First function five fluorescent microsphere joint-detection device and preparation method thereofTechnical field
The present invention relates to medical treatment detection device field, in particular to a kind of first function five joint-detection devices and its preparationMethod utilizes human thyroid element (T4), three in fluorescence immune chromatography technology quantitative detection clinical samples (whole blood/serum/plasma)Iodine thyronine (T3), thyrotropic hormone (TSH), free T3, detection device of free T4 and preparation method thereof, can be realSensitive, special, the quick detection of existing thyroid function marker.
Background technique
Thyroid disease is a kind of common disease clinically.Such disease mainly includes hypothyroidism and thyroid glandHyperfunction.Hypothyroidism is otherwise known as " first subtracts ", this disease is mostly because the thyroid hormone total amount of thyroid gland is very fewCaused, the main clinical manifestation of this patient is the symptoms such as cold, memory and appetite stimulator, anemia.Thyroid functionIt is hyperfunction to be otherwise known as " hyperthyroidism ", this disease be then due to thyroid gland thyroid hormone total amount excessively caused by, this patient masterThe clinical manifestation wanted is honey stomach, tachycardia, impatience etc., this disease often brings apparent damage to the body of patient.It is relatedResearch point out, subtract the thyroid function of patient with reasonable, effective Evaluation of detection methods hyperthyroidism and first, for Clinics and PracticesEqual hyperthyroidism subtracts with first to have very important significance.Clinically, mostly using five joint inspection methods of first function to the first shape of these patientsGland function is measured, and " first function five " refers to thyroxine (T4), trilute (T3), thyrotropic hormone(TSH), free T3, free T4.Five joint inspection methods of first function have very important effect in terms of assessing thyroid function, this inspectionSensibility and specificity of the survey method in Diagnosis of Thyroid Diseases, which is higher than, individually detects first function five.
Radioimmunology (radioimmunoassay, RIA) is using first in radioiodine rubidium marking antibody test serumShape parathyrine is traditional detection method.Utilization due to detecting radiating immuning analysis technology starts from the 1960s, so farHas more than 40 years history, domestic still some clinical labororatories continue to use this method measurement serum FT 3, FT4, TSH at present.RIA is to utilizeRadioisotope labeling antigen or antibody form the principle of antigen antibody complex then with tested antibody or antigen bindingCome the analytic approach detected.Radiating immuning analysis technology since foundation, due to its measurement high sensitivity, high specificity,Precision is good, and can be measured to antigen haptens, is usually used in various hormones, trace of albumin, tumor markers and medicineThe detection of the micro substances such as object.It is at present still base pair since most of inspection projects have the offer of RIA or IRMA kitThe main means of ultra-micro substance measurement.But exist be easy to produce radioactive pollution in the detection process, testing result is putThe deficiencies of decaying of penetrating property element etc. influences, and the porous sample-adding process of detection process needs manual operations, and accuracy is relatively low.
Trivalent rare earth ion and its chelating with unique fluorescent characteristic is utilized in time-resolved fluoroimmunoassay (TRFIA)Object replaces fluorescent material, enzyme, isotope, chemiluminescent substance etc., labelled antibody/antigen, to antigen-antibody reaction by tracerAfter generation, with the fluorescence intensity in TRFIA detector measurement reaction product, according to product fluorescence intensity and relative intensity of fluorescenceRatio, judges the concentration of analyte in reaction system, to reach quantitative analysis.Time-resolved fluoroimmunoassay (TRFIA) byIn its Low background, the features such as highly sensitive and specificity, fluorescence lifetime is long, "dead" isotopic contamination, from 80 years 20th centuryIt is rapidly developed after registering for the first time for Pettersson and Eskola et al., and is widely used in clinical disease diagnosis.
Fluorescence immune chromatography detection in, fluorescence antibody be quenched and antibody peridium concentration influence experimental result.ProteinIt is bonded to capture reagent of the nitrocellulose filter (NC film) as sample to be tested.Since testing result depends entirely on capture reagentReach good adsorption effect on film, thus protein is uniform on film, good absorption to colloidal gold testing result veryIt is important.If the protein content combined on NC film is insufficient or protein binding power is not strong enough, with regard to will appear considerable problem, examiningIt surveys in the detection line of result clearly.If the protein content combined on film is too low, detection line colour developing is weaker in the resultAnd detection sensitivity reduces.If albumen cannot firmly be adsorbed in NC film, occurred before NC film in protein adsorptionDiffusion, it is wider so as to cause detection line, colour developing is weaker rather than bright-coloured and clear, make testing result be difficult to explain.In extreme itemUnder part, if the physisorption of albumen and NC film is too weak, the Protein Detection object and surfactant solution flowed through may be incited somebody to actionFixed albumen is washed off from NC film, so that display is wider or unsharp detection line at all, it is difficult to explain testing result.
Summary of the invention
The present invention provides first function five fluorescent microsphere joint-detection devices and preparation method thereof, is deposited with solving the prior artFluorescence antibody be easily quenched and NC film adhesion protein amount is insufficient, binding force is not strong problem.Human thyroid prepared by the present inventionElement (T4), trilute (T3), thyrotropic hormone (TSH), free T3, free T4 joint-detection device, can be realSensitive, special, the quick detection of existing thyroid function marker, improves and carries out thyroid function to Patients With Various Thyroid DisordersRight combination determines, can quickly, accurately carry out early warning and the state of an illness risk judgment of thyroid disease.
The technical solution adopted by the present invention is that a kind of first function five fluorescent microsphere joint-detection devices, are by following fiveKind detection device composition:
Detection device one: sample pad one, immunofluorescence are crosslinked thyronine T4 antibody glass fibre membrane, nitrocelluloseFilm, absorption pad are respectively adhered on plastic plate, and the both ends of the nitrocellulose filter are crosslinked first shape with absorption pad, immunofluorescence respectivelyParathyrine T4 antibody glass fibre membrane overlap joint, the other end and sample of the immunofluorescence crosslinking thyronine T4 antibody glass fibre membraneProduct pad one overlaps；Detection line T is set on the nitrocellulose filterT4With nature controlling line C, detection line TT4Mark thyronine T4Antibody；Specking sheep anti-mouse igg polyclonal antibody on the nature controlling line C；
Detection device two: sample pad one, immunofluorescence are crosslinked trilute T3 antibody glass fibre membrane, nitric acidCellulose membrane, absorption pad are respectively adhered on plastic plate, and the both ends of the nitrocellulose filter are handed over absorption pad, immunofluorescence respectivelyJoin trilute T3 antibody glass fibre membrane overlap joint, the immunofluorescence is crosslinked trilute T3 antibodyThe other end and sample pad one of glass fibre membrane overlap；Detection line T is set on the nitrocellulose filterT3With nature controlling line C, the inspectionSurvey line TT3Mark trilute T3 antibody；Specking sheep anti-mouse igg polyclonal antibody on the nature controlling line C；
Detection device three: sample pad one, immunofluorescence crosslinking thyrotropic hormone TSH antibody glass fibre membrane, nitric acid are fineThe plain film of dimension, absorption pad are respectively adhered on plastic plate, and the both ends of the nitrocellulose filter are crosslinked with absorption pad, immunofluorescence respectivelyThyrotropic hormone TSH antibody glass fibre membrane overlap joint, the immunofluorescence are crosslinked thyrotropic hormone TSH antibody glass fibreThe other end and sample pad one of film overlap；Detection line T is set on the nitrocellulose filterTSHWith nature controlling line C, detection line TTSHMark thyrotropic hormone TSH antibody；Specking sheep anti-mouse igg polyclonal antibody on the nature controlling line C；
Detection device four: sample pad two, immunofluorescence are crosslinked trilute T3 antibody glass fibre membrane, nitric acidCellulose membrane, absorption pad are respectively adhered on plastic plate, and the both ends of the nitrocellulose filter are handed over absorption pad, immunofluorescence respectivelyJoin trilute T3 antibody glass fibre membrane overlap joint, the immunofluorescence is crosslinked trilute T3 antibodyThe other end and sample pad two of glass fibre membrane overlap；Detection line T is set on the nitrocellulose filterT3With nature controlling line C, the inspectionSurvey line TTSHMark trilute T3 antibody；Specking sheep anti-mouse igg polyclonal antibody on the nature controlling line C；
Detection device five: sample pad two, immunofluorescence are crosslinked thyronine T4 antibody glass fibre membrane, nitrocelluloseFilm, absorption pad are respectively adhered on plastic plate, and the both ends of the nitrocellulose filter are crosslinked first shape with absorption pad, immunofluorescence respectivelyParathyrine T4 antibody glass fibre membrane overlap joint, the other end and sample of the immunofluorescence crosslinking thyronine T4 antibody glass fibre membraneProduct pad two overlaps；Detection line T is set on the nitrocellulose filterT4With nature controlling line C, detection line TT4Mark thyronine T4Antibody；Specking sheep anti-mouse igg polyclonal antibody on the nature controlling line C.
