CN104422765A - Test bar and method for quantitatively detecting micromolecular compound in sample - Google Patents
Test bar and method for quantitatively detecting micromolecular compound in sample Download PDFInfo
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- CN104422765A CN104422765A CN201310391061.0A CN201310391061A CN104422765A CN 104422765 A CN104422765 A CN 104422765A CN 201310391061 A CN201310391061 A CN 201310391061A CN 104422765 A CN104422765 A CN 104422765A
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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Abstract
The invention provides a test bar and a method for quantitatively detecting a micromolecular compound in a sample. The detection is performed by adopting the test bar containing fluorescence emulsion particles and a micromolecular compound antibody on a binding pad and using a time resolution fluorescence immunochromatographic assay, and a specific value is compared with a standard curve, so that whether the micromolecular compound exists or not is determined or the content of the micromolecular compound is detected. The test bar can be used for quantitatively detecting the micromolecular compound in the sample, can be used for greatly eliminating the non-specific combination so that the signal to noise ratio of a detection signal is remarkably improved, and test bar has the advantages of rapidness, high sensitivity, high specificity and the like.
Description
Technical field
The present invention relates to detection field.Particularly, the present invention relates to a kind of test-strips and the method that detect sample small molecular compound.
Background technology
Immuno-chromatographic assay technology (immunochromatographic test) is also called immune flow measurement and detects (lateral flow immunoassay, LFIA), is a Fast Detection Technique of rising the eighties.Through development for many years, technique is widely used in clinical diagnosis, the field such as illicit drugs inspection and food security.
Detect most of micromolecular compound, usually adopt A competitive inhibition method to detect.In immune chromatography method, the comlete antigen of small-molecule substance is fixed on the detection zone (solid phase antigen) on nitrocellulose membrane, the monoclonal antibody (labelled antibody) of the anti-small-molecule substance that the small-molecule substance (free antigen) in measuring samples solution marks with solid phase antigen competition binding collaurum or color latex microballoon.If the small-molecule substance contained in measuring samples, will the combination of labelled antibody and immobilized antigen be suppressed, suppress to form colour band in the detection zone of nitrocellulose filter.If detection zone forms colour band after measuring, then result is negative, and testing sample is not containing small-molecule substance to be measured; Otherwise do not form colour band, then result is positive, detects sample and contains small-molecule substance to be measured.
Because collaurum or the detection of color latex microballoon mark differentiate result by naked eyes, at present can only as the qualitative or semiquantitative detection of one using the detection method of collaurum or color micro-sphere mark.For a long time; people attempt using reading instrument to carry out quantitatively to immunochromatography detector bar; but; because collaurum or coloured latex particle are using the depth of color as the signal detected; the accuracy of quantitative detection and sensitivity are all difficult to reach requirement; also there are some using magnetic particle or common fluorescent microsphere as mark, realize quantitatively, achieving good effect by the power detecting magnetic or fluorescence.But still there are some problems.Mainly in the material that detects of immunochromatography, nitrocellulose membrane etc. all can send fluorescence in various degree under exciting light effect, detect sample such as blood, urine sample and saliva etc. simultaneously and all containing albumen, nucleic acid, carbohydrate, all there is fluorescent effect, therefore cause the background of detection higher, affect the accuracy of testing result.
Therefore, this area needs exploitation fast, accurately, efficiently, quantitatively detect the method for micromolecular compound compound easily.
Summary of the invention
Object of the present invention is just to provide a kind of method of quantitative detection sample small molecular compound, quick, efficient, easy, accurate.
In a first aspect of the present invention, a kind of test-strips is provided, comprise sample pad, pad, reaction film, absorption pad and backing, described reaction film is arranged detection zone and quality control region, described pad comprises antibody complex, described antibody complex comprises fluorescent latex particles and micromolecular compound antibody, and described micromolecular compound antibody coupling is in described fluorescent latex particles.
In another preference, the molecular weight of described micromolecular compound is 1-10000Da.
In another preference, described micromolecular compound is drugs and Psychopathic Drugs, includes but not limited to: heroin, morphine, amphetamine, meth, MDMA, ketamine, cocaine, Sauteralgyl, fentanyl, C16H25NO2, cannabis, buprenorphine, methadone, codeine, caffeine, ephedrine, d-pseudo-ephedrine, stable, triazolam, BZO, barbital, tricyclic antidepressants Remeron.
