CN104049083A - Direct competitive enzyme immunoassay method based on nano-gold plasma excimer - Google Patents
Direct competitive enzyme immunoassay method based on nano-gold plasma excimer Download PDFInfo
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- CN104049083A CN104049083A CN201410307017.1A CN201410307017A CN104049083A CN 104049083 A CN104049083 A CN 104049083A CN 201410307017 A CN201410307017 A CN 201410307017A CN 104049083 A CN104049083 A CN 104049083A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
Abstract
The invention discloses a direct competitive enzyme immunoassay method based on a nano-gold plasma excimer, and immunoassay visible detection on small-molecule compounds can be realized. The direct competitive enzyme immunoassay method comprises the steps of competitively combining a test sample possibly containing the small-molecule compounds and haptens labeled with catalase with small-molecule compound antibodies covering a solid phase, decomposing hydrogen peroxide through the combined enzyme-labeled antigens, reducing chloroauric acid into nano-gold through the hydrogen peroxide under a certain buffering condition, and detecting the small-molecule compounds in the sample by observing the color or the light absorption intensity of a developing liquid. The method disclosed by the invention has the advantages of high sensitivity, simplicity in operation, low cost, visible result distinguishing and the like, and the shortcomings of the conventional small-molecule compound enzyme-linked immunoassay detection technology are overcome.
Description
Technical field
The present invention relates to a kind of detection method of micromolecular compound, particularly a kind of competitive ELISA analytical approach based on nm of gold plasmon, belongs to immunology detection analysis field.
Background technology
Enzyme-linked immuno assay technology is a kind of very ripe solid-phase immunoassay pattern, has obtained application for many years in clinical analysis of diagnosis,, nearly ten years, also in the fields such as environmental monitoring, the survey of food safety hazard quality testing, obtains a wide range of applications meanwhile.In the detection to micromolecular compound, enzyme-linked immuno assay mainly adopts two kinds of patterns, indirect competitive and direct competition method.In two kinds of detecting patterns, need respectively the haptens of second antibody and the enzyme labeling of enzyme labeling, they are the effects of amplifying as the signal of immuning adsorpting analysis.In environmental monitoring and food hazardous material detect, enzyme-linked immuno assay the most frequently used enzyme molecule be horseradish peroxidase (HRP) enzyme, its most frequently used substrate is tetramethyl benzidine (TMB) and hydrogen peroxide (H
2o
2), under the catalysis of HRP, they change blue material into, and under the effect of strong acid, blue product can change yellow into, detects the optical absorption intensity of product by microplate reader, can realize the detection to target micromolecular compound.
At present, in many fields such as environmental monitoring, the analyses of food hazardous material, all more highly sensitive immune analysis method is had to urgent demand, therefore, improve the detection performance of traditional enzyme-linked immune analytic method, there is important using value.Much research adopts new detecting pattern, as chemiluminescence, electrochemiluminescence, compared with traditional enzyme-linked immune analytic method, they have sensitiveer detectability, but these immune analysis methods need more expensive equipment and reagent, therefore, testing cost significantly improves, and this has greatly hindered its application in many fields such as environmental monitoring, the analyses of food hazardous material.
Summary of the invention
For the deficiencies in the prior art, the object of the invention is to propose a kind of competitive ELISA analytical approach based on nm of gold plasmon, to realize highly sensitive, the detection cheaply to micromolecular compound.
For realizing aforementioned goal of the invention, the present invention has adopted following technical scheme:
A direct competitive enzyme immunoassay method based on nm of gold plasmon, comprising:
Test sample is mixed with the haptens of hydrogen peroxide enzyme labeling, and the micromolecular compound antibody competition of the haptens that makes the micromolecular compound that may contain in described test sample and hydrogen peroxide enzyme labeling on being coated on solid phase carrier be combined,
Remove the micromolecular compound of not being combined with described micromolecular compound antibody and the haptens of hydrogen peroxide enzyme labeling, and add hydrogen peroxide, gold chloride and in order to form the auxiliary reagent of selected buffer system, observe or detect again color or the optical absorption intensity of the hybrid reaction system forming, realize the detection to contained micromolecular compound in test sample;
Wherein, in described selected buffer system, the haptens that is incorporated into the described hydrogen peroxide enzyme labeling on described micromolecular compound antibody can decomposition of hydrogen peroxide, and hydrogen peroxide can reduce described gold chloride and forms nano Au particle.
