CN106645043A - Kit and method for fast quantitatively detecting small molecule compound - Google Patents
Kit and method for fast quantitatively detecting small molecule compound Download PDFInfo
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- CN106645043A CN106645043A CN201510731013.0A CN201510731013A CN106645043A CN 106645043 A CN106645043 A CN 106645043A CN 201510731013 A CN201510731013 A CN 201510731013A CN 106645043 A CN106645043 A CN 106645043A
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Abstract
The invention provides a kit and method for fast quantitatively detecting a small molecule compound. Particularly, the kit comprises a test strip and an independently placed fluorescent latex particle-antibody conjugate, wherein a to-be-tested sample and the conjugate are mixed and then added to the test strip, and are detected through a time-resolved fluorescent immunochromatography method, thereby determining the presence of the small molecule compound or detecting the content of the small molecule compound. The kit has excellent stability and relatively high sensitivity and specificity and is suitable for detection of various samples.
Description
Technical field
The present invention relates to detection field, in particular it relates to a kind of examination of Quantitative detection micromolecular compound
Agent box and method.
Background technology
The detection method of conventional micromolecular compound, including chemical staining method, immunochromatographic assays, height
Effect liquid phase chromatogram detection method (HPLC) and makings coupled HPLC detection method (GC/MS) method etc..Wherein, chemistry is aobvious
Color method needs a large amount of detection samples, and sensitivity and precision is not high；Afterwards both have higher sensitivity
And precision, but detection have high demands, it is difficult to promote.
Immunochromatographic assays (Immunochromatographic test) are that for rising the eighties is fast
Fast detection technique.Through development for many years, technique has been widely used for clinical diagnosis, illicit drugs inspection
With the field such as food security.In immune chromatography method, the comlete antigen of small-molecule substance is fixed on into nitric acid
Detection zone (solid phase antigen) on tunica fibrosa, the small-molecule substance (free antigen) in measuring samples solution with it is solid
Monoclonal antibody (the mark of the anti-small-molecule substance of phase antigenic competition association colloid gold or color latex microballoon mark
Note antibody).If the small-molecule substance contained in measuring samples, labelled antibody will be suppressed with immobilized antigen
With reference to suppression forms colour band in the detection zone of nitrocellulose filter.If detection zone forms colour band after measure,
Then result is feminine gender, and testing sample does not contain small-molecule substance to be measured；Conversely, not forming colour band, then result is
The positive, detection sample contains small-molecule substance to be measured.Because collaurum or the mark detection of color latex microballoon are
Differentiate result by naked eyes, the detection method for marking using collaurum or color micro-sphere at present can only be used as one kind
Qualitative or semiquantitative detection.
Fluorescent quantitation immunochromatography technique, is immunofluorescence technique (Immunofluorescence technique)
Combine a kind of quantitative measurement technology of development innovation with Traditional immunochromatographic technology.The technology is with fluorescent microsphere
As mark, by detecting that fluorescence signal realizes the quantitative of testing result.
But, because nitrocellulose membrane etc. can send in the case where light action is excited in the material of immunochromatography detection
Different degrees of fluorescence, while detecting that sample such as blood, urine sample and saliva etc. contain albumen, nucleic acid, sugar
Class is respectively provided with fluorescent effect, therefore and the background that causes to detect is higher, affect the accuracy of testing result.For
Overcome the problems referred to above, have researcher to develop time-resolved fluoroimmunoassay chromatographic technique, to eliminate sample in it is glimmering
Interference of stimulative substance, exciting light and the film to detecting itself.
But, although time-resolved fluoroimmunoassay chromatography is favorably improved the accuracy of detection, but due to a variety of
Reason, larger deviation and fluctuation are still occurred in quantitative determination, cause testing result to meet reality
Need.Especially for micromolecular compound (such as morphine, crystal methamphetamine drugs), quantitative determination result
Accuracy it is on the one hand closely related with the action of drug containment, also for the important references of the measurement of penalty of criminal
Factor.
Therefore, this area is in the urgent need to developing quick, quantitative and accurate (deviation is little) detection small molecule
The new method of compound.
The content of the invention
It is an object of the invention to provide the kit and method of a kind of Quantitative detection micromolecular compound.
It is described in a first aspect of the present invention, there is provided a kind of kit for detecting micromolecular compound
Kit includes：
A () test-strips, the test-strips include backing and the sample pad on backing, reaction film and suction
Pad is received, wherein being provided with detection zone and quality control region on the reaction film；And
(b) first container, and be independently arranged in first container and with the test-strips and (or put
Put) antibody complex, wherein the antibody complex comprising fluorescent latex particles and specificity be directed to little point
The antibody of sub- compound, the specificity is directed to the antibody coupling of micromolecular compound in the fluorescent latex particles；
Wherein, described detection zone is fixed with the comlete antigen of the micromolecular compound.
In another preference, in described test-strips, the sample pad and reaction film be direct neighbor or
Contact.
In another preference, by the liquid flow direction of sample, include sample in described test-strips successively
Pad, reaction film and absorption pad.
