CN106568949B - A kind of small haptens detection method based on direct competitive fluoroimmunoassay - Google Patents
A kind of small haptens detection method based on direct competitive fluoroimmunoassay Download PDFInfo
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Abstract
The present invention provides a kind of small haptens detection methods based on direct competitive fluoroimmunoassay, this method substitutes conventional zymophore using the fluorescent microsphere for being embedded with quantum dot and is coupled with small haptens, and direct competive ELISA is executed using coupled product as competition antigen.In the technical solution, fluorescent microsphere has embedded a large amount of quantum dot by polymer support, thus has higher luminous intensity, can effectively improve the sensitivity of detection.Further, since quantum dot is wrapped in the inside of microballoon, be affected by the external environment small, avoid to a certain extent fluorescence be quenched and the coagulation of microballoon.Simultaneously as quantum dot microsphere has relatively large grain size, therefore simplicity is isolated and purified compared with quantum dot, the slow-speed of revolution (<10000rpm) centrifugation can be achieved with the separation of the microballoon in conventional soln；Moreover, larger grain size can reduce affinity excessively high between competition antigen and coated antibody to a certain extent, to promote the sensitivity of detection.
Description
Technical field
The present invention relates to Antigen Detection Techniques fields, further to the improvement of direct competive ELISA method, and in particular to one
Small haptens detection method of the kind based on direct competitive fluoroimmunoassay.
Background technology
Immunoassay is the trace analysis based on specific recognition and invertibity association reaction between antigen and antibody
Method.Immunoassay is applicable not only to the detection of macromolecular compound (such as protein, nucleic acid, bacterium), is also applied for small molecule
Compound (such as mycotoxin, pesticide, veterinary drug, environmental hormone, violated food additives and physiological activity chemical substance)
It measures.Since immunoassay has, high specificity, high sensitivity, simple, quick, expense is low and is sieved suitable for live batch samples
The advantages that selecting, it is answered extensively in the analysis fields such as environmental monitoring, food safety detection, clinical diagnosis and bioanalysis
With.
There are one of form of the enzyme linked immunosorbent assay as immunoassay technical conditions to require low, easy to carry, operation
Easily and economically, term of validity length, high sensitivity, high specificity, can be achieved mass detection, often in the form of kit occur and
The advantages that being easily commercialized has become and is most widely used and develops the most ripe biological detection and analytical technology.Now,
Common enzyme linked immunosorbent assay includes mainly two major classes：One kind is Double antibody sandwich-ELISA, such method is
It is widely used in macromolecular antigen of the detection containing multiple antigenic determinants, such as albumen, microorganism and cell.Relative to tool
There are the macromolecular antigen of multiple antigenic determinants, micromolecular compound often only single because of its molecular weight smaller (being less than 6000)
Antigenic determinant can not be detected using Double antibody sandwich-ELISA.Competitive enzyme-linked immune absorption method is detection
One of most common method of small haptens, there are mainly two types of detection patterns：Competitive ELISA absorption method and indirectly
Competitive enzyme-linked immune absorption method.Wherein, competitive ELISA absorption method is because of its simple and quick inspection in small haptens
Important role is played in survey.However, there are two apparent disadvantages for traditional competitive ELISA absorption method tool：One,
Cause sensitivity relatively low as signal output using horseradish peroxidase or the colour developing of alkaline phosphatase catalytic chemistry substrate；Two,
Enzyme-labelled antigen or the relatively high antigen that constitutes competition of affinity of competition antigen and antibody are difficult to be competed by target analytes, to
Cause sensitivity relatively low.Therefore, improving the sensitivity of detection signal or reduce competition antigen can be effective to the affinity of antibody
Improve the sensitivity of traditional competitive ELISA absorption method in ground.
In traditional competitive ELISA absorption method, the preparation that competes antigen be by by small haptens with carry
Body protein (such as horseradish peroxidase, alkaline phosphatase) is coupled.Since the size of carrier protein is smaller, constitute competition antigen
It is higher with the affinity of corresponding antibodies, it can not be competed by object.Relative to carrier protein, nano particle has the ruler of bigger
Very little and weight, at that same temperature, Brownian movement is slower.It can be with as the carrier of small haptens using nano particle
Synthesize the lower competition antigen of affinity, to be easier to be competed by target analytes, and then it is sensitive to obtain higher detection
Degree.In addition, substituting albumen as the carrier of competition antigen using nano material, traditional chemical synthesis or biology are effectively evaded
The limitation of synthetic antigen analog, such as it is complicated for operation it is cumbersome, time-consuming and laborious and contingency is big.It is excellent based on above-mentioned technology
The antigen vectors of gesture, nano particle have received widespread attention, however in terms of the selection of carrier, both it is contemplated that molecular level
Coupling effect, while should also have the good characteristics of luminescence.In recent years, quantum dot is with its wide excitation, narrow transmitting, stoke
This displacement is widely applied with excellent optical characteristics such as resistance to photobleachings in immune analysis greatly, has researcher to attempt profit
Conventional carrier HRP, ALP are replaced with quantum dot, however finds that the combination effect of quantum dot and antigen is undesirable in practical study, phase
The luminescent properties answered are difficult to ensure, simultaneously because quantum dot nature is unstable, therefore it is easily affected by environment and fluorescence occurs
It is quenched, further, since on the one hand the grain size of quantum dot is still relatively small, therefore there is a problem of isolating and purifying inconvenience, it is another
Aspect can still result in the phenomenon that affinity is excessively high between competition antigen and antibody.
Invention content
The present invention is directed to the technological deficiencies for the prior art, provide a kind of based on the small of direct competitive fluoroimmunoassay
Molecule haptens detection method, in the direct competive ELISA method to solve the prior art due to chromogenic substrate luminescent properties are bad
Lead to the technical problem that detection sensitivity is relatively low.
Another technical problem to be solved by the present invention is that in the direct competive ELISA method of the prior art because competitive antigen with
The affinity of antibody is excessively high and causes detection sensitivity relatively low.
The invention solves another technical problem be to by quantum dot substitute zymophore direct competive ELISA method
In, the luminescent properties of quantum dot are difficult to be guaranteed.