In another preference, described micromolecular compound is the objectionable impurities in food, include but not limited to: Clenizole Hydrochloride, Ractopamine, salbutamol, melamine, malachite green, tonyred, aflatoxin B1, aflatoxins M1, organophosphorus, insecticide, herbicide, various hormone is as progesterone, estriol, stosterone etc.
In another preference, described micromolecular compound is microbiotic residual in food, includes but not limited to: penicillin, ampicillin, Amoxicillin, cephalo, gentamicin, tetracycline, sulfa antibiotics, furans microbiotic.
In another preference, described fluorescent latex particles is the emulsion particle of lanthanide series metal and/or lanthanide chelates bag quilt.
In another preference, described pad also comprises rabbit igg or mouse IgG.
In another preference, described quality control region comprises goat anti-rabbit igg or sheep anti-mouse igg.
In another preference, described detection zone comprises the conjugate of micromolecular compound and carrier protein.
In another preference, described carrier protein is human serum albumins, bovine serum albumin(BSA), ovalbumin.
A second aspect of the present invention, provides a kind of detection kit, comprising:
(a) test-strips, described test-strips comprises sample pad, pad, detection zone, reaction film, quality control region, absorption pad and backing;
(b) antibody complex, described antibody complex comprises fluorescent latex particles and micromolecular compound antibody, and described micromolecular compound antibody coupling is in described fluorescent latex particles; With
(c) container.
A third aspect of the present invention, provides the purposes of the test-strips described in first aspect, for the preparation of the kit detecting potable water, beverage, whole blood, blood plasma, serum, urine, sweat, tear or saliva small molecular compound.
A fourth aspect of the present invention, provides a kind of method detecting micromolecular compound, comprises step:
(1) determinand sample is added to the sample pad of the test pieces described in first aspect;
(2) adopt time-resolved fluoroimmunoassay chromatography method to measure the fluorescence intensity of the detection zone of described test pieces and the fluorescence intensity of quality control region, thus determine whether micromolecular compound exists, or thus be scaled the content of micromolecular compound.
In another preference, in step (2), by the ratio of the fluorescence intensity of test section and the fluorescence intensity of quality control region, and typical curve compares, thus determines the content of micromolecular compound.
In another preference, described detection micromolecular compound is qualitative, sxemiquantitative or quantitatively detects micromolecular compound.
A fifth aspect of the present invention, provides a kind of fluorescence measuring device of quantitative detection determinand, comprising:
Test pieces described in (a) first aspect; With
B () is for the detecting device of fluorescence intensity.
Should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and can combining mutually between specifically described each technical characteristic in below (eg embodiment), thus form new or preferred technical scheme.As space is limited, tiredly no longer one by one to state at this.
Accompanying drawing explanation
Fig. 1 shows the structural representation of a kind of immuno-chromatographic test paper strip (plate) of the present invention,
Wherein, each mark is as follows: 1 is sample pad, and 2 is pad, and 3 is detection zone, and 4 is reaction film, and 5 is quality control region (check plot), and 6 is absorption pad, and 7 is backing.
Fig. 2 shows the present invention and adopts immuno-chromatographic test paper strip to carry out the principle schematic detected.
Fig. 3 is sample concentration and T/C value graph of a relation, and wherein, X-axis is sample concentration, and Y-axis is T/C value, i.e. T line fluorescent value/C line fluorescent value.
Embodiment
The present inventor is through extensive and deep research, and unexpected discovery selects fluorescent latex particles to mark the antibody of micromolecular compound, binding time resolved fluorometric immunochromatography technique, can the specific micromolecular compound of Quantitative detection immediately.With POCT (the Point of Care Test of existing collaurum and common fluorescence, bedside diagnosis or immediately detect) method compares, the inventive method can eliminate fluorescent material, exciting light and film itself in sample to the interference detected, the sensitivity of detection and/or specificity are all significantly increased.
Term
Immunochromatography technique
Immunochromatography technique (immunochromatography) is a kind of Fast Detection Technique set up beginning of the nineties late 1980s.Because immunochromatography technique must not carry out being separated of binding label and free label, thus simple to operate, quick, be applicable to very much the use of Site Detection.