A kind of preparation method of first function five fluorescent microsphere joint-detection devices, including the following steps:
(a) preparation of immunofluorescence microballoon takes 100ul to contain the microsphere suspension liquid for 1% admittedly, dilutes 10 times with ultrapure water,That is 1000ul is added in EP pipe, is taken the n-hydroxysuccinimide liquid (NHS) of 40ul to be added in microsphere suspension liquid and is mixed, soMicrosphere suspension liquid is added in 1- (3- the dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride liquid (EDC) that 40ul is followed by addedIt is mixed in liquid, is reacted 0.5 hour under room temperature, by the suspension ultrasonic echography after reaction, hang the microballoon on tube wall againIt floats in aqueous solution, is then centrifuged microsphere suspension liquid, 8000~14000r/min of centrifugal condition, 15~25min outwell supernatantLiquid is added 1ml ultrapure water, is then uniformly dispersed with ultrasonic echography；
(b) immunofluorescence microballoon is crosslinked thyronine T4 antibody, trilute T3 antibody, thyroid respectivelyHormone TSH antibody, the microsphere suspension liquid after taking 1ml to activate, ultrasonic disperse is uniform, and antibody is then added dropwise respectively while stirring；AddAfter entering antibody, after reacting 1~1.5min, then by ultrasound 25~35 seconds on ultrasonic wave, then reacts again 1~1.5 hour, add ox bloodPure protein B SA is carried out closing 1~1.5 hour respectively, and the microballoon closed is centrifuged respectively, speed be 8000~Buffer is added separately in the immunofluorescence microballoon after centrifugation by 14000r/min, 15~20min, keeps each microballoon dispersion equalIt is even, for use；
(c) the immunofluorescence microballoon adaptive immune fluorescent microsphere solution for using buffer dilution step (b), uses immunofluorescenceMicrospheres solution is sprayed at fiberglass packing respectively, and immunofluorescence crosslinking thyronine T4 antibody glass fibre membrane is made respectively, exempts fromEpidemic disease fluorescence is crosslinked trilute T3 antibody glass fibre membrane and immunofluorescence is crosslinked thyrotropic hormone TSH antibody glassGlass tunica fibrosa；
(d) after pre-processing nitrocellulose filter with polyvinyl alcohol treatment fluid, specking is in conjunction with graphite nanoparticles respectivelyThyronine T4 antibody, trilute T3 antibody, thyrotropic hormone TSH antibody, thyronine T4 antibody, threeIodine thyronine T3 antibody is detection line T, and specking goat anti-rabbit igg antibody is as nature controlling line, each detection line specking amount 1ul/Each nitrocellulose filter is made in cm；
(e) preparation of five kinds of detection devices, wherein
Prepare detection device one:
Immunofluorescence prepared by pretreated sample pad one, step (c) is crosslinked thyronine T4 antibody glass fibre membrane,The thyronine T4 antibody of nitrocellulose filter and its specking in conjunction with graphite nanoparticles of step (d) preparation is detection lineTT4, specking goat anti-rabbit igg antibody as nature controlling line, absorption pad is successively pasted on plastic plate respectively, and detection reagent is made in cuttingItem finally will test reagent strip and be respectively charged into plastic shell；
Prepare detection device two:
Immunofluorescence prepared by pretreated sample pad one, step (c) is crosslinked trilute T3 antibody glassGlass tunica fibrosa, triiodo thyroid gland original ammonia of the nitrocellulose filter and its specking of step (d) preparation in conjunction with graphite nanoparticlesSour T3 antibody is detection line TT3, specking goat anti-rabbit igg antibody as nature controlling line, absorption pad is successively pasted on plastic plate respectively,Detection reagent item is made in cutting, finally will test reagent strip and is respectively charged into plastic shell；
Prepare detection device three:
Immunofluorescence prepared by pretreated sample pad one, step (c) is crosslinked thyrotropic hormone TSH antibody glass fibersFilm is tieed up, thyrotropic hormone TSH of the nitrocellulose filter and its specking of step (d) preparation in conjunction with graphite nanoparticles is anti-Body is detection line TTSH, specking goat anti-rabbit igg antibody as nature controlling line, absorption pad is successively pasted on plastic plate respectively, cutting systemDetection reagent item is obtained, reagent strip is finally will test and is respectively charged into plastic shell；
Prepare detection device four:
Trilute T3 antibody glass fibre membrane prepared by pretreated sample pad two, step (c), step(d) the trilute T3 antibody of nitrocellulose filter and its specking in conjunction with graphite nanoparticles prepared is inspectionSurvey line TT3, specking goat anti-rabbit igg antibody as nature controlling line, absorption pad is successively pasted on plastic plate respectively, cutting be made detectionReagent strip finally will test reagent strip and be respectively charged into plastic shell；
Prepare detection device five:
Thyronine T4 antibody glass fibre membrane prepared by pretreated sample pad two, step (c), step (d) preparationThe thyronine T4 antibody of nitrocellulose filter and its specking in conjunction with graphite nanoparticles be detection line TT4, specking goat-antiRabbit igg antibody is successively pasted on plastic plate respectively as nature controlling line, absorption pad, and detection reagent item is made in cutting, finally will inspectionTest agent item is respectively charged into plastic shell.
The step (b), (c) ball buffer by Tris-HCL liquid, sucrose, trehalose, bovine serum albumin(BSA) BSA groupAt, pH value 8.5, wherein Tris-Hcl concentration is 0.02mol/L, and sucrose concentration is 5~20%, and trehalose concentration is 1~5%,Bovine serum albumin(BSA) BSA concentration is 0.5~1%.
Thyronine T4 antibody of the step (d) in conjunction with graphite nanoparticles, trilute T3 antibody,The preparation method of thyrotropic hormone TSH antibody is: using graphite as carrier, taking 200 μ L, concentration 1mg/mL graphite nanoparticlesSolution and 25 μ L, 40 μm of ol/L thyronine T4s of concentration, trilute T3, thyrotropic hormone TSH antibody-solutionsBe added in ultrapure water, make end reaction system 1mL, after mixing well, setting shaking table temperature be 25 DEG C, revolving speed be 200~Under the conditions of 300r/m, above-mentioned mixed solution is protected from light 2~3h of shake culture in shaking table respectively, each mixed solution after reaction is usedUltracentrifuge distinguishes thorough centrifuge washing 3~4 times under 13 000r/m speed conditions, using ultrapure water, removes supernatant respectivelyExcessive unreacted thyronine T4 antibody, trilute T3 antibody, thyrotropic hormone TSH antibody in liquid,Gained sediment is that graphite-thyronine T4 antibody probe compound, graphite-trilute T3 antibody probe are multipleObject, graphite-thyrotropic hormone TSH antibody probe compound are closed, is settled to 1mL with ultrapure water, and store up in 4 DEG C of conditionsIt deposits.
