CN102495205A - Quantum dot labeled antibody multicomponent non-competitive homogeneous immunoassay method for small-molecule organic matter - Google Patents
Quantum dot labeled antibody multicomponent non-competitive homogeneous immunoassay method for small-molecule organic matter Download PDFInfo
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- CN102495205A CN102495205A CN201110399385XA CN201110399385A CN102495205A CN 102495205 A CN102495205 A CN 102495205A CN 201110399385X A CN201110399385X A CN 201110399385XA CN 201110399385 A CN201110399385 A CN 201110399385A CN 102495205 A CN102495205 A CN 102495205A
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Abstract
The invention discloses a quantum dot labeled antibody multicomponent non-competitive homogeneous immunoassay method for a small-molecule organic matter, and relates to a quantum dot labeled antibody multicomponent non-competitive homogeneous immunoassay technology for the small-molecule organic matter. Fluorescent light emits high-efficiency water-soluble quantum dots with different wavelengths to label antibodies resisting different target analytes. Different quantum dot labeled antibodies are dispersed into a phosphate buffering solution, and a sample solution containing the corresponding target analytes is added into the phosphate buffering solution and uniformly mixed with the phosphate buffering solution, so that non-competitive homogeneous immunoassay reaction is performed; respective labeled samples, samples and a blank contrast system which are subjected to reaction and balanced are directly subjected to fluorescent scanning or multi-wavelength detection under the excitation of ultraviolet light with the same wavelength; and the high-sensitivity quantum dot labeled antibody multicomponent non-competitive homogeneous immunoassay technology for quickly detecting various target small-molecule organic matters is constructed according to a rule that a ratio of the characteristic fluorescent light intensity of the blank contrast system to the characteristic fluorescent light intensity of a sample system is in direct proportion to the concentration of the corresponding target analytes in a certain range.
Description
Technical field
The present invention relates to the non-competing homogeneous immunoassay technology of quantum dot-labeled antibody polycomponent of small organic molecule (agricultural chemicals, medicine, environment incretion interferent).
Background technology
Agricultural chemicals, medicine, the residual of environment incretion interferent exceed standard in environment and the agricultural and animal products, and environment and environmental organism are constituted certain threat, influence food security and human health.Environmental enhancement and food safety monitoring need efficiently analytical technology fast.
The how residual detection method of having reported at present of agricultural chemicals, medicine, environment incretion interferent mainly contains chromatography and application of gas chromatorgraphy/mass method.Adopt these methods to need expensive instrument and equipment, specialized laboratory and well-trained specialized personnel; Sample pre-treatments requires height, process is complicated, speed is slow; The detection cost is high, and specificity is not strong, is difficult to adapt to the requirement of a large amount of samples and field quick detection.Existing small organic molecule multi-component immunity analytical; Usually the different parts that is employed in polystyrene micropore plate is different antibodies or antigen fixedly, carries out different immune responses through spatial discrimination, is reflected at solid phase and liquid interface and carries out; Realize Separation of Solid and Liquid, complicated operation through mode of washing; Or adopt different labels, like mark different antibodies or antigens such as enzyme, luciferins, reaction conditions and detection means are difficult to compatibility.Therefore, truly small organic molecule multi-component immunity analytical technology waits to set up.
Summary of the invention
The objective of the invention is to analyze the antibody of small organic molecule (agricultural chemicals, medicine, environment incretion interferent) as the anti-different target of namo fluorescence probe mark, set up a kind of high specificity, highly sensitive, simple and effective, be used for the non-competing polycomponent homogeneous immunoassay technology of quantum dot-labeled antibody of plurality of target analyte fast detecting with the efficient water soluble quantum dot of different fluorescent emission wavelength.
