CN101526534A - Fluorescence immune chromatography test paper and preparing method and application thereof - Google Patents
Fluorescence immune chromatography test paper and preparing method and application thereof Download PDFInfo
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Abstract
The invention relates to fluorescence immune chromatography test paper which is formed by mutually and sequentially overlapping a sample pad, a combination pad, an antibody carrying film and water absorbing paper on a lining board with adhesive, wherein the combination pad is coated with a fluorescence material antibody 1 composite, a fluorescence-marked material is fluorescein granules or fluorescence material converted by rare earth, the antibody carrying film is a cellulose nitrate film or a nylon film and is respectively connected with an antibody 2 and the T line and the C line of a secondary antibody, the fluorescence material is fluorescein granules or the fluorescence granules converted by the rare earth, and the fluorescein granules are selected from isosulfocyanic acid fluorescein or tetramethyl isosulfocyanic acid rhodamine or tetraethyl rhodamine, and the surface of the fluorescence-marked material is aminated and combined with the antibody by cross-linking reaction. The fluorescence quantitative measuring system is used for detecting the fluorescence strength of the areas of the T line and the C line, and the quantitative detection is completed by the standard curve of the antigen concentration. The invention is suitable for the immune detection of tumor marked objects of urinal fiber-connected protein, and the like.
Description
Technical field
The invention belongs to field of medical examination, particularly relate to a kind of fluorescence immune chromatography and detect test paper.
Background technology
Tumour is high incidence and the high mortality disease that threatens human health, and the big quantity research and the data of preventing and treating confirm, early diagnosis and early treatment are anti-curing oncomas and the effective way that reduces mortality ratio.For this reason, people have imagined multiple measure and approach, to early stage asymptomatic tumor patient, tumor marker usually as can early diagnosis one of the method for tumour.Therefore, constantly seek the focus that new tumor marker and detection method thereof have become academia's research.
At present, mainly still tumor marker is detected clinically by immuno analytical method, as euzymelinked immunosorbent assay (ELISA) (ELISA), radioimmunology (RIA), chemoluminescence method (CLIA) etc., but traditional immune analysis method needs special instrument and equipment, and expense is more expensive.The development of immune chromatography test paper technology has remedied this deficiency to a certain extent, and it is simple to operate, and is accurate and visual, for individual, the conventional quick diagnosis detection of family provide possibility.It as immunoreactive solid phase carrier takes place, makes the antigen or the antibodies of wrapping quilt on antibody in the testing sample or antigen and the film through infiltration and capillary syphonic effect, by label spike reaction result with microporous barrier.Current common marker material mainly comprises radioactive isotope, enzyme, collaurum and luminescent marking material, and wherein luminescent marking material comprises fluorescence, chemiluminescence, bioluminescent material etc. again.But above-mentioned material all has its limitation: radioactive isotope easily decays, and has radiocontamination; Enzyme self property instability is subject to the interference of biological sample; Collaurum is difficult to carry out multiple, quantitative test; Chemiluminescence is an instantaneous light emission, the detecting instrument costliness; Then there is quenching phenomenon in fluorescent material, and making time resolution detect can't carry out.
Therefore, improve or seek new marker material and now become the focus that people pay close attention to.There is the photobleaching phenomenon in fluorescent dye commonly used, causes the fluorescence signal instability, by the quenching phenomenon that has not only overcome it is wrapped up on its surface, and has realized the surface-functionalized of fluorescent grain.SiO
2High with stability, bonding power is strong, and advantages such as the easy functionalization in surface become first-selection naturally.This patent has prepared SiO
2Fluorescein particle-the antibody complex and the fluorescence immune chromatography test paper of parcel, and be applied to fluorescence immune chromatography technology for detection tumor markers, mention 10-100 doubly than the sensitivity of immune golden chromatographic test paper, further expanded its application in biomedical sector.