In another preference, the test-strips are made up of sample pad, reaction film, absorption pad and backing.
In another preference, described antibody complex is located in independent packaging or container.
In another preference, described test-strips are not provided with land.
In another preference, described test-strips are not contained little with described for specificity when unused
Antibody or the antibody complex that molecular compound is combined.
In another preference, the first described container is independent and closing packaging or reagent cup.
In another preference, also contain in the first described container
In another preference, the molecular weight of the micromolecular compound is 1-1000Da, preferably
5-800Da, is more preferably 10-500Da.
In another preference, described micromolecular compound is morphine.
In another preference, the fluorescent latex particles are that lanthanide series metal and/or lanthanide chelates are coated
Emulsion particle.
In another preference, the quality control region includes SA, and the SA specifically binds to spy
Antibody of the opposite sex for micromolecular compound.
In another preference, described SA is many anti-or antiserums.
In another preference, described specificity is mouse antibody for the antibody of micromolecular compound, and institute
The SA stated is rabbit anti-mouse igg antibody or sheep anti-mouse igg antibody.
In another preference, described specificity is rabbit antibody for the antibody of micromolecular compound, and institute
The SA stated is goat anti-rabbit igg antibody.
In another preference, the comlete antigen of described micromolecular compound is micromolecular compound and carrier egg
White conjugate.
In another preference, described carrier protein includes BSA albumen.
In a second aspect of the present invention, there is provided a kind of method of detection micromolecular compound, including step：
(1) determinand sample is mixed with an antibody complex, obtains first and process mixture, wherein
The antibody complex is directed to the antibody of micromolecular compound, and institute comprising fluorescent latex particles and specificity
The antibody coupling that specificity is stated for micromolecular compound is in the fluorescent latex particles；
(2) the first described process mixture is added to the sample pad of a test-strips, wherein, the test-strips
Sample pad, reaction film and absorption pad including backing and on backing, wherein being provided with the reaction film
Detection zone and quality control region；Described detection zone is fixed with the comlete antigen of the micromolecular compound；
(3) using the fluorescence intensity of the time-resolved fluoroimmunoassay chromatography method measurement detection zone for testing piece
And the fluorescence intensity of quality control region, so that it is determined that micromolecular compound whether there is, and/or it is defined as small molecule
The content of compound.
In another preference, described micromolecular compound includes morphine.
In another preference, described sample include the aqueous solution, beverage, whole blood, blood plasma, serum, urine,
Sweat, tear or saliva.
It is in step (3), the fluorescence intensity of test section is strong with the fluorescence of quality control region in another preference
The ratio of degree, and calibration curve is compared, so that it is determined that the content of micromolecular compound.
In another preference, the fluorescent latex particles are that lanthanide series metal and/or lanthanide chelates are coated with
Emulsion particle.
It is described in a third aspect of the present invention, there is provided a kind of fluorescence measuring device of quantitative determination determinand
Device include：
Kit described in (a) first aspect present invention；With
B () is used for the detector of fluorescence intensity.
It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and below (such as enforcement
Example) in can be combined with each other between each technical characteristic for specifically describing, so as to constitute new or preferred skill
Art scheme.As space is limited, here is no longer tired out one by one and is stated.
Description of the drawings
Fig. 1 shows the structural representation of immuno-chromatographic test paper strip (plate) in a kind of embodiment of the invention,
Wherein, each mark is as follows：1 is sample pad, and 3 is detection zone, and 4 is reaction film, and 5 is that quality control region is (right
According to area), 6 is absorption pad, and 7 is backing.
Specific embodiment
The present inventor, by screening in a large number and groping, has found unexpectedly first through extensively in-depth study
It is found that the kit and method of a kind of new Quantitative detection micromolecular compound.Experiment shows, during detection
First by testing sample and the independent fluorescent latex particles-antibody coupling matter mixing placed, then the mixture is added
To test-strips, and detected by time-resolved fluoroimmunoassay chromatography method, the spirit of detection can be significantly improved
Sensitivity, specificity and stability, complete on this basis the present invention.
Specifically, the present inventor has found that the structure of existing chromatography detector bar is for quantitative inspection through research
The fluctuation (or deviation) for surveying result causes large effect.One main influence factor is to combine pad part
(or land).If the emulsion particle of coupled antibody is coated on pad, it is dried.During detection
Latex discharges from pad just becomes one of the key factor for affecting detection accuracy.Additionally, emulsion particle
The material of the degree, the speed of release and/or pad of release, is all probably to cause quantified results ripple
The big factor of dynamic property.The present inventors have additionally discovered that, when processing pad, the processing mode of pad
And the degree of drying of latex is also difficult to be consistent, and fix or positioned at the detection antibody of the pad
The property of (such as the antibody coupling matter of fluorescent latex particles mark) easily changes, under causing long-time stability
Drop, so as to cause the fluctuation of testing result big.
Additionally, the experimental result of inventor is also shown that directly setting morphine etc. is little in detection zone (or detection line)
During molecular compound (standard items), the fluctuation for also resulting in testing result is big.
