CN107449906A - A kind of paper chromatography chemical luminescence detection method - Google Patents

Abstract

The invention discloses a kind of chemiluminescence detection combined complete and detection method, include thing specificity junction mixture, particulate, immobilon-p, chemiluminescent reactant, centrifugal device and luminescence detector to be checked, wherein described chemiluminescent reactant includes chemiluminescence enzyme, chemiluminescent substance, chemiluminescence enzyme reaction substrate and starts at least one of luminescence reagent；The particulate is directly and/or can form non-specific binding by the mode of being chemically crosslinked and protein and/or the chemiluminescent reactant and maintain stable particle；The immobilon-p is with the film quality material with protein non-specific binding characteristic；The centrifugal device is the structure that can centrifuge driving liquid phase chromatographic flow on the immobilon-p；The thing specificity junction mixture to be checked is at least one of antigen with specific binding capacity, antibody, Avidin, biotin and the like.The present invention has the characteristics of high sensitivity, detection time is short, has a good application prospect.

Description

A kind of paper chromatography chemical luminescence detection method

Technical field

The present invention relates to a kind of paper chromatography chemical luminescence detection method, belong to technical field of immunoassay.

Background technology

Immunology detection technology is measure antigen, antibody, immunocyte and the chemical composition of applied immunology principle design Deng laboratory facilities, the sample of medical diagnosis on disease and health detection can be carried out from human body and animal body and be used for by being widely used in Environment, Pharmaceutical Analysis, food and the sample of Industrial Analysis.Conventional has immune turbidity technology, solid-phase enzyme immunoassay technology, changes Learn luminescent detection techniques, immunofluorescence label technology, flow cytometry, colloidal gold technique etc..Immune turbidity technology, it is also referred to as immune Nephelometry is soluble antigen, antibody specific bond in the liquid phase, produces a certain size compound, forms the refraction or suction of light Receive, determine the transmitted light after this refraction or absorption or scattering light as unit of account, for quantitatively detecting, but detection sensitivity It is low, it is not suitable for trace detection.The enzyme of immobilization and antigen or antibody of the solid-phase enzyme immunoassay technology based on antigen or antibody Mark, is incorporated in the antigen of surface of solid phase carriers or antibody keeps its immunologic competence, and the enzyme conjugates of antigen or antibody was both protected Its immunologic competence is stayed, retains the activity of enzyme again, in measure, by inspection sample（Determine antibody or antigen therein）With enzyme mark Antigen or antibody react by the antigen or antibody of different step and surface of solid phase carriers, have high sensitivity, linear response The remarkable advantages such as scope is wide and easy to automate, but detect reaction time length and limit its use.Immunochemiluminescence is examined Survey technology is a kind of highly sensitive micro and Analytical Methods of Trace, have easy to operate, high sensitivity, linear response range wide and It is easy to automate to wait remarkable advantage, it is widely used in environment, clinic, Pharmaceutical Analysis, food and Industrial Analysis, and The luminescence reagent labelling technique of solid phase separation means and antigen or antibody based on antigen or antibody, but detect the reaction time it is long and Requirement height on detection device also influences its use.Immunofluorescence label technology, flow cytometry, colloidal gold technique are also conventional Detection technique be widely used, but have its corresponding deficiency, the detection reaction time is long or sensitivity, accuracy shortcoming are general All over existing deficiency.High sensitivity, quick, miniaturization, quantitative, automation is the hair of current clinical immunization detection technique product entirely Exhibition trend, but existing above-mentioned function can not be all realized simultaneously.

The content of the invention

It is an object of the invention to provide a kind of paper chromatography chemical luminescence detection method.The present invention has high sensitivity, detection The characteristics of time is short, the detection range of linearity is wide, easy to use.

A kind of paper chromatography chemiluminescence detection combined complete provided by the invention, is characterised by, the detection combination suit Containing thing specificity junction mixture, particulate, immobilon-p, chemiluminescent reactant, centrifugal device and luminescence detector to be checked, wherein Described chemiluminescent reactant includes chemiluminescence enzyme, chemiluminescent substance, chemiluminescence enzyme reaction substrate and started luminous At least one of reagent；The particulate is directly and/or can pass through the mode of being chemically crosslinked and protein and/or the chemistry Luminescence-producing reaction thing forms non-specific binding and maintains stable particle；The immobilon-p be with protein non-specific knot Close the film quality material of characteristic；The centrifugal device is the structure that can centrifuge driving liquid phase chromatographic flow on the immobilon-p； The thing specificity junction mixture to be checked is the antigen with specific binding capacity, antibody, Avidin, biotin and the like At least one of.