In addition, a class novel markings thing---the development that appears as immunochromatography technique of nanometer up-conversion luminescent material (UCP) provides new possibility in recent years.UCP is doped in the nano-scale particle that constitutes in the lattice of crystal by thulium.Because particular structure, UCP can be by infrared ray excited and the long-range visible light that is shorter than exciting light of transmitted wave is promptly gone up and transmitted light.This peerless character make UCP serve as a mark thing compare with the conventional tag thing have no background interference, no cancellation, safe in utilization, be fit to significant advantages such as multiple analysis and quantitative test, guaranteed that UCP is in the medium-altitude susceptibility of testing process, stability, security and dirigibility.Chinese food health magazine 2007,19 (1): 41-44 discloses a kind of method of utilizing the converting phosphor immunochromatography to detect enterorrhagia Bacillus coil 0157, but the UCP particle that adopts wraps up, and the fluorescent quenching phenomenon easily takes place, and this detects and is qualitative detection, can not be used for quantitatively.The detection that the UCP material is applied to urinate the fibronectin tumor markers does not appear in the newspapers.
Summary of the invention
Technical matters to be solved
Technical matters to be solved by this invention provides a kind of fluorescence immune chromatography and detects test paper, wrap up with the UCP particle that overcomes the prior art employing, the fluorescent quenching phenomenon easily takes place, and utilize the immune detection of UCP particle to be qualitative detection, can not be used for quantitatively, and the low defective of detection sensitivity.
Technical scheme
One of technical scheme of the present invention provides a kind of fluorescence immune chromatography test paper, by forming at the sample pad, pad, antibody carrier film, the thieving paper that have mutual overlap joint on the liner plate of bonding agent successively, wherein, scribble fluorescence labeling material-antibody 1 compound on the described pad, described antibody carrier film is nitrocellulose filter or nylon membrane, be respectively the T line and the C line that are connected with antibody 2 and second antibody on the antibody carrier film, it is characterized in that, the surface of described fluorescence labeling material is through silication parcel and amination, and by cross-linking reaction and antibodies; The fluorescence intensity in described T line and C line zone is corresponding with the typical curve of standard antigen concentration respectively in detection, finishes detection by quantitative.
One of preferred version of above-mentioned fluorescence immune chromatography test paper is that described fluorescence labeling material is to change fluorescent material on fluorescein particle or the rare earth.
Two of the preferred version of above-mentioned fluorescence immune chromatography test paper is that described fluorescein particle is fluorescein isothiocynate or TRITC or RB 200.
Three of the preferred version of above-mentioned fluorescence immune chromatography test paper is that changeing fluorescent grain on the described rare earth is the alkaline earth sulfide that europium mixes or erbium mixes or thulium mixes.
The preferred version of above-mentioned each fluorescence immune chromatography test paper is that described antibody 1 and antibody 2 are the antibody of alpha-fetoprotein or urine fibronectin.
Two of technical scheme of the present invention provides a kind of preparation method of fluorescence immune chromatography test paper, in turn includes the following steps:
1) surface siliconization of commentaries on classics fluorescent grain on the rare earth: getting on the 5-50mg rare earth changes fluorescent grain, add 300-800mL 35-70% ethanolic solution, sonicated mixes reaction system, 15-50 ℃ of stirred in water bath to balance, again to wherein adding 0.1-1mL ethyl orthosilicate (TEOS), continue reaction 3-10 hour, centrifuge washing;
2) change the surface amination of fluorescent grain on the rare earth: getting on the rare earth of above-mentioned surface siliconization changes fluorescent grain, add the mixed solution that 1: 3～3: 1 (V/V) methyl alcohol of 30～100mL and glycerine are formed, ultrasonic dispersion, then to wherein adding 10-1000 μ L N-(2-amino-ethyl)-3-TSL 8330,15-50 ℃ of constant temperature stirred 5～10 hours down, use the ethanol centrifuge washing, kept dry;
3) change the fluorescent grain surface on the rare earth and connect antibody: change fluorescent grain on the rare earth that the surface connection is amino and be dispersed in the phosphate buffer (PB), add a certain amount of glutaraldehyde, reacted 2-5 hour, the centrifugal glutaraldehyde of removing, again be dispersed in the phosphate buffer, add antibody 1 then, reacted 1-5 hour, centrifuge washing finally is kept in the phosphate buffer that contains 0.1%BSA and 0.1%Tween 20;
4) assembling of fluorescence immune chromatography test paper: will change fluorescent grain-antibody 1 composite coated on the rare earth on pad, antibody 2 and second antibody are connected in formation T line and C line on the antibody carrier film with wire respectively, and are having the bonding sample pad in mutual overlap joint ground, pad, antibody carrier film, thieving paper on the liner plate of bonding agent.