Time resolved fluoro-immunoassay
Time resolved fluoro-immunoassay (time resolved fluoro immunoassay, TRFIA) being the novel nonradioactive labeling's immuno analytical method of one founded on the basis of conventional fluorescent immunoassay early 1980s, is trace analysis the sensitiveest at present.
The acquisition of the detection signal of surveyed area in test-strips, by the light signal that the detection signal in test-strips passes over, by optical concentration device, light-dividing device, with optical beam path shaping etc., obtain the fluorescence hot spot of about 615nm ± 10nm, this fluorescence hot spot is delivered to optical sensor photomultiplier, obtains the sensed light signal of surveyed area in test-strips.By control signal read the time be 10 microseconds to 400 microseconds, obtain one deduction autofluorescent background detection signal.By photoelectric signal transformation, light signal is converted to electric signal and sends the process of calculating control assembly to, finally complete detection.
Fluorescein-labelled different from traditional, its tracer used is lanthanide series and the chelate thereof with unique fluorescent characteristic, effectively can get rid of the interference of sample natural fluorescence, have highly sensitive, the feature such as high specificity, good stability and no radioactivity pollute, sensitivity is up to 10
-19, comparatively radiommunoassay (RIA) exceeds 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 the Time-resolved fluorescence assay technology based on immunochromatography technique, on the basis of time-resolved fluorescence immunoassay instrument, time resolved fluoro-immunoassay and immunochromatography technique are combined, eliminate loaded down with trivial details application of sample, washing step, reagent stability is good, simple to operate, detection speed is fast, highly sensitive, in situ quantitation can be widely used in detect, also can be used as POCT (Point of CareTest, bedside diagnosis or immediately detection) analyser.
Lanthanide series
Up to the present, oneself has 5 kinds of lanthanide series to be used to TRFIA, and that wherein commonly uses has Eu, Tb and Sm, and Eu (europium element) is element most widely used in labelled antigen antibody.Lanthanide series is under free state, and fluorescence signal is very faint, is only the energy transferring of intermolecular resonant energy level, and the probability that ground state emission fluorescence is returned in radiationless transition is very little, but its chelate can emitting fluorescence under the exciting of ultraviolet source.Compared with traditional fluorescein-labelled thing, lanthanide series has wider exciting light bands of a spectrum and narrower emission band, fluorescence duration time is long, and the Stocks displacement of fluorescence spectrum is comparatively large, utilizes spectrally resolved technology and TIME RESOLVED TECHNIQUE effectively can get rid of the interference of exciting light and non-specific fluorescence.
Emulsion particle
In this article, term " emulsion particle ", " latex beads " can exchange use; " fluorescent microsphere ", " fluorescent particle ", " fluorescent latex particles ", " fluorescent latex microballoon " can exchange use.
In area of medical diagnostics, the diversification developing into complexity today from latex agglutination test the earliest detects.Using emulsion particle as the label of antigen-antibody, mainly due to the characteristic of emulsion particle itself, it can carry out the surface-functionalized modification of various ways, density changes and the change (such as: color change, fluorescence or magnetic etc.) of specific properties, makes latex become a pith in detection.
The diameter of latex beads is generally 100-300nm, is 150-200nm best.
In the present invention, preferred time-resolved fluorescence emulsion particle has the fluorescent latex particles of emission wavelength at 610-620nm, so that detect.Preferred emulsion particle is the fluorescent latex particles containing lanthanide series, preferably, containing Europium chelate.Latex beads used in the present invention is not particularly limited, and can select latex beads that is commercially available or that prepare by conventional method.
Adopt the inner emulsion particle containing europium, namely in conjunction with the fluorescent particle of Europium chelate.This is in conjunction with the fluorescent particle of Europium chelate, under ultraviolet excitation, sends the red fluorescence of 610-620nm, can be used as antibody labeling.
Fluorescent particle labelled antibody (antibody complex)
In the present invention, the antibody through fluorescent latex particles mark is list/polyclonal antibody.Preferably, described fluorescent latex particles wraps up through glycosaminoglycan gel.
After mark, antibody is coupled to fluorescent latex particles.Certainly, also can be considered that emulsion particle is coupled and have antibody.
Preferably, antibody covalent coupling is in fluorescent latex particles.