The invention solves another technical problem be to by quantum dot substitute zymophore direct competive ELISA method
In, the chemical stability of quantum dot is relatively low.
The invention solves another technical problem be when using quantum dot microsphere as antigenic mark carrier to small molecule
When haptens executes enzyme linked immunosorbent assay detection, the biological activity for being labeled antigen declines.
The invention solves another technical problem be when using quantum dot microsphere as antigenic mark carrier to small molecule
When haptens executes enzyme linked immunosorbent assay detection, antigen is lived in antigen-quantum dot microsphere compound during long-term keep
Property decline.
To realize that the above technical purpose, the present invention use following technical scheme：
A kind of small haptens detection method based on direct competitive fluoroimmunoassay, this method belong to direct competitive
ELISA method, wherein the carrier being coupled with small haptens is the fluorescent microsphere for the quantum dot for being marked with carboxyl modified.
Preferably, this approach includes the following steps：
1) coated antibody on ELISA Plate；
2) fluorescent microsphere for the quantum dot for being marked with carboxyl modified is prepared；
3) step 2) products therefrom and small haptens are coupled to get to competition antigen；
4) solution to be measured and the competition antigen are added to step 1) and are coated in the ELISA Plate of antibody, antigen-antibody knot
Reaction is closed, the fluorescence intensity of ELISA Plate is then detected.
Preferably, the fluorescent microsphere for the quantum dot that the step 2) label has is to be prepared by the following method
's：Using chloroform as solvent, a concentration of 15~25mg/mL of quantum dot, a concentration of 25~35mg/ of PMMA of oleic acid moieties modification are prepared
The mixed solution of a concentration of 15~25mg/mL of mL, PMAO keeps 25~35min, will then take sodium dodecyl sulfate aqueous solution
A concentration of 3~the 4mg/mL of quantum dot modified to wherein oleic acid moieties is mixed with the mixed solution, goes to dechlorinate after mixing
Imitative, separation of solid and liquid takes solid phase to get to the fluorescent microsphere for the quantum dot for being marked with carboxyl modified.On this basis further preferably
's：Described be uniformly mixed can be realized under the conditions of ultrasonic vibration；The separation of solid and liquid can be realized by centrifuging；
Separation of solid and liquid can use milli-Q water after taking solid phase, and washing times can be that three times, the product after washing can be in ultra-pure water
In in 4 DEG C preservation.
Preferably, the quantum dot is CdSe/ZnS quantum dots.
Preferably, the excitation wavelength of the quantum dot is 450nm, launch wavelength 620nm.
Preferably, the removal of chloroform is realized using revolving.
Preferably, step 3) specifically includes following operation：It is marked with carboxyl modified using bovine serum albumin(BSA) coating
Product and small haptens are coupled to get to competition antigen by the fluorescent microsphere of quantum dot.
Preferably, the fluorescent microsphere of the quantum dot for being marked with carboxyl modified using bovine serum albumin(BSA) coating, packet
Include following steps：The fluorescent microsphere and 1- ethyls-(3- diformazans of the quantum dot of carboxyl modified will be marked in phosphate buffer
Base aminopropyl) phosphinylidyne diimine, bovine serum albumin(BSA) mixing, until the quality volume fraction of bovine serum albumin(BSA) is in solution
0.8%~1.2%, 25~35min is reacted in 35~39 DEG C, then adds 1- ethyls-(3- dimethylaminopropyls) phosphinylidyne two
Imines 3~5 times reacts 25~35min after adding every time in 35~39 DEG C, adds separation of solid and liquid after being fully completed and takes solid phase, washes
It is dissolved in sodium bicarbonate solution after washing.In above optimal technical scheme, in order to obtain high repeatability, bovine serum albumin
Use saturation flags in vain.It is further preferred on this basis：The number added can be 4 times；The phosphate buffer
Initial concentration can be 0.04~0.06mol/L, pH can be 5.8~6.2；The 1- ethyls-(3- bis- added is repeated every time
Dimethylaminopropyl) amount of phosphinylidyne diimine can be equal；Milli-Q water can be used after taking solid phase, washing times can be three
Secondary (washing step is for removing extra BSA)；The pH of the sodium bicarbonate solution can be 8.2~8.6；It is dissolved in carbonic acid
It can be in 4 DEG C of preservations after in hydrogen sodium solution.
Preferably, the separation of solid and liquid in step 3) is to centrifuge 8~12min with the rotating speed of 12000~15000rpm.
Preferably, the coupling being marked between the fluorescent microsphere and small haptens of the quantum dot of carboxyl modified is profit
It is realized with active ester method.
Preferably, marking the fluorescent microsphere for the quantum dot having and small haptens even through BSA is coated
The coupling of connection is realized using active ester method.
It, can be anti-by controlling small molecule half to obtain the competitive antigen of different affinity in above technical scheme
The former molar ratio with the bovine serum albumin(BSA) on the coated quantum dot microsphere of bovine serum albumin(BSA) realizes, specific dosage and operation
Condition can carry out adaptability selection according to the general technology common sense of active ester method；Certainly, small haptens and bovine serum albumin
Specifically label mole can be than being respectively 5 between bovine serum albumin(BSA) on white coated quantum dot microsphere:1、1:1、1:5、1:
10、1:20, preferably 1:10.
Preferably, the condition of the antigen-antibody reaction is 25~35min of reaction at 35~39 DEG C.
Preferably, the addition of competitive antigen and sample to be tested is 40~60 holes μ L/ in step 4)；More optimizedly
50 holes μ L/.
Preferably, after antigen-antibody reaction, first with the 0.01M phosphate buffer detersive enzymes containing 0.05% polysorbas20
Target three times, then with the phosphate buffer of 0.01M washs ELISA Plate 1 time, then fluorescence intensity.