For this purpose, applicant optimizes the structure of detection reagent, pad is on the one hand eliminated, on the other hand existed
Quality control region (or nature controlling line) arranges SA (such as the antibody coupling matter for fluorescent latex particles mark
SA), and it is related to the comlete antigen of the micromolecular compound in detection zone (or detection line), so
The accuracy of testing result not only can be significantly improved, deviation is reduced, even and if the detection reagent is long-term
Storage (>=2 years) can still keep stable, and with higher accuracy and sensitivity.
Term
Morphine
As used herein, " morphine " includes morphine and the like, and the chemical name of morphine is 17- methyl
- 4,5 α-oxymorphines of epoxy -7,8- two are muttered -3,6 salmefamols.Morphine (Morphine, MOP) is opium class
The important component part of drugs, the content in opium be 4%-21%, average 10% or so.Its derivative salt
Sour morphine is clinically conventional anesthetic, there is extremely strong analgesic activity, is used for wound, operation, burn
Etc. the severe pain for causing, the angina pectoris that myocardial infarction causes is also used for, is alternatively arranged as analgesia, antibechic and antidiarrheic；
The diacetate esters of morphine are otherwise known as heroin.The easy habituation of morphine so that long-term smoker is no matter from body
Or it is psychological all to produce serious dependence to morphine, cause serious toxicomania, so as to itself and
Society causes greatly harm.Morphine is that 21 century global range abuses one of most commonly used drugs.
Immunochromatography technique
Immunochromatography technique (Immunochromatography) is to build beginning of the nineties late 1980s
A kind of vertical Fast Detection Technique.Because immunochromatography technique is not necessary to be combined label and free label
Separation, it is thus simple to operate, quick, be especially suitable for Site Detection and be used.
Time resolved fluoro-immunoassay
Time resolved fluoro-immunoassay (Time resolved fluoro immunoassay, TRFIA) is
A kind of new nonradioactive labeling that early 1980s found on the basis of conventional fluorescent immunoassay
Immuno analytical method, is current most sensitive trace analysis.
The acquisition of the detection signal of detection zone in test-strips, the detection signal in test-strips is passed over
Optical signal, by optical concentration device, light-dividing device, and optical beam path shaping etc., obtain 615nm ± 10nm
The fluorescence hot spot of left and right, by this fluorescence hot spot optical pickocff photomultiplier is delivered to, and is obtained in test-strips
The detection optical signal of detection zone.By control signal read access time be 10 microseconds to 400 microseconds, obtain
The detection signal of one deduction autofluorescent background.By photoelectric signal transformation, electric signal biography is converted optical signals to
Give calculating control unit to process, be finally completed detection.
Different from traditional fluorescein mark, the tracer used by it is the group of the lanthanides unit with unique fluorescent characteristic
Element and its chelate, can effectively exclude the interference of sample natural fluorescence, with sensitivity height, high specificity,
The features such as good stability and "dead" pollution, sensitivity is up to 10-19, it is high compared with radiommunoassay (RIA)
Go out 3 orders of magnitude.Application in clinical immunoassay test and scientific research is more and more extensive.
Time-resolved fluoroimmunoassay chromatographic technique
Time-resolved fluoroimmunoassay chromatographic technique is based on the Time-resolved fluorescence assay skill of immunochromatography technique
Art, is on the basis of time-resolved fluorescence immunoassay instrument, by time resolved fluoro-immunoassay and immunity
Chromatographic technique combines, and eliminates loaded down with trivial details sample-adding, washing step, and reagent stability is good, simple to operate,
Detection speed is fast, and sensitivity is high, can be widely applied in situ quantitation detection, also can be used as POCT (Point of
Care Test, immediately bedside diagnosis or detection) analyzer.
Lanthanide series
At present, oneself has 5 kinds of lanthanide series to be used for TRFIA, has Eu, Tb and Sm wherein conventional, and
Eu (europium element) is again most widely used element in labelled antigen antibody.Lanthanide series under free state,
Fluorescence signal is very faint, only the energy transmission of intermolecular resonant energy level, and ground state emission is returned in radiationless transition
The probability very little of fluorescence, but its chelate can launch fluorescence under the exciting of ultraviolet source.With traditional fluorescence
Plain label is compared, and lanthanide series has wider excitation spectrum band and narrower emission band, and fluorescence is held
The continuous time is long, and the Stocks displacements of fluorescence spectrum are larger, using spectrally resolved technology and time resolution skill
Art can effectively exclude the interference of exciting light and non-specific fluorescence.
Emulsion particle
As used herein, term " emulsion particle ", " latex beads " can be with used interchangeably；" fluorescence is micro-
Ball ", " fluorescent particle ", " fluorescent latex ", " fluorescent latex particles ", " fluorescent latex microballoon "
Can be with used interchangeably.
In area of medical diagnostics, from earliest latex agglutination test complicated diversification detection today is developed into.
Using emulsion particle as the label of antigen-antibody, mainly due to the characteristic of emulsion particle itself, it can be with
The surface-functionalized modification of various ways is carried out, density change and the change of specific properties are (such as：Color change,
Fluorescence or magnetic etc.) so that latex becomes a pith in detection.
