CN109799355A

CN109799355A - First function five fluorescent microsphere joint-detection device and preparation method thereof

Info

Publication number: CN109799355A
Application number: CN201910109523.2A
Authority: CN
Inventors: 李志兴; 赵旻; 李欣
Original assignee: Jilin Shuangzheng Bioengineering Co Ltd
Current assignee: Jilin Shuangzheng Bioengineering Co Ltd
Priority date: 2019-02-06
Filing date: 2019-02-06
Publication date: 2019-05-24

Abstract

The present invention relates to a kind of first function five fluorescent microsphere joint-detection devices and preparation method thereof, belong to medical treatment detection device field.Sample pad, immunofluorescence cross-linking antibody glass fibre membrane, nitrocellulose filter, absorption pad are respectively adhered on plastic plate, the both ends of the nitrocellulose filter are overlapped with absorption pad, immunofluorescence cross-linking antibody glass fibre membrane respectively, and the immunofluorescence cross-linking antibody glass fibre membrane other end and sample pad overlap；Detection line T and nature controlling line C is set on the nitrocellulose filter；Detection line marks Thyroxine antibody, trilute antibody, thyrotropic hormone antibody respectively, specking sheep anti-mouse igg polyclonal antibody on nature controlling line C.Advantage is that thyroxine, trilute and thyrotropic hormone antibody are coated on nitrocellulose membrane film, high specificity, T3 antibody in sample, T4 antibody, TSH antibody, free T3 antibody, free T4 antibody can be detected simultaneously, it is easy to operate, it is practical.

Description

First function five fluorescent microsphere joint-detection device and preparation method thereof

Technical 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 preparation Method 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 real Sensitive, 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 gland Hyperfunction.Hypothyroidism is otherwise known as " first subtracts ", this disease is mostly because the thyroid hormone total amount of thyroid gland is very few Caused, the main clinical manifestation of this patient is the symptoms such as cold, memory and appetite stimulator, anemia.Thyroid function It 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 master The clinical manifestation wanted is honey stomach, tachycardia, impatience etc., this disease often brings apparent damage to the body of patient.It is related Research point out, subtract the thyroid function of patient with reasonable, effective Evaluation of detection methods hyperthyroidism and first, for Clinics and Practices Equal 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 patients Gland 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 inspection Sensibility 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 serum Shape parathyrine is traditional detection method.Utilization due to detecting radiating immuning analysis technology starts from the 1960s, so far Has 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 utilize Radioisotope labeling antigen or antibody form the principle of antigen antibody complex then with tested antibody or antigen binding Come 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 medicine The 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 kit The main means of ultra-micro substance measurement.But exist be easy to produce radioactive pollution in the detection process, testing result is put The 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 tracer After generation, with the fluorescence intensity in TRFIA detector measurement reaction product, according to product fluorescence intensity and relative intensity of fluorescence Ratio, judges the concentration of analyte in reaction system, to reach quantitative analysis.Time-resolved fluoroimmunoassay (TRFIA) by In its Low background, the features such as highly sensitive and specificity, fluorescence lifetime is long, "dead" isotopic contamination, from 80 years 20th century It 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.Protein It is bonded to capture reagent of the nitrocellulose filter (NC film) as sample to be tested.Since testing result depends entirely on capture reagent Reach good adsorption effect on film, thus protein is uniform on film, good absorption to colloidal gold testing result very It 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, examining It 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 result And detection sensitivity reduces.If albumen cannot firmly be adsorbed in NC film, occurred before NC film in protein adsorption Diffusion, 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 item Under 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 action Fixed 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 art Fluorescence antibody be easily quenched and NC film adhesion protein amount is insufficient, binding force is not strong problem.Human thyroid prepared by the present invention Element (T4), trilute (T3), thyrotropic hormone (TSH), free T3, free T4 joint-detection device, can be real Sensitive, special, the quick detection of existing thyroid function marker, improves and carries out thyroid function to Patients With Various Thyroid Disorders Right 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 five Kind detection device composition:

Detection device one: sample pad one, immunofluorescence are crosslinked thyronine T4 antibody glass fibre membrane, nitrocellulose Film, absorption pad are respectively adhered on plastic plate, and the both ends of the nitrocellulose filter are crosslinked first shape with absorption pad, immunofluorescence respectively Parathyrine T4 antibody glass fibre membrane overlap joint, the other end and sample of the immunofluorescence crosslinking thyronine T4 antibody glass fibre membrane Product pad one overlaps；Detection line T is set on the nitrocellulose filter_T4With nature controlling line C, detection line T_T4Mark thyronine T4 Antibody；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 acid Cellulose membrane, absorption pad are respectively adhered on plastic plate, and the both ends of the nitrocellulose filter are handed over absorption pad, immunofluorescence respectively Join trilute T3 antibody glass fibre membrane overlap joint, the immunofluorescence is crosslinked trilute T3 antibody The other end and sample pad one of glass fibre membrane overlap；Detection line T is set on the nitrocellulose filter_T3With nature controlling line C, the inspection Survey line T_T3Mark 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 fine The 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 respectively Thyrotropic hormone TSH antibody glass fibre membrane overlap joint, the immunofluorescence are crosslinked thyrotropic hormone TSH antibody glass fibre The other end and sample pad one of film overlap；Detection line T is set on the nitrocellulose filter_TSHWith nature controlling line C, detection line T_TSH Mark 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 acid Cellulose membrane, absorption pad are respectively adhered on plastic plate, and the both ends of the nitrocellulose filter are handed over absorption pad, immunofluorescence respectively Join trilute T3 antibody glass fibre membrane overlap joint, the immunofluorescence is crosslinked trilute T3 antibody The other end and sample pad two of glass fibre membrane overlap；Detection line T is set on the nitrocellulose filter_T3With nature controlling line C, the inspection Survey line T_TSHMark 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, nitrocellulose Film, absorption pad are respectively adhered on plastic plate, and the both ends of the nitrocellulose filter are crosslinked first shape with absorption pad, immunofluorescence respectively Parathyrine T4 antibody glass fibre membrane overlap joint, the other end and sample of the immunofluorescence crosslinking thyronine T4 antibody glass fibre membrane Product pad two overlaps；Detection line T is set on the nitrocellulose filter_T4With nature controlling line C, detection line T_T4Mark thyronine T4 Antibody；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, so Microsphere suspension liquid is added in 1- (3- the dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride liquid (EDC) that 40ul is followed by added It 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 again It floats in aqueous solution, is then centrifuged microsphere suspension liquid, 8000~14000r/min of centrifugal condition, 15~25min outwell supernatant Liquid is added 1ml ultrapure water, is then uniformly dispersed with ultrasonic echography；

(b) immunofluorescence microballoon is crosslinked thyronine T4 antibody, trilute T3 antibody, thyroid respectively Hormone TSH antibody, the microsphere suspension liquid after taking 1ml to activate, ultrasonic disperse is uniform, and antibody is then added dropwise respectively while stirring；Add After 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 blood Pure 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 equal It is even, for use；

(c) the immunofluorescence microballoon adaptive immune fluorescent microsphere solution for using buffer dilution step (b), uses immunofluorescence Microspheres solution is sprayed at fiberglass packing respectively, and immunofluorescence crosslinking thyronine T4 antibody glass fibre membrane is made respectively, exempts from Epidemic disease fluorescence is crosslinked trilute T3 antibody glass fibre membrane and immunofluorescence is crosslinked thyrotropic hormone TSH antibody glass Glass tunica fibrosa；

(d) after pre-processing nitrocellulose filter with polyvinyl alcohol treatment fluid, specking is in conjunction with graphite nanoparticles respectively Thyronine T4 antibody, trilute T3 antibody, thyrotropic hormone TSH antibody, thyronine T4 antibody, three Iodine 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 line T_T4, 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 cutting Item 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 glass Glass tunica fibrosa, triiodo thyroid gland original ammonia of the nitrocellulose filter and its specking of step (d) preparation in conjunction with graphite nanoparticles Sour T3 antibody is detection line T_T3, 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 fibers Film 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 T_TSH, specking goat anti-rabbit igg antibody as nature controlling line, absorption pad is successively pasted on plastic plate respectively, cutting system Detection 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 inspection Survey line T_T3, specking goat anti-rabbit igg antibody as nature controlling line, absorption pad is successively pasted on plastic plate respectively, cutting be made detection Reagent 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) preparation The thyronine T4 antibody of nitrocellulose filter and its specking in conjunction with graphite nanoparticles be detection line T_T4, specking goat-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, finally will inspection Test 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 group At, 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 nanoparticles Solution and 25 μ L, 40 μm of ol/L thyronine T4s of concentration, trilute T3, thyrotropic hormone TSH antibody-solutions Be 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 used Ultracentrifuge distinguishes thorough centrifuge washing 3~4 times under 13 000r/m speed conditions, using ultrapure water, removes supernatant respectively Excessive 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 multiple Object, graphite-thyrotropic hormone TSH antibody probe compound are closed, is settled to 1mL with ultrapure water, and store up in 4 DEG C of conditions It deposits.