Further, the described direct competitive enzyme immunoassay method based on nm of gold plasmon specifically can comprise the steps:
(1) antibody of micromolecular compound is dissolved in coated damping fluid, and is applied on solid phase carrier, hatch at 37 DEG C, then with confining liquid sealing, thereby micromolecular compound antibody is coated on solid phase carrier;
(2) haptens of hydrogen peroxide enzyme labeling and the test sample that may contain micromolecular compound are mixed in phosphate buffer, be applied on step (1) described solid phase carrier after treatment, hatch rear washing in 37 DEG C;
(3) on step (2) described solid phase carrier after treatment, applying hydrogen peroxide;
(4) to apply the nitrite ion that contains gold chloride on step (3) described solid phase carrier after treatment, after at room temperature fully reacting, observe or detect again color or the optical absorption intensity of the hybrid reaction system forming, realize the detection to contained micromolecular compound in test sample.
In a better embodiment, be by detecting the optical absorption intensity of described hybrid reaction system for visible ray, realize the detection to contained micromolecular compound in test sample.
Wherein, described visible light wavelength is preferably 540 nm.
Further, the described direct competitive enzyme immunoassay method based on nm of gold plasmon specifically can comprise the steps:
(1) coated antibody
After the antibody of micromolecular compound is diluted respectively with coated damping fluid, add in each hole of ELISA Plate (also as solid phase carrier), hatch a period of time (for example 2h) for 37 DEG C, seal with confining liquid;
(2) competitive reaction
By the enzyme-labelled antigen (haptens of hydrogen peroxide enzyme labeling, also can be called for short CAT enzyme-labelled antigen) dilution after, add in each hole of ELISA Plate, add respectively again the micromolecular compound of variable concentrations, hatch a period of time (for example 1h) for 37 DEG C, then put washing more than 2 times, more than washing with water once with cleansing solution;
(3) add hydrogen peroxide
Add H
2o
2, 25 DEG C of reactions a period of time (for example 1h);
(4) colour developing
In the each hole of ELISA Plate in above-mentioned steps (3), add respectively nitrite ion, vibration mixes, for example, after reaction a period of time (15min), and the color of observation hybrid reaction system or detect visible absorption intensity.
Further, can to adopt concentration be the sodium carbonate buffer that 0.05 M, pH value are 9.6 to described coated damping fluid.
Further, described confining liquid can adopt phosphate (PBS) buffer solution that interpolation 0.2wt% BSA, concentration are 0.01M.
Further, described enzyme-labelled antigen can be adopt that the method coupling hydrogen peroxidases (CAT) such as carbodiimide method, diazonium method, glutaraldehyde method and micromolecular compound or derivatives thereof obtain compound.
Further, in the PBS damping fluid that is 0.01M every liter of aforementioned concentration, can comprise sodium chloride 8 g, potassium dihydrogen phosphate 0.24 g, sodium hydrogen phosphate 3.62 g, potassium chloride 0.2g.
Further, described cleansing solution (PBST) can adopt add 0.05wt% Tween-20, concentration is the phosphate buffer that 0.01M, pH value are 7.4.
Further, described nitrite ion can be by superoxol, 5mM chlorauric acid solution, 2mM sodium citrate solution and 2mM MES(2-(N-morpholine) ethyl sulfonic acid) solution etc. is mixed to form.