Technical scheme of the present invention is: with the excitation wave length and width, the fluorescent emission wavelength is narrow and do not disturb mutually, the different water-soluble quantum dots of fluorescence quantum efficiency height, good stability, surperficial tool pendant carboxylic group are as namo fluorescence probe, adopts the carbodlimide method anti-different target analyte of mark (agricultural chemicals, medicine, environment incretion interferent) antibody respectively; Different quantum dot-labeled antibody is diluted to the mixed solution of debita spissitudo with phosphate buffer; Add the mixing standard specimen that contains corresponding target analytes; The panimmunity reaction takes place in homogeneous system simultaneously; With testing sample and the blank standard specimen that replaces of target analytes, sample and blank reaction system are set under the similarity condition, quantum dot-labeled antibody combines the back that fluorescent quenching takes place with corresponding target analytes; The reaction system that will reach balance is directly carried out the detection of fluorescent scanning or multi-wavelength under same excitation wavelength, calculate the characteristic fluorescence intensity F of each quantum dot-labeled antibody in the blank system respectively
0Characteristic fluorescence intensity F with each corresponding quantum dot labelled antibody in the standard specimen system
SRatio F
0/ F
SOn the basis of condition optimizing, according to F
0/ F
SThe rule that (the cancellation degree of characteristic fluorescence) and the concentration of corresponding target analytes are proportional within the specific limits and the F of testing sample system
0/ F
S, the content of each target analytes is set up the non-competing homogeneous immunoassay method of quantum dot-labeled antibody polycomponent that high sensitivity fast detecting plurality of target is analyzed small organic molecule (agricultural chemicals, medicine, environment incretion interferent) in the calculating testing sample.
The present invention fully utilizes that immunoassay high specificity, quantum dot fluorescence excellent performance, homogeneous phase immune response speed are fast, panimmunity be reflected in the homogeneous system carry out simultaneously, equilibration time is short, directly carry out advantage such as fluoroscopic examination behind the molecular balance.Compare with the routine immunization analytical technology; The non-competing homogeneous immunoassay technology of the quantum dot-labeled antibody polycomponent that the present invention set up can be analyzed small organic molecule (agricultural chemicals, medicine, environment incretion interferent) thing to plurality of target simultaneously and carry out qualitative and quantitative detection, has saved Separation of Solid and Liquid and chromogenic reaction step; Not only high specificity, highly sensitive, favorable reproducibility; And simple and effective more, be about 1/4 of conventional single component ELISA detection time, be development and innovation to existing small organic molecule immuno analytical method; Do not see similar report so far as yet, have very high development and application values.
In addition; Efficient water soluble quantum dot according to the invention in 200～350nm scope with same wavelength ultraviolet excitation; Launch half-peak breadth less than 30nm, non-interfering characteristic fluorescence, the interval of the maximum fluorescence wavelength of different quantum dots>50nm, fluorescence quantum efficiency is higher than 60%.
Description of drawings
Fig. 1 is the non-competing homogeneous immunoassay fluorescence spectrum of quantum dot-labeled anti-pesticide antibody polycomponent figure.
Fig. 2 is the non-competing homogeneous immunoassay fluorescence spectrum of quantum dot-labeled anti-drug antibodies polycomponent figure.
Fig. 3 is the relation curve that variable concentrations chloromycetin and quantum dot-labeled chloramphenicol resistance antibody fluorescence intensity change.
Embodiment
One, the non-competing homogeneous immunoassay technology implementation example 1 of quantum dot-labeled anti-pesticide antibody polycomponent
With the fluorescent emission wavelength is efficient water soluble quantum dot employing carbodlimide method (the Greg T. Hermanson of 545nm, 625nm, 705nm; Bioconjugate Techniques; 2ed 2008; P495-496) with anti-uniconazole P antibody, anti-cypermethrin antibody, anti-carbofuran antibody covalent coupling; Prepare quantum dot-labeled anti-uniconazole P antibody, the fluorescent emission wavelength that the fluorescent emission wavelength is about 545nm respectively and be about the quantum dot-labeled anti-cypermethrin antibody of 625nm and the quantum dot-labeled anti-carbofuran antibody that the fluorescent emission wavelength is about 705nm; Behind ultrafiltration purification, be settled to 1 μ moL/L with pH8.3, the 0.05mol/L borate buffer solution dissolving that contains 0.5g/L sodium azide and 1mol/L betaine; 4 ℃ of preservations are subsequent use, face with preceding and respectively said quantum dot-labeled anti-uniconazole P antibody, quantum dot-labeled anti-cypermethrin antibody and quantum dot-labeled anti-carbofuran antibody mixed with equal-volume after 100 times, 200 times and 50 times of the PBS of 0.02mol/L, the pH7.0 dilutions.