The detection method that detection combination described in a kind of claim 1 is set with, it is characterised in that：The detection method is as follows （A）-（F）In it is any：

（A）Comprise the following steps：

1）The particulate is marked with the thing specificity junction mixture to be checked and the chemiluminescence enzyme simultaneously；

2）Reacted with the particulate after the sample containing thing to be checked and the mark, the thing to be checked by with after the mark Particulate on the thing specificity junction mixture to be checked combine, and then form " chemiluminescence enzyme-particulate-thing to be checked specificity knot Compound-thing to be checked " compound, i.e. compound 1；

3）Coating can be treated by forming the second of specific binding with the thing to be checked on compound 1 on the immobilon-p Examine thing specificity junction mixture；

4）With the liquid phase containing the compound 1, by chromatography, to flow through coated second thing to be checked on the immobilon-p special Property conjugate, form " chemiluminescence enzyme-particulate-thing to be checked specificity junction mixture-thing-the second to be checked thing to be checked specific binding Thing " compound, i.e. compound 2, and be captured and be fixed on the immobilon-p；

5）Using centrifugal chromatography, cleaned with cleaning fluid described in the compound 1 being not associated with the immobilon-p and residual Chemiluminescence enzyme；

6）The immobilon-p after cleaning is placed in the chemiluminescence enzyme reaction substrate liquid and reacted, and with the luminous detection Chemiluminescence enzyme reaction substrate described in the chemiluminescence enzymatic that device detection is indirectly fixed on the immobilon-p produces Luminous value, to detect the content of the thing to be checked；

（B）Comprise the following steps：

1）The particulate described in the thing specific binding substance markers to be checked；

2）The specificity junction mixture of the thing specificity junction mixture to be checked is marked with the chemiluminescence enzyme, forms " chemiluminescence The specificity junction mixture of enzyme-thing to be checked specificity junction mixture ", i.e. chemiluminescence enzyme marker；

3）Reacted with the particulate after the sample containing thing to be checked and the mark, the thing to be checked by with after the mark Particulate on the thing specificity junction mixture to be checked combine, and then form " particulate-thing to be checked specificity junction mixture-thing to be checked " Compound, i.e. compound 3；

4）The described second thing specificity junction mixture to be checked is coated with the immobilon-p；

5）With the liquid phase containing the compound 3, by chromatography, to flow through coated second thing to be checked on the immobilon-p special Property conjugate, formed " particulate-thing to be checked specificity junction mixture-thing-the second to be checked thing specificity junction mixture to be checked " compound, i.e., Compound 4, and be captured and be fixed on the immobilon-p；

6）With the liquid phase containing the chemiluminescence enzyme marker by chromatography flow through be trapped in it is compound on the immobilon-p Thing 4, by being combined with the thing specificity junction mixture to be checked, formed " the chemiluminescence enzyme marker-compound of compound 4 ", i.e., Compound 5, and be captured and be fixed on the immobilon-p；

6）Using centrifugal chromatography, the chemiluminescence enzyme marker that is not associated with the immobilon-p and residual is cleaned with cleaning fluid The chemiluminescence enzyme stayed；

7）The immobilon-p after cleaning is placed in the chemiluminescence enzyme reaction substrate liquid and reacted, and with the luminous detection The chemiluminescence enzyme reaction substrate described in the chemiluminescence enzymatic that device detection is indirectly fixed on the immobilon-p Caused luminous value, to detect the content of the thing to be checked；

（C）Comprise the following steps：

1）The particulate is marked with the thing specificity junction mixture to be checked and the chemiluminescence enzyme simultaneously；

2）Reacted with the particulate after the sample containing thing to be checked and the mark, the thing to be checked by with after the mark Particulate on the thing specificity junction mixture to be checked combine, the thing to be checked by with the thing specificity junction mixture knot to be checked Close, and then form " chemiluminescence enzyme-particulate-thing to be checked specificity junction mixture-thing to be checked " compound, i.e., described compound 1；

3）The second thing specificity junction mixture to be checked is marked with intermediate material A；

4）Coating can form the intermediate material B of specific binding with the intermediate material A on the immobilon-p；

5）The the second thing specificity junction mixture to be checked marked with the liquid phase containing the compound 1 with the intermediate material A is carried out Reaction, formed " chemiluminescence enzyme-particulate-thing to be checked specificity junction mixture-thing-the second to be checked thing specificity junction mixture to be checked-in Between substance A " compound, i.e. compound 6；

6）The coated intermediate material B on the immobilon-p is flowed through by chromatography with the liquid phase containing the compound 6, formed " chemiluminescence enzyme-particulate-thing to be checked specificity junction mixture-thing-the second to be checked thing specificity junction mixture-intermediate material A- to be checked Intermediate material B " compounds, i.e. compound 7, and be captured and be fixed on the immobilon-p；