Further, the described direct competitive enzyme immunoassay method based on nm of gold plasmon also comprises:
A, after the micromolecular compound standard model of getting a series of variable concentrations mixes with the haptens of the hydrogen peroxide enzyme labeling of fixing consumption respectively, micromolecular compound antibody competition on being coated on solid phase carrier is combined, remove the micromolecular compound of not being combined with described micromolecular compound antibody and the haptens of hydrogen peroxide enzyme labeling, and add hydrogen peroxide, gold chloride and in order to form the auxiliary reagent of selected buffer system, observe again or detect color or the optical absorption intensity of final the formed hybrid reaction system of each micromolecular compound standard model, and then according to corresponding relation Criterion colorimetric card or typical curve between the concentration of described micromolecular compound standard model and color or the visible absorption intensity of final obtained corresponding hybrid reaction system,
B, after sample to be tested is mixed with the haptens of hydrogen peroxide enzyme labeling, micromolecular compound antibody competition on being coated on solid phase carrier is combined, remove the haptens of the hydrogen peroxide enzyme labeling of not being combined with described micromolecular compound antibody and the micromolecular compound that may exist, and add hydrogen peroxide, gold chloride and in order to form the auxiliary reagent of selected buffer system, observe again or detect color or the optical absorption intensity of final formed hybrid reaction system, and compare with described standard color comparison card or typical curve, and then qualitative or quantitatively record the concentration of contained micromolecular compound in sample to be tested.
Should the direct competitive enzyme immunoassay method based on nm of gold plasmon be a kind of method of visual detection micromolecular compound of the formation of the enzyme linked immunological based on nm of gold plasmon, its haptens according to hydrogen peroxide enzyme labeling (being called for short CAT enzyme-labelled antigen or enzyme-labelled antigen) is coated on the micromolecular compound antibody (abbreviation insolubilized antibody) on solid phase carrier with micromolecular compound competitive binding, wherein, do not exist or when less existence at micromolecular compound, most enzyme-labelled antigens are combined with insolubilized antibody and are fixed, and in can the follow-up hydrogen peroxide adding of more decomposition, in the time of micromolecular compound existence or more existence, less enzyme-labelled antigen is combined with insolubilized antibody, and only can decompose a small amount of follow-up hydrogen peroxide adding, and the reducible follow-up gold chloride adding of the hydrogen peroxide not being degraded forms golden nanometer particle, because of the difference of residual quantities of hydrogen peroxide, can produce and obtain color difference or the different golden nanometer particle of the depth, and then, by color or the optical absorption intensity of observation nitrite ion, can realize the qualitative of micromolecular compound or quantitatively detect.
Compared with prior art, the present invention at least tool has the following advantages: should can realize the highly sensitive detection to contained micromolecular compound in sample by the direct competitive enzyme immunoassay method based on nm of gold plasmon, test result can adopt the conventional equipment such as naked eyes or microplate reader, spectrophotometer to detect, with low cost, easy operating, has made up the deficiency of existing detection micromolecular compound technology.
Brief description of the drawings
Fig. 1 is the fundamental diagram of a kind of direct competitive enzyme immunoassay method based on nm of gold plasmon in the present invention's one typical embodiments;
Fig. 2 is that in the embodiment of the present invention 1, concentration gradient is respectively 0 ng/mL, 0.03 ng/mL, 0.1 ng/mL, 0.3 ng/mL, 1 ng/mL, 3ng/mL, 10 ng/mL, the reaction solution figure of the methyltestosterone standard items of 30ng/mL;
Fig. 3 is that in the embodiment of the present invention 1, concentration gradient is respectively 0 ng/mL, 0.03 ng/mL, 0.1 ng/mL, 0.3 ng/mL, 1 ng/mL, 3ng/mL, 10 ng/mL, the reaction optical absorption intensity canonical plotting of the methyltestosterone standard items of 30ng/mL.