In above technical scheme, the fluorescence intensity detected is i.e. for reacting sample to be tested small molecular haptens
Content passes through above method using the small haptens standard solution of known concentration and distribution gradient in practical operation
Drawing percentage fluorescence rate, (percentage fluorescence rate (%)=F/F0 × 100%, wherein F0 are that the fluorescence of first standard (0 standard) is strong
Angle value, F are the average value of the fluorescence intensity level of standard items or sample) --- the standard curve of small haptens concentration, then profit
Corresponding percentage fluorescence rate is calculated with the fluorescence intensity of sample to be tested, small point of sample to be tested is then calculated from standard curve
Sub- haptens content.Concrete operation method can carry out adaptability selection according to the general technology common sense of the art.Above-mentioned ladder
The titer for spending the small haptens of distribution, can select 1.5 respectively, 1,0.75,0.4,0.2,0.1,0.05,0pg/mL.
In above technical scheme, each reagent can be reused before use prior to equilibrium at room temperature 30min or more.It is described
PMAO is maleic anhydride/1- octadecene alternate copolymers, and the PMMA is polymethyl methacrylate, and the two can be from market
It buys.The quantum dot of the oleic acid moieties modification can be prepared according to the ordinary skill in the art.
The present invention is suitable for the quantitative detection of small haptens, such as mycotoxin, pesticide, veterinary drug, environmental hormone, violated
Food additives and chemical substance etc. with physiological activity, are especially suitable for the trace detection of target analytes.Sample
Processing is according to conventional treatment method.
It is had the advantages that using technical solution of the present invention：
1, the present invention is embedded single fluorescence quantum into polymer microballoon by microemulsion method, has been prepared and has been shone
The higher fluorescence quantum microballoon of intensity, the more corresponding quantum dot of luminous intensity improve 2800 times；Further, since quantum dot
It is wrapped in the inside of microballoon, small by external environment (solvent, heat, electricity, magnetic etc.) influence, property is more steady under the protection of shell structure
It is fixed, avoid to a certain extent fluorescence be quenched and the coagulation of microballoon.Meanwhile the grain size of quantum dot microsphere is about tens to arrive
Hundreds of nanometers, isolate and purify compared with quantum dot simplicity, the slow-speed of revolution (<10000rpm) centrifugation can be achieved with the microballoon in conventional soln point
From.
2, the method for the present invention prepares high luminous quantum dot fluorescence microballoon by using oil-soluble quantum dot and is used to replace to measure
Son point is directly used in conventional immunological fluorescent marker, greatly increases the intensity of fluorescence signal output, is conducive to improve detection
Sensitivity.
3, the method for the present invention substitutes small albumen particle as small molecule half by using the quantum dot microsphere of grain size bigger
The coupling carrier of antigen can obtain the competition antigen of different affinity, and affinity variation range is wider, help to improve straight
Connect the detection sensitivity of competition immune analysis.
4, the technology of the present invention substitutes the chemical colour reaction signal of traditional enzymatic by using the luminous quantum dot microsphere of height, subtracts
The step of having lacked enzymatic, therefore operation is simpler, detection time is shorter.
The present invention provides a kind of small haptens detection method based on direct competitive fluoroimmunoassay, this method
Conventional zymophore is substituted using the fluorescent microsphere (Quantum dot beads, QBs) for being embedded with quantum dot and small molecule half is anti-
Original coupling executes direct competive ELISA using coupled product as competition antigen.In the technical solution, fluorescent microsphere is poly- by height
Object carrier has embedded a large amount of quantum dot, thus has higher luminous intensity, can effectively improve the sensitivity of detection.This
Outside, small by external environment (solvent, heat, electricity, magnetic etc.) influence since quantum dot is wrapped in the inside of microballoon, in the guarantor of shell structure
The lower property of shield is more stable, avoid to a certain extent fluorescence be quenched and the coagulation of microballoon.Meanwhile the grain of quantum dot microsphere
Diameter is about tens to hundreds of nanometers, isolate and purify compared with quantum dot simplicity, the slow-speed of revolution (<10000rpm) centrifugation can be achieved with conventional molten
Microballoon separation in liquid；Moreover, because fluorescent microsphere has larger grain size, therefore competition antigen can be reduced to a certain extent
The excessively high affinity between coated antibody, to promote the sensitivity of detection.
Description of the drawings
Fig. 1 is the principle schematic of the method for the present invention；
Fig. 2 is the standard curve that the fluorescence immunoassay credit of Aflatoxins M1 direct competitive is analysed in the embodiment of the present invention 1；
Fig. 3 is the standard curve that the fluorescence immunoassay credit of parathion direct competitive is analysed in the embodiment of the present invention 1；
Fig. 4 is the standard curve that the fluorescence immunoassay credit of Enrofloxacin direct competitive is analysed in the embodiment of the present invention 1；
Fig. 5 is the standard curve that the fluorescence immunoassay credit of 19- nortestosterones direct competitive is analysed in the embodiment of the present invention 1；
Fig. 6 is the standard curve that the fluorescence immunoassay credit of melamine direct competitive is analysed in the embodiment of the present invention 1；
Fig. 7 is that the standard that the fluorescence immunoassay credit of 1,25- dihydroxyvitamin Ds direct competitive is analysed in the embodiment of the present invention 1 is bent
Line.
Specific implementation mode
The specific implementation mode of the present invention will be described in detail below.In order to avoid excessive unnecessary details,
It will not be described in detail in following embodiment to belonging to well known structure or function.
Approximating language used in following embodiment can be used for quantitative expression, show in the feelings for not changing basic function
Quantity is allowed to have certain variation under condition.Therefore, it is not limited to this accurately with the modified numerical value of the language such as " about ", " left and right " institute
Numerical value itself.In some embodiments, the range for allowing its modified numerical value in positive and negative 10 (10%) " about " is indicated
Interior variation, for example, what " about 100 " indicated can be any numerical value between 90 to 110.In addition, " the about first numerical value arrives
In the statement of second value ", the first and second numerical value two values are at about corrected.In some cases, approximating language
It may be related with the precision of measuring instrument.
In addition to being defined, technical and scientific term used in following embodiment has and fields technology people of the present invention
The identical meanings that member is commonly understood by.
Test reagent consumptive material used in following embodiment is unless otherwise specified conventional biochemical reagent；The experiment
Method is unless otherwise specified conventional method；Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result
It is averaged；% in following embodiment is unless otherwise instructed mass percentage.