The preparation method's of above-mentioned fluorescence immune chromatography test paper preferred version is that described antibody 1 and antibody 2 are the antibody of alpha-fetoprotein or urine fibronectin.
Three of technical scheme of the present invention provides the preparation method of another kind of fluorescence immune chromatography test paper, in turn includes the following steps:
1) surface siliconization of fluorescein particle: in ethanol system, add 0.1-10mg fluorescein isothiocynate or TRITC or RB 200 respectively, with 0.1-10 μ L N-(2-amino-ethyl)-3-TSL 8330,15-50 ℃ of constant temperature stirred 3～10 hours, add 0.1-10mL water, 0.1-10mL 15%wt ammoniacal liquor, 0.1-10mL ethyl orthosilicate (TEOS) more successively, continue reaction 3-10 hour, washing obtains the fluorescein particle of surface siliconization;
2) surface amination of fluorescein: the fluorescein particle of getting above-mentioned surface siliconization, add 10～50mL1: the mixed solution that 3～3: 1 (V/V) methyl alcohol and glycerine are formed, ultrasonic dispersion, add 10-500 μ L N-(2-amino-ethyl)-3-TSL 8330 then, 15-50 ℃ of constant temperature stirred 5～10 hours, with ethanol washing, drying, obtain the fluorescein particle of surface amination;
3) the fluorescein particle surface connects antibody: with the fluorescein particle dispersion of above-mentioned surface amination in phosphate buffer, add glutaraldehyde reaction 2-5 hour, the centrifugal glutaraldehyde of removing, again be dispersed in the fluorescein particle in the phosphate buffer, add antibody 1, reacted 1-5 hour, the washing back obtains fluorescein particle-antibody 1 compound, is kept in the phosphate buffer that contains 0.1%BSA and 0.1%Tween20;
4) assembling of fluorescence immune chromatography test paper: with fluorescein particle-antibody 1 composite coated on pad, antibody 2 and second antibody are connected in formation T line and C line on the antibody carrier film with wire respectively, and are having the bonding sample pad in mutual overlap joint ground, pad, antibody carrier film, thieving paper on the liner plate of bonding agent.
Four of technical scheme of the present invention provides a kind of method of utilizing above-mentioned fluorescence immune chromatography test paper detection by quantitative antigen, in turn includes the following steps:
I. the antigen standard items are mixed with the concentration series standard items of 2～10 times of gradients;
Ii. above-mentioned concentration series standard items are carried out immunochromatography with above-mentioned test paper respectively, utilize the fluorescent quantitation spectrometer to detect the fluorescence intensity in T line and C line zone respectively, make the typical curve of antigen concentration;
Iii. determined antigen carries out immunochromatography with above-mentioned test paper, utilizes above-mentioned typical curve to try to achieve antigen concentration.
The preferred version of the method for above-mentioned fluorescence immune chromatography test paper detection by quantitative antigen is that described antigen is alpha-fetoprotein or urine fibronectin.
Above-mentioned fluorescence immune chromatography test paper also can be used for the antigen qualitative detection, and method is as follows:
The sample pad end of test strips is inserted analyte sample fluid, take out observations under the exciting of ultraviolet or infrared light behind the 10-30min.With the quality control band place fluorescence is arranged all if detect band, illustrate and contain object in the testing sample, positive result detects and is with the strong more object content of fluorescence high more; If have only Quality Control to have fluorescence, illustrating does not have object, negative result in the testing sample; All do not have fluorescence if detect band with the quality control band place, illustrate that detection is invalid.
Beneficial effect
The present invention carries out the fluorescence labeling material-antibody complex of silication parcel and then has prepared fluorescence immune chromatography test paper by preparing the surface.Rare earth up-conversion luminescent material and fluorescein particle surface are carried out the silication parcel, overcome the defective of the easy cancellation of fluorescein.Use fluorescence immune chromatography technology detection by quantitative tumor markers, overcome the shortcoming that gold test strip can not quantitative measurement, and the sensitivity of this immune chromatography test paper reaches 1-1.5ng/mL, improved 10-100 doubly than gold test strip.