Elder generation disposes the mother liquor that contains uniconazole P, cypermethrin and each 1mg/mL of carbofuran standard specimen with methyl alcohol, is diluted to 0.02mol/L, pH7.0 PBS to contain uniconazole P, cypermethrin and carbofuran standard specimen standard specimen 10～10 respectively again
-4The mixing standard specimen series of μ g/mL with testing sample and the blank standard specimen that replaces of target analytes, is provided with sample and blank under the similarity condition.
In 100 μ L standard specimens, testing sample and blank solution, add isopyknic said quantum dot-labeled antibody mixed liquor, the at room temperature about 10～15min of hybrid reaction respectively.Behind the molecular balance in scanning (seeing shown in Figure 1) under the 235nm excitation wavelength or detect near the maximum characteristic fluorescence intensity 545nm, 625nm and the 705nm respectively.
Optimize reaction medium, reactant concentration than, temperature of reaction and correlated conditions such as time, excitation wavelength and fluorescent emission wavelength, select that the quantum dot labelled antibody is fast with corresponding target analytes immune response speed, susceptibility by force, good stability, few, the blank system fluorescence intensity (F of quantum dot-labeled antibody consumption
0) with the fluorescence intensity (F that contains corresponding target analysis objects system
S) ratio is high, background interference is few condition combination.
On this basis, according to F
0/ F
SThe rule that (degree of quantum dot-labeled antibody fluorescent quenching) and corresponding target analyte concentration are directly proportional within the specific limits and the F of each sample
0/ F
SThe content of uniconazole P, cypermethrin and carbofuran in the calculation sample; Uniconazole P in the testing sample, cypermethrin and carbofuran are carried out the qualitative, quantitative fast detecting, thereby set up the non-competing homogeneous immunoassay technology of quantum dot-labeled antibody polycomponent of uniconazole P, cypermethrin and carbofuran.
Two, the non-competing homogeneous immunoassay technology implementation example 2 of quantum dot-labeled anti-drug antibodies polycomponent
With the fluorescent emission wavelength is efficient water soluble quantum dot employing carbodlimide method (the Greg T. Hermanson of 545nm, 625nm, 705nm; Bioconjugate Techniques; 2ed 2008; P495-496) with chloramphenicol resistance antibody, anti-diethylstilbestrol antibody, anti-clenbuterol antibody covalent coupling; Prepare the quantum dot-labeled anti-clenbuterol antibody that quantum dot-labeled chloramphenicol resistance antibody that the fluorescent emission wavelength is about 545nm, quantum dot-labeled anti-diethylstilbestrol antibody that the fluorescent emission wavelength is about 625nm and fluorescent emission wavelength are about 705nm respectively; Quantum dot-labeled antibody behind ultrafiltration purification is settled to 1 μ moL/L with pH8.3, the 0.05mol/L borate buffer solution dissolving that contains 0.5g/L sodium azide and 1mol/L betaine, and 4 ℃ of preservations are subsequent use.Face with preceding and respectively said quantum dot-labeled chloramphenicol resistance antibody, quantum dot-labeled anti-diethylstilbestrol antibody and quantum dot-labeled anti-clenbuterol antibody are mixed with equal-volume after 100 times, 200 times and 50 times of the PBS of 0.02mol/L, the pH7.0 dilutions.
Contain the mother liquor of chloromycetin, diethylstilbestrol and each 1mg/mL of Clenbuterol standard specimen earlier with the methyl alcohol configuration, be diluted to chloride mycin, diethylstilbestrol and Clenbuterol standard specimen 1～10 respectively with 0.02mol/L, pH7.0 PBS again
-5The mixing standard specimen series of μ g/mL with testing sample and the blank standard specimen that replaces of target analytes, is provided with sample and blank under the similarity condition.
In 100 μ L standard specimens, testing sample and blank solution, add isopyknic said quantum dot-labeled antibody mixed liquor, the at room temperature about 10～15min of hybrid reaction respectively.Behind the molecular balance in scanning (seeing shown in Figure 2) under the 235nm excitation wavelength or detect near the maximum characteristic fluorescence intensity 545nm, 625nm and the 705nm respectively.
Optimize reaction medium, reactant concentration than, temperature of reaction and correlated conditions such as time, excitation wavelength and fluorescent emission wavelength, select that the quantum dot labelled antibody is fast with corresponding target analytes immune response speed, susceptibility by force, good stability, few, the blank system fluorescence intensity (F of quantum dot-labeled antibody consumption
0) with the fluorescence intensity (F that contains corresponding target analytes reaction system
S) ratio is high, background interference is few condition combination.