Polyvinyl alcohol treatment fluid in the step (d) is made into 1% after being mixed by polyvinyl alcohol with Triton X-100, warp 0.22 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 blood Protein B SA, casein, surfactant Tween-80 composition, wherein Tris-HCL liquid concentration is 0.1mol/L, bovine serum albumin White 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 sample Two 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, surface Activating agent Tween-80 composition, wherein Tris-HCL liquid concentration is 0.1mol/L, and bovine serum albumin(BSA) BSA concentration is 1%, junket egg White 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 surface Ankyrin.Polyvinyl alcohol hydrogel organizes similar water content, elasticity modulus, low-friction coefficient with human body natural due to having And the features such as higher mechanical strength, hole abundant leakage network structure, good biocompatibility, have in field of biomedicine It is widely applied.Under normal condition, the quantity of binding antibody is limited on the NC film of unit area, is handled using polyvinyl alcohol Later, 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, cause Albumen 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 two The 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 effect Another approach of sensitivity.Graphite nanoparticles have preferable stability, easily prepared, biocompatibility is preferable and lower The 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 technology With.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 antibody Burl closes, and closing, centrifugal purification removes unbonded antibody, then redissolves and arrives certain proportion, then draws film, such a stone Black particle can be in conjunction with multiple antibody, to increase coated antibody efficiency, sensitivity can also be greatly improved.Again since graphite is received Rice 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 alcohol Reason 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 dissociated T3 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 step On the basis of guaranteeing the release completely of immunofluorescence microballoon, the sensitivity of reaction is effectively raised, under same threshold value, can also drop The 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 had Effect.

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 sample Figure；

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 shown It 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 with Absorption pad 104, immunofluorescence crosslinking thyroxine (T4) antibody glass fibre membrane 102 overlap, and the immunofluorescence is crosslinked first shape The other end and sample pad 1 of parathyrine (T4) antibody glass fibre membrane 102 overlap；It is arranged on the nitrocellulose filter 103 Detection line T_T4With nature controlling line C, detection line T_T4Mark thyroxine (T4) antibody；Specking sheep anti mouse on the nature controlling line C IgG polyclonal antibody；

Detection device 2 200: sample pad 1, immunofluorescence are crosslinked trilute (T3) antibody glass fibers Dimension film 202, nitrocellulose filter 203, absorption pad 204 are respectively adhered on plastic plate 205, and the two of the nitrocellulose filter 203 End is overlapped with absorption pad 204, immunofluorescence crosslinking trilute (T3) antibody glass fibre membrane 202 respectively, described Immunofluorescence is crosslinked the other end of trilute (T3) antibody glass fibre membrane 202 and sample pad 1 overlaps；Institute State setting detection line T on nitrocellulose filter 203_T3With nature controlling line C, detection line T_T3It 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 membrane 302, nitrocellulose filter 303, absorption pad 304 are respectively adhered on plastic plate 305, the both ends point of the nitrocellulose filter 303 It is not overlapped with absorption pad 304, immunofluorescence crosslinking thyrotropic hormone (TSH) antibody glass fibre membrane 302, the immunofluorescence The other end and sample pad 1 for being crosslinked thyrotropic hormone (TSH) antibody glass fibre membrane 302 overlap；The cellulose nitrate Detection line T is set on plain film 303_TSHWith nature controlling line C, detection line T_TSHMark thyrotropic hormone (TSH) antibody；The matter Control 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 fibers Dimension film 402, nitrocellulose filter 403, absorption pad 404 are respectively adhered on plastic plate 405, and the two of the nitrocellulose filter 403 End is overlapped with absorption pad 404, immunofluorescence crosslinking trilute (T3) antibody glass fibre membrane 402 respectively, described Immunofluorescence is crosslinked the other end of trilute (T3) antibody glass fibre membrane 402 and sample pad 2 401 overlaps；Institute State setting detection line T on nitrocellulose filter 403_T3With nature controlling line C, detection line T_TSHIt 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 with Absorption pad 504, immunofluorescence crosslinking thyroxine (T4) antibody glass fibre membrane 502 overlap, and the immunofluorescence is crosslinked first shape The other end and sample pad 2 501 of parathyrine (T4) antibody glass fibre membrane 502 overlap；Inspection is set on the nitrocellulose filter 3 Survey line T_T4With nature controlling line C, detection line T_T4Mark thyroxine (T4) antibody；Specking sheep anti-mouse igg on the nature controlling line C Polyclonal antibody.