Polyvinyl alcohol treatment fluid in the step (d) is made into 1% after being mixed by polyvinyl alcohol with Triton X-100, warp0.22 μm of membrane filtration, it is spare.
Pretreated detection device a sample pad one, two sample pad one of detection device, detection device in the step (e)Three sample pads one use one 200ul/cm of sample pad treatment fluid, and the sample pad treatment fluid one is pure by Tris-HCL liquid, ox bloodProtein B SA, casein, surfactant Tween-80 composition, wherein Tris-HCL liquid concentration is 0.1mol/L, bovine serum albuminWhite BSA concentration is 1%, and casein concentration is 0.2%, surfactant Tween-80 concentration is 1%；
Four sample pad two of pretreated detection device, five sample pad two of detection device in the step (e) use sampleTwo 200ul/cm for the treatment of fluid is padded, the sample pad treatment fluid two is by Tris-HCL liquid, bovine serum albumin(BSA) BSA, casein, surfaceActivating agent Tween-80 composition, wherein Tris-HCL liquid concentration is 0.1mol/L, and bovine serum albumin(BSA) BSA concentration is 1%, junket eggWhite concentration is 0.2%, surfactant concentration is 1% and anti-thyroglobulin antibody 10ug/ml composition.
Containing multiple hydroxyls for coupling in the structure of polyvinyl alcohol of the present invention, activation process is simple, facilitates NC film surfaceAnkyrin.Polyvinyl alcohol hydrogel organizes similar water content, elasticity modulus, low-friction coefficient with human body natural due to havingAnd the features such as higher mechanical strength, hole abundant leakage network structure, good biocompatibility, have in field of biomedicineIt is widely applied.Under normal condition, the quantity of binding antibody is limited on the NC film of unit area, is handled using polyvinyl alcoholLater, binding antibody quantity on the NC film of unit area can be allowed to increase, and then higher detection sensitivity may be implemented.With the site of NC film suction-operated can occur for Triton X-100, after albumen coating, the combination water of albumen can be absorbed, causeAlbumen is more hydrophobic, and the combination of such albumen and NC film is stronger, and the coating efficiency of albumen is significantly improved by hydrophobic effect.To sum up, poly vinyl alcohol and Triton X-100 combination formula can improve NC in terms of charge effect and hydrophobic effect twoThe coating efficiency of memebrane protein.
For the present invention on the basis of improving NC film to protein adsorption, inquiring into albumen is also to improve colloidal gold to NC film adsorption effectAnother approach of sensitivity.Graphite nanoparticles have preferable stability, easily prepared, biocompatibility is preferable and lowerThe advantages such as immunogenicity, it is relatively broad in field of biomedicine research.But it not yet has been reported that and answers in fluorescence immune chromatography technologyWith.The present invention will first draw antibody elder generation and the Nano graphite of film by influence of the graphite nanoparticles to NC film coated antibodyBurl closes, and closing, centrifugal purification removes unbonded antibody, then redissolves and arrives certain proportion, then draws film, such a stoneBlack particle can be in conjunction with multiple antibody, to increase coated antibody efficiency, sensitivity can also be greatly improved.Again since graphite is receivedRice grain is colorless and transparent, so will not influence colour developing, only increases the coating efficiency of antibody by its bigger surface area,Improve the sensitivity of fluorescence immune chromatography experiment.
The present invention is in order to improve the sensitivity of fluorescence immune chromatography technology, to nitrocellulose filter polyvinyl alcoholReason liquid is pre-processed, and by the antibody for being coated with NC in conjunction with graphite nanoparticles, has achieved the purpose that improve test paper sensitivity.
The beneficial effects of the present invention are:
1, structure of the detecting device of the invention is novel, and thyroxine (T4), trilute (T3) and TSH is anti-Body is coated on nitrocellulose membrane film, high specificity, can be detected T3 antibody in sample simultaneously, T4 antibody, TSH antibody, be dissociatedT3 antibody, free T4 antibody, and the complexity without increasing production operation.
It 2,, can by cooperating suitable fluorescent microsphere buffer and sample pad treatment fluid in immune colloid gold preparation stepOn the basis of guaranteeing the release completely of immunofluorescence microballoon, the sensitivity of reaction is effectively raised, under same threshold value, can also dropThe dosage of low immune microsphere, save the cost.
3, free T3, free T4 sample pad treatment fluid can be effectively blocked with protein binding T3, T4, and formula is simple, be hadEffect.
4, the present invention pre-processes nitrocellulose filter, is modified the antibody of coating nitrocellulose filter,Improve Test paper sensitivity, specificity.
5, detection device of the invention is easy to operate, does not need professional's operation.It is practical.
Detailed description of the invention
Fig. 1 is the structural schematic diagram of the detection device one of thyronine T4 antibody in present invention detection sample；
Fig. 2 is the structural representation of the detection device two of trilute (T3) antibody in present invention detection sampleFigure；
Fig. 3 is the structural schematic diagram of the detection device three of thyrotropic hormone (TSH) antibody in present invention detection sample；
Fig. 4 is that the structure of the detection device four of free triiodothyronine (T3) antibody in present invention detection sample is shownIt is intended to；
Fig. 5 is the structural schematic diagram of the detection device five of free thyroxine (T4) antibody in present invention detection sample.
Specific embodiment
Embodiment 1
A kind of first function five fluorescent microsphere joint-detection devices, are made of following five kinds of detection devices:
Detection device 1: sample pad 1, immunofluorescence crosslinking thyroxine (T4) antibody glass fibre membrane 102,Nitrocellulose filter 103, absorption pad 104 are respectively adhered on plastic plate 105, the both ends of the nitrocellulose filter 103 respectively withAbsorption pad 104, immunofluorescence crosslinking thyroxine (T4) antibody glass fibre membrane 102 overlap, and the immunofluorescence is crosslinked first shapeThe other end and sample pad 1 of parathyrine (T4) antibody glass fibre membrane 102 overlap；It is arranged on the nitrocellulose filter 103Detection line TT4With nature controlling line C, detection line TT4Mark thyroxine (T4) antibody；Specking sheep anti mouse on the nature controlling line CIgG polyclonal antibody；
Detection device 2 200: sample pad 1, immunofluorescence are crosslinked trilute (T3) antibody glass fibersDimension film 202, nitrocellulose filter 203, absorption pad 204 are respectively adhered on plastic plate 205, and the two of the nitrocellulose filter 203End is overlapped with absorption pad 204, immunofluorescence crosslinking trilute (T3) antibody glass fibre membrane 202 respectively, describedImmunofluorescence is crosslinked the other end of trilute (T3) antibody glass fibre membrane 202 and sample pad 1 overlaps；InstituteState setting detection line T on nitrocellulose filter 203T3With nature controlling line C, detection line TT3It marks trilute (T3)Antibody；Specking sheep anti-mouse igg polyclonal antibody on the nature controlling line C；
Detection device 3 300: sample pad 1, immunofluorescence are crosslinked thyrotropic hormone (TSH) antibody glass fibre membrane302, nitrocellulose filter 303, absorption pad 304 are respectively adhered on plastic plate 305, the both ends point of the nitrocellulose filter 303It is not overlapped with absorption pad 304, immunofluorescence crosslinking thyrotropic hormone (TSH) antibody glass fibre membrane 302, the immunofluorescenceThe other end and sample pad 1 for being crosslinked thyrotropic hormone (TSH) antibody glass fibre membrane 302 overlap；The cellulose nitrateDetection line T is set on plain film 303TSHWith nature controlling line C, detection line TTSHMark thyrotropic hormone (TSH) antibody；The matterControl specking sheep anti-mouse igg polyclonal antibody on line C；
Detection device 4 400: sample pad 2 401, immunofluorescence are crosslinked trilute (T3) antibody glass fibersDimension film 402, nitrocellulose filter 403, absorption pad 404 are respectively adhered on plastic plate 405, and the two of the nitrocellulose filter 403End is overlapped with absorption pad 404, immunofluorescence crosslinking trilute (T3) antibody glass fibre membrane 402 respectively, describedImmunofluorescence is crosslinked the other end of trilute (T3) antibody glass fibre membrane 402 and sample pad 2 401 overlaps；InstituteState setting detection line T on nitrocellulose filter 403T3With nature controlling line C, detection line TTSHIt marks trilute (T3)Antibody；Specking sheep anti-mouse igg polyclonal antibody on the nature controlling line C；
Detection device 5 500: sample pad 2 501, immunofluorescence crosslinking thyroxine (T4) antibody glass fibre membrane 502,Nitrocellulose filter 503, absorption pad 504 are respectively adhered on plastic plate 505, the both ends of the nitrocellulose filter 503 respectively withAbsorption pad 504, immunofluorescence crosslinking thyroxine (T4) antibody glass fibre membrane 502 overlap, and the immunofluorescence is crosslinked first shapeThe other end and sample pad 2 501 of parathyrine (T4) antibody glass fibre membrane 502 overlap；Inspection is set on the nitrocellulose filter 3Survey line TT4With nature controlling line C, detection line TT4Mark thyroxine (T4) antibody；Specking sheep anti-mouse igg on the nature controlling line CPolyclonal antibody.
