CN104977409A - Preparation of fluorescent microsphere immunochromatographic test strip and quantitative detection method - Google Patents
Preparation of fluorescent microsphere immunochromatographic test strip and quantitative detection method Download PDFInfo
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- 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 discloses a preparation method of a fluorescent microsphere immunochromatographic test strip and a quantitative detection method. The fluorescent microsphere immunochromatographic test strip is prepared by taking light emitting nanoparticles prepared by a soap-free emulsion polymerization method as a label and is prepared through an immunochromatographic technology; and then, the fluorescent microsphere immunochromatographic test strip is prepared into a detection card comprising a sample cushion, a glass fibre film, a nitrocellulose film and a piece of absorbent paper, wherein a detection line and a quality control line are fixed on the nitrocellulose film. The quantitative detection method disclosed by the invention comprises the following steps: stimulating by adopting an optimal excitation light source of a fluorescent microsphere, enabling emitted fluorescence to pass through a light filter, collecting, gathering and multiplying an emission spectrum by using a CCD scanning technology or an optical fibre technology, converting into a numerical signal, multiplying the tested fluorescence intensity of a detection line by a correction coefficient, substituting the corrected fluorescence intensity in a pre-set standard curve in a fluoroanalyzer, and then, automatically calculating through the fluoroanalyzer so as to obtain the concentration of an object to be detected in a sample. The detection method disclosed by the invention is high in sensitivity, accurate in quantification and convenient to operate.
Description
Technical field
The present invention relates to field of biological medicine, specifically relate to a kind of preparation method and quantitative detecting method of fluorescent micro-ball immune chromatography test paper strip.
Technical background
In biological and medical science detect, often use immunoassay, it comprises the methods such as radiommunoassay, enzyme-linked immuno assay and immunochromatography.Radiommunoassay and enzyme-linked immunosorbent assay need expensive equipment, the operating personnel of specialty and the operation steps of complexity, are difficult to obtain testing result fast.Immunochromatographic method because of at the bottom of simple to operate, quick and cost, and is usually used in fast qualitative or half-quantitative detection, example hydrochloric acid Clenbuterol, hepatitis B surface antigen, aflatoxin and metakentrin isocolloid gold test paper strip.The incidence of disease of the common transmittable disease such as current China acquired immune deficiency syndrome (AIDS), hepatitis B, the third liver, syphilis constantly rises, and causes safely very big hidden danger to society and people's lives.And based on immunochromatography technique product because of easy and simple to handle, need visual inspection or simple instrument just can show result fast and only, can be used as the preliminary screening protocol of disease or Illegal addition detection.Therefore, the product developed based on immunochromatography technique is conducive to alleviating the situation that the common transmittable disease incidences such as current China angiocardiopathy, acquired immune deficiency syndrome (AIDS), hepatitis B, the third liver and syphilis rise day by day.
Immunochromatography technique is the immunoassay formats of a kind of uniqueness coming across the initial stage eighties, it usually with fibre strip chromatographic material for solid phase, make sample solution swimming on chromatography strip by capillary action, and make the immune response that the determinand in sample and chromatographic material occur for the acceptor (as antibody or antigen) of determinand high special, high-affinity simultaneously.In chromatography process, immune complex is by enrichment or the certain area (detection zone) being trapped in chromatographic material, the label (as collaurum) can estimated by enzyme reaction or directly utilize the experimental phenomena intuitively (as colour developing) seen and obtain measurement result.Free label (label be not namely combined with determinand) then crosses detection zone, reaches the object be automatically separated with binding label.The common trace particle of immunochromatography technique has collaurum, latex, electroselenium, gelatin etc., wherein uses the most successful label to be collaurum.But colloidal gold immunochromatographimethod technology still has the following disadvantages:
(1) colloid gold label process is Electrostatic Absorption process, belongs to physisorption, in the liquid phase less stable, and the protein molecular on oneself mark easily comes off again.
(2) only have when gold grain reaches a certain amount of, naked eyes just can tell test strip and background difference, and thus detection sensitivity is not high.