(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, so Microsphere suspension liquid is added in 1- (3- the dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride liquid (EDC) that 40ul is followed by added It 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 again It is floating to be then centrifuged microsphere suspension liquid, centrifugal condition 8000r/min, 15min in aqueous solution, supernatant is outwelled, 1ml is added Then ultrapure water is uniformly dispersed with ultrasonic echography；

(b) immunofluorescence microballoon is crosslinked thyroxine (T4) antibody respectively, trilute (T3) antibody, promotees first Shape 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 buffered Liquid 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 immunofluorescence Microspheres 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 respectively Thyroxine (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 line Line 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 fibre Film, thyroxine (T4) antibody of nitrocellulose filter and its specking in conjunction with graphite nanoparticles of step (d) preparation are inspection Survey line T_T4, specking goat anti-rabbit igg antibody as nature controlling line, absorption pad is successively pasted on plastic plate respectively, cutting be made detection Reagent 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) antibody Glass fibre membrane, triiodo thyroid gland of the nitrocellulose filter and its specking of step (d) preparation in conjunction with graphite nanoparticles are former Propylhomoserin (T3) antibody is detection line T_T3, specking goat anti-rabbit igg antibody as nature controlling line, absorption pad is successively pasted onto plastic plate respectively On, 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 Tunica fibrosa, thyrotropic hormone of the nitrocellulose filter and its specking of step (d) preparation in conjunction with graphite nanoparticles (TSH) antibody is detection line T_TSH, specking goat anti-rabbit igg antibody as nature controlling line, absorption pad is successively pasted onto plastic plate respectively On, 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), step Suddenly trilute (T3) antibody of the nitrocellulose filter and its specking of (d) preparation in conjunction with graphite nanoparticles For detection line T_T3, specking goat anti-rabbit igg antibody as nature controlling line, absorption pad is successively pasted on plastic plate respectively, cutting be made Detection 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) system Standby thyroxine (T4) antibody of nitrocellulose filter and its specking in conjunction with graphite nanoparticles is detection line T_T4, specking Goat 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, finally It 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 group At pH value 8.5, wherein Tris-Hcl concentration is 0.02mol/L, and sucrose concentration 5%, trehalose concentration 1%, ox blood is pure Protein B SA concentration is 0.5%；

Thyroxine (T4) antibody, trilute (T3) of the step (d) in conjunction with graphite nanoparticles Antibody, thyrotropic hormone (TSH) antibody preparation method be: using graphite as carrier, take 200 μ L, concentration 1mg/mL graphite Nanoparticles 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 molten Liquid ultracentrifuge distinguishes thorough centrifuge washing 3 times under 13000r/m speed conditions, using ultrapure water, removes supernatant respectively Excessive unreacted thyroxine (T4) antibody, trilute (T3) antibody, thyrotropic hormone (TSH) in liquid Antibody, 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, 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, warp 0.22 μm of membrane filtration, it is spare；

Embodiment 2

(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, so Microsphere suspension liquid is added in 1- (3- the dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride liquid (EDC) that 40ul is followed by added It 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 again It is floating to be then centrifuged microsphere suspension liquid, centrifugal condition 11000r/min, 20min in aqueous solution, supernatant is outwelled, 1ml is added Then ultrapure water is uniformly dispersed with ultrasonic echography；

(b) immunofluorescence microballoon is crosslinked thyroxine (T4) antibody respectively, trilute (T3) antibody, promotees first Shape 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 pure Protein 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；

(e) preparation of five kinds of detection devices, wherein

Prepare detection device one:

Prepare detection device two:

Prepare detection device three:

Prepare detection device four:

Prepare detection device five:

The step (b), (c) ball buffer by Tris-HCL liquid, sucrose, trehalose, bovine serum albumin(BSA) BSA group At pH value 8.5, wherein Tris-Hcl concentration is 0.02mol/L, sucrose concentration 12%, trehalose concentration 3%, cow's serum Albumin BSA concentration is 0.8%；

Embodiment 3

(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, so Microsphere suspension liquid is added in 1- (3- the dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride liquid (EDC) that 40ul is followed by added It 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 again It is floating to be then centrifuged microsphere suspension liquid, centrifugal condition 14000r/min, 25min in aqueous solution, supernatant is outwelled, 1ml is added Then ultrapure water is uniformly dispersed with ultrasonic echography；

(b) immunofluorescence microballoon is crosslinked thyroxine (T4) antibody respectively, trilute (T3) antibody, promotees first Shape 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 pure Protein 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；

(e) preparation of five kinds of detection devices, wherein

Prepare detection device one:

Prepare detection device two:

Prepare detection device three:

Prepare detection device four:

Prepare detection device five:

The step (b), (c) ball buffer by Tris-HCL liquid, sucrose, trehalose, bovine serum albumin(BSA) BSA group At pH value 8.5, wherein Tris-Hcl concentration is 0.02mol/L, sucrose concentration 20%, trehalose concentration 5%, cow's serum Albumin BSA concentration is 1%；

Thyroxine (T4) antibody, trilute (T3) of the step (d) in conjunction with graphite nanoparticles Antibody, thyrotropic hormone (TSH) antibody preparation method be: using graphite as carrier, take 200 μ L, concentration 1mg/mL graphite Nanoparticles 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 reaction Each 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 first Shape glandular hormone (TSH) antibody, gained sediment are graphite-thyroxine (T4) antibody probe compound, graphite-triiodo first shape Gland original ammonia acid (T3) antibody probe compound, graphite-thyrotropic hormone (TSH) antibody probe compound, with ultrapure water by its It is settled to 1mL, and in 4 DEG C of condition storages；

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 most Low 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 Sigma Company

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,, warp 0.22 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-up Water 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 respectively Fluorescent microsphere Test paper, testing process compare fluorescence after nitrocellulose filter is untreated and processing referring to test paper specification Indicator 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 processing Influence of the caudacoria to fluorescence quenching capability, the results are shown in Table 1.The results show that treated nitrocellulose filter is in solution impregnation Aspect will be substantially better than untreated film, especially when concentration is lower, improve the sensitivity of reaction, show that fluorescence quenching capability drops It 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 situations Afterwards 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 processing Acid 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 super Fast centrifuge is under 13 000r/m speed conditions, using the thorough centrifuge washing of ultrapure water 4 times, remove supernatant in it is excessive not The T4 serum thyroid hormones antibody of reaction.Gained sediment is graphite-T4 serum thyroid hormones antibody probe compound, with super Pure 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 respectively Body prepares T4 serum thyroid hormones antibody test test paper according to above-described embodiment process flow, and testing process is referring to test paper explanation Book, 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 situation Judgement processing caudacoria the results are shown in Table 3 to protein adsorption ability.The results show that graphite nanoparticles modification group especially concentration compared with When 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 situations After 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 field For 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.

Claims

1. a kind of first function five fluorescent microsphere joint-detection devices, it is characterised in that: be made of following five kinds of detection devices:

Detection device one: sample pad one, immunofluorescence are crosslinked thyronine T4 antibody glass fibre membrane, nitrocellulose filter, suction It receives pad and is respectively adhered on plastic plate, the both ends of the nitrocellulose filter are crosslinked thyroxine with absorption pad, immunofluorescence respectively T4 antibody glass fibre membrane overlap joint, the other end and sample pad of the immunofluorescence crosslinking thyronine T4 antibody glass fibre membrane One overlap joint；Detection line T is set on the nitrocellulose filter_T4With nature controlling line C, detection line T_T4Mark thyronine T4 antibody； 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, cellulose nitrate Plain film, absorption pad are respectively adhered on plastic plate, and the both ends of the nitrocellulose filter are crosslinked three with absorption pad, immunofluorescence respectively Iodine thyronine T3 antibody glass fibre membrane overlap joint, the immunofluorescence are crosslinked trilute T3 antibody glass The other end and sample pad one of tunica fibrosa overlap；Detection line T is set on the nitrocellulose filter_T3With nature controlling line C, the detection line T_T3Mark trilute T3 antibody；Specking sheep anti-mouse igg polyclonal antibody on the nature controlling line C；