Embodiment
The present invention mainly provides a kind of direct competitive enzyme immunoassay method based on nm of gold plasmon, its technical scheme mainly comprises: coated micromolecular compound antibody (abbreviation insolubilized antibody) on the combination solid phase carrier that the haptens (hereinafter to be referred as enzyme-labelled antigen) of the test sample micromolecular compound that may contain and hydrogen peroxide enzyme labeling can be competed, the enzyme-labelled antigen decomposition of hydrogen peroxide of solid phase carrier combination, under certain buffer condition, hydrogen-peroxide reduction gold chloride is nm of gold (also claiming golden nanometer particle), by color or the optical absorption intensity of observation nitrite ion, realize the detection to contained micromolecular compound in test sample.
In a typical embodiments, a kind of direct competitive enzyme immunoassay method based on nm of gold plasmon can comprise the following steps:
(1) prepare Ban Kang Yuan – CAT enzyme-labelled antigen;
(2) the micromolecular compound antibody of solid phase carrier (for example microwell plate) is coated;
(3) competitive reaction;
(4) add hydrogen peroxide;
(5) colour developing.
Wherein, described micromolecular compound can be selected from but be not limited to methyltestosterone, Clenbuterol, gentamicin etc.
Consulting shown in Fig. 1 is the process principle figure of the direct competitive enzyme immunoassay method of a kind of typical case based on nm of gold plasmon in the present invention.
Further, among a typical case study on implementation, the method can comprise the steps:
(1) coated antibody
The sodium carbonate buffer of 0.05 M, pH 9.6 for micromolecular compound antibody prepared by immunity is diluted to respectively 2-10 μ g/mL, add in ELISA Plate, every hole adds 100 μ L, hatch 2h for 37 DEG C, use is added with the 0.01M of 0. 2% bovine serum albumin(BSA) (BSA), the phosphate buffer of pH7.4 seals as confining liquid;
(2) competitive reaction
By the enzyme-labelled antigen enzyme mark diluted with carbodiimide method, diazonium method, glutaraldehyde method coupling, be diluted to 5-20 μ g/mL, join in ELISA Plate, every hole adds 50 μ L, adds micromolecular compound standard items simultaneously, as concentration is respectively 0 ng/mL, 0.03 ng/mL, 0.1 ng/mL, 0.3 ng/mL, 1 ng/mL, 3ng/mL, 10 ng/mL, 30ng/mL, every hole adds 50 μ L, hatch 1h for 37 DEG C, then be placed on shaking table and wash 2 times with PBST cleansing solution, wash once with ultrapure water, each 3min;
Described cleansing solution PBST: be the 0.01M that contains 0.05% Tween-20, the phosphate buffer of pH7.4;
Described enzyme mark dilution: containing the PBST solution of 0.1% gelatin;
(3) add hydrogen peroxide
Adding concentration is the H of 400 μ M
2o
2, every hole adds 100 μ L, 25 DEG C of reaction 1h.
(4) colour developing
In the each hole of ELISA Plate in above-mentioned (3), add respectively 6 μ L 5mM gold chlorides, 6 μ L 2mM sodium citrates, 88 μ L MES buffer solution (2mM), vibration mixes, after reaction 15min, observe colour developing result, or detect the 540 optical absorption intensity (A of nm place
540).
As follows in conjunction with aforementioned typical case study on implementation brief description principle of work of the present invention:
Be coated with the antibody of same amount in each hole of ELISA Plate, add to be measured containing after micromolecular compound sample and enzyme-labelled antigen, the micromolecular compound in testing sample and enzyme-labelled antigen the vie each other coated antibody of limited solid phase surface combination with it.Because the antibody in each hole is all identical with the enzyme-labelled antigen content adding, so in the time that the contained micromolecular compound concentration of testing sample is high, the enzyme-labelled antigen of being combined with insolubilized antibody will reduce.When reaction reaches balance, supernatant is poured out, and wash with cleansing solution, therefore only have enzyme-labelled antigen and the little molecular targets thing of being combined with insolubilized antibody to be combined in ELISA Plate micropore, then add hydrogen peroxide, enzyme-labelled antigen decomposition of hydrogen peroxide in ELISA Plate, adds substrate (gold chloride, sodium citrate, MES damping fluid) again after reaction.In the time that testing concentration is high, reaction solution is darker, takes on a red color, and detects the light absorption value (OD value) at 540nm place by microplate reader, and light absorption value is higher; Otherwise in the time that testing concentration is low, reaction solution is more shallow, be light blue, the OD value surveyed is low.According to done typical curve, can extrapolate the concentration height of contained micromolecular compound in testing sample.