A kind of preparation method of first function five fluorescent microsphere joint-detection devices, including the following steps:
(a) preparation of immunofluorescence microballoon takes 100ul to contain the microsphere suspension liquid for 1% admittedly, dilutes 10 times with ultrapure water,That is 1000ul is added in EP pipe, is taken the n-hydroxysuccinimide liquid (NHS) of 40ul to be added in microsphere suspension liquid and is mixed, soMicrosphere suspension liquid is added in 1- (3- the dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride liquid (EDC) that 40ul is followed by addedIt is mixed in liquid, is reacted 0.5 hour under room temperature, by the suspension ultrasonic echography after reaction, hang the microballoon on tube wall againIt is floating to be then centrifuged microsphere suspension liquid, centrifugal condition 8000r/min, 15min in aqueous solution, supernatant is outwelled, 1ml is addedThen ultrapure water is uniformly dispersed with ultrasonic echography；
(b) immunofluorescence microballoon is crosslinked thyroxine (T4) antibody respectively, trilute (T3) antibody, promotees firstShape glandular hormone (TSH) antibody, the microsphere suspension liquid after taking 1ml to activate, ultrasonic disperse is uniform, is then added dropwise respectively while stirring anti-Body；After antibody is added, after reacting 1min, then by ultrasound 25 seconds on ultrasonic wave, then reacts again 1 hour, add bovine serum albumin(BSA)BSA carries out closing 1 hour respectively, the microballoon closed is centrifuged respectively, speed 8000r/min, 15min will be bufferedLiquid is added separately in the immunofluorescence microballoon after centrifugation, so that each microballoon is uniformly dispersed, for use；
(c) the immunofluorescence microballoon adaptive immune fluorescent microsphere solution for using buffer dilution step (b), uses immunofluorescenceMicrospheres solution is sprayed at fiberglass packing respectively, respectively be made immunofluorescence crosslinking thyroxine (T4) antibody glass fibre membrane,Immunofluorescence is crosslinked trilute (T3) antibody glass fibre membrane and immunofluorescence crosslinking thyrotropic hormone (TSH)Antibody glass fibre membrane；
(d) after pre-processing nitrocellulose filter with polyvinyl alcohol treatment fluid, specking is in conjunction with graphite nanoparticles respectivelyThyroxine (T4) antibody, trilute (T3) antibody, thyrotropic hormone (TSH) antibody, thyroxine(T4) antibody, trilute (T3) antibody are detection line T, and specking goat anti-rabbit igg antibody is each to detect as nature controlling lineLine specking amount 1ul/cm, is made each nitrocellulose filter；
(e) preparation of five kinds of detection devices, wherein
Prepare detection device one:
Immunofluorescence prepared by pretreated sample pad one, step (c) is crosslinked thyroxine (T4) antibody glass fibreFilm, thyroxine (T4) antibody of nitrocellulose filter and its specking in conjunction with graphite nanoparticles of step (d) preparation are inspectionSurvey line TT4, specking goat anti-rabbit igg antibody as nature controlling line, absorption pad is successively pasted on plastic plate respectively, cutting be made detectionReagent strip finally will test reagent strip and be respectively charged into plastic shell；
Prepare detection device two:
Immunofluorescence prepared by pretreated sample pad one, step (c) is crosslinked trilute (T3) antibodyGlass fibre membrane, triiodo thyroid gland of the nitrocellulose filter and its specking of step (d) preparation in conjunction with graphite nanoparticles are formerPropylhomoserin (T3) antibody is detection line TT3, specking goat anti-rabbit igg antibody as nature controlling line, absorption pad is successively pasted onto plastic plate respectivelyOn, detection reagent item is made in cutting, finally will test reagent strip and is respectively charged into plastic shell；
Prepare detection device three:
Immunofluorescence prepared by pretreated sample pad one, step (c) is crosslinked thyrotropic hormone (TSH) antibody glassTunica fibrosa, thyrotropic hormone of the nitrocellulose filter and its specking of step (d) preparation in conjunction with graphite nanoparticles(TSH) antibody is detection line TTSH, specking goat anti-rabbit igg antibody as nature controlling line, absorption pad is successively pasted onto plastic plate respectivelyOn, detection reagent item is made in cutting, finally will test reagent strip and is respectively charged into plastic shell；
Prepare detection device four:
Trilute (T3) antibody glass fibre membrane prepared by pretreated sample pad two, step (c), stepSuddenly trilute (T3) antibody of the nitrocellulose filter and its specking of (d) preparation in conjunction with graphite nanoparticlesFor detection line TT3, specking goat anti-rabbit igg antibody as nature controlling line, absorption pad is successively pasted on plastic plate respectively, cutting be madeDetection reagent item finally will test reagent strip and be respectively charged into plastic shell；
Prepare detection device five:
Thyroxine (T4) antibody glass fibre membrane prepared by pretreated sample pad two, step (c), step (d) systemStandby thyroxine (T4) antibody of nitrocellulose filter and its specking in conjunction with graphite nanoparticles is detection line TT4, speckingGoat anti-rabbit igg antibody is successively pasted on plastic plate respectively as nature controlling line, absorption pad, and detection reagent item is made in cutting, finallyIt will test reagent strip and be respectively charged into plastic shell；
The step (b), (c) ball buffer by Tris-HCL liquid, sucrose, trehalose, bovine serum albumin(BSA) BSA groupAt pH value 8.5, wherein Tris-Hcl concentration is 0.02mol/L, and sucrose concentration 5%, trehalose concentration 1%, ox blood is pureProtein B SA concentration is 0.5%；
Thyroxine (T4) antibody, trilute (T3) of the step (d) in conjunction with graphite nanoparticlesAntibody, thyrotropic hormone (TSH) antibody preparation method be: using graphite as carrier, take 200 μ L, concentration 1mg/mL graphiteNanoparticles solution and 25 μ L, 40 μm of ol/L thyroxine (T4) of concentration, trilute (T3), thyrotropic hormone(TSH) antibody-solutions are added in ultrapure water, make end reaction system 1mL, and after mixing well, setting shaking table temperature is 25 DEG C,Under the conditions of revolving speed is 200r/m, above-mentioned mixed solution is protected from light shake culture 2h in shaking table respectively, each mixing after reaction is moltenLiquid ultracentrifuge distinguishes thorough centrifuge washing 3 times under 13000r/m speed conditions, using ultrapure water, removes supernatant respectivelyExcessive unreacted thyroxine (T4) antibody, trilute (T3) antibody, thyrotropic hormone (TSH) in liquidAntibody, gained sediment are graphite-thyroxine (T4) antibody probe compound, graphite-trilute (T3)Antibody probe compound, graphite-thyrotropic hormone (TSH) antibody probe compound, are settled to 1mL with ultrapure water, andIn 4 DEG C of condition storages；
Polyvinyl alcohol treatment fluid in the step (d) is made into 1% after being mixed by polyvinyl alcohol with Triton X-100, warp0.22 μm of membrane filtration, it is spare；
Pretreated detection device a sample pad one, two sample pad one of detection device, detection device in the step (e)Three sample pads one use one 200ul/cm of sample pad treatment fluid, and the sample pad treatment fluid one is pure by Tris-HCL liquid, ox bloodProtein B SA, casein, surfactant Tween-80 composition, wherein Tris-HCL liquid concentration is 0.1mol/L, bovine serum albuminWhite BSA concentration is 1%, and casein concentration is 0.2%, surfactant Tween-80 concentration is 1%；
Four sample pad two of pretreated detection device, five sample pad two of detection device in the step (e) use sampleTwo 200ul/cm for the treatment of fluid is padded, the sample pad treatment fluid two is by Tris-HCL liquid, bovine serum albumin(BSA) BSA, casein, surfaceActivating agent Tween-80 composition, wherein Tris-HCL liquid concentration is 0.1mol/L, and bovine serum albumin(BSA) BSA concentration is 1%, junket eggWhite concentration is 0.2%, surfactant concentration is 1% and anti-thyroglobulin antibody 10ug/ml composition.