This research combines with the double antibodies sandwich immunochromatography technique by fluorescein particle or up-conversion luminescent material mark, detects the content of tumor marker, has the sensitivity height, and accuracy is good, can realize advantages such as quantitative test.And, filled up technological gap to the quantitative immune detection of urine fibronectin (Fn).Monitoring for early diagnosis, prognosis judgement and the therapeutic process of tumour all has far-reaching influence.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, as the molecular cloning operation manual, or the condition of advising according to manufacturer.All inorganic chemical reagents and organic solvent are available from Shanghai chemical reagent factory; FITC and AEAPS are available from the great Bioisystech Co., Ltd in Shanghai; infrared up-conversion phosphor (UCP) is available from Shanghai Keyan Opto-electrical Technology Co., Ltd, ShangHai HuaMing GaoNa tombar thite New Materials Co., Ltd and Shanghai Kerune Phosphor Technology Co., Ltd.; mouse anti AFP monoclonal antibody and mouse anti FN monoclonal antibody be available from my actor playing a martial role in Chinese operas's thing Science and Technology Ltd. of Shanghai, but the sheep anti-mouse igg antibody auspicious bio tech ltd product that is the Shanghai history.
Embodiment 1: prepare hud typed FITC-alpha-fetoprotein (AFP) antibody complex
The surface siliconization of fluorescein particle: in ethanol system, add 0.1-10mg fluorescein isothiocynate FITC and 0.1-10 μ L N-(2-amino-ethyl)-3-TSL 8330 respectively, 15-50 ℃ of constant temperature stirred 3～10 hours, add 0.1-10mL water, 0.1-10mL 15%wt ammoniacal liquor, 0.1-10mL ethyl orthosilicate (TEOS) more successively, continue reaction 3-10 hour, washing obtains the fluorescein particle of surface siliconization.
The surface amination of fluorescein: the fluorescein particle of getting above-mentioned surface siliconization, add 10～50mL1: the mixed solution that 3～3: 1 (V/V) methyl alcohol and glycerine are formed, ultrasonic dispersion, add 10-500 μ LN-(2-amino-ethyl)-3-TSL 8330 then, 15-50 ℃ of constant temperature stirred 5～10 hours, with ethanol washing, drying, obtain the fluorescein particle of surface amination.
Utilizing, the hud typed FITC of process surface amination is connected with AFP antibody:
1, gets among the PB (pH=7.2) that the amidized hud typed FITC of 2mg joins 2mL0.03mol/L, fully mixing.
2, the glutaraldehyde that adds 500 μ L25% in above-mentioned system, reaction is 3 hours under the room temperature.
3, the unnecessary glutaraldehyde of centrifugal flush away is dispersed among the 5mL 0.03mol/L PB again.
4, again to wherein adding 100 μ L mouse anti AFP monoclonal antibodies, 4 ℃ were reacted 3 hours.
5,4 ℃ of centrifuge washings for several times, be stored in 0.03mol/L PB (contain 0.1%BSA, 0.1%Tween20) in.
Embodiment 2: preparation double-antibody sandwich AFP Detection of antigen test paper
1, the preparation of sample pad: select for use cellulose membrane as the sample pad material, be cut into the band of 1.5 * 30.0cm specification, (0.03mol/L PB (pH=7.2) contains 5mg/mLBSA to put it into the sample pad confining liquid, 0.1%Triton X-100) soaks 30min, 37 ℃ of dry for standby in.
2, the preparation of pad: select for use the plain film of glass fibre as the pad material, it is cut into the band of 1.0 * 30.0cm specification, the hud typed FITC-AFP antibody complex of preparation among the embodiment 1 is centrifugal, in precipitum, add 0.03mol/LPB (pH=7.2), contain 1% sucrose, 1%BSA), abundant mixing, be added on this band 37 ℃ of oven dry.
3, the preparation of NC: the NC film is cut into the band of 2.5 * 30.0cm specification, with point sample instrument diverse location specking AFP monoclonal antibody and sheep anti-mouse igg antibody respectively on the NC film, as detecting band and quality control band, 37 ℃ of oven dry.