The diameter of latex beads is generally 100-300nm, is most preferably 150-200nm.
In the present invention, it is preferred to time-resolved fluorescence emulsion particle be in 610-620nm with launch wavelength
Fluorescent latex particles, in order to detect.A kind of preferred emulsion particle is the fluorescent latex containing lanthanide series
Particulate, it is preferred that containing Europium chelate.The latex beads that can be used for the present invention is not particularly limited, can be with
From latex beads that is commercially available or being prepared with conventional method.
Using the internal emulsion particle containing europium, i.e., with reference to the fluorescent particle of Europium chelate.This combines Europium chelate
Fluorescent particle, under ultraviolet excitation, send the red fluorescence of 610-620nm, can be used to as anti-
Body tag.
Antibody complex
" antibody complex ", " fluorescent latex particles-antibody coupling matter " and " fluorescence is micro- as used herein
Grain labelled antibody " is used interchangeably.
In the present invention, the antibody of Jing fluorescent latex particles mark is single multi- clonal antibody.After labeled, resist
Body is coupled to fluorescent latex particles.Certainly, also can be considered that emulsion particle is coupled to antibody.
It is preferred that antibody covalent coupling is in fluorescent latex particles.
More preferably, by antibody coupling in fluorescent latex particles be by antibody pass through fluorescent latex particles surface active
Carboxyl, hydroxyl or aldehyde radical and covalent coupling in fluorescent latex particles.
Wherein, a diameter of 100-300nm of the fluorescent latex microballoon, preferably 150-200nm.
Additionally, the antibody is 1 with the part by weight of the emulsion particle:2～1:50, preferably 1:5～
1:25。
More preferably, the antibody and the part by weight of the emulsion particle are 1：8～12.
Detection kit
The invention provides one kind can be used to detect drinking water, beverage, whole blood, blood plasma, serum, urine, sweat
The detection kit of specific micromolecular compound in liquid, tear, saliva equal samples.
The kit includes：
A () test-strips, the test-strips include backing and the sample pad on backing, reaction film and absorption
Pad, wherein being provided with detection zone and quality control region on the reaction film；And
(b) first container, and be independently arranged in first container and with the test-strips and (or put
Put) antibody complex, wherein the antibody complex comprising fluorescent latex particles and specificity be directed to little point
The antibody of sub- compound, the specificity is directed to the antibody coupling of micromolecular compound in the fluorescent latex particles；
Wherein, described detection zone is fixed with the comlete antigen of the micromolecular compound；
It is preferred that the specificity adopts the fluorescent latex particles mark containing europium for the antibody of micromolecular compound
Note.
A kind of preferred test-strips are using the immunochromatography effluent piece or immuno-chromatographic test paper strip of effluent principle.
In the present invention, term " flow measurement piece ", " test-paper ", " test strips ", " test paper plate ",
" chromatography strip ", " test-strips " have identical implication, can be with used interchangeably.
The structure of currently preferred immunochromatography effluent piece (or test-strips) as shown in figure 1, including：
Sample pad 1, detection zone (or detection line) 3, reaction film 4, check plot (or quality control region, control line, Quality Control
Line) 5, absorption pad 6, backing 7.
Wherein, the length of backing 7 is identical with test-strips.
Sample pad 1 is located at one end of test-strips, and absorption pad 6 is located at the other end of test-strips.
Detection zone 3 (also referred to as detection line) is located between sample pad 1 and absorption pad 6, and usual detection zone 3 is arranged
On reaction film 4, sample pad 1 and adsorptive pads 6 are connected by the reaction film.Preferably, the reaction
Be additionally provided with quality control region 5 (also referred to as nature controlling line) on film 4, quality control region 5 be located at detection zone 3 and absorption pad 6 it
Between.
Wherein, the detection zone is fixed with the comlete antigen of micromolecular compound, and the quality control region is fixed with goat-anti
Mouse IgG, is analyzed respectively by the fluorescence signal to two regions, obtains the quantitative target of detectable substance.
The manufacture method of test-strips, it is preferable that respectively pass through sample pad 1, reaction film 4, absorption pad 6
Adhesive is pasted on backing 7, obtains final product the test-strips.
In the present invention, each element (or component) of the test-strips can select the existing material in this area
Make.
Backing 7 can be made with any stable, non-porous material, and its intensity should be enough to supporting material and glue
Each element thereon.Because many measure water are used as dispersive medium, therefore backing 7 is preferably
It is substantially water-impervious.In a preference, backing 7 is used made by polymer film, is more preferably to use
(such as PVC offset plates) made by polychloroethylene film.
Sample pad 1 can be made with any absorbent material.The example of material that can be used includes：Cellulose, nitre
Acid cellulose, cellulose acetate, glass fibre, nylon, polyelectrolyte ion exchange membrane, propylene copolymer
/ nylon and polyether sulfone.