Embodiment 2
A kind of first function five fluorescent microsphere joint-detection devices, are made of following five kinds of detection devices:
Detection device 1: sample pad 1, immunofluorescence crosslinking thyroxine (T4) antibody glass fibre membrane 102,Nitrocellulose filter 103, absorption pad 104 are respectively adhered on plastic plate 105, the both ends of the nitrocellulose filter 103 respectively withAbsorption pad 104, immunofluorescence crosslinking thyroxine (T4) antibody glass fibre membrane 102 overlap, and the immunofluorescence is crosslinked first shapeThe other end and sample pad 1 of parathyrine (T4) antibody glass fibre membrane 102 overlap；It is arranged on the nitrocellulose filter 103Detection line TT4With nature controlling line C, detection line TT4Mark thyroxine (T4) antibody；Specking sheep anti mouse on the nature controlling line CIgG polyclonal antibody；
Detection device 2 200: sample pad 1, immunofluorescence are crosslinked trilute (T3) antibody glass fibersDimension film 202, nitrocellulose filter 203, absorption pad 204 are respectively adhered on plastic plate 205, and the two of the nitrocellulose filter 203End is overlapped with absorption pad 204, immunofluorescence crosslinking trilute (T3) antibody glass fibre membrane 202 respectively, describedImmunofluorescence is crosslinked the other end of trilute (T3) antibody glass fibre membrane 202 and sample pad 1 overlaps；InstituteState setting detection line T on nitrocellulose filter 203T3With nature controlling line C, detection line TT3It marks trilute (T3)Antibody；Specking sheep anti-mouse igg polyclonal antibody on the nature controlling line C；
Detection device 3 300: sample pad 1, immunofluorescence are crosslinked thyrotropic hormone (TSH) antibody glass fibre membrane302, nitrocellulose filter 303, absorption pad 304 are respectively adhered on plastic plate 305, the both ends point of the nitrocellulose filter 303It is not overlapped with absorption pad 304, immunofluorescence crosslinking thyrotropic hormone (TSH) antibody glass fibre membrane 302, the immunofluorescenceThe other end and sample pad 1 for being crosslinked thyrotropic hormone (TSH) antibody glass fibre membrane 302 overlap；The cellulose nitrateDetection line T is set on plain film 303TSHWith nature controlling line C, detection line TTSHMark thyrotropic hormone (TSH) antibody；The matterControl specking sheep anti-mouse igg polyclonal antibody on line C；
Detection device 4 400: sample pad 2 401, immunofluorescence are crosslinked trilute (T3) antibody glass fibersDimension film 402, nitrocellulose filter 403, absorption pad 404 are respectively adhered on plastic plate 405, and the two of the nitrocellulose filter 403End is overlapped with absorption pad 404, immunofluorescence crosslinking trilute (T3) antibody glass fibre membrane 402 respectively, describedImmunofluorescence is crosslinked the other end of trilute (T3) antibody glass fibre membrane 402 and sample pad 2 401 overlaps；InstituteState setting detection line T on nitrocellulose filter 403T3With nature controlling line C, detection line TTSHIt marks trilute (T3)Antibody；Specking sheep anti-mouse igg polyclonal antibody on the nature controlling line C；
Detection device 5 500: sample pad 2 501, immunofluorescence crosslinking thyroxine (T4) antibody glass fibre membrane 502,Nitrocellulose filter 503, absorption pad 504 are respectively adhered on plastic plate 505, the both ends of the nitrocellulose filter 503 respectively withAbsorption pad 504, immunofluorescence crosslinking thyroxine (T4) antibody glass fibre membrane 502 overlap, and the immunofluorescence is crosslinked first shapeThe other end and sample pad 2 501 of parathyrine (T4) antibody glass fibre membrane 502 overlap；Inspection is set on the nitrocellulose filter 3Survey line TT4With nature controlling line C, detection line TT4Mark thyroxine (T4) antibody；Specking sheep anti-mouse igg on the nature controlling line CPolyclonal antibody.