4, the assembling of immuno-chromatographic test paper strip: thieving paper, NC film, pad, sample pad are attached on the base plate that has bonding agent successively, and are cut into the wide test strips of 4cm.
Embodiment 3: prepare double-antibody sandwich AFP Detection of antigen test paper with the fluorescein isothiocynate FITC among TRITC TRITC and the RB 200 RB200 alternate embodiment 1-2 respectively.
Embodiment 4: quantitative immune detection AFP antigen
1,20 parts of clinical Serum AFP antigen samples of fluorescence immune chromatography test paper bar detection by quantitative of utilizing embodiment 1-3 to make:
I. the AFP antigen standard items of 1mg/mL are mixed with the concentration series standard items of 10 times of gradients;
Ii. above-mentioned concentration series standard items are carried out immunochromatography with test paper respectively, utilize the fluorescent quantitation spectrometer to detect the fluorescence intensity in T line and C line zone respectively, make the typical curve of antigen concentration;
Iii. the sample pad end with test strips inserts analyte sample fluid, take out behind the 15min, and observations under uviol lamp, the qualitative detection result of acquisition sample: in 20 duplicate samples serum, clinical coincidence rate is 95%;
Iv. detect the T line of 20 duplicate samples serum and the fluorescence intensity in C line zone with fluorescence detector, utilize above-mentioned typical curve to try to achieve antigen concentration, detection by quantitative result: in 20 duplicate samples serum, clinical coincidence rate is 100%.
2, utilize AFP antigen standard items to be mixed with the concentration series of 10 times of gradients, analyze the sensitivity of this test paper monitoring system with fluorescence detector after immunochromatography detects, the result reaches 1ng/mL.
Embodiment 5: preparation UCP-urine fibronectin (FN) antibody complex
Change the surface siliconization of fluorescent grain (going up of alkaline earth sulfide that europium mixes or ShangHai HuaMing GaoNa tombar thite New Materials Co., Ltd transforms fluorescent powder) on the rare earth: getting on the 5-50mg rare earth changes fluorescent grain, add 300-800mL 35-70% ethanolic solution, sonicated mixes reaction system, 15-50 ℃ of stirred in water bath to balance, again to wherein adding 0.1-1mL ethyl orthosilicate (TEOS), continue reaction 3-10 hour, centrifuge washing;
Change the surface amination of fluorescent grain on the rare earth: getting on the rare earth of above-mentioned surface siliconization changes fluorescent grain, add the mixed solution that 1: 3～3: 1 (V/V) methyl alcohol of 30～100mL and glycerine are formed, ultrasonic dispersion, then to wherein adding 10-1000 μ L N-(2-amino-ethyl)-3-TSL 8330,15-50 ℃ of constant temperature stirred 5～10 hours down, use the ethanol centrifuge washing, kept dry;
Utilizing, the UCP of process surface amination is connected with FN antibody:
1, gets among the PB (pH=7.2) that the amidized UCP of 5mg joins 3mL0.03mol/L, fully mixing.
2, the glutaraldehyde that adds 250 μ L25% in above-mentioned system, reaction is 3 hours under the room temperature.
3, the unnecessary glutaraldehyde of centrifugal flush away is dispersed among the 5mL 0.03mol/L PB again.
4, again to wherein adding 150 μ L mouse anti FN monoclonal antibodies, 4 ℃ were reacted 3 hours.
5,4 ℃ of centrifuge washings for several times, be stored in 0.03mol/L PB (contain 0.1%BSA, 0.1%Tween20) in.
Embodiment 6: preparation double-antibody sandwich FN Detection of antigen test paper
1, the preparation of sample pad: select for use cellulose membrane as the sample pad material, be cut into the band of 1.5 * 30.0cm specification, (0.03mol/L PB (pH=7.2) contains 5mg/mLBSA to put it into the sample pad confining liquid, 0.1%Triton X-100) soaks 30min, 37 ℃ of dry for standby in.