More preferably, by antibody coupling in fluorescent latex particles be by antibody by the carboxyl of fluorescent latex particles surface active or hydroxyl and covalent coupling in fluorescent latex particles.
Wherein, the diameter of described fluorescent latex microballoon is 100-300nm, is preferably 150-200nm.
In addition, the part by weight of described antibody and described emulsion particle, for being 1:2 ~ 1:50, is preferably 1:5 ~ 1:25.
In a preference, the part by weight of described antibody and described emulsion particle is 1:8 ~ 12.
Immunochromatography effluent sheet (or test strips or test-paper)
In the present invention, a kind of immuno-chromatographic test paper strip is provided.
A kind of preferred test strips is the immunochromatography effluent sheet or the immuno-chromatographic test paper strip that utilize effluent principle.
In the present invention, term " flow measurement sheet ", " test-paper ", " test strips ", " test paper plate ", " chromatography strip ", " test-strips " have identical implication, can exchange use.
The structure of the present invention's preferred immunochromatography effluent sheet (or test-strips) as shown in Figure 1, comprising sample pad 1, pad 2, detection zone (or detection line) 3, reaction film 4, check plot (or quality control region, control line, nature controlling line) 5, absorption pad 6, backing 7.
The length of backing 7 is identical with test-strips.
Sample pad 1 is positioned at one end of test-strips, and absorption pad 6 is positioned at the other end of test-strips.
Pad 2, between sample pad 1 and absorption pad 6, at the near-end of sample pad 1 and the far-end of absorption pad 6, closes on sample pad 1.
Detection zone 3 (also claiming detection line) is between pad 2 and absorption pad 6, and usual detection zone 3 is arranged on reaction film 4, connects pad 2 and adsorptive pads 6 by described reaction film.Preferably, described reaction film 4 is also provided with quality control region 5 (also claiming nature controlling line), quality control region 5 is between detection zone 3 and absorption pad 6.
The manufacture method of test-strips, preferably, is pasted on sample pad 1, pad 2, reaction film 4, absorption pad 6 on backing 7 by bonding agent respectively, obtains described test-strips.
In the present invention, each element (or assembly) of described test-strips can select the existing material in this area to make.
Backing 7 can be made with any material that is stable, atresia, and its intensity should be enough to supporting material and sticky each element thereon.Because much mensuration uses water as dispersive medium, therefore backing 7 is preferably substantially fluid-tight.In a preference, backing 7 is made with polymer film, more preferably makes (as PVC offset plate) with polychloroethylene film.
Sample pad 1 can be made with any absorbent material.Spendable example of material comprises: cellulose, cellulose nitrate, cellulose acetate, glass fibre, nylon, polyelectrolyte ion exchange membrane, propylene copolymer/nylon and polyethersulfone.
Pad 2 or reaction film 4 can be made with any material, as long as this material has enough factor of porosity thus allows in surface and the inner capillarity that fluid occurs.Pad 2 or reaction film 4 should have enough factor of porosity, thus allow the particle moving scribbling antibody or antigen.Pad 2 or reaction film 4 also can be soaked (such as, have water wettability for waterborne liquid, have hydrophobicity for organic solvent) by containing liquid used in the sample of analysis thing to be detected.By such as in U.S. Patent No. 4,340,482 or No.4,618, the method (these methods describe and hydrophobic surface is transformed into water-wetted surface) described in 533, can change its hydrophobicity thus make it have water wettability for use in waterborne liquid.The example of material that can be used for manufacturing pad 2 or reaction film 4 includes but not limited to: polymer PET, cellulose, cellulose nitrate, cellulose acetate, glass fibre, nylon, polyelectrolyte ion exchange membrane, propylene copolymer/nylon and polyethersulfone (polyethersulfone).In a preference, pad 2 is made with polymer PET, and reaction film 4 is made with cellulose nitrate.
Absorption pad 6 can be made by the material absorbed as the liquid of sample and damping fluid with any.The receptivity of absorption pad 6 should be enough large, to absorb the liquid being added into test-strips.The example being applicable to the material of absorption pad 6 comprises cellulose and absorbent filter.
For the ease of understanding the present invention, provide the Cleaning Principle of immune chromatograph testing strip of the present invention.Should be understood that protection scope of the present invention not by impact or the restriction of this principle.