In following tests, the configuration method of phosphate buffer (PBS, 0.05M, pH 7.4) is as follows：NaCl 40g,
Na2HPO413.5g KH2PO41.0g, KCl 1.0g are dissolved in 1L ultra-pure waters.With 0.1M NaOH tune pH value to 8.0~9.0.
Involved mouse IgG class monoclonal antibodies in experiment：Aspergillus flavus resisting toxin M1 monoclonal antibodies, anti-parathion
Monoclonal antibody, anti-enrofloxacin monoclonal antibody, anti-19- nortestosterones monoclonal antibody, anti-melamine monoclonal antibody
And anti-1,25- dihydroxyvitamin D monoclonal antibodies, it is provided by Wuxi Zhongde Bore Bioisystech Co., Ltd, this experiment
Involved all small haptens are purchased from Sigma companies.
Embodiment 1
1, the preparation of the quantum dot fluorescence microballoon of carboxyl modified
Quantum dot after the modification of 10mg oleic acid moieties (excitation wavelength of the quantum dot is 450nm, launch wavelength 620nm)
It is dissolved in the chloroform of 0.5mL, adds the polymethyl methacrylate (PMMA) and 10mg maleic anhydrides/18 carbon of 1- of 15mg
Alkene alternate copolymer (PMAO), the mixed liquor is re-dissolved in the sodium dodecyl sulfate aqueous solution of 2.5mL most after half an hour
Final concentration about 3.3mg/mL, mixed liquor mixing under ultrasound condition remove this non-pole of chloroform after mixing with the method for revolving
The quantum dot fluorescence microballoon of water-soluble carboxyl modified is obtained after property organic solvent by way of centrifugation by quantum dot fluorescence microballoon
Separate, the quantum dot fluorescence microballoon after separation with milli-Q water three times.Quantum dot fluorescence microballoon after washing is again molten
Solution 4 DEG C of preservations in ultra-pure water.
2, the preparation of the coated quantum dot microsphere of bovine serum albumin(BSA)
The quantum dot fluorescence microballoon of water-soluble carboxyl modified is dissolved in pH 6.0, in 0.05mol/L phosphate buffers,
After suitable 1- ethyls-(3- dimethylaminopropyls) phosphinylidyne diimine is added, the cow's serum that quality volume fraction is 1% is added
Albumin, 37 DEG C are reacted half an hour, and 1- ethyls-(3- dimethylaminopropyls) phosphinylidyne two that equivalent is continuously added after half an hour is sub-
Amine, repetition are added four times, which is saturated coated quantum dot fluorescence microballoon and centrifuges 10min at 14000rpm,
Extra seralbumin is removed three times with milli-Q water after removing supernatant, and the quantum dot fluorescence microballoon after centrifugation is redissolved in pH
In 8.4 sodium bicarbonate solution, 4 DEG C of preservations.
3, detection result verification test
This section executes this hair using the prepared coated quantum dot microsphere of bovine serum albumin(BSA) of the 1st, 2 sections as experimental raw
Bright method, to verify this method to small-molecule substances such as mycotoxin, pesticide, veterinary drug, environmental hormone, violated food additives
Detection result.
The method of the present invention by following steps for when detecting small haptens content, implementing sample pre-treatments, then
It is detected with detection method, analysis result.
1) sample pre-treatments：The small haptens standard items bought are diluted to corresponding concentration gradient, concentration according to
Required actually detected limit determines；It is spare that the antigen sample diluted is put into 4 DEG C of refrigerators.Target analysis in different samples to be checked
The purification process of object is completed referring in particular to national standard.
2) mycotoxin, pesticide, veterinary drug, environmental hormone, violated food additives are detected with detection method
And the content of chemical substances with physiological activity.
3) analysis result.
With the standard items 1.5 of 8 various concentrations of above-mentioned preparation, 1,0.75,0.4,0.2,0.1,0.05,0pg/mL.
Excitation wavelength is 450nm, and launch wavelength is fluorescence intensity at 620nm.
The percentage fluorescence rate of the calculating of percentage fluorescence rate, standard items or sample is equal to the fluorescence intensity level of standard items or sample
Average value remove in first standard (0 standard), i.e. percentage fluorescence rate (%)=F/F0× 100%, wherein F0For first standard
The fluorescence intensity level of (0 standard), F are the average value (diplopore) of the fluorescence intensity level of standard items or sample.
Using percentage fluorescence rate as ordinate, it is that abscissa draws standard curve with small haptens concentration (g/mL), asks
Linear equation.When carrying out actual sample detection, by the fluorescent value (F/F of sample0× 100%) value substitutes into standard curve,
The concentration of corresponding sample is read from standard curve, it is sample small molecular haptens to be multiplied by its corresponding extension rate
Actual concentrations.
3.1 pairs of aflatoxin Ms1Test experience
The Protein G of 20 μ g/mL is added per 100 μ L of hole in ELISA Plate, and 4 DEG C overnight, with containing 0.05% polysorbas20
The PBS washing ELISA Plates of 0.01M washed once with the PBS of 0.01M afterwards three times, and the aspergillus flavus resisting toxin M of 0.1 μ g/mL is added1It is single
100 μ L of clonal antibody are per hole, and the PBS washings ELISA Plate of 37 DEG C of incubations 120min, the 0.01M containing 0.05% polysorbas20 are three times
It washed once afterwards with the PBS of 0.01M, the 340 μ L of bovine serum albumin(BSA) of 10mg/mL be added per hole, 37 DEG C of incubation 120min have
The PBS washing ELISA Plates of the 0.01M of 0.05% polysorbas20 washed once with the PBS of 0.01M afterwards three times, and various concentration is added
Aflatoxin M1, it is added in ELISA Plate per 50 μ L of hole, 50 μ L aflatoxin Ms is added1It is coated to be coupled bovine serum albumin(BSA)
Quantum dot microsphere (competition antigen), after 37 DEG C are incubated 40min, is washed with the 0.01M phosphate buffers containing 0.05% polysorbas20
ELISA Plate washs ELISA Plate 1 time with the phosphate buffer of 0.01M afterwards three times, is surveyed with SpectraMax i3x multi-function microplate readers
Determine ELISA Plate fluorescence intensity.Solution to be checked is added to per 50 μ L of hole in the ELISA Plate for being coated with antibody, while it is competing that 50 μ L are added
Antigen is striven, 37 DEG C are incubated the fluorescence intensity measured after forty minutes per hole, by being updated to standard curve acquisition after calculating average value
Aflatoxin M in measuring samples1Concentration.Specific experiment result is as follows：Linear standard curve be y=-25.92ln (x)+
17.158 R2=0.9984, see attached drawing 2.By standard curve calculate this method half-inhibition concentration IC50(i.e. F/F0×
100%=50%) it is 0.28pg/mL.