(3) different matrix of materials effects is obvious, and background interference is very large.
(4) colloidal gold immunochromatographimethod technology is generally used for qualitative or half-quantitative detection.
The sensitivity of colloidal gold immunochromatographimethod technology for detection is lower, can only survey the qualitative or sxemiquantitative of thing in reality detects to detection, cannot accurate quantitative analysis.At present, oneself has Patents to report with fluorescent nano particles is that label carries out immunochromatography detection, as publication number be CN1645146A patent discloses the immune chromatography method of a kind of fluorescent rare earth nanometer particle as label and the preparation of test strip thereof.Publication number be CN1866012A patent discloses one quantitatively, immunologic detection method and special purpose device thereof fast, the method is by fluorescent material huge legendary turtle compound Eu
3+, sm
3+, Tb
3+, Dy
3+be combined with organic polymer nanoparticle, be prepared into fluorescent particle, undertaken quantitatively by fluoroscopic examination.Method disclosed in these patents can improve the sensitivity that immunochromatography detects, but the fluorescent nano particle of report exists certain defect, as dyestuff easily reveal, anti-solution interference performance difference etc., thus range of application is restricted to a great extent.
Summary of the invention
A kind of method that the object of this invention is to provide preparation method that is highly sensitive, easy and fluorescent micro-ball immune chromatography test paper strip fast and quantitatively detect.The fluorescent microsphere that the present invention uses is not by the impact of external environment, and thus fluorescent stabilization, provides good condition for being undertaken by fluorescence quantitatively detecting.
For achieving the above object, the technical solution used in the present invention is: a kind of preparation method of fluorescent micro-ball immune chromatography test paper strip, comprises the following steps:
(1) preparation of fluorescent microsphere pad: take 0.009g potassium persulfate, 0.004g NaHCO
3be dissolved in 7ml deionized water water, after the fluorescent dye of certain mass being dissolved in 2ml absolute ethyl alcohol and 1ml styrene, join in aqueous solution, shake up, logical nitrogen 5min, puts into after 70 DEG C of waters bath with thermostatic control jolt 24h, takes out, centrifugal (10000rpm × 10min) isolated polymkeric substance uses ethanol, deionized water centrifuge washing 3 times respectively, namely obtains polystyrene fluorescent microsphere.Come labelled antibody, antigen or other specific binding material with the fluorescent microsphere of preparation, and be sprayed on glass fibre membrane.
(2) preparation of nitrocellulose filter (NC film): antibody, antigen or other specific binding material bag are made detection zone by nitrocellulose filter, by another kind of antibody or antigen coatedly make quality control region to nitrocellulose filter.
(3) assembling and shearing: paste with overlapping successively in adhesive base: filter paper, sample pad, fluorescent microsphere pad, nitrocellulose filter and thieving paper, and cut into proper width and namely become immuno-chromatographic test paper strip.
Described fluorescent microsphere diameter is 30-150nm
Described reactive group is-CHO ,-COOH ,-OH ,-NH: or-SH
Described fluorescent material is that organic nail compound, fluorescein isothiocynate, rhodamine isothiocyanate, 6-shuttle base fluorescein phthalein amine are cruel, fluorescence alkane derivatives class, 1,8-Cai two phthalimide class, Coumarins organic fluorescent dye or its alloy and quantum dot
Another technical scheme of the present invention provides the method preparing fluorescent microsphere immunochromatographydetection detection card with fluorescent micro-ball immune chromatography test paper strip.
One or multiple immuno-chromatographic test paper strips are fixed on end card side by side, then compress at test strips surface face card, namely become immunochromatographydetection detection card.End card and face card are generally all selected as plastic clip, and end card can make the sample pad in test strips, fluorescent microsphere pad, nitrocellulose filter and thieving paper combine closely, and face card can protect test paper, makes it not be damaged.Reserved well and view window on the card of face, the position of well is corresponding with the sample pad of test strips, and the position of view window is corresponding with the NC film of test strips.