As shown in Figure 2, the present invention adopts the principle of competition law immunochromatography, the comlete antigen of small-molecule substance is fixed on the detection zone (solid phase antigen) on nitrocellulose membrane, if not containing specific micromolecular compound in sample, the micromolecular compound antibody of fluorescent latex mark will along reaction film (as nitrocellulose membrane) flow forward, when arriving detection line position, fluorescent latex labelled antibody is fixed on Small molecular on reaction film and carrier protein couplet thing is caught, by exciting of 365nm ultraviolet light, red 615nm fluorescent bands is formed at detection zone T line position.If containing certain density small-molecule substance in measuring samples, will the combination of Fluorophotometry labelled antibody and immobilized antigen, the fluorescent bands in the detection zone of nitrocellulose filter will weaken.The concentration of the small-molecule substance contained in the power of fluorescent bands and measuring samples is certain linear relationship, gathers fluorescence signal and counts, thus calculate the concentration of the small-molecule substance contained in measuring samples by photomultiplier.The present invention arranges sheep anti-mouse igg in the quality control region of reaction film (as nitrocellulose membrane) and resists more, no matter in measuring samples whether containing micromolecular compound to be measured, fluorescent latex labelled antibody pre-coated on pad can form a fluorescence quality control band with the many anti-bindings of the sheep anti-mouse igg of quality control region, this fluorescent bands judges the chromatography process whether standard that whether lost efficacy of normal and check-out console, while also as the parameter controlling difference between batch in quantitative test.
In a preferred embodiment of the invention, by using the fluorescent latex particles of rare earth element Europium chelate bag quilt, the monoclonal antibody of mark micromolecular compound forms antibody complex, be fixed on pad, detection zone on nitrocellulose membrane and quality control region, wrap by the micromolecular compound of variable concentrations and carrier protein couplet thing, sheep anti-mouse igg respectively, by being analyzed respectively the fluorescence signal in two regions.Obtain the quantitative target detecting thing.
Detection kit and detection method
Present invention also offers the detection kit that can be used for detecting specific micromolecular compound in potable water, beverage, whole blood, blood plasma, serum, urine, sweat, tear, saliva equal samples.Described kit comprises: a container, and is positioned at test pieces and the antibody complex of container, and described antibody complex is the micromolecular compound antibody of fluorescent latex particles mark.
Wherein, described micromolecular compound antibody adopts the fluorescent latex particles containing europium to mark.Preferably, described micromolecular compound antibody adopts the fluorescent latex particles containing europium of gel parcel to mark.
Pick-up unit
Pick-up unit of the present invention can comprise: test-strips, detecting device, light source, light transmitting fiber and computing machine.The operation instruction of a detection method can also be comprised.Wherein the principle of work of test-strips is described above, any Time-resolved fluorescence assay method pick-up unit all used in the present invention that can be used for fluorescence intensity.
Major advantage of the present invention has:
(1) the invention provides a kind of novel test pieces.
(2) the invention provides a kind of detection method adopting above-mentioned test pieces, solve colloidal gold chromatographic and quantitatively forbidden, the shortcoming that common fluorescent chromatography detection background is too high, considerably improve sensitivity and the specificity of detection, reduce false positive rate.And described method is quick, easy, with low cost, quantitatively accurately.
(3) present invention also offers a kind of pick-up unit, described device, based on above-mentioned detection method, can be widely used in quantitative detection field.
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition, the people such as such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the condition that manufacturer advises.Unless otherwise indicated, otherwise number percent and number are percentage by weight and parts by weight.
Embodiment 1
The preparation of fluorescent latex mark ketamine antibody (antibody complex)
Get 100 μ l10% surfaces with the europium latex beads (200nm, purchased from American ThermoFisher company) of activated carboxyl, add 900 μ l MES(0.05M, pH6.1) washing, centrifugal 13000 turns 15 minutes, abandon supernatant.Add 1ml marking fluid (0.05M MES pH6.1,2.0mg/ml N-hydroxy-succinamide NHS, and 0.5mg/ml EDC.HCl(1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides EDC)) resuspended, ultrasonic, react 1 hour under room temperature.The latex activated centrifugal 15 minutes with 13000rpm, abandons supernatant.Resuspended with 1ml0.05M MES buffer pH6.1.Add ketamine antibody 80 μ g/ml after ultrasonic, mixing, at room temperature react 2 hours.Latex after mark centrifugal 15 minutes with 13000rpm, abandons supernatant.Add 1ml0.05M MES buffer pH6.14%BSA to close, ultrasonic, at room temperature close 2 hours.After having closed, centrifugal 13000rpm, 15 minutes, abandons supernatant.With the resuspended emulsion particle of dilution.Use front ultrasonic.