Above method is not limited to aflatoxin M1Detection, can be also used for the detection of other mycotoxins, Huang Qu
Mould toxin B1/B2/G1/G2, ochratoxin, vomitoxin, zearalenone, fumonisins, T2 toxin etc..
The test experience of 3.2 pairs of parathion
The Protein G of 20 μ g/mL is added per 100 μ L of hole in ELISA Plate, and 4 DEG C overnight, with containing 0.05% polysorbas20
The PBS washing ELISA Plates of 0.01M washed once with the PBS of 0.01M afterwards three times, and the anti-parathion monoclonal that 0.1 μ g/mL are added is anti-
100 μ L of body per hole, use afterwards three times by the PBS washing ELISA Plates of 37 DEG C of incubations 120min, the 0.01M containing 0.05% polysorbas20
The PBS of 0.01M washed once, and the 340 μ L of bovine serum albumin(BSA) of 10mg/mL be added per hole, 37 DEG C of incubation 120min have 0.05%
Polysorbas20 0.01M PBS washing ELISA Plate washed once afterwards with the PBS of 0.01M three times, be added various concentration to sulphur
Phosphorus is added to per 50 μ L of hole in ELISA Plate, and the coated quantum dot microsphere (competition of 50 μ L parathion coupling bovine serum albumin(BSA) is added
Antigen), after 37 DEG C are incubated 40min, wash ELISA Plate with the 0.01M phosphate buffers containing 0.05% polysorbas20 and use afterwards three times
The phosphate buffer washing ELISA Plate of 0.01M 1 time, it is strong to measure ELISA Plate fluorescence with SpectraMax i3x multi-function microplate readers
Degree.Solution to be checked is added to per 50 μ L of hole in the ELISA Plate for being coated with antibody, while 50 μ L are added and compete antigen, 37 DEG C of incubations
The fluorescence intensity per hole is measured after forty minutes, by being updated to parathion in standard curve acquisition measuring samples after calculating average value
Concentration.Specific experiment result is as follows：Linear standard curve is y=-24.94ln (x)+15.374, R2=0.9965, see attached drawing 3.
By standard curve calculate this method half-inhibition concentration IC50(i.e. F/F0× 100%=50%) it is 0.25pg/mL.
Above method is not limited to the detection of parathion, can be also used for the detection of other kinds of pesticide, such as other
Organophosphor or organo-chlorine pesticide, carbamate chemicals for agriculture etc..
The test experience of 3.3 pairs of Enrofloxacins
The Protein G of 20 μ g/mL is added per 100 μ L of hole in ELISA Plate, and 4 DEG C overnight, with containing 0.05% polysorbas20
The PBS washing ELISA Plates of 0.01M washed once with the PBS of 0.01M afterwards three times, and the anti-enrofloxacin monoclonal of 0.1 μ g/mL is added
100 μ L of antibody per hole, use afterwards three times by the PBS washing ELISA Plates of 37 DEG C of incubations 120min, the 0.01M containing 0.05% polysorbas20
The PBS of 0.01M washed once, and the 340 μ L of bovine serum albumin(BSA) of 10mg/mL be added per hole, 37 DEG C of incubation 120min have 0.05%
Polysorbas20 0.01M PBS washing ELISA Plate washed once afterwards with the PBS of 0.01M three times, the En Nuosha of various concentration is added
Star is added to per 50 μ L of hole in ELISA Plate, and it is (competing that the 50 μ L Enrofloxacins coupling coated quantum dot microsphere of bovine serum albumin(BSA) is added
Strive antigen), after 37 DEG C are incubated 40min, wash ELISA Plate with the 0.01M phosphate buffers containing 0.05% polysorbas20 and use afterwards three times
The phosphate buffer washing ELISA Plate of 0.01M 1 time, it is strong to measure ELISA Plate fluorescence with SpectraMax i3x multi-function microplate readers
Degree.Solution to be checked is added to per 50 μ L of hole in the ELISA Plate for being coated with antibody, while 50 μ L are added and compete antigen, 37 DEG C of incubations
The fluorescence intensity per hole is measured after forty minutes, by being updated to En Nuosha in standard curve acquisition measuring samples after calculating average value
Star concentration.Specific experiment result is as follows：Linear standard curve is y=-21.94ln (x)+20.74, R2=0.9905, see attached drawing
4.By standard curve calculate this method half-inhibition concentration IC50(i.e. F/F0× 100%=50%) it is 0.26pg/mL.
Above method is not limited to the detection of Enrofloxacin, the detection of other kinds of veterinary drug is can be also used for, such as other
Fluoroquinolones, sulfamido, beta receptor excitant and tetracycline medication etc.