Reaction film 4 can be made with any material, as long as the material has enough porositys to allow on surface
With the internal capillarity that fluid occurs.Reaction film 4 should have enough porositys, anti-so as to allow to scribble
The particle movement of body or antigen.Reaction film 4 can also be by liquid profit used in the sample containing analyte to be detected
Wet (for example, for waterborne liquid has hydrophily, for organic solvent has hydrophobicity).For example, by
Method described in United States Patent (USP) No.4,340,482 or No.4,618,533 (these methods describe by
Hydrophobic surface is transformed into water-wetted surface), thus it is possible to vary its hydrophobicity so as to make it have hydrophily for use in
Waterborne liquid.The example of material that can be used to manufacture reaction film 4 is included but is not limited to：Polymer PET, cellulose,
Celluloid, cellulose acetate, glass fibre, nylon, polyelectrolyte ion exchange membrane, propylene copolymerization
Thing/nylon and polyether sulfone (polyethersulfone).In a preference, reaction film 4 is to use nitre
Made by acid cellulose film.
Absorption pad 6 can be made with any can absorption as the material of sample and the liquid of buffer solution.Absorption pad
6 absorbability is sufficiently large, to absorb the liquid added to test-strips.Suitable for the material of absorption pad 6
The example of material includes cellulose and absorbent filter.
For the ease of understanding the present invention, the Cleaning Principle of kit of the present invention is given.It should be understood that the present invention
Protection domain is not affected or restriction by the principle.
The comlete antigen of small-molecule substance is fixed on nitric acid by the present invention using the principle of competition law immunochromatography
Detection zone (solid phase antigen) on tunica fibrosa.Premixing sample to be tested and the independently fluorescent latex particles of placement-
Antibody coupling matter, by the mixture sample pad of test-strips is added to, if do not contained in sample specific little point
Sub- compound, the fluorescent latex particles-antibody coupling matter will forward flow along reaction film (such as nitrocellulose membrane)
Dynamic, when reaching detection line position, fluorescent particle labelled antibody is fixed on the small molecule on reaction film and carrier
Protein conjugate is captured, and by exciting for 365nm ultraviolet lights, in detection zone T line positions redness is formed
615nm fluorescent bands.If containing certain density small-molecule substance in measuring samples, it will Fluorophotometry
The combination of particulate labelled antibody and immobilized antigen, will subtract in the fluorescent bands of the detection zone of nitrocellulose filter
It is weak.The concentration of the small-molecule substance contained in the power and measuring samples of fluorescent bands is in certain linear pass
System, by photomultiplier gather fluorescence signal counted, so as to calculate measuring samples in contain it is little
The concentration of molecular substance.It is many that the present invention arranges sheep anti-mouse igg in the quality control region of reaction film (such as nitrocellulose membrane)
It is anti-, no matter whether contain micromolecular compound to be measured, fluorescent particle labelled antibody total energy and matter in measuring samples
The many anti-bindings of sheep anti-mouse igg in control area form a fluorescence quality control band, and the fluorescent bands are to judge chromatography process
The standard that whether normal and detection plate fails, while also serving as the calculating of control difference between batch in quantitative analysis
Index.
Detection means
The detection means of the present invention can include：Test-strips, detector, light source, optical fiber and computer.
The operation instruction of a detection method can also be included.Wherein the operation principle of test-strips is as described above, any
The Time-resolved fluorescence assay method that can be used for fluorescence intensity is used equally to the detection means of the present invention.
Main advantages of the present invention include：
A the stabilization of kit of () present invention is good, sensitivity and accuracy are high.
B the kit specificity of () present invention is good.
C the kit of () present invention can be used for the detection of several samples.
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate
The present invention rather than restriction the scope of the present invention.The experiment side of unreceipted actual conditions in the following example
Method, generally according to normal condition, or according to the condition proposed by manufacturer.Unless otherwise indicated, otherwise
Percentage and number are calculated by weight.
Material
Monoclonal antibodies against morphine and morphine comlete antigen (Morphine-BSA) and many grams of sheep anti-mouse igg
Grand antibody leads to bio tech ltd purchased from Shanghai eight；Emulsion particle containing europium
(Carboxylate-Modified Dyed Microparticles, Fluoro-MAX, PART NO:
93470520010150) particle diameter is 0.199nm, purchased from ThermoFisher Scientific companies.
EDC (1- ethyl -3- (3- dimethyl aminopropyls)-carbodiimides), NHS (N-hydroxy-succinamide) and little
Bovine serum albumin(BSA) (BSA) is Sigma Products；Nitrocellulose filter is Millipore companies product
Product, aperture is 8 μm；Glass fibre membrane is Millipore Products；Morphine reference substance is Zhong Jiansuo states
Family's arcotic laboratory provides；Time-resolved fluorescence instrument is by the limited public affairs of Shanghai Yi Sibai biotechnologies
Department provides.
Embodiment 1
The preparation of fluorescent latex particles-antibody coupling matter
Take the μ L of 1% fluorescent latex microballoon 250 plus 1000 μ L 0.05M 2- (N- morpholines) ethyl sulfonic acids
(MES) (ph6.0) mix.15000rpm is centrifuged 20 minutes, supernatant is abandoned, with 1000 μ L 0.05M MES
Resuspended, ultrasonic (50W, 1 second working time, 1 second interval time), afterwards 15000rpm was centrifuged 20 minutes.