Below in conjunction with embodiment and accompanying drawing, technical scheme of the present invention is done to more detailed explaining.
embodiment 1the present embodiment relates to the detection method of methyltestosterone, comprises the steps:
(1) prepare methyltestosterone Yan Sheng Wu – CAT enzyme-labelled antigen
Methyltestosterone, by ethyloic azanol half hydrochloride oximate, is obtained to derivant 3-ethyloic oxime-methyltestosterone (3-CMO-MT), then adopt carbodiimide method by 3-CMO-MT and the coupling of CAT enzyme.Specific as follows:
Get 3-CMO-MT 2.5mg, NHS 1.5mg, EDCHCl 2.5mg, join in 1mL DMF, room temperature lucifuge is shaken 2 h.Separately get 27mg hydrogen peroxidase, be dissolved in 1.5mL containing in the PBS of 20% DMF liquid, 4 DEG C of precoolings.3-CMO-MT activating solution is dropwise joined in CAT solution, and constantly stir, 4 DEG C of reaction 2 h; After reaction finishes, reactant liquor is first dialysed with the PBS of 20% DMF, finally with 0.01M PBS dialysis 2 days ,-20 DEG C of freezing preservations.
(2) preparation of coated plate
Adopt the carbonate buffer solution of 0.05M pH 9.6 as coated diluted methyltestosterone antibody, antibody concentration is about 2-10 μ g/mL, then in each hole of coated plate, add 100 μ L coating buffers, 37 DEG C of baking ovens are placed 2h or 4 DEG C and are spent the night, with PBST cleansing solution washing 3min, on thieving paper, pat dry at every turn, repeat 3 times.Then add containing the phosphate buffer of 0.2%BSA and seal as confining liquid, 37 DEG C of baking oven incubation 2h or 4 DEG C spend the night, and take out and wash 3 times with above-mentioned cleansing solution.
(3) competitive reaction
Respectively by the methyltestosterone standard solution of a series of concentration gradients, as 0 ng/mL, 0.03 ng/mL, 0.1 ng/mL, 0.3 ng/mL, 1 ng/mL, 3 ng/mL, 10 ng/mL, 30ng/mL, or join respectively in coated plate micropore separately every hole 50 μ L through the sample solution of pre-treatment.Again by enzyme-labelled antigen dilution for methyltestosterone derivant-CAT enzyme-labelled antigen (containing 0.1% gelatin, containing the phosphate buffered solution of 0.05% Tween-20, pH 7.4, PBST) be diluted to respectively 5-20 μ g/mL, add in ELISA Plate, every hole adds 50 μ L, 37 DEG C of constant temperature ovens are hatched 1h, then with PBST cleansing solution washing 2 times, wash once 3min at every turn with ultrapure water.
(4) add hydrogen peroxide
Add hydrogen peroxide, be diluted to 400 μ M with 2mM pH 6.5 MES buffer solution, every hole adds 100 μ L, is placed in 25 DEG C of light tight constant temperature ovens and reacts 1h.
(5) colour developing
In the each hole of ELISA Plate in above-mentioned (4), add respectively 6 μ L 5mM gold chlorides, 6 μ L 2mM sodium citrates, 88 μ L 2mM MES buffer solution, on 25 DEG C of constant temperature oscillators, vibrate and mix, after reaction 15min, observe colour developing result and take pictures, or detecting the 540 optical absorption intensity (A of nm place
540).