The test experience of 3.4 pairs of 19- nortestosterones
The Protein G of 20 μ g/mL is added per 100 μ L of hole in ELISA Plate, and 4 DEG C overnight, with containing 0.05% polysorbas20
The PBS washing ELISA Plates of 0.01M washed once with the PBS of 0.01M afterwards three times, and the anti-19- nortestosterones list of 0.1 μ g/mL is added
100 μ L of clonal antibody are per hole, and the PBS washings ELISA Plate of 37 DEG C of incubations 120min, the 0.01M containing 0.05% polysorbas20 are three times
It washed once afterwards with the PBS of 0.01M, the 340 μ L of bovine serum albumin(BSA) of 10mg/mL be added per hole, 37 DEG C of incubation 120min have
The PBS washing ELISA Plates of the 0.01M of 0.05% polysorbas20 washed once with the PBS of 0.01M afterwards three times, and various concentration is added
19- nortestosterones are added to per 50 μ L of hole in ELISA Plate, and it is coated that 50 μ L19- nortestosterones coupling bovine serum albumin(BSA) is added
Quantum dot microsphere (competition antigen), after 37 DEG C are incubated 40min, is washed with the 0.01M phosphate buffers containing 0.05% polysorbas20
ELISA Plate washs ELISA Plate 1 time with the phosphate buffer of 0.01M afterwards three times, is surveyed with SpectraMax i3x multi-function microplate readers
Determine ELISA Plate fluorescence intensity.Solution to be checked is added to per 50 μ L of hole in the ELISA Plate for being coated with antibody, while it is competing that 50 μ L are added
Antigen is striven, 37 DEG C are incubated the fluorescence intensity measured after forty minutes per hole, by being updated to standard curve acquisition after calculating average value
19- nortestosterones concentration in measuring samples.Specific experiment result is as follows：Linear standard curve be y=-28.39ln (x)+
20.294 R2=0.9802, see attached drawing 5.By standard curve calculate this method half-inhibition concentration IC50(i.e. F/F0×
100%=50%) it is 0.35pg/mL.
Above method is not limited to the detection of 19- nortestosterones, can be also used for the detection of other kinds of environmental hormone,
Such as sex hormone (progesterone, testosterone), steroid derivatives.
The test experience of 3.5 pairs of melamines
The Protein G of 20 μ g/mL is added per 100 μ L of hole in ELISA Plate, and 4 DEG C overnight, with containing 0.05% polysorbas20
The PBS washing ELISA Plates of 0.01M washed once with the PBS of 0.01M afterwards three times, and the anti-melamine monoclonal of 0.1 μ g/mL is added
100 μ L of antibody per hole, use afterwards three times by the PBS washing ELISA Plates of 37 DEG C of incubations 120min, the 0.01M containing 0.05% polysorbas20
The PBS of 0.01M washed once, and the 340 μ L of bovine serum albumin(BSA) of 10mg/mL be added per hole, 37 DEG C of incubation 120min have 0.05%
Polysorbas20 0.01M PBS washing ELISA Plate washed once afterwards with the PBS of 0.01M three times, the melamine of various concentration is added
Amine is added to per 50 μ L of hole in ELISA Plate, and it is (competing that the 50 μ L melamines coupling coated quantum dot microsphere of bovine serum albumin(BSA) is added
Strive antigen), after 37 DEG C are incubated 40min, wash ELISA Plate with the 0.01M phosphate buffers containing 0.05% polysorbas20 and use afterwards three times
The phosphate buffer washing ELISA Plate of 0.01M 1 time, it is strong to measure ELISA Plate fluorescence with SpectraMax i3x multi-function microplate readers
Degree.Solution to be checked is added to per 50 μ L of hole in the ELISA Plate for being coated with antibody, while 50 μ L are added and compete antigen, 37 DEG C of incubations
The fluorescence intensity per hole is measured after forty minutes, by being updated to melamine in standard curve acquisition measuring samples after calculating average value
Amine concentration.Specific experiment result is as follows：Linear standard curve is y=-17.11ln (x)+42.003, R2=0.9777, see attached drawing
6.By standard curve calculate this method half-inhibition concentration IC50(i.e. F/F0× 100%=50%) it is 0.63pg/mL.
Above method is not limited to the detection of melamine, can be also used for the detection of other violated food additives.3.6
To the test experience of 1,5- dihydroxyvitamin Ds
The Protein G of 20 μ g/mL is added per 100 μ L of hole in ELISA Plate, and 4 DEG C overnight, with containing 0.05% polysorbas20
The PBS washing ELISA Plates of 0.01M washed once with the PBS of 0.01M afterwards three times, and anti-1, the 25- dihydroxy dimension of 0.1 μ g/mL is added
Raw 100 μ L of element D monoclonal antibodies are per hole, 37 DEG C of incubation 120min, the PBS detersive enzymes of the 0.01M containing 0.05% polysorbas20
Target washed once with the PBS of 0.01M afterwards three times, the 340 μ L of bovine serum albumin(BSA) of 10mg/mL be added per hole, 37 DEG C are incubated
120min has the PBS washing ELISA Plates of the 0.01M of 0.05% polysorbas20 to washed once afterwards with the PBS of 0.01M three times, is added
1, the 25- dihydroxyvitamin Ds of various concentration are added to per 50 μ L of hole in ELISA Plate, and 50 μ L1,25- dihydroxyvitamins are added
D is coupled the coated quantum dot microsphere of bovine serum albumin(BSA) (competition antigen), after 37 DEG C are incubated 40min, with containing 0.05% polysorbas20
0.01M phosphate buffers washing ELISA Plate wash ELISA Plate 1 time with the phosphate buffer of 0.01M afterwards three times, use
SpectraMax i3x multi-function microplate readers measure ELISA Plate fluorescence intensity.By solution to be checked per 50 μ L of hole be added to be coated with it is anti-
In the ELISA Plate of body, while 50 μ L are added and compete antigen, 37 DEG C are incubated the fluorescence intensity measured after forty minutes per hole, pass through calculating
It is updated to standard curve after average value and obtains 1,25- dihydroxyvitamin D concentration in measuring samples.Specific experiment result is as follows：
Linear standard curve is y=-24.3ln (x)+26.373, R2=0.9844, see attached drawing 7.The party is calculated to obtain by standard curve
The half-inhibition concentration IC of method50(i.e. F/F0× 100%=50%) it is 0.38pg/mL.
Above method is not limited to the detection of 1,5- dihydroxyvitamin Ds, can be also used for other kinds of physiological activity
The detection of chemical substance.
