CN102175846A - Fluorescence polarization immunoassay method of bisphenol A - Google Patents
Fluorescence polarization immunoassay method of bisphenol A Download PDFInfo
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- CN102175846A CN102175846A CN2010106046744A CN201010604674A CN102175846A CN 102175846 A CN102175846 A CN 102175846A CN 2010106046744 A CN2010106046744 A CN 2010106046744A CN 201010604674 A CN201010604674 A CN 201010604674A CN 102175846 A CN102175846 A CN 102175846A
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Abstract
A fluorescence polarization immunoassay method of bisphenol A belongs to the technical field of fluorescence polarization immunoassay. In the invention, diphenolic acid is used as a hapten coupling FITC (fluorescein isothiocyanate) fluorescein derivative to compound a fluorescent marker; a bisphenol A is used as the standard substance; a bisphenol A polyclonal antibody is used as the antibody; and a fluorescence polarization immunoassay method of bisphenol A is established. In the invention, the fluorescence polarization immunoassay method of bisphenol A is established, so that a fast and efficient detection method is supplied for detecting the residuals of bisphenol A. The method provided by the invention is simple and fast; the cost is low, and the result is reliable. The sensitivity is 2ng/mL, and the linear range is 20-800ng/mL. The high specificity and affinity of the immunoreaction enable the fluorescence polarization immunoassay method to have quite high selectivity and sensitivity.
Description
Technical field
The present invention relates to a kind of fluorescence polarization immunoassay detection method of bisphenol-A, belong to the fluorescence polarization technical field of immunoassay.
Background technology
Bisphenol-A (Bisphenol A, BPA) formal name used at school 2, and 2-two (4-hydroxy phenyl) propane is from packaging material for food newfound a kind of " destroy endocrine chemical substance (Endocrine Disrupting Chemicals, EDC) ".BPA is synthetic in acid medium by phenol, acetone, is important Organic Chemicals, is mainly used in the manufacturing macromolecular material, at aspects such as container inner wall coating, packaging materials for food important purposes is arranged.In producing and living, BPA can enter human body by approach such as skin, respiratory tract, alimentary canals, and BPA all has the medium tenacity pungency to skin, respiratory tract, alimentary canal and cornea.Human mainly by the edible food that contains BPA material packing, the use baby bottles, dentistry filling material and sealable tank etc. are taken in BPA.BPA has the effect of DNA oxidative damage when low dosage.Therefore residual in its packaging material for food also more and more caused attention both domestic and external.Though existing these instrument detecting methods have advantages such as highly sensitive, applied widely, sample pre-treatments complexity, required instrument costliness, the cost height, detection time is long, therefore is necessary to study high specificity, the highly sensitive while is cheap, immunoassay technology that can be fast and convenient.In recent years, the domestic and international research of having carried out the bisphenol-A immune analysis method.But so far, the domestic report that does not still use fluorescence polarization immunoassay technology for detection bisphenol-A, design the analog diphenolic acid that has synthesized with bisphenol-A is that haptens has synthesized fluorescein-labelled thing for this reason.
Summary of the invention
The object of the present invention is to provide the residual fluorescence polarization immunologic detection method of a kind of fast detecting bisphenol-A, and higher sensitivity and specificity are arranged.
Technical scheme of the present invention: a kind of fluorescence polarization immunoassay detection method of bisphenol-A, utilize diphenolic acid as the synthesizing fluorescently labeled thing of hapten conjugation FITC fluorescein derivative, and be standard items with the bisphenol-A, resist with bisphenol-A is antibody more, sets up the fluorescence polarization immunoassay detection method of bisphenol-A; Step is as follows:
(1) preparation of FITC fluorescein derivative EDF:
(2) preparation of EDF label:
N-hydroxy-succinamide NHS 9.2mg, dicyclohexylcarbodiimide DCC16.4mg and diphenolic acid 1.4mg are dissolved among the DMF of 2.0mL, stir under the room temperature and spend the night;
Add the derivant EDF 2-3mg of the fluorescein-labelled thing of FITC in reaction solution, lucifuge stirring reaction 3h makes the EDF label under the room temperature;
(3) purifying of EDF label:
With CHCl
3/ MeOH=1:1 is a developping solution, carries out thin-layer chromatography under the room temperature, separation and purification EDF label, and ultraviolet transilluminator is observed down, selects R
f=0.56 band is with 300 μ L methanol extractions;
(4) determining of EDF label working concentration:
With the EDF label of 0.025M, pH8.0 borate buffer solution dilution variable concentrations, last polarimeter detection intensity of polarization light, with 10 times of intensity of polarization light to blank solution as its working concentration; Determine to choose the 1:1500 dilution as working concentration; Blank solution is selected 0.025M, pH8.0 borate buffer solution for use;
(5) determining of the bisphenol-A working concentrations that resist more:
In each test tube, add 500 μ L successively and be diluted to different dilute concentrations: 1:200 with 0.025M, pH8.0 borate buffer solution, 1:400,1:800,1:1600,1:3200,1:6400, how anti-the bisphenol-A of 1:12800 is, and add the EDF label of 500 μ L successively, hatches 5min under the room temperature, last polarimeter detects the polarization light value, determines to choose the working concentration of 1:800 dilution as antibody;
(6) competition:
Bisphenol-A is diluted to 1,5,25,125,625,3125,15625 with ultrapure water, and the 78125ng/mL series concentration adds respectively in the different test tubes, and other establishes a ultrapure water blank, 50 μ L/ pipe; Add the EDF label of 1500 times of 500 μ L dilutions then in each test tube, the bisphenol-A that adds 800 times of 500 μ L dilutions at last respectively resists under room temperature more hatches 5min, and last polarimeter detects the polarization light value;
(7) standard of setting up bisphenol-A suppresses logarithmic curve: with measured detection polarization light value is ordinate, is the standard inhibition logarithmic curve that bisphenol-A is set up in the horizontal ordinate mapping with the bisphenol A concentration;
(8) detection of actual sample: with actual sample set by step the method for (6) detect, the polarization light value that records suppresses the logarithmic curve contrast with the standard of bisphenol-A, determines the bisphenol-A residual concentration of sample.
More detailed step is:
Main solution preparation
1) preparation 0.025M, pH8.0 borate buffer solution:
Na
2B
4O
710H
2O 9.534 g add ultrapure water and are diluted to 980 mL, transfer to pH8.0 with 2M NaOH, add ultrapure water and are settled to 1000 mL.
The step of fluorescence polarization immunoassay detection method is as follows:
The methanol solution that in advance standard items of bisphenol-A is mixed with 1mg/mL is as the work mother liquor, and is stand-by 4 ℃ of preservations.Preparation borate buffer solution (0.025mol/L, pH8.0) is prepared serial reaction liquid based on this, and is how anti-in order to dilute fluorescein-labelled thing and bisphenol-A.
Determining of a, fluorescein-labelled thing working concentration: the fluorescein-labelled thing of dilution variable concentrations, last machine testing intensity of polarization light, with 10 times of intensity of polarization light to blank solution (0.025M, pH8.0 borate buffer solution) as its working concentration.Experimental result is chosen 1:5000 as working concentration.
Determining of the working concentration of b, bisphenol-A antibody: in each test tube, add 500 μ L are diluted to different dilute concentrations with 0.025M, pH8.0 borate buffer solution bisphenol-A antibody (1:200 successively, 1:400,1:800,1:1600,1:3200,1:6400,1:12800), and add the fluorescein-labelled thing of 500 μ L successively, hatch 5min under the room temperature, last machine testing polarization light value.Experimental result is chosen the working concentration of 1:800 as antibody.
C, competition:
Bisphenol-A is diluted to 1,5,25,125,625,3125,15625 with ultrapure water, and the 78125ng/mL series concentration adds respectively in the different test tubes, and other establishes a ultrapure water blank, 50 μ L/ pipe.The fluorescein-labelled thing that adds 1500 times of 500 μ L dilutions then in each test tube, how anti-the bisphenol-A that adds 800 times of 500 μ L dilutions at last respectively is, hatches 5min under room temperature.
D, mensuration: go up machine testing polarization light value.
Beneficial effect of the present invention: the present invention has set up bisphenol-A fluorescence polarization immunologic detection method, for the bisphenol-A residue detection provides a kind of detection means rapidly and efficiently, because employing is polyclonal antibody and portable fluorescence polarization instrument, expense is lower, fast, convenient, reliable.Sensitivity is 2ng/mL, and the range of linearity is 20-800ng/mL.Immunoreactive high specific and high-affinity make the fluorescence polarization immunity have high selectivity and sensitivity, and sample pre-treatment process is simple, and is easy to operate, fast.
Description of drawings
Fig. 1 suppresses logarithmic curve with the standard of the bisphenol-A that the fluorescence polarization instrument is set up.
Specific embodiments
Further specify the present invention by the following examples.
One, instrument:
The KFLOW water purification machine, Kai Folong company,
AB104-N type electronic analytical balance,
Portable fluorescence polarization instrument, SENTARY 100,
Can debug pipettor, Thermo Labsystems company,
Turbine mixer, Shanghai Hu Xi instrumental analysis factory.
Two, reagent:
The bisphenol-A polyclonal antibody, the laboratory self-control,
Other reagent are analytical reagent.