A kind of preparation method of first function five fluorescent microsphere joint-detection devices, including the following steps:
(a) preparation of immunofluorescence microballoon takes 100ul to contain the microsphere suspension liquid for 1% admittedly, dilutes 10 times with ultrapure water,That is 1000ul is added in EP pipe, is taken the n-hydroxysuccinimide liquid (NHS) of 40ul to be added in microsphere suspension liquid and is mixed, soMicrosphere suspension liquid is added in 1- (3- the dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride liquid (EDC) that 40ul is followed by addedIt is mixed in liquid, is reacted 0.5 hour under room temperature, by the suspension ultrasonic echography after reaction, hang the microballoon on tube wall againIt is floating to be then centrifuged microsphere suspension liquid, centrifugal condition 11000r/min, 20min in aqueous solution, supernatant is outwelled, 1ml is addedThen ultrapure water is uniformly dispersed with ultrasonic echography；
(b) immunofluorescence microballoon is crosslinked thyroxine (T4) antibody respectively, trilute (T3) antibody, promotees firstShape glandular hormone (TSH) antibody, the microsphere suspension liquid after taking 1ml to activate, ultrasonic disperse is uniform, is then added dropwise respectively while stirring anti-Body；After antibody is added, after reacting 1.2min, then by ultrasound 30 seconds on ultrasonic wave, then reacts again 1.2 hours, add ox blood pureProtein B SA carries out closing 1.2 hours respectively, the microballoon closed is centrifuged respectively, speed 11000r/min, 18min,Buffer is added separately in the immunofluorescence microballoon after centrifugation, so that each microballoon is uniformly dispersed, for use；
(c) the immunofluorescence microballoon adaptive immune fluorescent microsphere solution for using buffer dilution step (b), uses immunofluorescenceMicrospheres solution is sprayed at fiberglass packing respectively, respectively be made immunofluorescence crosslinking thyroxine (T4) antibody glass fibre membrane,Immunofluorescence is crosslinked trilute (T3) antibody glass fibre membrane and immunofluorescence crosslinking thyrotropic hormone (TSH)Antibody glass fibre membrane；
(d) after pre-processing nitrocellulose filter with polyvinyl alcohol treatment fluid, specking is in conjunction with graphite nanoparticles respectivelyThyroxine (T4) antibody, trilute (T3) antibody, thyrotropic hormone (TSH) antibody, thyroxine(T4) antibody, trilute (T3) antibody are detection line T, and specking goat anti-rabbit igg antibody is each to detect as nature controlling lineLine specking amount 1ul/cm, is made each nitrocellulose filter；
(e) preparation of five kinds of detection devices, wherein
Prepare detection device one:
Immunofluorescence prepared by pretreated sample pad one, step (c) is crosslinked thyroxine (T4) antibody glass fibreFilm, thyroxine (T4) antibody of nitrocellulose filter and its specking in conjunction with graphite nanoparticles of step (d) preparation are inspectionSurvey line TT4, specking goat anti-rabbit igg antibody as nature controlling line, absorption pad is successively pasted on plastic plate respectively, cutting be made detectionReagent strip finally will test reagent strip and be respectively charged into plastic shell；
Prepare detection device two:
Immunofluorescence prepared by pretreated sample pad one, step (c) is crosslinked trilute (T3) antibodyGlass fibre membrane, triiodo thyroid gland of the nitrocellulose filter and its specking of step (d) preparation in conjunction with graphite nanoparticles are formerPropylhomoserin (T3) antibody is detection line TT3, specking goat anti-rabbit igg antibody as nature controlling line, absorption pad is successively pasted onto plastic plate respectivelyOn, detection reagent item is made in cutting, finally will test reagent strip and is respectively charged into plastic shell；
Prepare detection device three:
Immunofluorescence prepared by pretreated sample pad one, step (c) is crosslinked thyrotropic hormone (TSH) antibody glassTunica fibrosa, thyrotropic hormone of the nitrocellulose filter and its specking of step (d) preparation in conjunction with graphite nanoparticles(TSH) antibody is detection line TTSH, specking goat anti-rabbit igg antibody as nature controlling line, absorption pad is successively pasted onto plastic plate respectivelyOn, detection reagent item is made in cutting, finally will test reagent strip and is respectively charged into plastic shell；
Prepare detection device four:
Trilute (T3) antibody glass fibre membrane prepared by pretreated sample pad two, step (c), stepSuddenly trilute (T3) antibody of the nitrocellulose filter and its specking of (d) preparation in conjunction with graphite nanoparticlesFor detection line TT3, specking goat anti-rabbit igg antibody as nature controlling line, absorption pad is successively pasted on plastic plate respectively, cutting be madeDetection reagent item finally will test reagent strip and be respectively charged into plastic shell；
Prepare detection device five:
Thyroxine (T4) antibody glass fibre membrane prepared by pretreated sample pad two, step (c), step (d) systemStandby thyroxine (T4) antibody of nitrocellulose filter and its specking in conjunction with graphite nanoparticles is detection line TT4, speckingGoat anti-rabbit igg antibody is successively pasted on plastic plate respectively as nature controlling line, absorption pad, and detection reagent item is made in cutting, finallyIt will test reagent strip and be respectively charged into plastic shell；
The step (b), (c) ball buffer by Tris-HCL liquid, sucrose, trehalose, bovine serum albumin(BSA) BSA groupAt pH value 8.5, wherein Tris-Hcl concentration is 0.02mol/L, sucrose concentration 12%, trehalose concentration 3%, cow's serumAlbumin BSA concentration is 0.8%；
Thyroxine (T4) antibody, trilute (T3) of the step (d) in conjunction with graphite nanoparticlesAntibody, thyrotropic hormone (TSH) antibody preparation method be: using graphite as carrier, take 200 μ L, concentration 1mg/mL graphiteNanoparticles solution and 25 μ L, 40 μm of ol/L thyroxine (T4) of concentration, trilute (T3), thyrotropic hormone(TSH) antibody-solutions are added in ultrapure water, make end reaction system 1mL, and after mixing well, setting shaking table temperature is 25 DEG C,Under the conditions of revolving speed is 200r/m, above-mentioned mixed solution is protected from light shake culture 2h in shaking table respectively, each mixing after reaction is moltenLiquid ultracentrifuge distinguishes thorough centrifuge washing 3 times under 13000r/m speed conditions, using ultrapure water, removes supernatant respectivelyExcessive unreacted thyroxine (T4) antibody, trilute (T3) antibody, thyrotropic hormone (TSH) in liquidAntibody, gained sediment are graphite-thyroxine (T4) antibody probe compound, graphite-trilute (T3)Antibody probe compound, graphite-thyrotropic hormone (TSH) antibody probe compound, are settled to 1mL with ultrapure water, andIn 4 DEG C of condition storages；
Polyvinyl alcohol treatment fluid in the step (d) is made into 1% after being mixed by polyvinyl alcohol with Triton X-100, warp0.22 μm of membrane filtration, it is spare；
Pretreated detection device a sample pad one, two sample pad one of detection device, detection device in the step (e)Three sample pads one use one 200ul/cm of sample pad treatment fluid, and the sample pad treatment fluid one is pure by Tris-HCL liquid, ox bloodProtein B SA, casein, surfactant Tween-80 composition, wherein Tris-HCL liquid concentration is 0.1mol/L, bovine serum albuminWhite BSA concentration is 1%, and casein concentration is 0.2%, surfactant Tween-80 concentration is 1%；
Four sample pad two of pretreated detection device, five sample pad two of detection device in the step (e) use sampleTwo 200ul/cm for the treatment of fluid is padded, the sample pad treatment fluid two is by Tris-HCL liquid, bovine serum albumin(BSA) BSA, casein, surfaceActivating agent Tween-80 composition, wherein Tris-HCL liquid concentration is 0.1mol/L, and bovine serum albumin(BSA) BSA concentration is 1%, junket eggWhite concentration is 0.2%, surfactant concentration is 1% and anti-thyroglobulin antibody 10ug/ml composition.