On this basis, according to F
0/ F
SThe rule that (degree that reflects quantum dot-labeled antibody fluorescent quenching) and corresponding target analyte concentration are directly proportional within the specific limits and the F of each sample
0/ F
SThe content of chloromycetin, diethylstilbestrol and Clenbuterol in the calculation sample; Chloromycetin in the testing sample, diethylstilbestrol and Clenbuterol are carried out the qualitative, quantitative fast detecting, thereby set up the non-competing homogeneous immunoassay technology of quantum dot-labeled antibody polycomponent of chloromycetin, diethylstilbestrol and Clenbuterol.
The relation curve that variable concentrations chloromycetin and quantum dot-labeled chloramphenicol resistance antibody fluorescence intensity change, as shown in Figure 3, F
0/ F
SBe directly proportional within the specific limits with the concentration of chloromycetin.
Claims (2)
1. the non-competing homogeneous immunoassay method of quantum dot-labeled antibody polycomponent of a small organic molecule; It is characterized in that: high with excitation wave length and width, fluorescence quantum efficiency, the fluorescent emission wavelength is different and do not disturb mutually, different IPs shell structure water-soluble quantum dot that good stability, surface have pendant carboxylic group is as namo fluorescence probe, adopts the antibody of the anti-different target analysis of carbodlimide method mark small organic molecule; The quantum dot-labeled antibody of difference is disposed mixed solution with the phosphate buffer dilution; Add the mixing standard specimen that contains corresponding target analytes; The panimmunity reaction takes place in homogeneous system simultaneously; With testing sample and the blank standard specimen that replaces of target analytes, sample and blank reaction system are set under the similarity condition; The reaction system that will reach balance is directly carried out the detection of fluorescent scanning or multi-wavelength under same excitation wavelength, calculate the characteristic fluorescence intensity F of each quantum dot-labeled antibody in the blank system respectively
0Characteristic fluorescence intensity F with each corresponding quantum dot labelled antibody in the standard specimen system
SRatio; On the basis of condition optimizing, according to F
0/ F
SWith the proportional within the specific limits rule of the concentration of corresponding target analytes and the F of testing sample system
0/ F
S, the content of each target analytes is set up the non-competing polycomponent homogeneous immunoassay method of quantum dot-labeled antibody that high sensitivity fast detecting plurality of target is analyzed small organic molecule in the calculating testing sample.
2. according to the non-competing homogeneous immunoassay method of quantum dot-labeled antibody polycomponent of the said small organic molecule of claim 1; It is characterized in that said efficient water soluble quantum dot in 200～350nm scope with same wavelength excitation; The emission half-peak breadth is less than 30nm, non-interfering characteristic fluorescence; The interval of the maximum fluorescence wavelength of different quantum dots>50nm, fluorescence quantum efficiency is higher than 60%.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111505273A (en) * | 2020-04-10 | 2020-08-07 | 蓝怡科技集团股份有限公司 | Non-competitive chemiluminescence immunoassay kit for digoxin and application thereof |
| CN113899898A (en) * | 2021-10-15 | 2022-01-07 | 江苏省农业科学院 | Carbofuran immunodetection method based on reverse fluorescence enhanced double-channel signal and antibody |
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| CN1515909A (en) * | 2003-08-27 | 2004-07-28 | 魏景艳 | Quantum point marker sandwich immunodetection method and its diagnosis kit |
| CN1945331A (en) * | 2006-10-20 | 2007-04-11 | 邹明强 | Method for preparing and using reagent for simultaneously detecting multiple small molecular compounds |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111505273A (en) * | 2020-04-10 | 2020-08-07 | 蓝怡科技集团股份有限公司 | Non-competitive chemiluminescence immunoassay kit for digoxin and application thereof |
| CN113899898A (en) * | 2021-10-15 | 2022-01-07 | 江苏省农业科学院 | Carbofuran immunodetection method based on reverse fluorescence enhanced double-channel signal and antibody |
| CN113899898B (en) * | 2021-10-15 | 2023-12-19 | 江苏省农业科学院 | Carbofuran immunodetection method and antibody based on reverse fluorescence enhanced double-channel signal |
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