thiosulfonic acid HEPES solution, promptly is prepared into the DTPA chelating agent solution after the dissolving.Taking by weighing 0.25g BSA, to be dissolved in 5mL, 0.1mol/L, pH value be the BSA solution that is prepared into 50mg/mL among 7.4 the HEPES.The first DTPA chelating agent solution of adding 520 μ L in the reactor adds the copper ion solution of 12 μ L again.At room temperature, reacting more than the 1h formation haptens reaction solution on the magnetic stirring apparatus.The 0.1mol/L, the pH value that almost add the BSA solution of 500 μ L and 200 μ L in the haptens reaction solution simultaneously are 9.7 HEPES, regulate pH value to 9.0.After reaction is spent the night, protein complex is carried out separation and purification with the Centricon-30 ultra-filtration centrifuge tube.Promptly get the heavy metal copper artificial antigen.

(2) evaluation of heavy metal copper artificial antigen: artificial antigen adopts gel electrophoresis to identify its coupling result, utilizes standard protein to obtain typical curve, calculates its coupling ratio.

Claims

1. the synthetic method of a heavy metal copper artificial antigen is characterized in that with diethylene triamine pentacetic acid (DTPA) DTPA be sequestrant, with cupric ion and carrier proteins bovine serum albumin BSA coupling;

(1) the heavy metal copper artificial antigen is synthetic:

The cupric chloride that takes by weighing 0.147g is dissolved in the 1mL concentrated nitric acid and is prepared into copper ion solution;

Take by weighing in DTPA to the 1.5mL centrifuge tube of 6mg, add the KOH solution of the 10mol/L of 10 μ L earlier, add the hydroxyethyl piperazine second thiosulfonic acid HEPES solution of p H 9.7, the 0.1mol/L of 990 μ L again, promptly be prepared into the DTPA chelating agent solution after the dissolving;

Taking by weighing 0.25g bovine serum albumin BSA, to be dissolved in 5mL, 0.1mol/L, pH value be the BSA solution that is prepared into 50mg/mL in 7.4 the HEPES solution;

The DTPA chelating agent solution that adds earlier 520 μ L in the reactor adds the copper ion solution of 12 μ L again, at room temperature more than reaction 1h on the magnetic stirring apparatus, and formation haptens reaction solution;

The 0.1mol/L, the pH value that almost add the BSA solution of 500 μ L and 200 μ L in the haptens reaction solution simultaneously are 9.7 HEPES solution, regulate pH value to 9.0; With the Centricon-30 ultra-filtration centrifuge tube protein complex is carried out separation and purification after reaction is spent the night, promptly get the heavy metal copper artificial antigen;