Present invention also offers one and utilize fluorescent microsphere immunochromatographydetection detection card quantitative detecting method, comprise the following steps:
(1) drawing standard curve: the standard solution (concentration more than 5) preparing a series of variable concentrations (x), with several the immune detection card examination criteria product solution of same batch, obtain detection line fluorescence intensity (y) and standard nature controlling line fluorescence intensity C
0, with the concentration of standard solution for horizontal ordinate, detection line fluorescence intensity is ordinate, draws a typical curve y=ax+b, records this typical curve and standard nature controlling line fluorescence intensity C in fluorescence analyser
0.
(2) add sample to be checked in the well of immunochromatographydetection detection card, after reaction 3-20min, test card is put into detection window.
(3) fluorescent microsphere being trapped in detection zone and quality control region, under lamp source excitation, sends strong fluorescence.
(4) fluorescence launched is after filtering veiling glare, and the optical signalling will gathered by focusing system, send into photomultiplier, light signal is enhanced, then through signal conversion element, obtains the fluorescence intensity of detection line and nature controlling line.
(5) nature controlling line fluorescence intensity built-in in the nature controlling line fluorescence intensity of acquisition and fluorescence analyser is corrected, obtain correction coefficient when this sample to be checked measures, eliminate the matrix interference effect detected in sample.
(6) the detection line fluorescence values of acquisition is multiplied by correction coefficient, and institute's value is substituted into the typical curve be arranged in fluorescence analyser, namely obtain the concentration of determinand in sample, above process calculates acquisition automatically by the built-in analysis software of fluorescence analyser.
Correction principle described in the present invention is as follows:
Nature controlling line fluorescence intensity during drawing standard curve is C
0, and nature controlling line fluorescence intensity when actual sample detects is C, detection line fluorescence intensity is T, because nature controlling line difference and substrate concentration to be checked have nothing to do, only relevant with testing conditions, therefore can be used for the fluorescence intensity T of detection line when being corrected to drawing standard curve
0.The fluorescence intensity T of detection line
0by following formulae discovery out:
By corrected value T
0substitute into typical curve y=x+b, can draw the concentration of determinand in sample, above process calculates acquisition automatically by the built-in analysis software of analyser.
Immune response on immuno-chromatographic test paper strip described in the present invention comprises two kinds of patterns: sandwich mode and competitive mode.
(a) sandwich mode: can be used for detecting in sample exist pathogen, microorganism and large molecular antibody, antigen etc.; Comprise double-antibody sandwich and double antigens sandwich, both principles are identical, are now described for double-antibody sandwich.
In the preparation process of immuno-chromatographic test paper strip, first the determinand specific antibody A (specific binding is in the antibody in determinand A site) that fluorescent microsphere marks is sprayed on glass fibre membrane and makes fluorescent microsphere pad; Then determinand specific antibody B (specific binding is in determinand B site) is fixed on as detection zone on nitrocellulose filter, two anti-(can with the antibody of antibody A specific binding) be fixed on nitrocellulose filter as quality control band.
When sample pad adding sample, first determinand combines with the fluorescent microsphere-antibody A on fluorescent microsphere pad, forms immune complex (fluorescent microsphere-antibody A-determinand); Then under capillary action, the fluid matrix that immune complex and free fluorescent microsphere-antibody A are followed in sample enters NC film together; When through detection zone, B site on determinand in immune complex is combined and forms fluorescent microsphere-antibody A-determinand-antibody B compound by the antibody B in detection zone, and be fixed in detection zone, and free fluorescent microsphere-antibody A is not combined with antibody B, continue flowing under capillary action, when through quality control band, merge with two resistive connections and be fixed on quality control band.Detect the fluorescence intensity of detection zone and quality control band with fluorescence analyser, the fluorescence intensity wherein in detection zone is directly proportional to testing concentration.