Embodiment 2
A kind of small haptens detection method based on direct competitive fluoroimmunoassay, this method include following step
Suddenly：
1) coated antibody on ELISA Plate；
2) fluorescent microsphere for the quantum dot for being marked with carboxyl modified is prepared；
3) step 2) products therefrom and small haptens are coupled to get to competition antigen；
4) solution to be measured and the competition antigen are added to step 1) and are coated in the ELISA Plate of antibody, antigen-antibody knot
Reaction is closed, the fluorescence intensity of ELISA Plate is then detected.
On the basis of above technical scheme, meet the following conditions：
Step 2) the fluorescent microsphere for marking the quantum dot having is prepared by the following method：With chloroform
For solvent, quantum dot a concentration of 15mg/mL, PMMA a concentration of 25mg/mL, the PMAO for preparing oleic acid moieties modification are a concentration of
The mixed solution of 15mg/mL, keep 25min, will then take sodium dodecyl sulfate aqueous solution mixed with the mixed solution to
The wherein a concentration of 3mg/mL of quantum dot of oleic acid moieties modification, removes chloroform after mixing, separation of solid and liquid take solid phase to get to
It is marked with the fluorescent microsphere of the quantum dot of carboxyl modified.
The quantum dot is CdSe/ZnS quantum dots.
The excitation wavelength of the quantum dot is 450nm, launch wavelength 620nm.
The removal of chloroform is realized using revolving.
Step 3) specifically includes following operation：The glimmering of the quantum dot of carboxyl modified is marked with using bovine serum albumin(BSA) coating
Product and small haptens are coupled to get to competition antigen by light microballoon.
The fluorescent microsphere of the quantum dot that carboxyl modified is marked with using bovine serum albumin(BSA) coating, including following step
Suddenly：The fluorescent microsphere and 1- ethyls-(3- dimethylaminos third of the quantum dot of carboxyl modified will be marked in phosphate buffer
Base) phosphinylidyne diimine, bovine serum albumin(BSA) mixing, until in solution bovine serum albumin(BSA) quality volume fraction be 0.8%%, in
35 DEG C of reaction 25min, then add 1- ethyls-(3- dimethylaminopropyls) phosphinylidyne diimine 3 times, in 35 DEG C after adding every time
25min is reacted, separation of solid and liquid after being fully completed is added and takes solid phase, be dissolved in sodium bicarbonate solution after washing.
The separation of solid and liquid is to centrifuge 8min with the rotating speed of 12000rpm.
The condition of the antigen-antibody reaction is reaction 25min at 35 DEG C.
Embodiment 3
A kind of small haptens detection method based on direct competitive fluoroimmunoassay, this method include following step
Suddenly：
1) coated antibody on ELISA Plate；
2) fluorescent microsphere for the quantum dot for being marked with carboxyl modified is prepared；
3) step 2) products therefrom and small haptens are coupled to get to competition antigen；
4) solution to be measured and the competition antigen are added to step 1) and are coated in the ELISA Plate of antibody, antigen-antibody knot
Reaction is closed, the fluorescence intensity of ELISA Plate is then detected.
On the basis of above technical scheme, meet the following conditions：
Step 2) the fluorescent microsphere for marking the quantum dot having is prepared by the following method：With chloroform
For solvent, quantum dot a concentration of 25mg/mL, PMMA a concentration of 35mg/mL, the PMAO for preparing oleic acid moieties modification are a concentration of
The mixed solution of 25mg/mL, keep 35min, will then take sodium dodecyl sulfate aqueous solution mixed with the mixed solution to
The wherein a concentration of 4mg/mL of quantum dot of oleic acid moieties modification, removes chloroform after mixing, separation of solid and liquid take solid phase to get to
It is marked with the fluorescent microsphere of the quantum dot of carboxyl modified.
The removal of chloroform is realized using revolving.
Step 3) specifically includes following operation：The glimmering of the quantum dot of carboxyl modified is marked with using bovine serum albumin(BSA) coating
Product and small haptens are coupled to get to competition antigen by light microballoon.
The fluorescent microsphere of the quantum dot that carboxyl modified is marked with using bovine serum albumin(BSA) coating, including following step
Suddenly：The fluorescent microsphere and 1- ethyls-(3- dimethylaminos third of the quantum dot of carboxyl modified will be marked in phosphate buffer
Base) phosphinylidyne diimine, bovine serum albumin(BSA) mixing, until in solution bovine serum albumin(BSA) quality volume fraction be 1.2%, in 39
DEG C reaction 35min, then adds 1- ethyls-(3- dimethylaminopropyls) phosphinylidyne diimine 5 times, anti-in 39 DEG C after adding every time
35min is answered, separation of solid and liquid after being fully completed is added and takes solid phase, be dissolved in sodium bicarbonate solution after washing.
The separation of solid and liquid is to centrifuge 12min with the rotating speed of 15000rpm.
The condition of the antigen-antibody reaction is reaction 35min at 39 DEG C.
Embodiment 4
A kind of small haptens detection method based on direct competitive fluoroimmunoassay, this method include following step
Suddenly：
1) coated antibody on ELISA Plate；
2) fluorescent microsphere for the quantum dot for being marked with carboxyl modified is prepared；
3) step 2) products therefrom and small haptens are coupled to get to competition antigen；
4) solution to be measured and the competition antigen are added to step 1) and are coated in the ELISA Plate of antibody, antigen-antibody knot
Reaction is closed, the fluorescence intensity of ELISA Plate is then detected.
On the basis of above technical scheme, meet the following conditions：
The quantum dot is CdSe/ZnS quantum dots.
The condition of the antigen-antibody reaction is reaction 35min at 39 DEG C.
Step 3) specifically includes following operation：The glimmering of the quantum dot of carboxyl modified is marked with using bovine serum albumin(BSA) coating
Product and small haptens are coupled to get to competition antigen by light microballoon.
Embodiment 5
A kind of small haptens detection method based on direct competitive fluoroimmunoassay, this method belong to direct competitive
ELISA method, wherein the carrier being coupled with small haptens is the fluorescent microsphere for the quantum dot for being marked with carboxyl modified.
The embodiments of the present invention have been described in detail above, but content is only the preferred embodiment of the present invention,
It is not intended to limit the invention.All all any modification, equivalent and improvement etc. done in the application range of the present invention, should all
It is included within protection scope of the present invention.