Supernatant is abandoned, with 0.05M MES 500 μ L are diluted to, ultrasound.Add the NHS (10mg/ml) of 38.5 μ L
React 15 minutes after mixing with 5 μ L EDC (15mg/ml).The morphine monoclonal antibody for adding 0.15mg is mixed
It is even.React 2 hours under lucifuge room temperature on shaking table.25 μ L 0.1M monoethanolamines are added to react 30 minutes to seal
Close the uncombined carboxyl of microsphere surface.13000rpm abandons supernatant after being centrifuged 10 minutes, is sealed with 500 μ L caseins
Close liquid to react 1 hour.15000rpm is centrifuged 10 minutes, collects the fluorescent latex microballoon casein of mark
Confining liquid is diluted to 250 μ L, 2-8 DEG C of preservation after ultrasound.
Embodiment 2
The preparation of cryodesiccated fluorescent latex particles-antibody coupling matter
Will the upper antibody of mark fluorescent latex particles solution and freeze-drying process liquid (Borax0.1M, BSA1%,
S172%, sucrose 5%, trehalose 5%) mixing, mixed solution is added into 25 μ L/ holes in 96 orifice plates, put
Enter -80 DEG C of refrigerator freezings 30 minutes.96 orifice plates after by freezing are put into and open in advance and be cooled to
In -35 DEG C of vacuum freeze drier, temperature-gradient method is simultaneously vacuum dried, and is taken out after 30 minutes 2 hours,
The emulsion particle after freeze-drying is vacuum-packed with vacuum packing machine, 2-8 DEG C of guarantor is positioned over after packaging
Deposit.
Embodiment 3
The Seal treatment of sample pad
Sample pad adopts glass fibre, is to reduce the non-specific binding and increase stability in detection, uses table
Face activating agent and polymer carry out Seal treatment to sample pad.Specifically, with containing the Hes of 0.2%Tween 20
The pH 8.0 of 0.5%PVA, 0.1M Tris buffer solution aqueous solution soaking absorbent filters, 37 DEG C are dried 16 hours.
Embodiment 4
The preparation of immune chromatograph testing strip
Detection line and nature controlling line are drawn respectively with point film machine on nitrocellulose filter, and detection line is morphine
(0.8mg/mL), nature controlling line (C lines) is to resist (1.0 sheep anti-mouse igg after purification to-BSA (comlete antigen) more
Mg/mL), 18-24 hours after 37 DEG C of dryings.Successively cellulose nitrate film and sample pad are pasted on into PVC
Hold on material.The reagent strip of 4mm is cut into cutting machine, is placed in plastic casing, well alignment detection galley proof
Product pad position, is assembled into detection kit after compression, add drier sealing preserve.
When being detected, the morphine in sample is glimmering with the morphine-BSA competition bindings being fixed on nitrocellulose membrane
Light emulsion particle-antibody coupling matter, the fluorescence of detection zone (T) is strong and weak related to the morphine content in sample, leads to
The fluorescent value that time resolution instrument reads the fluorescent bands of observation window detection zone (T) position is crossed, is passed through
The standard items of variable concentrations can draw the correlation curve and computing formula of fluorescent value and morphine concentration, thus may be used
To carry out quantitatively to the morphine in sample exactly.
Embodiment 5
The preparation of calibration curve
Cryodesiccated fluorescent particle antibody coupling matter is taken out from hermetic bag, with liquid-transfering gun plus 100 μ L not
With the morphine standard items of concentration in hole, solution piping and druming is mixed with liquid-transfering gun, take 50 μ L and instill well
It is interior, 100 μ L flushing liquors are added after 10 minutes, then wait 5 minutes, agent plate is inserted in detector and is read
Take fluorescent value.According to the morphine standard items and fluorescent value of variable concentrations, by four parametric methods calibration curve is prepared.
As a result show, the regression equation by four parameter fittings is：Y=(A-D)/[1+ (X/C) ^B]+D, its
Middle A=19973, B=1.14506, C=1.83069, D=-1408.93, R2=0.99488.
Embodiment 6
Sample detection methods
Method mixes 100 μ L testing samples and fluorescent particle antibody coupling matter with the methods described of embodiment 5
Detected after conjunction, obtained fluorescent value, by the computing formula of calibration curve, morphine is contained in calculating sample
Amount.
Embodiment 7
Specificity analysis
Specific test, to the various common drugs, poisonous substance and the medicine that provide, in certain concentration range
Carry out cross-over experiment.
A) drugs, poisonous substance and the drug standards tester for selecting is as shown in the table：
Note：K is thousand
B) the freeze thawing urine using No Poison detects each cross jamming thing (standard items) to negative sample as negative control
The interference of product, i.e., have positive reaction when negative sample is detected, it is T line readings to show as instrument readings reaction
Decline (relative to negative control instrument readings).
C) each standard concentration of no cross reaction is determined by experiment, the possibility that acquisition can be used to assess occurs
The concentration of cross reaction.