(b) competitive mode: can be used for detecting the small molecule antigens existed in sample
In the preparation process of immuno-chromatographic test paper strip, fluorescent microsphere is combined with determinand specific antibody A, and be sprayed on glass fibre membrane and make fluorescent microsphere pad; The antigen identical with determinand (all containing antibody A can the A site of specific binding) is fixed on as detection zone on NC film, two anti-(can with the antibody of determinand specific antibody A specific binding) be fixed on NC film as quality control band.When sample pad adding sample, the determinand in sample is combined with fluorescent microsphere-antibody A, forms complex fluorescence microballoon-antibody A-determinand; Under capillary action, the fluid matrix in compound and free fluorescent microsphere-antibody A and sample-work entering NC film; When through detection zone, the free antigen of fluorescent microsphere-antibody A in detection zone is combined and forms immune complex and be fixed in detection zone, and the complex fluorescence microballoon-antigen of antibody A-determinand not in detection zone is combined, continue flowing under capillary action; When through quality control band, antibody A and two anti-generation immune responses, and fluorescent microsphere one antibody A-determinand is fixed on quality control band.Detect the fluorescence intensity of detection zone and quality control band with fluorescence analyser, wherein the fluorescence intensity of detection zone and testing concentration are inversely proportional to.
Advantage of the present invention is as follows:
1. after fluorescent material is excited by specific wavelength, there is Stokes shift effect, can at larger wavelength emission light, therefore fluorescence is not by the interference from exciting light background, sensitivity is much higher than UV-VIS spectrophotometry, and its sensitivity is 10-1000 times by conventional dyes and coloured label substance detecting method.Compared with fluorescence labeling detection method and this two kinds of methods, also have easy and simple to handle, detect the advantages such as quick, cheap.
2. fluorescent microsphere is a kind of nucleocapsid double structure Nano particles of silicon dioxide, organic dyestuff is positioned at kernel, not by the impact of external environment, thus fluorescent stabilization, good condition is provided, the shortcomings such as the strong dyestuff overcoming conventional fluorescent microballoon is easily revealed, anti-solution interference performance difference for being undertaken by fluorescence quantitatively detecting, add fluorescent stability and fluorescence lifetime, expand the scope and kind that can detect thing.
3. fluorescent microsphere surface easily modification activities group, can adopt chemical conjugation methods mark one to remember antibody or antigen, form the stable bond of antibody or antigen and microballoon.
4. after being collected by the emission spectrum after filtration by CCD scanning technique or optical fiber technology, carry out photoelectricity and analog/digital conversion, then through software process, finally with numerical monitor testing concentration, thus realize quantitatively detecting.
Accompanying drawing explanation
Fig. 1 is the structural representation of immuno-chromatographic test paper strip
Fig. 2 is the structural representation of immunochromatographydetection detection card
Fig. 3 is fluorescent micro-ball immune chromatography test paper Cleaning Principle figure
Fig. 4 is a kind of Cleaning Principle and structural representation thereof of simple and easy fluorescence detector
As shown in Figure 1, the formation of this immuno-chromatographic test paper strip is: in adhesive base 18, overlap the glass fibre membrane 13 of ground stickup filter paper 11, sample pad 12, the antibody being coated with fluorescent microsphere mark or antigen, nitrocellulose filter 14 and thieving paper 17 successively, on nitrocellulose filter 14, wherein have the skilful quality control region 16 with being coated with another kind of antibody or antigen in the detection zone of coated antibody or antigen.
As shown in Figure 2, this immunochromatographydetection detection card is single deck tape-recorder type, is fixed on end card is formed by an immuno-chromatographic test paper strip, and concrete structure comprises end card 21, face card 22, well 23, view window 24, NC film 25, nature controlling line 26, detection line 270.
As shown in Figure 1,2 and 3, Cleaning Principle is as follows: in sample pad 12, drip sample, the fluorescent microsphere labelled antibody of spraying on sample dissolution glass fibre membrane 13 or antigen, by capillary action swimming forward on tunica fibrosa, the test substance simultaneously in sample and Fluorescent microsphere marker are reacted; Reaction solution, when detection zone is skilful, is combined with the encrusting substance of detection zone, and is enriched in this detection zone; Test strips is put into detection window 41, fluorescent microsphere 43 is under light source 42 excites, and the fluorescence 44 of transmitting, by after monochromatic filter 35 filtration, can observe a fluorescent bands clearly by watch window 36 in detection zone 15.