Claims (6)
1. a kind of small haptens detection method based on direct competitive fluoroimmunoassay, it is characterised in that including following step
Suddenly：
1) coated antibody on ELISA Plate；
2) using chloroform as solvent, prepare oleic acid moieties modification a concentration of 15~25mg/mL of quantum dot, PMMA a concentration of 25~
The mixed solution of a concentration of 15~25mg/mL of 35mg/mL, PMAO keeps 25~35min, will then take dodecyl sodium sulfate
Aqueous solution mixes a concentration of 3~4mg/mL of quantum dot modified to wherein oleic acid moieties with the mixed solution, after mixing
Chloroform is removed, separation of solid and liquid takes solid phase to get to the fluorescent microsphere for the quantum dot for being marked with carboxyl modified；
3) fluorescent microsphere of the quantum dot of carboxyl modified is marked with using bovine serum albumin(BSA) coating, product and small molecule half is anti-
Original coupling to get to competition antigen；
4) solution to be measured and the competition antigen are added to step 1) and are coated in the ELISA Plate of antibody, antigen-antibody combines anti-
It answers, then detects the fluorescence intensity of ELISA Plate；
In step 3), the fluorescent microsphere of the quantum dot that carboxyl modified is marked with using bovine serum albumin(BSA) coating, including with
Lower step：The fluorescent microsphere and 1- ethyls-(3- dimethylaminos of the quantum dot of carboxyl modified will be marked in phosphate buffer
Base propyl) phosphinylidyne diimine, bovine serum albumin(BSA) mixing, until in solution bovine serum albumin(BSA) quality volume fraction be 0.8%
~1.2%, 25~35min is reacted in 35~39 DEG C, then adds 1- ethyls-(3- dimethylaminopropyls) phosphinylidyne diimine 3
~5 times, 25~35min is reacted in 35~39 DEG C after adding every time, separation of solid and liquid after being fully completed is added and takes solid phase, it is molten after washing
Solution is in sodium bicarbonate solution.
2. a kind of small haptens detection method based on direct competitive fluoroimmunoassay according to claim 1,
It is characterized in that the quantum dot is CdSe/ZnS quantum dots.
3. a kind of small haptens detection method based on direct competitive fluoroimmunoassay according to claim 1,
It is characterized in that the excitation wavelength of the quantum dot is 450nm, launch wavelength 620nm.
4. a kind of small haptens detection method based on direct competitive fluoroimmunoassay according to claim 1,
It is characterized in that the removal of chloroform is realized using revolving.
5. a kind of small haptens detection method based on direct competitive fluoroimmunoassay according to claim 1,
It is characterized in that it is to centrifuge 8~12min with the rotating speed of 12000~15000rpm to be separated by solid-liquid separation described in step 3).
6. a kind of small haptens detection method based on direct competitive fluoroimmunoassay according to claim 1,
It is characterized in that the condition of the antigen-antibody reaction is 25~35min of reaction at 35~39 DEG C.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101893623A (en) * | 2010-06-22 | 2010-11-24 | 上海师范大学 | Rapid detection method employing ultrasensitive quantum dot microsphere immunity-chromatograph test paper strips |
| CN102253214A (en) * | 2011-07-06 | 2011-11-23 | 清华大学深圳研究生院 | Quantum dot-based method for detecting ciprofloxacin by immunofluorescence and special kit |
| CN104991070A (en) * | 2015-06-25 | 2015-10-21 | 浙江大学 | Method for detecting fungaltoxin by using quantum dot cation exchange signal amplification technology |
| CN105602545A (en) * | 2015-12-24 | 2016-05-25 | 天津大学 | Preparation method of monodisperse quantum dot micro spheres with optical gain property |
| CN105759045A (en) * | 2016-03-18 | 2016-07-13 | 南昌大学 | Method for detecting aflatoxin B1 |
| CN105842441A (en) * | 2016-03-18 | 2016-08-10 | 南昌大学 | Detection method for ochratoxin A |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100119697A1 (en) * | 2006-05-10 | 2010-05-13 | 3M Innovative Properties Company | Compositions and coatings containing fluorescent, inorganic nanoparticles |
-
2016
- 2016-11-02 CN CN201610944123.XA patent/CN106568949B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101893623A (en) * | 2010-06-22 | 2010-11-24 | 上海师范大学 | Rapid detection method employing ultrasensitive quantum dot microsphere immunity-chromatograph test paper strips |
| CN102253214A (en) * | 2011-07-06 | 2011-11-23 | 清华大学深圳研究生院 | Quantum dot-based method for detecting ciprofloxacin by immunofluorescence and special kit |
| CN104991070A (en) * | 2015-06-25 | 2015-10-21 | 浙江大学 | Method for detecting fungaltoxin by using quantum dot cation exchange signal amplification technology |
| CN105602545A (en) * | 2015-12-24 | 2016-05-25 | 天津大学 | Preparation method of monodisperse quantum dot micro spheres with optical gain property |
| CN105759045A (en) * | 2016-03-18 | 2016-07-13 | 南昌大学 | Method for detecting aflatoxin B1 |
| CN105842441A (en) * | 2016-03-18 | 2016-08-10 | 南昌大学 | Detection method for ochratoxin A |
Non-Patent Citations (5)
| Title |
|---|
| 双亲性高分子PMAO超声乳化法改性量子点的制备;董春红等;《天津市生物医学工程学会第三十三届学术年会论文集》;20130401;第3页 * |
| 无机/有机掺杂CdTe/PMMA的合成;金丽等;《吉林化工学院院报》;20130115;第30卷(第1期);第24-27页 * |
| 量子点制备与表面修饰;刘富华等;《化学通报》;20130918;第76卷(第9期);第822-827页 * |
| 量子点标记免疫技术在食品中小分子有害物检测中的应用;戴尽波;《食品科学》;20160513;第19卷(第37期);第296-301页 * |
| 量子点荧光微球免疫层析试纸条定量检测玉米中的黄曲霉毒素B1;任美玲;《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》;20160315(第03期);第1-60页 * |
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