Three, step
1.FITC the preparation of fluorescein derivative (EDF), step is as follows:
Dropwise the FITC drips of solution is added and contain in the triethylamine solution of ethylenediamine-hydrochloride.
2.EDF the preparation of label, step is as follows:
N-hydroxy-succinamide (NHS) (9.2mg, 80 μ mol), dicyclohexylcarbodiimide (DCC) (16.4mg, 80 μ mol), and diphenolic acid (1.4mg, 4.8 μ mol) is dissolved among the DMF of 2.0mL.Stir under the room temperature and spend the night.
Add the derivant EDF2-3mg of the fluorescein-labelled thing of FITC in reaction solution, lucifuge stirring reaction 3h makes the EDF label under the room temperature.
3, the purifying of EDF label:
With CHCl
3/ MeOH=1:1 is a developping solution, carries out thin-layer chromatography under the room temperature, separation and purification EDF label.Ultraviolet transilluminator is observed down, selects R
f=0.56 band.With 300 μ L methanol extractions.
4, determining of EDF label working concentration:
With the EDF label of 0.025M, pH8.0 borate buffer solution dilution variable concentrations, last machine testing intensity of polarization light, with 10 times of intensity of polarization light to blank solution (0.025M, pH8.0 borate buffer solution) as its working concentration.Experimental result is chosen 1:1500 as working concentration.
5, determining of the working concentration of bisphenol-A antibody:
In each test tube, add 500 μ L successively and be diluted to the bisphenol-A antibody (1:200 of different dilute concentrations with 0.025M, pH8.0 borate buffer solution, 1:400,1:800,1:1600,1:3200,1:6400,1:12800), and add the EDF label of 500 μ L successively, and hatch 5min under the room temperature, last polarimeter detects the polarization light value.Experimental result is chosen the working concentration of 1:800 as antibody.
6, competition:
Bisphenol-A is diluted to 1,5,25,125,625,3125,15625 with ultrapure water, and the 78125ng/mL series concentration adds respectively in the different test tubes, and other establishes a ultrapure water blank, 50 μ L/ pipe.Add the EDF label of 1500 times of 500 μ L dilutions then in each test tube, the bisphenol-A that adds 800 times of 500 μ L dilutions at last respectively resists under room temperature more hatches 5min.Last polarimeter detects the polarization light value.
Test findings is as follows:
1, typical curve: the range of linearity of the antibody test that the present invention obtained is to be 20～800ng/mL.
2, sensitivity: sensitivity is the concentration of the pairing standard items of gained 90% maximum polarization light value, i.e. IC
10Be 2ng/mL.
Claims (1)
1. the fluorescence polarization immunoassay detection method of a bisphenol-A, it is characterized in that utilizing diphenolic acid as the synthesizing fluorescently labeled thing of hapten conjugation FITC fluorescein derivative, and be standard items with the bisphenol-A, be antibody with bisphenol-A is how anti-, set up the fluorescence polarization immunoassay detection method of bisphenol-A; Step is as follows:
(1) preparation of FITC fluorescein derivative EDF:
Measure the 200mg ethylenediamine-hydrochloride respectively, 500 μ L triethylamines are dissolved in the methyl alcohol of 50mL, must contain the triethylamine solution of ethylenediamine-hydrochloride;
Mixed dissolution, stirring reaction 10h under the room temperature lucifuge filters to isolate precipitation then, and drying gets FITC fluorescein derivative EDF, keeps in Dark Place;
(2) preparation of EDF label:
N-hydroxy-succinamide NHS 9.2mg, dicyclohexylcarbodiimide DCC16.4mg and diphenolic acid 1.4mg are dissolved among the DMF of 2.0mL, stir under the room temperature and spend the night;
Add the derivant EDF 2-3mg of the fluorescein-labelled thing of FITC in reaction solution, lucifuge stirring reaction 3h makes the EDF label under the room temperature;
(3) purifying of EDF label:
With CHCl
3/ MeOH=1:1 is a developping solution, carries out thin-layer chromatography under the room temperature, separation and purification EDF label, and ultraviolet transilluminator is observed down, selects R
f=0.56 band is with 300 μ L methanol extractions;
(4) determining of EDF label working concentration:
With the EDF label of 0.025M, pH8.0 borate buffer solution dilution variable concentrations, last polarimeter detection intensity of polarization light, with 10 times of intensity of polarization light to blank solution as its working concentration; Determine to choose the 1:1500 dilution as working concentration; Blank solution is selected 0.025M, pH8.0 borate buffer solution for use;