As shown in Figure 4, the detecting step that quantitatively detects of fluorescent microsphere immunochromatographydetection detection card is as follows:
(1) in the well 23 of test card, add measuring samples, after reaction 10min, put it into detection window 41.
(2) fluorescent microsphere 43 that is trapped of detection zone and quality control region is under best excitation source 42 excites, and sends strong fluorescence.
(3) fluorescence 44 launched is after CCD scanning system or fibre system 45 converge, pipe 46 is assembled through photoelectricity, send into photomultiplier 47, light signal is enhanced, again after signal conversion element 48 and software analysis 49 process, in sample, the concentration of determinand exports the display screen display of 410 in data.
Embodiment
Embodiment:
1, the preparation of rhodamine B n-octyl-polystyrene fluorescent microsphere
Take 0.009g potassium persulfate, 0.004gNaHCO
3be dissolved in 7ml deionized water water, after 1mg rhodamine B n-octyl is dissolved in 2ml absolute ethyl alcohol and 1ml styrene, join in aqueous solution, shake up, logical nitrogen 5min, puts into after 70 DEG C of waters bath with thermostatic control jolt 24h, takes out, centrifugal (10000rpm × 10min) isolated polymkeric substance uses ethanol, deionized water centrifuge washing 3 times respectively, namely obtains rhodamine B n-octyl-polystyrene fluorescent microsphere.
2, the preparation (EDC method) of the fluorescent microsphere of CRP monoclonal antibody S1 is marked
Get 1mg and wrap up the fluorescent microsphere of rhodamine B n-octyl fluorescein at the centrifugal 10-15min of 1000 × g, collecting precipitation, regulate microballoon concentration to be OD with the borate buffer solution of 0.01M pH4.8
450=0.2.Then 90 μ L50mg/mL are added to ethyl-N, N-dimethyl propyl carbodiimide (EDC), 150 μ L5mg/mL nitrogen are bluffed through base and are clapped phthalimide (NHS), vibration mixing, after incubated at room 10-30min, the centrifugal 5-15min of 1000 × g, the precipitation borate buffer solution of 0.01M pH4.8 dissolves, and regulates microballoon concentration to be OD
450.For 0.2-1.1-10 μ LCRP monoclonal antibody C will be added in 0.1mL fluorescent microsphere
1after abundant mixing, stirring at room temperature reaction 3h, after using centrifugal 3 times of milli-Q water respectively, the PBS solution (wherein comprising 5% sucrose and 0.05%Tween-20) of precipitation 0.01M pH7.2 is the mark CRP monoclonal antibody C prepared after redissolving and being precipitated to initial volume
1fluorescent microsphere.
3, the preparation of fluorescent microsphere pad
With BIODOT Dispensing System, CRP monoclonal antibody C will be marked
1fluorescent microsphere be sprayed on glass fibre membrane (30 × 0.8cm) according to the amount of 4 μ L/cm, 25 DEG C of vacuum drying 1-2h, are put in dry environment for subsequent use.
4, the preparation of nitrocellulose filter (NC film)
CRP monoclonal antibody C is regulated with 0.01M pH7.4PBS (phosphate buffer wherein comprises 5% sucrose and 0.05%Tween-20)
2concentration be 0.5mg/mL, gained solution spraying is formed detection line on NC film; Regulate the concentration of anti-mouse antibody to be 0.5mg/mL with 0.01M pH7.2PBS (phosphate buffer wherein comprises 5% sucrose and 0.05%Tween-20), gained solution spraying is formed quality control region on NC film.The spray film amount in twoth district is 0.74 μ L/cm, and twoth district are separated by 5mm, quality control region distance NC film one end 2mm, and 37 DEG C of oven dry save backup after spending the night under drying at room temperature environment.