Testing result shows, adopts the method for the present embodiment, the methyltestosterone (Fig. 2) of distinguishable 0.1 nM of naked eyes, and utilize spectrophotometer can reach 0.03 nM(Fig. 3 to the detectability of methyltestosterone).
embodiment 2the present embodiment relates to the detection method of Clenbuterol, comprises the steps:
(1) prepare Ke Lunteluo – CAT enzyme-labelled antigen
Adopt diazonium method by clenobuterol hydrochloride (CL) and the coupling of CAT enzyme, specific as follows:
With 0.1 mol/L HCl solution dissolving 2.5mg clenobuterol hydrochloride, be positioned in 4 DEG C of refrigerators and stir.After 20min, to the continuous NaNO that adds 0.2mol/L for three times in beaker
2solution, each 20uL, interval 25s.4 DEG C of stirring at low speed approximately 1 hour are colourless to solution.The whether excessive available starches potassium iodide starch paper test of sodium nitrite.The CAT that takes 39.4mg is dissolved in the borate buffer solution of 20mM.Under the condition of ice bath, the CL having activated is dropwise joined in CAT solution, in dropping process, to constantly stir and use 1mol/L NaOH to regulate pH value to maintain between 8.0 ~ 8.5.After being added dropwise to complete, stir lower reaction 6 hours in 4 DEG C, 300r/min.Finally with 0.01M PBS dialysis 2 days.After having dialysed, 4 DEG C, 5000rpm are centrifugal, and 10min removes precipitation, by supernatant packing be stored in-20 DEG C for subsequent use.
(2) preparation of coated plate
Employing concentration is that the carbonate buffer solution of 0.05M, pH 9.6 is as coated diluted antibody of clenbuteral, antibody concentration is about 2-10 μ g/mL, then in each hole of coated plate, add 100 μ L coating buffers, 37 DEG C of baking ovens are placed 2h or 4 DEG C and are spent the night, with PBST cleansing solution washing 3min, on thieving paper, pat dry at every turn, repeat 3 times.Then add containing the phosphate buffer of 0.2%BSA and seal as confining liquid, 37 DEG C of baking oven incubation 2h or 4 DEG C spend the night, and take out and wash 3 times with above-mentioned cleansing solution.
(3) competitive reaction
Respectively by the Clenbuterol standard solution of a series of concentration gradients, as 0 ng/mL, 0.03 ng/mL, 0.1 ng/mL, 0.3 ng/mL, 1 ng/mL, 3 ng/mL, 10 ng/mL, 30ng/mL, or join respectively in coated plate micropore separately every hole 50 μ L through the sample solution of pre-treatment.Again by enzyme-labelled antigen dilution for Clenbuterol-CAT enzyme-labelled antigen (containing 0.1% gelatin, containing the phosphate buffered solution of 0.05% Tween-20, pH 7.4, PBST) be diluted to respectively 5-20 μ g/mL, add in ELISA Plate, every hole adds 50 μ L, 37 DEG C of constant temperature ovens are hatched 1h, then with PBST cleansing solution washing 2 times, wash once 3min at every turn with ultrapure water.
(4) add hydrogen peroxide
Add hydrogen peroxide, be diluted to 400 μ M with 2mM pH 6.5 MES buffer solution, every hole adds 100 μ L, is placed in 25 DEG C of light tight constant temperature ovens and reacts 1h.
(5) colour developing
In the each hole of ELISA Plate in above-mentioned (4), add respectively 6 μ L 5mM gold chlorides, 6 μ L 2mM sodium citrates, 88 μ L 2mM MES buffer solution, on 25 DEG C of constant temperature oscillators, vibrate and mix, after reaction 15min, observe colour developing result and take pictures, or detecting the 540 optical absorption intensity (A of nm place
540).