Embodiment 2
The preparation of immunochromatographiassay assay reagent plate
(1) preparation of pad (label pad)
Antibody complex prepared by embodiment 1, mouse IgG and containing 0.5% surfactant Tetronic-1037(purchased from BASF) pH be 7.4 10mM phosphate buffered solution mix, be mixed with the solution of 0.5mg/ml concentration, be coated on equably on glass fibre element paper or polyester film, coating weight is 50 μ l/cm
2, vacuum drying.Prepare pad.
(2) detection zone and quality control region
Detection zone (also referred to as detection line): be dissolved in by ketamine bSA (BSA) conjugate in the 0.01M pH7.3 phosphate buffer containing 10% sucrose, specking is on cellulose nitrate reaction film, and coating weight is 10 μ l/cm
2; Then 15 ~ 35 DEG C of dryings 20 hours, detection zone is made;
Quality control region (also referred to as control line or check plot): be dissolved in by sheep anti-mouse igg in the 0.01MpH7.3 phosphate buffer containing 10% sucrose, concentration is 0.8mg/ml.Specking is on nitrocellulose membrane, and coating weight is 10 μ l/cm2; Then 15 ~ 35 DEG C of dryings 20 hours, quality control region is made.
(3) preparation of sample pad
Sample pad material is all-glass paper or polyester film, and soak sample pad with the sample pad treating fluid prepared, the amount of sample treatment liquid is 100ul/cm
2, then dry at 37 DEG C, consisting of of sample pad treating fluid: pH is the 10mM phosphate buffered solution of 7.4, containing 0.5%S9(surfactant Tetronic-1037), 1%PVP, 0.2%EDTA and 0.5%BSA.
(4) test-strips assembling
By conventional method, by following assembly by being assembled into detection reagent strip shown in Fig. 1:
1. sample pad: through sample pad treating fluid immersion treatment
2. pad: the all-glass paper of coated antibody compound and mouse IgG
3. detection line: nitrocellulose filter wraps by ketamine bSA (BSA) conjugate
4. reaction film
5. nature controlling line: nitrocellulose filter wraps and is resisted by sheep anti-mouse igg more
6. absorption pad (absorbent filter)
7. backing (PVC)
Embodiment 3
Detect
(1) drawing standard curve
Preparation 1.0ml concentration is the PBS solution of the ketamine of 1000ng/ml, get 500 μ l PBS and carry out the ketamine solution that doubling dilution obtains variable concentrations, as follows: 10000ng/ml, 5000ng/ml, 2500ng/ml, 1250ng/ml, 625ng/ml, 312.5ng/ml, 156ng/ml, 78ng/ml, 39ng/ml, 0ng/ml.Take PBS as blank.Get 200 μ l samples directly to add in sample pad, at room temperature react 5 minutes, then detector bar is inserted pick-up unit detect, obtain the fluorescent value of detection zone T line and quality control region C line respectively, calculate T/C ratio, result is as shown in table 1.
Table 1 fluoroscopic examination data
| Ketamine concentration (ng/ml) | T line fluorescent value | C line fluorescent value | T/C ratio |
| 10000 | 5017 | 17724 | 0.283063 |
| 5000 | 5914 | 17730 | 0.333559 |
| 2500 | 7187 | 17722 | 0.405541 |
| 1250 | 9607 | 17728 | 0.541911 |
| 625 | 13490 | 17752 | 0.759914 |
| 312.5 | 16565 | 17728 | 0.934398 |
| 156 | 17530 | 17749 | 0.987661 |
| 78 | 15723 | 17734 | 0.886602 |
| 39 | 18059 | 17723 | 1.018958 |
| 0 | 15836 | 17680 | 0.895701 |
Take sample concentration as X-axis, T/C ratio is Y-axis drawing, as shown in Figure 3, carries out four parameter Logistic regression fit analysis, obtains following regression equation (R
2=0.99180; ED
50=1265):
Y=(A-D)/[1+(X/C)^B]+D
In formula, A=0.96021, B=1.54081, C=1265, D=0.2225.