5, the preparation of fluorescent microsphere immunochromatographydetection detection card:
Assembling test strips: paste with overlapping successively on PVC base plate: (1) filter paper and sample pad, sample pad is a kind of glass fibre membrane through 5%Tween-20 process; (2) the CRP monoclonal antibody C of fluorescent microsphere mark is coated with
1fluorescent microsphere pad; (3) spraying CRP monoclonal antibody C is had
2as detection zone and the anti-mouse antibody nitrocellulose filter as quality control region; (4) thieving paper, cuts into the width of 4mm, namely becomes immuno-chromatographic test paper strip after having assembled.
An immuno-chromatographic test paper strip is fixed on plastic bottom card, and test paper surface face card compresses, and face is stuck in the sample pad of corresponding test strips and position reserved well and the view window respectively of NC film.Immunochromatographydetection detection card assembles in rear loading aluminium foil bag, and after adding drying agent, sealing is preserved, under drying at room temperature environment, at least can preserve 1 year.
6, fluorescent micro-ball immune chromatography quantitatively detects human serum CRP concentration:
The drafting of typical curve: CRP standard items are mixed with a series of concentration (more than 5), detect the standard solution of each concentration with several the immunochromatographydetection detection cards of same batch.With the fluorescence intensity of detection line for ordinate, CRP standard solution concentration is horizontal ordinate, draws a typical curve.Typical curve and corresponding standard nature controlling line fluorescence intensity are kept in fluorescence analyser.
The detection of sample:
(1) keep flat test card, test serum balance, to room temperature, is got its 50 μ L and is added in well, react 10min, test card is put into detection window under room temperature.
(2) fluorescent microsphere being trapped in detection zone and quality control region, under the exciting of best excitation source, sends strong fluorescence.
(3) fluorescence launched is after filtering veiling glare, and the optical signalling will gathered by lens system, send into photomultiplier, light signal is enhanced, then through signal conversion element, obtains the fluorescence values of detection line and nature controlling line.
(4) the built-in analysis software in fluorescence analyser corrects detection line fluorescence intensity, and corrected value is substituted into built-in typical curve, and automatically calculating CRP concentration in test serum is 3.02ng/mL.
Get 10 serum samples to be checked, detect with fluorescent microsphere immunochromatographydetection detection card of the present invention and ELISA respectively,
The testing result of two kinds of methods compared, result is as follows: unit is ng/mL
The related coefficient of two groups of data is r=0.9913, shows the testing result significant correlation of these two kinds of methods.Fluorescent microsphere immunochromatographydetection detection card of the present invention can be used to carry out Quantitative detection CRP.
Embodiment two
1, the preparation of rhodamine B methyl esters-polystyrene fluorescent microsphere
Take 0.009g potassium persulfate, 0.004g NaHCO
3be dissolved in 7ml deionized water water, after 1mg rhodamine B methyl esters is dissolved in 2ml absolute ethyl alcohol and 1ml styrene, join in aqueous solution, shake up, logical nitrogen 5min, puts into after 70 DEG C of waters bath with thermostatic control jolt 24h, takes out, centrifugal (10000rpm × 10min) isolated polymkeric substance uses ethanol, deionized water centrifuge washing 3 times respectively, namely obtains rhodamine B methyl esters-polystyrene fluorescent microsphere.
2, the preparation of the fluorescent microsphere of S100 monoclonal antibody is marked:
Get 1mg rhodamine B methyl esters-polystyrene fluorescent microsphere at the centrifugal 10-15min of 1000 × g, collecting precipitation, regulate microballoon concentration to be OD with the borate buffer solution of 0.01M pH4.8
450=0.2.Then 90 μ L50mg/mL are added to ethyl-N, N-dimethyl propyl carbodiimide (EDC), the light base glass of 150 μ L5mg/mL nitrogen claps phthalimide (NHS), vibration mixing, after incubated at room 10-30min, the centrifugal 5-15min of 1000 × g, the precipitation borate buffer solution of 0.01M pH4.8 dissolves, and regulates microballoon concentration to be OD
450for 0.2-1.1-20 μ g S100 monoclonal antibody will be added in 0.1mL fluorescent microsphere, after abundant mixing, stirring at room temperature reaction 6h, after using centrifugal 3 times of milli-Q water respectively, the PBS solution (wherein comprising 5% sucrose and 0.05%Tween-20) of precipitation 0.01M pH7.2 is the fluorescent microsphere of the S100 monoclonal antibody prepared after redissolving and being precipitated to initial volume.