D) concentration of generation cross reaction for assessment is obtained more than more than 100 times of test limit concentration, then may be used
Regard as meeting specific test requirement.
As a result it is as shown in the table：Kit of the present invention only reacts with m orphine sample, does not have with other materials
There is cross reaction, with very strong specificity.
Embodiment 8
Stability analysis
Destructive testing one month at a temperature of 45 DEG C of baking oven is placed in after detection kit is sealed.Method is with enforcement
The methods described of example 5, respectively with the destructive kit for processing and the kit not toasted, to containing different dense
The sample and negative sample of degree morphine is tested.
As a result show, 45 DEG C of the destructivenesses kits for processing month and the kit testing results do not toasted
Unanimously (deviation≤± 2%).Show that the detection kit has good stability.
Embodiment 9
Sensitivity analysis
Method is 0.5ng/mL, 1ng/mL, 2ng/mL with the method in embodiment 5, respectively detectable concentration
Morphine sample, each detectable concentration duplicate detection 5 times.Calculate the standard deviation (SD values) of testing result, put down
Average (AVG values) and the coefficient of variation (CV values), wherein, CV values should be within 10%, then it is believed that meeting spirit
Sensitivity test request.
As a result it is as shown in the table：The sensitivity of the morphine test-strips of the present invention is about 0.5ng/ml.Detection
CV<10%.
Embodiment 10
Analysis of the accuracy
Method is examined with kit of the present invention with the method in embodiment 5 to 10 real case samples
Survey, detection data is compared with the result of GC-MS, and coincidence rate is all higher than 95%.As a result show, this
Bright detection kit has very high accuracy.
Embodiment 11
Detection speed is tested
Ready test-strips (card) are inserted into detecting instrument, starts test and timing, test completes to be surveyed
Academic probation number, stops timing, obtains timing time, and timing time then can be regarded as meeting surveying in 10 minutes
Examination is required.
Experimental result：Detection time is respectively less than 5 minutes, and detection speed meets Site Detection and quick detection
Require.
Comparative example C1
The preparation and performance test of test-strips C1
1. prepare
The structure of test-strips C1 is similar with test-strips of the present invention, and its difference is that test-strips C1 are combined including one
Pad, the pad be located between sample pad 1 and absorption pad 6, sample pad 1 near-end and absorption pad 6 it is remote
End, closes on sample pad 1.Also, antibody complex is included on the pad, the antibody complex is included
Fluorescent latex particles and micromolecular compound antibody, the micromolecular compound antibody coupling is in fluorescence breast
Glue particulate.
2. accuracy
Using test-strips C1, for the real case sample in embodiment 10 is detected, detection data
It is compared with the result of GC-MS, coincidence rate is only 70%.This shows that the accuracy of detector bar C1 is not high,
Be not suitable for the high quantitative determination application of accuracy requirement.
3. stability
Method carries out stability test with embodiment 8 to test-strips C1.
As a result show, 45 DEG C of the destructivenesses kits for processing month and the kit testing results do not toasted
A certain degree of deviation (degree of deviation >=10%) is there occurs, additionally, after 45 DEG C of destructivenesses are processed two months, partially
Margin is further up into >=20%.This shows that the long-time stability of test-strips C1 are poor.
4. sensitivity
Method carries out sensitivity test with embodiment 9 to test-strips C1.
As a result show, the sensitivity of test-strips C1 is for about 3ng/ml, although can meet and generally require,
But sensitivity is far below test-strips of the present invention.
Comparative example C2
The preparation and performance test of test-strips C1
In comparative example C2, the structure of test-strips C2 is similar with test-strips of the present invention, and its difference is to examine
Survey area and be provided with morphine micromolecular compound, and be not provided with morphine comlete antigen.
Method carries out sensitivity test with embodiment 9 to test-strips C2.
As a result show, the sensitivity of test-strips C2 is that sensitivity is far below present invention survey more than 10ng/ml
Strip.
The all documents referred in the present invention are all incorporated as in this application reference, just as each document
It is individually recited as with reference to such.In addition, it is to be understood that after the above-mentioned instruction content for having read the present invention,
Those skilled in the art can make various changes or modifications to the present invention, and these equivalent form of values equally fall within this Shen
Please appended claims limited range.
Claims (10)
1. a kind of kit for detecting micromolecular compound, it is characterised in that the kit includes：
A () test-strips, the test-strips include backing and the sample pad on backing, reaction film and suction
Pad is received, wherein being provided with detection zone and quality control region on the reaction film；And
(b) first container, and be independently arranged in first container and with the test-strips and (or put
Put) antibody complex, wherein the antibody complex comprising fluorescent latex particles and specificity be directed to little point
The antibody of sub- compound, the specificity is directed to the antibody coupling of micromolecular compound in the fluorescent latex particles；
Wherein, described detection zone is fixed with the comlete antigen of the micromolecular compound.
2. kit as claimed in claim 1, it is characterised in that described micromolecular compound is morphine.
3. kit as claimed in claim 1, it is characterised in that described test-strips are not provided with land.