Detection device three: sample pad one, immunofluorescence are crosslinked thyrotropic hormone TSH antibody glass fibre membrane, nitrocellulose Film, absorption pad are respectively adhered on plastic plate, and the both ends of the nitrocellulose filter promote first with absorption pad, immunofluorescence crosslinking respectively Shape glandular hormone TSH antibody glass fibre membrane overlap joint, the immunofluorescence crosslinking thyrotropic hormone TSH antibody glass fibre membrane The other end and sample pad one overlap；Detection line T is set on the nitrocellulose filter_TSHWith nature controlling line C, detection line T_TSHLabel 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, cellulose nitrate Plain film, absorption pad are respectively adhered on plastic plate, and the both ends of the nitrocellulose filter are crosslinked three with absorption pad, immunofluorescence respectively Iodine thyronine T3 antibody glass fibre membrane overlap joint, the immunofluorescence are crosslinked trilute T3 antibody glass The other end and sample pad two of tunica fibrosa overlap；Detection line T is set on the nitrocellulose filter_T3With nature controlling line C, the detection line T_TSHMark 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, nitrocellulose filter, suction It receives pad and is respectively adhered on plastic plate, the both ends of the nitrocellulose filter are crosslinked thyroxine with absorption pad, immunofluorescence respectively T4 antibody glass fibre membrane overlap joint, the other end and sample pad of the immunofluorescence crosslinking thyronine T4 antibody glass fibre membrane Two overlap joints；Detection line T is set on the nitrocellulose filter_T4With nature controlling line C, detection line T_T4Mark thyronine T4 antibody； Specking sheep anti-mouse igg polyclonal antibody on the nature controlling line C.

2. a kind of preparation method of first function five fluorescent microsphere joint-detection devices as described in claim 1, which is characterized in that Include 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, i.e., 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, then Microsphere suspension liquid liquid is added in 1- (3- the dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride liquid (EDC) for being subsequently added into 40ul Middle mixing reacts 0.5 hour under room temperature, by the suspension ultrasonic echography after reaction, the microballoon on tube wall is made to suspend again In aqueous solution, then microsphere suspension liquid is centrifuged, 8000~14000r/min of centrifugal condition, 15~25min outwell supernatant Liquid is added 1ml ultrapure water, is then uniformly dispersed with ultrasonic echography；

(b) immunofluorescence microballoon is crosslinked thyronine T4 antibody, trilute T3 antibody, thyrotropic hormone respectively TSH antibody, the microsphere suspension liquid after taking 1ml to activate, ultrasonic disperse is uniform, and antibody is then added dropwise respectively while stirring；It is added anti- After body, after reacting 1~1.5min, then by ultrasound 25~35 seconds on ultrasonic wave, then reacts again 1~1.5 hour, add ox blood pure Protein B SA is carried out closing 1~1.5 hour respectively, and the microballoon closed is centrifuged respectively, and speed is 8000~14000r/ Buffer is added separately in the immunofluorescence microballoon after centrifugation, so that each microballoon is uniformly dispersed, for use by min, 15~20min；

(c) the immunofluorescence microballoon adaptive immune fluorescent microsphere solution for using buffer dilution step (b), with immunofluorescence microballoon Solution is sprayed at fiberglass packing respectively, respectively be made immunofluorescence crosslinking thyronine T4 antibody glass fibre membrane, be immunized it is glimmering Photo-crosslinking trilute T3 antibody glass fibre membrane and immunofluorescence are crosslinked thyrotropic hormone TSH antibody glass fibers Tie up film；