Similar in test result and embodiment 1, prove that the sensitivity of the present embodiment method is far away higher than traditional enzyme-linked immune analytic method.
embodiment 3the present embodiment relates to the detection method of gentamicin, comprises the steps:
(1) prepare the large Mei Su of Qing – CAT enzyme-labelled antigen
Adopt glutaraldehyde method by gentamicin and the coupling of CAT enzyme.Specific as follows:
Get gentamicin 3mg 0.5 mL 0.01M PBS and dissolve, get 2.0% glutaraldehyde 0.1 mL and join in above-mentioned solution, priming reaction 30min.Get after CAT 38.7 mg 1.2mL 0.01M PBS dissolving, the solution having activated is dropwise joined in CAT solution, stirring reaction spends the night.Finally with 0.01M PBS dialysis 2 days.After having dialysed, 4 DEG C, 5000rpm are centrifugal, and 10min removes precipitation, by supernatant packing be stored in-20 DEG C for subsequent use.
(2) preparation of coated plate
Adopt the carbonate buffer solution of 0.05M pH 9.6 as coated diluted gentamicin antibody, antibody concentration is about 2-10 μ g/mL, then in each hole of coated plate, add 100 μ L coating buffers, 37 DEG C of baking ovens are placed 2h or 4 DEG C and are spent the night, with PBST cleansing solution washing 3min, on thieving paper, pat dry at every turn, repeat 3 times.Then add containing the phosphate buffer of 0.2%BSA and seal as confining liquid, 37 DEG C of baking oven incubation 2h or 4 DEG C spend the night, and take out and wash 3 times with above-mentioned cleansing solution.
(3) competitive reaction
Respectively by the gentamicin standard solution of a series of concentration gradients, as 0 ng/mL, 0.03 ng/mL, 0.1 ng/mL, 0.3 ng/mL, 1 ng/mL, 3 ng/mL, 10 ng/mL, 30ng/mL, or join respectively in coated plate micropore separately every hole 50 μ L through the sample solution of pre-treatment.Again by enzyme-labelled antigen dilution for gentamicin-CAT enzyme-labelled antigen (containing 0.1% gelatin, containing the phosphate buffered solution of 0.05% Tween-20, pH 7.4, PBST) be diluted to respectively 5-20 μ g/mL, add in ELISA Plate, every hole adds 50 μ L, 37 DEG C of constant temperature ovens are hatched 1h, then with PBST cleansing solution washing 2 times, wash once 3min at every turn with ultrapure water.
(4) add hydrogen peroxide
Add hydrogen peroxide, be diluted to 400 μ M with 2mM pH 6.5 MES buffer solution, every hole adds 100 μ L, is placed in 25 DEG C of light tight constant temperature ovens and reacts 1h.
(5) colour developing
In the each hole of ELISA Plate in above-mentioned (4), add respectively 6 μ L 5mM gold chlorides, 6 μ L 2mM sodium citrates, 88 μ L 2mM MES buffer solution, on 25 DEG C of constant temperature oscillators, vibrate and mix, after reaction 15min, observe colour developing result and take pictures, or detecting the 540 optical absorption intensity (A of nm place
540).
Similar in test result and embodiment 1, prove that the sensitivity of the present embodiment method is far away higher than traditional enzyme-linked immune analytic method.
Above embodiment is only for illustrating content of the present invention; in addition; the present invention also has other embodiments, as long as those skilled in the art enlighten because inventing related technology, and adopts the technical scheme that is equal to replacement or equivalents formation all to drop in protection scope of the present invention.