(2) sample test
Adopt the urine specimen alternate standard serial solution that 5 different, repeat above-mentioned steps, T/C is substituted into regression equation, calculate the content obtaining and record ketamine in sample.In addition, adopt existing colloidal gold immunochromatographimethod method to detect identical sample with GC/MS method, result is as shown in table 2.
Table 2 serum sample testing result
Known after compared with the data adopting the data of time-resolved fluorescence chromatography method of the present invention detection gained and laboratory standard method GC/MS to survey, this method is consistent with GC/MS testing result, but this method is compared with collaurum method, having can the advantage of accurate quantitative analysis.
Embodiment 4
Accuracy compares
Get the positive urine that sucks ketamine personnel, reagent and gold-immunochromatographyreagent reagent for assay is detected with a collection of time-resolved fluorescence, detect for continuous 10 times, data are read respectively with time-resolved fluorescence reading apparatus and collaurum reading apparatus, the relatively CV(coefficient of variation of two kinds of methods) value, result is as shown in table 3.
The comparison of table 3 this method and collaurum testing result
Result display uses no matter detection method of the present invention is T line, C line or T/C, and its CV value is all below 10%, and the CV value of collaurum quantivative approach is 13.70%.Detection method of the present invention has higher accuracy.
The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention, and these equivalent form of values fall within the application's appended claims limited range equally after having read above-mentioned instruction content of the present invention.
Claims (10)
1. a test-strips, comprise sample pad, pad, reaction film, absorption pad and backing, it is characterized in that, described reaction film is arranged detection zone and quality control region, described pad comprises antibody complex, described antibody complex comprises fluorescent latex particles and micromolecular compound antibody, and described micromolecular compound antibody coupling is in described fluorescent latex particles.
2. test-strips as claimed in claim 1, it is characterized in that, the molecular weight of described micromolecular compound is 1-10000Da.
3. test-strips as claimed in claim 1, it is characterized in that, described fluorescent latex particles is the emulsion particle of lanthanide series metal and/or lanthanide chelates bag quilt.
4. test-strips as claimed in claim 1, it is characterized in that, described quality control region comprises goat anti-rabbit igg or sheep anti-mouse igg.
5. test-strips as claimed in claim 1, it is characterized in that, described detection zone comprises the conjugate of micromolecular compound and carrier protein.
6. a detection kit, is characterized in that, described kit comprises:
(a) test-strips, described test-strips comprises sample pad, pad, detection zone, reaction film, quality control region, absorption pad and backing;
(b) antibody complex, described antibody complex comprises fluorescent latex particles and micromolecular compound antibody, and described micromolecular compound antibody coupling is in described fluorescent latex particles; With
(c) container.
7. the purposes of test-strips as claimed in claim 1, is characterized in that, for the preparation of the kit detecting potable water, beverage, whole blood, blood plasma, serum, urine, sweat, tear or saliva small molecular compound.
8. detect a method for micromolecular compound, it is characterized in that, comprise step:
(1) determinand sample is added to the sample pad of the test pieces as described in any one of claim 1-5;
(2) adopt time-resolved fluoroimmunoassay chromatography method to measure the fluorescence intensity of the detection zone of described test pieces and the fluorescence intensity of quality control region, thus determine whether micromolecular compound exists, or thus be scaled the content of micromolecular compound.
9. method as claimed in claim 8, is characterized in that, in step (2), by the ratio of the fluorescence intensity of test section and the fluorescence intensity of quality control region, and typical curve compares, thus determines the content of micromolecular compound.
10. quantitatively detect a fluorescence measuring device for determinand, it is characterized in that, described device comprises:
(a) test pieces according to claim 1; With
B () is for the detecting device of fluorescence intensity.
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Address after: 200231, room 317, building 2A, 2715 Wu Long Road, Shanghai, Xuhui District Applicant after: Shanghai eight way biological Polytron Technologies Inc Address before: 200231, room 317, building 2A, 2715 Wu Long Road, Shanghai, Xuhui District Applicant before: SHANGHAI VENTURE BIOTECHNOLOGY CO., LTD. |
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