2, the preparation of pad: select for use the plain film of glass fibre as the pad material, it is cut into the band of 1.0 * 30.0cm specification, the UCP-FN antibody complex of preparation among the embodiment 3 is centrifugal, in precipitum, add 0.03mol/LPB (pH=7.2), contain 1% sucrose, 1%BSA), abundant mixing, be added on this band 37 ℃ of oven dry.
3, the preparation of NC: the NC film is cut into the band of 2.5 * 30.0cm specification, with point sample instrument diverse location specking FN monoclonal antibody and sheep anti-mouse igg antibody respectively on the NC film, as detecting band and quality control band, 37 ℃ of oven dry.
4, the assembling of immuno-chromatographic test paper strip: thieving paper, NC film, pad, sample pad are attached on the base plate that has bonding agent successively, and are cut into the wide test strips of 4cm.
Embodiment 7: the alkaline earth sulfide that mixes with the europium among the alkaline earth sulfide alternate embodiment 5-6 that erbium mixes or thulium mixes prepares double-antibody sandwich FN Detection of antigen test paper respectively.
Embodiment 8: detect urine fibronectin (FN) antibody complex
1,16 parts of clinical serum FN antigen samples of UCP fluorescence immune chromatography test paper bar detection by quantitative of utilizing embodiment 5-7 to make:
I. the FN antigen standard items of 200ng/mL are mixed with the concentration series standard items of 2 times of gradients;
Ii. above-mentioned concentration series standard items are carried out immunochromatography with test paper respectively, utilize the fluorescent quantitation spectrometer to detect the fluorescence intensity in T line and C line zone respectively, make the typical curve of antigen concentration;
Iii. the sample pad end with test strips inserts analyte sample fluid, take out behind the 15min, and observations under uviol lamp, the qualitative detection result of acquisition sample: in 16 duplicate samples serum, clinical coincidence rate is 94%;
Iv. detect the T line of 16 duplicate samples serum and the fluorescence intensity in C line zone with fluorescence detector, utilize above-mentioned typical curve to try to achieve antigen concentration, detection by quantitative result: in 16 duplicate samples serum, clinical coincidence rate is 100%.
2, utilize FN antigen standard items to be mixed with the concentration series of 2 times of gradients, analyze the sensitivity of this test paper monitoring system with fluorescence detector after immunochromatography detects, the result reaches 1.5ng/mL.
Claims (10)
1. fluorescence immune chromatography test paper, by forming at the sample pad, pad, antibody carrier film, the thieving paper that have mutual overlap joint on the liner plate of bonding agent successively, wherein, scribble fluorescence labeling material-antibody 1 compound on the described pad, described antibody carrier film is nitrocellulose filter or nylon membrane, be respectively the T line and the C line that are connected with antibody 2 and second antibody on the antibody carrier film, it is characterized in that, the surface of described fluorescence labeling material is through silication parcel and amination, and by cross-linking reaction and antibodies; The fluorescence intensity in described T line and C line zone is corresponding with the typical curve of standard antigen concentration respectively in detection, finishes detection by quantitative.
2. fluorescence immune chromatography test paper according to claim 1 is characterized in that, described fluorescence labeling material is to change fluorescent material on fluorescein particle or the rare earth.
3. fluorescence immune chromatography test paper according to claim 1 is characterized in that, described fluorescein particle is fluorescein isothiocynate or TRITC or RB 200.
4. fluorescence immune chromatography test paper according to claim 1 is characterized in that, changeing fluorescent grain on the described rare earth is the alkaline earth sulfide that europium mixes or erbium mixes or thulium mixes.
5. according to the described fluorescence immune chromatography test paper of one of claim 1-4, it is characterized in that described antibody 1 and antibody 2 are the antibody of alpha-fetoprotein or urine fibronectin.