3, the preparation of fluorescent microsphere pad:
With BloDoT Dispensing System, be sprayed on glass fibre membrane (30 × 0.8cm) by the fluorescent microsphere of mark S100 monoclonal antibody according to the amount of 4 μ L/cm, 25 DEG C of vacuum drying 1-2h, are put in dry environment for subsequent use.
4, the preparation of nitrocellulose filter (NC film):
With 0.01M pH7.4PBs, (phosphate buffer, wherein comprises 5% sucrose and 0.05%Tween-20 regulates the concentration of S100 to be 0.8mg/mL, and gained solution spraying is formed detection line on NC film; Regulate the concentration of anti-mouse antibody to be 0.5mg/mL with 0.01MpH7.2PBS (phosphate buffer wherein comprises 5% sucrose and 0.05% Tween-20), gained solution spraying is formed quality control region on NC film.The spray film amount in twoth district is 0.74 μ L/cm, and twoth district are separated by 5mm, quality control region distance NC film one end 2mm, and 37 DEG C of oven dry save backup after spending the night under drying at room temperature environment.
5, the preparation of fluorescent microsphere immunochromatographydetection detection card:
Assembling test strips: paste with overlapping successively on PVC base plate: (1) filter paper and sample pad, sample pad is a kind of glass fibre membrane through 5% polysorbas20 process; (2) the fluorescent microsphere pad of the S100 monoclonal antibody of fluorescent microsphere mark is coated with; (3) spraying S100 is had as detection zone and the anti-mouse antibody nitrocellulose filter as quality control region; (4) thieving paper, cuts into the width of 4mm, namely becomes immuno-chromatographic test paper strip after having assembled.
An immuno-chromatographic test paper strip is fixed on plastic bottom card, and test paper surface face card compresses, and face is stuck in the sample pad of corresponding test strips and position reserved well and the view window respectively of NC film.Immunochromatographydetection detection card assembles in rear loading aluminium foil bag, and after adding drying agent, sealing is preserved, under drying at room temperature environment, at least can preserve 1 year.
6, fluorescent micro-ball immune chromatography quantitatively detects human serum S100 concentration:
The drafting of typical curve: S100 standard items are mixed with a series of concentration (more than 5), detect the standard solution of each concentration with several the immunochromatographydetection detection cards of same batch.With the fluorescence intensity of detection line for ordinate, S100 standard solution concentration is horizontal ordinate, draws a typical curve.Typical curve and corresponding standard nature controlling line fluorescence intensity are kept in fluorescence analyser.
The detection of sample:
(1) keep flat test card, test serum balance, to room temperature, is got its 50 μ L and is added in well, after reacting 10min, test card is put into detection window under room temperature.
(2) fluorescent microsphere being trapped in detection zone and quality control region, under the exciting of best excitation source, sends strong fluorescence.
(3) fluorescence launched is after filtering veiling glare, and the optical signalling will gathered by lens system, send into photomultiplier, light signal is enhanced, then through signal conversion element, obtains the fluorescence values of detection line and nature controlling line.
(4) the built-in analysis software in fluorescence analyser corrects detection line fluorescence intensity, and corrected value is substituted into built-in typical curve, and automatically calculating S100 concentration in test serum is 5.9ng/mL.
Get 10 serum samples to be checked, detect with fluorescent microsphere immunochromatographydetection detection card of the present invention and ELISA respectively,
The testing result of two kinds of methods compared, result is as follows: unit is ng/mL
The related coefficient of two groups of data is r=0.9750, shows the testing result significant correlation of these two kinds of methods.Of the present invention
Fluorescent microsphere immunochromatographydetection detection card can be used to carry out Quantitative detection S100.