(d) after nitrocellulose filter being pre-processed with polyvinyl alcohol treatment fluid, first of the difference specking in conjunction with graphite nanoparticles Shape parathyrine T4 antibody, trilute T3 antibody, thyrotropic hormone TSH antibody, thyronine T4 antibody, triiodo first Shape gland original ammonia acid T3 antibody is detection line T, and specking goat anti-rabbit igg antibody is as nature controlling line, each detection line specking amount 1ul/cm, system Obtain 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 thyronine T4 antibody glass fibre membrane, step (d) the thyronine T4 antibody of nitrocellulose filter and its specking in conjunction with graphite nanoparticles prepared is detection line T_T4, spray Point goat anti-rabbit igg antibody is as nature controlling line, and absorption pad is successively pasted on plastic plate respectively, and detection reagent item is made in cutting, most After 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 glass fibers Tie up film, trilute T3 of the nitrocellulose filter and its specking of step (d) preparation in conjunction with graphite nanoparticles Antibody is detection line T_T3, specking goat anti-rabbit igg antibody as nature controlling line, absorption pad is successively pasted on plastic plate respectively, cutting Detection reagent item is made, finally will test reagent strip and be 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 fibre Film, thyrotropic hormone TSH antibody of the nitrocellulose filter and its specking of step (d) preparation in conjunction with graphite nanoparticles For detection line T_TSH, specking goat anti-rabbit igg antibody as nature controlling line, absorption pad is successively pasted on plastic plate respectively, cutting be made Detection reagent item finally will test reagent strip and be 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 the nitrocellulose filter of preparation and its specking in conjunction with graphite nanoparticles is detection line T_T3, 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 cutting Item 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), the nitre of step (d) preparation The thyronine T4 antibody of acid cellulose film and its specking in conjunction with graphite nanoparticles is detection line T_T4, specking goat 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, finally will test reagent Item is respectively charged into plastic shell.

3. a kind of preparation method of first function five fluorescent microsphere joint-detection devices, feature exist according to claim 2 It is made of in the ball buffer of: the step (b), (c) Tris-HCL liquid, sucrose, trehalose, bovine serum albumin(BSA) BSA, pH value 8.5, wherein Tris-Hcl concentration is 0.02mol/L, and sucrose concentration is 5~20%, and trehalose concentration is 1~5%, and ox blood is pure Protein B SA concentration is 0.5~1%.

4. a kind of preparation method of first function five fluorescent microsphere joint-detection devices, feature exist according to claim 2 In: thyronine T4 antibody, trilute T3 antibody, rush first of the step (d) in conjunction with graphite nanoparticles The preparation method of shape glandular hormone TSH antibody is: using graphite as carrier, taking 200 μ L, concentration 1mg/mL graphite nanoparticles solution It is added with 25 μ L, 40 μm of ol/L thyronine T4s of concentration, trilute T3, thyrotropic hormone TSH antibody-solutions Into ultrapure water, make end reaction system 1mL, after mixing well, setting shaking table temperature is 25 DEG C, and revolving speed is 200~300r/m Under the conditions of, above-mentioned mixed solution is protected from light 2~3h of shake culture in shaking table respectively, each mixed solution after reaction hypervelocity from Scheming distinguishes thorough centrifuge washing 3~4 times under 13 000r/m speed conditions, using ultrapure water, removes mistake in supernatant respectively Unreacted thyronine T4 antibody, the trilute T3 antibody, thyrotropic hormone TSH antibody of amount, gained are heavy Starch be graphite-thyronine T4 antibody probe compound, graphite-trilute T3 antibody probe compound, Graphite-thyrotropic hormone TSH antibody probe compound is settled to 1mL with ultrapure water, and in 4 DEG C of condition storages.

5. a kind of preparation method of first function five fluorescent microsphere joint-detection devices, feature exist according to claim 2 It is made into 1% after: the polyvinyl alcohol treatment fluid in the step (d) is mixed by polyvinyl alcohol with Triton X-100, through 0.22 μ M membrane filtration, it is spare.

6. a kind of preparation method of first function five fluorescent microsphere joint-detection devices, feature exist according to claim 2 In: pretreated detection device a sample pad one, two sample pad one of detection device, three sample of detection device in the step (e) Product pad one uses one 200ul/cm of sample pad treatment fluid, and the sample pad treatment fluid one is by Tris-HCL liquid, bovine serum albumin(BSA) BSA, casein, surfactant Tween-80 composition, wherein Tris-HCL liquid concentration is 0.1mol/L, bovine serum albumin(BSA) 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) is using at sample pad Two 200ul/cm of liquid is managed, the sample pad treatment fluid two is by Tris-HCL liquid, bovine serum albumin(BSA) BSA, casein, surface-active Agent Tween-80 composition, wherein Tris-HCL liquid concentration is 0.1mol/L, and bovine serum albumin(BSA) BSA concentration is 1%, and casein is dense Degree is 0.2%, surfactant concentration is 1% and anti-thyroglobulin antibody 10ug/ml composition.