Claims (5)
1. the direct competitive enzyme immunoassay method based on nm of gold plasmon, is characterized in that comprising:
Test sample is mixed with the haptens of hydrogen peroxide enzyme labeling, and the micromolecular compound antibody competition of the haptens that makes the micromolecular compound that may contain in described test sample and hydrogen peroxide enzyme labeling on being coated on solid phase carrier be combined,
Remove the micromolecular compound of not being combined with described micromolecular compound antibody and the haptens of hydrogen peroxide enzyme labeling, and add hydrogen peroxide, gold chloride and in order to form the auxiliary reagent of selected buffer system, observe or detect again color or the optical absorption intensity of the hybrid reaction system forming, realize the detection to contained micromolecular compound in test sample;
Wherein, in described selected buffer system, the haptens that is incorporated into the described hydrogen peroxide enzyme labeling on described micromolecular compound antibody can decomposition of hydrogen peroxide, and hydrogen peroxide can reduce described gold chloride and forms nano Au particle.
2. the direct competitive enzyme immunoassay method based on nm of gold plasmon according to claim 1, is characterized in that specifically comprising the steps:
(1) antibody of micromolecular compound is dissolved in coated damping fluid, and is applied on solid phase carrier, hatch at 37 DEG C, then with confining liquid sealing, thereby micromolecular compound antibody is coated on solid phase carrier;
(2) haptens of hydrogen peroxide enzyme labeling and the test sample that may contain micromolecular compound are mixed in phosphate buffer, be applied on step (1) described solid phase carrier after treatment, hatch rear washing in 37 DEG C;
(3) on step (2) described solid phase carrier after treatment, applying hydrogen peroxide;
(4) to apply the nitrite ion that contains gold chloride on step (3) described solid phase carrier after treatment, after at room temperature fully reacting, observe or detect again color or the optical absorption intensity of the hybrid reaction system forming, realize the detection to contained micromolecular compound in test sample.
3. according to the direct competitive enzyme immunoassay method based on nm of gold plasmon described in claim 1 or 2, it is characterized in that comprising: by detecting the optical absorption intensity of described hybrid reaction system for visible ray, realize the detection to contained micromolecular compound in test sample, described visible light wavelength is 540 nm.
4. according to the direct competitive enzyme immunoassay method based on nm of gold plasmon described in claim 1 or 2, characterized by further comprising:
A, after the micromolecular compound standard model of getting variable concentrations mixes with the haptens of the hydrogen peroxide enzyme labeling of fixing consumption respectively, micromolecular compound antibody competition on being coated on solid phase carrier is combined, remove the micromolecular compound of not being combined with described micromolecular compound antibody and the haptens of hydrogen peroxide enzyme labeling, and add hydrogen peroxide, gold chloride and in order to form the auxiliary reagent of selected buffer system, observe again or detect color or the optical absorption intensity of final the formed hybrid reaction system of each micromolecular compound standard model, and then according to corresponding relation Criterion colorimetric card or typical curve between the concentration of described micromolecular compound standard model and color or the visible absorption intensity of final obtained corresponding hybrid reaction system,
B, after sample to be tested is mixed with the haptens of hydrogen peroxide enzyme labeling, micromolecular compound antibody competition on being coated on solid phase carrier is combined, remove the haptens of the hydrogen peroxide enzyme labeling of not being combined with described micromolecular compound antibody and the micromolecular compound that may exist, and add hydrogen peroxide, gold chloride and in order to form the auxiliary reagent of selected buffer system, observe again or detect color or the optical absorption intensity of final formed hybrid reaction system, and compare with described standard color comparison card or typical curve, and then qualitative or quantitatively record the concentration of contained micromolecular compound in sample to be tested.
5. according to the direct competitive enzyme immunoassay method based on nm of gold plasmon described in claim 1,2 or 4, it is characterized in that described micromolecular compound comprises methyltestosterone, Clenbuterol, gentamicin.
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| CN107607702A (en) * | 2017-07-21 | 2018-01-19 | 上海科华生物工程股份有限公司 | The preparation method and applications of sterols haptens alkaline phosphatase enzyme conjugate |
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| CN112798780A (en) * | 2019-11-13 | 2021-05-14 | 中粮集团有限公司 | Method for quantum dot-labeled direct competition fluoroimmunoassay detection of catalase |
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