Claims (8)
1. a preparation method for fluorescent micro-ball immune chromatography test paper strip, comprises the following steps:
(1) preparation of fluorescent microsphere pad: take 0.009g potassium persulfate, 0.004g NaHCO
3be dissolved in 7ml deionized water water, after the fluorescent dye of certain mass being dissolved in 2ml absolute ethyl alcohol and 1ml styrene, join in aqueous solution, shake up, logical nitrogen 5min, puts into after 70 DEG C of waters bath with thermostatic control jolt 24h, takes out, centrifugal (10000rpm × 10min) isolated polymkeric substance uses ethanol, deionized water centrifuge washing 3 times respectively, namely obtains polystyrene fluorescent microsphere.Come labelled antibody, antigen or other specific binding material with the fluorescent microsphere of preparation, and be sprayed on glass fibre membrane.
(2) preparation of nitrocellulose filter: antibody, antigen or other specific binding material bag are made detection zone by nitrocellulose filter, by another kind of antibody or antigen coatedly make quality control region to nitrocellulose filter.
(3) assembling and shearing: paste filter paper, sample pad, fluorescent microsphere pad, nitrocellulose filter and thieving paper successively with overlapping in adhesive base, and cut into proper width and namely become immuno-chromatographic test paper strip.
2. the preparation method of fluorescent micro-ball immune chromatography test paper strip according to claim 1, is characterized in that: described fluorescent microsphere diameter is 30-150nm.
3. the preparation method of fluorescent micro-ball immune chromatography test paper strip according to claim 1, is characterized in that: described reactive group is-CHO ,-COOH ,-OH ,-NH or-SH.
4. the preparation method of fluorescent micro-ball immune chromatography test paper strip according to claim 1, it is characterized in that: described fluorescent material is that organic nail compound, fluorescein isothiocynate, rhodamine isothiocyanate, 6-shuttle base fluorescein phthalein amine are cruel, fluorescence alkane derivatives class, 1,8-Cai two phthalimide class, Coumarins organic fluorescent dye or its alloy and quantum dot.
5. the method for fluorescent microsphere immunochromatographydetection detection card is prepared with the fluorescent micro-ball immune chromatography test paper strip described in any one of claim 1-4, one or multiple immuno-chromatographic test paper strips is it is characterized in that to be fixed on side by side on end card, test paper surface face card compresses, reserved well and view window on the card of face, the position of well is corresponding with the sample pad of test strips, and the position of view window is corresponding with the nitrocellulose filter of test strips.
6. the preparation method of fluorescent microsphere immunochromatographydetection detection card according to claim 5, is characterized in that: described end card and face card material are plastics.
7., with fluorescent microsphere immunochromatographydetection detection card quantitative detecting method according to claim 5, comprise the following steps:
(1) drawing standard curve: the standard solution preparing a series of concentration, detecting its fluorescence intensity with several the immunochromatographydetection detection cards of same batch, take fluorescence intensity as ordinate, and standard solution concentration is horizontal ordinate, draw a typical curve, and stored in fluorescence analyser.
(2) add sample to be checked in the well of immunochromatographydetection detection card, after reaction 3-20min, test card is put into detection window.
(3) fluorescent microsphere being trapped in detection zone and quality control region, under lamp source excitation, sends strong fluorescence.
(4) fluorescence launched is after filtering veiling glare, and the optical signalling will gathered by focusing system, send into photomultiplier, light signal is enhanced, then through signal conversion element, obtains the fluorescence intensity of detection line and nature controlling line.
(5) nature controlling line fluorescence intensity built-in in the nature controlling line fluorescence intensity of acquisition and fluorescence analyser is corrected, obtain correction coefficient when this sample to be checked measures.
(6) the detection line fluorescence values of acquisition is multiplied by correction coefficient, and institute's value is substituted into the typical curve be arranged in fluorescence analyser, namely obtain the concentration of determinand in sample.
8. quantitative detecting method according to claim 7, is characterized in that: immune response pattern is sandwich mode or competitive mode.
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