(5) determining of the bisphenol-A working concentrations that resist more:
In each test tube, add 500 μ L successively and be diluted to different dilute concentrations: 1:200 with 0.025M, pH8.0 borate buffer solution, 1:400,1:800,1:1600,1:3200,1:6400, how anti-the bisphenol-A of 1:12800 is, and add the EDF label of 500 μ L successively, hatches 5min under the room temperature, last polarimeter detects the polarization light value, determines to choose the working concentration of 1:800 dilution as antibody;
(6) competition:
Bisphenol-A is diluted to 1,5,25,125,625,3125,15625 with ultrapure water, and the 78125ng/mL series concentration adds respectively in the different test tubes, and other establishes a ultrapure water blank, 50 μ L/ pipe; Add the EDF label of 1500 times of 500 μ L dilutions then in each test tube, the bisphenol-A that adds 800 times of 500 μ L dilutions at last respectively resists under room temperature more hatches 5min, and last polarimeter detects the polarization light value;
(7) standard of setting up bisphenol-A suppresses logarithmic curve: with measured detection polarization light value is ordinate, is the standard inhibition logarithmic curve that bisphenol-A is set up in the horizontal ordinate mapping with the bisphenol A concentration;
(8) detection of actual sample: with actual sample set by step the method for (6) detect, the polarization light value that records suppresses the logarithmic curve contrast with the standard of bisphenol-A, determines the bisphenol-A residual concentration of sample.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102590493A (en) * | 2012-02-17 | 2012-07-18 | 南开大学 | Kit for diethyl phthalate fluorescence polarization immunoassay |
| CN102621297A (en) * | 2012-03-12 | 2012-08-01 | 南开大学 | Fluorescence polarization immunoassay detection method for sarafloxacin |
| CN103848898A (en) * | 2014-02-17 | 2014-06-11 | 南昌大学 | Antigenic mimic epitope Ph3 of bisphenol A and application thereof |
| CN105136755A (en) * | 2015-08-11 | 2015-12-09 | 中国农业大学 | Fluorescence polarization immunoassay method for detection of erythromycin |
| CN105158457A (en) * | 2015-08-11 | 2015-12-16 | 中国农业大学 | Fluorescence polarization immunoassay method for detecting SM2 (sulfamethazine) on basis of egg yolk antibodies |
| CN105352930A (en) * | 2015-12-01 | 2016-02-24 | 深圳市绿诗源生物技术有限公司 | Bisphenol A fluorescence polarization immunity analysis and detection method based on adopting AMF as fluorescein marker |
| CN106771127A (en) * | 2016-12-02 | 2017-05-31 | 安徽出入境检验检疫局检验检疫技术中心 | The rapid fluorescence labeling method of Ractopamine |
| CN107796937A (en) * | 2017-04-27 | 2018-03-13 | 广东工业大学 | The FPIA method and its application of glycocholic acid in a kind of detection serum |
| CN112230001A (en) * | 2020-10-10 | 2021-01-15 | 河南农业大学 | Visual immunoassay method for detecting aspergillus flavus M1 |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102590493A (en) * | 2012-02-17 | 2012-07-18 | 南开大学 | Kit for diethyl phthalate fluorescence polarization immunoassay |
| CN102621297A (en) * | 2012-03-12 | 2012-08-01 | 南开大学 | Fluorescence polarization immunoassay detection method for sarafloxacin |
| CN103848898A (en) * | 2014-02-17 | 2014-06-11 | 南昌大学 | Antigenic mimic epitope Ph3 of bisphenol A and application thereof |
| CN103848898B (en) * | 2014-02-17 | 2016-01-27 | 南昌大学 | The antigenic epitope Ph3 of dihydroxyphenyl propane and application thereof |
| CN105136755A (en) * | 2015-08-11 | 2015-12-09 | 中国农业大学 | Fluorescence polarization immunoassay method for detection of erythromycin |
| CN105158457A (en) * | 2015-08-11 | 2015-12-16 | 中国农业大学 | Fluorescence polarization immunoassay method for detecting SM2 (sulfamethazine) on basis of egg yolk antibodies |
| CN105352930A (en) * | 2015-12-01 | 2016-02-24 | 深圳市绿诗源生物技术有限公司 | Bisphenol A fluorescence polarization immunity analysis and detection method based on adopting AMF as fluorescein marker |
| CN106771127A (en) * | 2016-12-02 | 2017-05-31 | 安徽出入境检验检疫局检验检疫技术中心 | The rapid fluorescence labeling method of Ractopamine |
| CN107796937A (en) * | 2017-04-27 | 2018-03-13 | 广东工业大学 | The FPIA method and its application of glycocholic acid in a kind of detection serum |
| CN112230001A (en) * | 2020-10-10 | 2021-01-15 | 河南农业大学 | Visual immunoassay method for detecting aspergillus flavus M1 |
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