6. the preparation method of a fluorescence immune chromatography test paper in turn includes the following steps:
1) surface siliconization of commentaries on classics fluorescent grain on the rare earth: getting on the 5-50mg rare earth changes fluorescent grain, add 300-800mL 35-70% ethanolic solution, sonicated mixes reaction system, 15-50 ℃ of stirred in water bath to balance, again to wherein adding the 0.1-1mL ethyl orthosilicate, continue reaction 3-10 hour, centrifuge washing;
2) change the surface amination of fluorescent grain on the rare earth: getting on the rare earth of above-mentioned surface siliconization changes fluorescent grain, add the mixed solution that 1: 3～3: 1 (VN) methyl alcohol of 30～100mL and glycerine are formed, ultrasonic dispersion, then to wherein adding 10-1000 μ L N-(2-amino-ethyl)-3-TSL 8330,15-50 ℃ of constant temperature stirred 5～10 hours down, use the ethanol centrifuge washing, kept dry;
3) change the fluorescent grain surface on the rare earth and connect antibody: change fluorescent grain on the rare earth that the surface connection is amino and be dispersed in the phosphate buffer, add a certain amount of glutaraldehyde, reacted 2-5 hour, the centrifugal glutaraldehyde of removing, again be dispersed in the phosphate buffer, add antibody 1 then, reacted 1-5 hour, centrifuge washing finally is kept in the phosphate buffer that contains 0.1%BSA and 0.1%Tween 20;
4) assembling of fluorescence immune chromatography test paper: will change fluorescent grain-antibody 1 composite coated on the rare earth on pad, antibody 2 and second antibody are connected in formation T line and C line on the antibody carrier film with wire respectively, and are having the bonding sample pad in mutual overlap joint ground, pad, antibody carrier film, thieving paper on the liner plate of bonding agent.
7. the preparation method of fluorescence immune chromatography test paper according to claim 6 is characterized in that, described antibody 1 and antibody 2 are the antibody of alpha-fetoprotein or urine fibronectin.
8. the preparation method of a fluorescence immune chromatography test paper in turn includes the following steps:
1) surface siliconization of fluorescein particle: in ethanol system, add 0.1-10mg fluorescein isothiocynate or TRITC or RB 200 respectively, with 0.1-10 μ L N-(2-amino-ethyl)-3-TSL 8330,15-50 ℃ of constant temperature stirred 3～10 hours, add 0.1-10mL water, 0.1-10mL 15%wt ammoniacal liquor, 0.1-10mL ethyl orthosilicate (TEOS) more successively, continue reaction 3-10 hour, washing obtains the fluorescein particle of surface siliconization;
2) surface amination of fluorescein: the fluorescein particle of getting above-mentioned surface siliconization, add 10～50mL1: the mixed solution that 3～3: 1 (VN) methyl alcohol and glycerine are formed, ultrasonic dispersion, add 10-500 μ L N-(2-amino-ethyl)-3-TSL 8330 then, 15-50 ℃ of constant temperature stirred 5～10 hours, with ethanol washing, drying, obtain the fluorescein particle of surface amination;
3) the fluorescein particle surface connects antibody: with the fluorescein particle dispersion of above-mentioned surface amination in phosphate buffer, add glutaraldehyde reaction 2-5 hour, the centrifugal glutaraldehyde of removing, again be dispersed in the fluorescein particle in the phosphate buffer, add antibody 1, reacted 1-5 hour, the washing back obtains fluorescein particle-antibody 1 compound, is kept in the phosphate buffer that contains 0.1%BSA and 0.1%Tween20;
4) assembling of fluorescence immune chromatography test paper: with fluorescein particle-antibody 1 composite coated on pad, antibody 2 and second antibody are connected in formation T line and C line on the antibody carrier film with wire respectively, and are having the bonding sample pad in mutual overlap joint ground, pad, antibody carrier film, thieving paper on the liner plate of bonding agent.
9. method of utilizing the described fluorescence immune chromatography test paper detection by quantitative of claim 1 antigen in turn includes the following steps:
I. the antigen standard items are mixed with the concentration series standard items of 2～10 times of gradients;
Ii. above-mentioned concentration series standard items are carried out immunochromatography with the described test paper of claim 1 respectively, utilize the fluorescent quantitation spectrometer to detect the fluorescence intensity in T line and C line zone respectively, make the typical curve of antigen concentration;
Iii. determined antigen carries out immunochromatography with the described test paper of claim 1, utilizes above-mentioned typical curve to try to achieve antigen concentration.
10. the method for fluorescence immune chromatography test paper detection by quantitative antigen according to claim 9 is characterized in that, described antigen is alpha-fetoprotein or urine fibronectin.
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