4. kit as claimed in claim 1, it is characterised in that the quality control region includes SA, institute
State SA and specifically bind to antibody of the specificity for micromolecular compound.
5. kit as claimed in claim 1, it is characterised in that described micromolecular compound resists completely
Originally it was the conjugate of micromolecular compound and carrier protein.
6. it is a kind of detection micromolecular compound method, it is characterised in that including step：
(1) determinand sample is mixed with an antibody complex, obtains first and process mixture, wherein
The antibody complex is directed to the antibody of micromolecular compound, and institute comprising fluorescent latex particles and specificity
The antibody coupling that specificity is stated for micromolecular compound is in the fluorescent latex particles；
(2) the first described process mixture is added to the sample pad of a test-strips, wherein, the test-strips
Sample pad, reaction film and absorption pad including backing and on backing, wherein being provided with the reaction film
Detection zone and quality control region；Described detection zone is fixed with the comlete antigen of the micromolecular compound；
(3) using the fluorescence intensity of the time-resolved fluoroimmunoassay chromatography method measurement detection zone for testing piece
And the fluorescence intensity of quality control region, so that it is determined that micromolecular compound whether there is, and/or determine small molecule
The content of compound.
7. method as claimed in claim 6, it is characterised in that described sample include the aqueous solution, beverage,
Whole blood, blood plasma, serum, urine, sweat, tear or saliva.
8. method as claimed in claim 6, it is characterised in that in step (3), by the glimmering of test section
The ratio of the fluorescence intensity of luminous intensity and quality control region, and calibration curve is compared, so that it is determined that small molecule
The content of compound.
9. method as claimed in claim 6, it is characterised in that the fluorescent latex particles be lanthanide series metal and
/ or the coated emulsion particle of lanthanide chelates.
10. a kind of fluorescence measuring device of quantitative determination determinand, it is characterised in that described device includes：
Kit described in (a) claim 1；With
B () is used for the detector of fluorescence intensity.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201510731013.0A CN106645043A (en) | 2015-10-30 | 2015-10-30 | Kit and method for fast quantitatively detecting small molecule compound |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510731013.0A CN106645043A (en) | 2015-10-30 | 2015-10-30 | Kit and method for fast quantitatively detecting small molecule compound |
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ID=58809594
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| Application Number | Title | Priority Date | Filing Date |
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| CN201510731013.0A Pending CN106645043A (en) | 2015-10-30 | 2015-10-30 | Kit and method for fast quantitatively detecting small molecule compound |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107037025A (en) * | 2017-06-24 | 2017-08-11 | 杭州微瑞科技有限公司 | The high quick fluorescent chromatographic device of group of the lanthanides and detection method |
| CN108802407A (en) * | 2018-05-29 | 2018-11-13 | 郑州左安检测科技有限公司 | A kind of test strips and its preparation method and application method of detection morphine |
| CN112698027A (en) * | 2020-12-14 | 2021-04-23 | 广东唯实生物技术有限公司 | Hapten immunochromatography detection reagent |
| CN114280312A (en) * | 2020-09-27 | 2022-04-05 | 河北特温特生物科技发展有限公司 | Whole blood separation membrane for immunofluorescence chromatography detection and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104090248A (en) * | 2013-12-24 | 2014-10-08 | 上海容晖生物科技有限公司 | Reagent for quantitative detection of Beta-receptor stimulant through Europium chelate latex time-resolved immunochromatographic assay |
| CN104422765A (en) * | 2013-08-30 | 2015-03-18 | 上海八通生物科技有限公司 | Test bar and method for quantitatively detecting micromolecular compound in sample |
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2015
- 2015-10-30 CN CN201510731013.0A patent/CN106645043A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104422765A (en) * | 2013-08-30 | 2015-03-18 | 上海八通生物科技有限公司 | Test bar and method for quantitatively detecting micromolecular compound in sample |
| CN104090248A (en) * | 2013-12-24 | 2014-10-08 | 上海容晖生物科技有限公司 | Reagent for quantitative detection of Beta-receptor stimulant through Europium chelate latex time-resolved immunochromatographic assay |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107037025A (en) * | 2017-06-24 | 2017-08-11 | 杭州微瑞科技有限公司 | The high quick fluorescent chromatographic device of group of the lanthanides and detection method |
| CN108802407A (en) * | 2018-05-29 | 2018-11-13 | 郑州左安检测科技有限公司 | A kind of test strips and its preparation method and application method of detection morphine |
| CN114280312A (en) * | 2020-09-27 | 2022-04-05 | 河北特温特生物科技发展有限公司 | Whole blood separation membrane for immunofluorescence chromatography detection and preparation method and application thereof |
| CN114280312B (en) * | 2020-09-27 | 2023-09-15 | 河北特温特生物科技发展有限公司 | Whole blood separation membrane for immunofluorescence chromatography detection and preparation method and application thereof |
| CN112698027A (en) * | 2020-12-14 | 2021-04-23 | 广东唯实生物技术有限公司 | Hapten immunochromatography detection reagent |
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