Embodiment 3
A kind of first function five fluorescent microsphere joint-detection devices, are made of following five kinds of detection devices:
Detection device 1: sample pad 1, immunofluorescence crosslinking thyroxine (T4) antibody glass fibre membrane 102,Nitrocellulose filter 103, absorption pad 104 are respectively adhered on plastic plate 105, the both ends of the nitrocellulose filter 103 respectively withAbsorption pad 104, immunofluorescence crosslinking thyroxine (T4) antibody glass fibre membrane 102 overlap, and the immunofluorescence is crosslinked first shapeThe other end and sample pad 1 of parathyrine (T4) antibody glass fibre membrane 102 overlap；It is arranged on the nitrocellulose filter 103Detection line TT4With nature controlling line C, detection line TT4Mark thyroxine (T4) antibody；Specking sheep anti mouse on the nature controlling line CIgG polyclonal antibody；
Detection device 2 200: sample pad 1, immunofluorescence are crosslinked trilute (T3) antibody glass fibersDimension film 202, nitrocellulose filter 203, absorption pad 204 are respectively adhered on plastic plate 205, and the two of the nitrocellulose filter 203End is overlapped with absorption pad 204, immunofluorescence crosslinking trilute (T3) antibody glass fibre membrane 202 respectively, describedImmunofluorescence is crosslinked the other end of trilute (T3) antibody glass fibre membrane 202 and sample pad 1 overlaps；InstituteState setting detection line T on nitrocellulose filter 203T3With nature controlling line C, detection line TT3It marks trilute (T3)Antibody；Specking sheep anti-mouse igg polyclonal antibody on the nature controlling line C；
Detection device 3 300: sample pad 1, immunofluorescence are crosslinked thyrotropic hormone (TSH) antibody glass fibre membrane302, nitrocellulose filter 303, absorption pad 304 are respectively adhered on plastic plate 305, the both ends point of the nitrocellulose filter 303It is not overlapped with absorption pad 304, immunofluorescence crosslinking thyrotropic hormone (TSH) antibody glass fibre membrane 302, the immunofluorescenceThe other end and sample pad 1 for being crosslinked thyrotropic hormone (TSH) antibody glass fibre membrane 302 overlap；The cellulose nitrateDetection line T is set on plain film 303TSHWith nature controlling line C, detection line TTSHMark thyrotropic hormone (TSH) antibody；The matterControl specking sheep anti-mouse igg polyclonal antibody on line C；
Detection device 4 400: sample pad 2 401, immunofluorescence are crosslinked trilute (T3) antibody glass fibersDimension film 402, nitrocellulose filter 403, absorption pad 404 are respectively adhered on plastic plate 405, and the two of the nitrocellulose filter 403End is overlapped with absorption pad 404, immunofluorescence crosslinking trilute (T3) antibody glass fibre membrane 402 respectively, describedImmunofluorescence is crosslinked the other end of trilute (T3) antibody glass fibre membrane 402 and sample pad 2 401 overlaps；InstituteState setting detection line T on nitrocellulose filter 403T3With nature controlling line C, detection line TTSHIt marks trilute (T3)Antibody；Specking sheep anti-mouse igg polyclonal antibody on the nature controlling line C；
Detection device 5 500: sample pad 2 501, immunofluorescence crosslinking thyroxine (T4) antibody glass fibre membrane 502,Nitrocellulose filter 503, absorption pad 504 are respectively adhered on plastic plate 505, the both ends of the nitrocellulose filter 503 respectively withAbsorption pad 504, immunofluorescence crosslinking thyroxine (T4) antibody glass fibre membrane 502 overlap, and the immunofluorescence is crosslinked first shapeThe other end and sample pad 2 501 of parathyrine (T4) antibody glass fibre membrane 502 overlap；Inspection is set on the nitrocellulose filter 3Survey line TT4With nature controlling line C, detection line TT4Mark thyroxine (T4) antibody；Specking sheep anti-mouse igg on the nature controlling line CPolyclonal antibody.
A kind of preparation method of first function five fluorescent microsphere joint-detection devices, including the following steps:
(a) preparation of immunofluorescence microballoon takes 100ul to contain the microsphere suspension liquid for 1% admittedly, dilutes 10 times with ultrapure water,That is 1000ul is added in EP pipe, is taken the n-hydroxysuccinimide liquid (NHS) of 40ul to be added in microsphere suspension liquid and is mixed, soMicrosphere suspension liquid is added in 1- (3- the dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride liquid (EDC) that 40ul is followed by addedIt is mixed in liquid, is reacted 0.5 hour under room temperature, by the suspension ultrasonic echography after reaction, hang the microballoon on tube wall againIt is floating to be then centrifuged microsphere suspension liquid, centrifugal condition 14000r/min, 25min in aqueous solution, supernatant is outwelled, 1ml is addedThen ultrapure water is uniformly dispersed with ultrasonic echography；
(b) immunofluorescence microballoon is crosslinked thyroxine (T4) antibody respectively, trilute (T3) antibody, promotees firstShape glandular hormone (TSH) antibody, the microsphere suspension liquid after taking 1ml to activate, ultrasonic disperse is uniform, is then added dropwise respectively while stirring anti-Body；After antibody is added, after reacting 1.5min, then by ultrasound 35 seconds on ultrasonic wave, then reacts again 1.5 hours, add ox blood pureProtein B SA carries out closing 1.5 hours respectively, the microballoon closed is centrifuged respectively, speed 4000r/min, 20min,Buffer is added separately in the immunofluorescence microballoon after centrifugation, so that each microballoon is uniformly dispersed, for use；
(c) the immunofluorescence microballoon adaptive immune fluorescent microsphere solution for using buffer dilution step (b), uses immunofluorescenceMicrospheres solution is sprayed at fiberglass packing respectively, respectively be made immunofluorescence crosslinking thyroxine (T4) antibody glass fibre membrane,Immunofluorescence is crosslinked trilute (T3) antibody glass fibre membrane and immunofluorescence crosslinking thyrotropic hormone (TSH)Antibody glass fibre membrane；
(d) after pre-processing nitrocellulose filter with polyvinyl alcohol treatment fluid, specking is in conjunction with graphite nanoparticles respectivelyThyroxine (T4) antibody, trilute (T3) antibody, thyrotropic hormone (TSH) antibody, thyroxine(T4) antibody, trilute (T3) antibody are detection line T, and specking goat anti-rabbit igg antibody is each to detect as nature controlling lineLine specking amount 1ul/cm, is made each nitrocellulose filter；
(e) preparation of five kinds of detection devices, wherein
Prepare detection device one:
Immunofluorescence prepared by pretreated sample pad one, step (c) is crosslinked thyroxine (T4) antibody glass fibreFilm, thyroxine (T4) antibody of nitrocellulose filter and its specking in conjunction with graphite nanoparticles of step (d) preparation are inspectionSurvey line TT4, specking goat anti-rabbit igg antibody as nature controlling line, absorption pad is successively pasted on plastic plate respectively, cutting be made detectionReagent strip finally will test reagent strip and be respectively charged into plastic shell；
Prepare detection device two:
Immunofluorescence prepared by pretreated sample pad one, step (c) is crosslinked trilute (T3) antibodyGlass fibre membrane, triiodo thyroid gland of the nitrocellulose filter and its specking of step (d) preparation in conjunction with graphite nanoparticles are formerPropylhomoserin (T3) antibody is detection line TT3, specking goat anti-rabbit igg antibody as nature controlling line, absorption pad is successively pasted onto plastic plate respectivelyOn, detection reagent item is made in cutting, finally will test reagent strip and is respectively charged into plastic shell；
Prepare detection device three:
Immunofluorescence prepared by pretreated sample pad one, step (c) is crosslinked thyrotropic hormone (TSH) antibody glassTunica fibrosa, thyrotropic hormone of the nitrocellulose filter and its specking of step (d) preparation in conjunction with graphite nanoparticles(TSH) antibody is detection line TTSH, specking goat anti-rabbit igg antibody as nature controlling line, absorption pad is successively pasted onto plastic plate respectivelyOn, detection reagent item is made in cutting, finally will test reagent strip and is respectively charged into plastic shell；
Prepare detection device four:
Trilute (T3) antibody glass fibre membrane prepared by pretreated sample pad two, step (c), stepSuddenly trilute (T3) antibody of the nitrocellulose filter and its specking of (d) preparation in conjunction with graphite nanoparticlesFor detection line TT3, specking goat anti-rabbit igg antibody as nature controlling line, absorption pad is successively pasted on plastic plate respectively, cutting be madeDetection reagent item finally will test reagent strip and be respectively charged into plastic shell；
Prepare detection device five:
Thyroxine (T4) antibody glass fibre membrane prepared by pretreated sample pad two, step (c), step (d) systemStandby thyroxine (T4) antibody of nitrocellulose filter and its specking in conjunction with graphite nanoparticles is detection line TT4, speckingGoat anti-rabbit igg antibody is successively pasted on plastic plate respectively as nature controlling line, absorption pad, and detection reagent item is made in cutting, finallyIt will test reagent strip and be respectively charged into plastic shell；
The step (b), (c) ball buffer by Tris-HCL liquid, sucrose, trehalose, bovine serum albumin(BSA) BSA groupAt pH value 8.5, wherein Tris-Hcl concentration is 0.02mol/L, sucrose concentration 20%, trehalose concentration 5%, cow's serumAlbumin BSA concentration is 1%；
Thyroxine (T4) antibody, trilute (T3) of the step (d) in conjunction with graphite nanoparticlesAntibody, thyrotropic hormone (TSH) antibody preparation method be: using graphite as carrier, take 200 μ L, concentration 1mg/mL graphiteNanoparticles solution and 25 μ L, 40 μm of ol/L thyroxine (T4) of concentration, trilute (T3), thyrotropic hormone(TSH) antibody-solutions are added in ultrapure water, make end reaction system 1mL, and after mixing well, setting shaking table temperature is 25 DEG C,Under the conditions of revolving speed is 200~300r/m, above-mentioned mixed solution is protected from light 2~3h of shake culture in shaking table respectively, after reactionEach mixed solution ultracentrifuge distinguishes thorough centrifuge washing 3~4 times under 13 000r/m speed conditions, using ultrapure water,Excessive unreacted thyroxine (T4) antibody in supernatant is removed respectively, trilute (T3) antibody, promotees firstShape glandular hormone (TSH) antibody, gained sediment are graphite-thyroxine (T4) antibody probe compound, graphite-triiodo first shapeGland original ammonia acid (T3) antibody probe compound, graphite-thyrotropic hormone (TSH) antibody probe compound, with ultrapure water by itsIt is settled to 1mL, and in 4 DEG C of condition storages；
Polyvinyl alcohol treatment fluid in the step (d) is made into 1% after being mixed by polyvinyl alcohol with Triton X-100, warp0.22 μm of membrane filtration, it is spare；
Pretreated detection device a sample pad one, two sample pad one of detection device, detection device in the step (e)Three sample pads one use one 200ul/cm of sample pad treatment fluid, and the sample pad treatment fluid one is pure by Tris-HCL liquid, ox bloodProtein B SA, casein, surfactant Tween-80 composition, wherein Tris-HCL liquid concentration is 0.1mol/L, bovine serum albuminWhite BSA concentration is 1%, and casein concentration is 0.2%, surfactant Tween-80 concentration is 1%；
Four sample pad two of pretreated detection device, five sample pad two of detection device in the step (e) use sampleTwo 200ul/cm for the treatment of fluid is padded, the sample pad treatment fluid two is by Tris-HCL liquid, bovine serum albumin(BSA) BSA, casein, surfaceActivating agent Tween-80 composition, wherein Tris-HCL liquid concentration is 0.1mol/L, and bovine serum albumin(BSA) BSA concentration is 1%, junket eggWhite concentration is 0.2%, surfactant concentration is 1% and anti-thyroglobulin antibody 10ug/ml composition.
It is detected by fluorescent quantitative detector, above-described embodiment detects T4 serum thyroid hormones minimum detected value: 65nmol/L, FT4 serum free thyroxine minimum detected value: 0.3pmol/L, T3 serum trilute minimum detected value:1.8nmol/L, FT3 free serum trilute minimum detected value: 2.0pmol/L, TSH serum thyroid-stimulating hormone are mostLow detected value: 0.3mU/L.
The present invention is further illustrated by experimental example below.
Experimental example 1
1, comparison of the polyvinyl alcohol treatment fluid to the anti-quenching ability of nitrocellulose filter
1.1 materials and methods
1.1 materials: nitrocellulose filter, aperture 4.5um are purchased from General Electric Company, and polyvinyl alcohol is purchased from SigmaCompany
1.2 nitrocellulose membrane processing methods
1.2.1 preparing polyvinyl alcohol treatment fluid
Prepare polyvinyl alcohol treatment fluid: it is 1% that polyvinyl alcohol and Triton X-100, which are configured to concentration by distilled water,, warp0.22 μm of membrane filtration, it is spare.
1.2.2 nitrocellulose membrane processing method
Nitrocellulose filter is put into polyvinyl alcohol treatment fluid and impregnates 1h, and oscillation is rocked at a slow speed, with distillation after taking-upWater cleans 3 times, finally dries in a vacuum drying oven.
1.3 experimental method
Untreated and processed nitrocellulose filter is prepared into first function five according to above-described embodiment process flow respectivelyFluorescent microsphere Test paper, testing process compare fluorescence after nitrocellulose filter is untreated and processing referring to test paper specificationIndicator difference.
1.4 results:
1.4.1 fluorescence intensity compares
Processing group and untreated fish group test paper are taken, measuring samples are separately added into, passes through observation fluorescence developing situation judgement processingInfluence of the caudacoria to fluorescence quenching capability, the results are shown in Table 1.The results show that treated nitrocellulose filter is in solution impregnationAspect will be substantially better than untreated film, especially when concentration is lower, improve the sensitivity of reaction, show that fluorescence quenching capability dropsIt is low, improve reaction sensitivity.
1 nitrocellulose filter fluorescence quenching capability of table compares
1.4.2 nitrocellulose membrane stability compares
Polyvinyl alcohol processing group and untreated fish group test paper are taken, judges to handle by 37 DEG C of Acceleration study observation colour developing situationsAfterwards on nitrocellulose filter adhesion protein stability, the results are shown in Table 2.2 result of table and 1 result of table it was found that, nitre after processingAcid cellulose film color change with 10 days before it is almost the same, stability is good.
2 nitrocellulose filter accelerated stability of table compares (37 DEG C are placed 10 days)
Experimental example 2:
2, graphite nanoparticles modify T4 serum thyroid hormones antibody
2.1 materials and methods
1.1 materials: nitrocellulose filter, aperture 4.5um are purchased from General Electric Company
2.2.1 graphite nanoparticles modify T4 serum thyroid hormones antibody
Using graphite as carrier, 200 μ L graphite nanoparticles (1mg/mL) solution and 25 μ T4 serum thyroid hormones antibody are taken(40 μm of ol/L) solution is added in ultrapure water, makes end reaction system 1mL.After mixing well, setting shaking table temperature is 25 DEG C,Under the conditions of revolving speed is 200r/m, above-mentioned mixed solution is protected from light shake culture 2h in shaking table.Mixed solution after reaction is used superFast centrifuge is under 13 000r/m speed conditions, using the thorough centrifuge washing of ultrapure water 4 times, remove supernatant in it is excessive notThe T4 serum thyroid hormones antibody of reaction.Gained sediment is graphite-T4 serum thyroid hormones antibody probe compound, with superPure water is settled to 1mL and in 4 DEG C of condition storage.
2.3 experimental method
The T4 serum thyroid hormones antibody that graphite nanoparticles are modified is resisted with unmodified T4 serum thyroid hormones respectivelyBody prepares T4 serum thyroid hormones antibody test test paper according to above-described embodiment process flow, and testing process is referring to test paper explanationBook, compare graphite nanoparticles processing and it is untreated to protein adsorption power and stability indicator difference.
2.4 result
2.4.1 protein adsorption power compares
Graphite nanoparticles processing group and untreated fish group test paper are taken, measuring samples are separately added into, passes through observation colour developing situationJudgement processing caudacoria the results are shown in Table 3 to protein adsorption ability.The results show that graphite nanoparticles modification group especially concentration compared withWhen low, the sensitivity of reaction is improved, shows that protein adsorption ability is remarkably reinforced, improves reaction sensitivity.
Protein adsorption ability is compared in the modification of 3 graphite nanoparticles of table
2.4.2 stability compares
Graphite nanoparticles processing group and untreated fish group test paper are taken, is judged by 37 DEG C of Acceleration study observation colour developing situationsAfter graphite modification on nitrocellulose filter adhesion protein stability, the results are shown in Table 4.4 result of table and 3 result of table it was found that,Almost the same before nitrocellulose filter color change was with 10 days after processing, stability is good.
4 accelerated stability of table compares (37 DEG C are placed 10 days)
The foregoing is merely preferred embodiments of the invention, are not intended to restrict the invention, for the technology of this fieldFor personnel, the invention may be variously modified and varied.All any modification, equivalent substitution, improvement and etc. made for the present invention,It should all be included in the protection scope of the present invention.