7）Using centrifugal chromatography, cleaned with cleaning fluid described in the compound 6 being not associated with the immobilon-p and residual Chemiluminescence enzyme；

8）The immobilon-p after cleaning is placed in the chemiluminescence enzyme reaction substrate liquid and reacted, and with the luminous detection Chemiluminescence enzyme reaction substrate described in the chemiluminescence enzymatic that device detection is indirectly fixed on the immobilon-p produces Luminous value, to detect the content of the thing to be checked；

（D）Comprise the following steps：

1）The particulate described in the thing specific binding substance markers to be checked；

2）The specificity junction mixture of the thing specificity junction mixture to be checked is marked with the chemiluminescence enzyme, forms " chemiluminescence The specificity junction mixture of enzyme-thing to be checked specificity junction mixture ", i.e., described chemiluminescence enzyme marker；

3）Reacted with the particulate after the sample containing thing to be checked and the mark, the thing to be checked by with after the mark Particulate on the thing specificity junction mixture to be checked combine, and then form " particulate-thing to be checked specificity junction mixture-thing to be checked " Compound, i.e., described compound 3；

4）The second thing specificity junction mixture to be checked is marked with the intermediate material A；

5）Coating can form the intermediate material B of specific binding with the intermediate material A on the immobilon-p；

6）Reacted with the second thing specificity junction mixture to be checked of the liquid phase containing the compound 3 and intermediate material A marks, Formed " particulate-thing to be checked specificity junction mixture-thing-the second to be checked thing specificity junction mixture to be checked-intermediate material A " compounds, That is compound 8；

7）The coated intermediate material B on the immobilon-p is flowed through by chromatography with the liquid phase containing the compound 8, formed " particulate-thing to be checked specificity junction mixture-thing-the second to be checked thing specificity junction mixture to be checked-intermediate material A- intermediate materials B " is multiple Compound, i.e. compound 9, and be captured and be fixed on the immobilon-p；

8）With the liquid phase containing the chemiluminescence enzyme marker by chromatography flow through be trapped in it is compound on the immobilon-p Thing 9, by being combined with thing specificity junction mixture to be checked, form " the specificity knot of chemiluminescence enzyme-thing to be checked specificity junction mixture Compound-the compound of compound 9 ", i.e. compound 10, and be captured and be fixed on the immobilon-p；

9）Using centrifugal chromatography, the chemiluminescence enzyme marker that is not associated with the immobilon-p and residual is cleaned with cleaning fluid The chemiluminescence enzyme stayed；

10）The immobilon-p after cleaning is placed in chemiluminescence enzyme reaction substrate liquid and reacted, and with the luminescence detector Detect and lighted caused by chemiluminescence enzyme reaction substrate described in the chemiluminescence enzymatic being indirectly fixed on the immobilon-p Value, to detect the content of the thing to be checked；

（E）Comprise the following steps：

1）The thing specificity junction mixture to be checked and chemiluminescent substance mark are connected on the particulate；

2）Reacted with the particulate after the sample containing thing to be checked and the mark, the thing to be checked by with after the mark Particulate on the thing specificity junction mixture to be checked combine, and then form " chemiluminescent substance-particulate-thing to be checked specificity Conjugate-thing to be checked " compound, i.e. compound 11；

3）The second thing specificity junction mixture to be checked is coated with the immobilon-p；

4）The coated second thing spy to be checked on the immobilon-p is flowed through by chromatography with the liquid phase containing the compound 11 Specific binding agent, form " chemiluminescent substance-particulate-thing to be checked specificity junction mixture-thing-the second to be checked thing specificity to be checked Conjugate " compound, i.e. compound 12, and be captured and be fixed on the immobilon-p；

5）Using centrifugal chromatography, described in cleaning fluid cleans the compound 11 being not associated with the immobilon-p and residual Chemiluminescent substance；

6）The immobilon-p after cleaning is placed in the starting luminescence reagent solution and reacted, and is examined with the luminescence detector Luminous value caused by the chemiluminescent substance decomposition being indirectly fixed on the immobilon-p is surveyed, to detect the thing to be checked Content；

（F）Comprise the following steps：

1）The particulate described in the thing specific binding substance markers to be checked；

2）The specificity junction mixture of the thing specificity junction mixture to be checked is marked with the chemiluminescent substance, forms " chemistry hair The specificity junction mixture of stimulative substance-thing to be checked specificity junction mixture ", i.e. chemiluminescent substance label；

3）Reacted with the particulate after the sample containing thing to be checked and the mark, the thing to be checked by with after the mark Particulate on the thing specificity junction mixture to be checked combine, and then form " particulate-thing to be checked specificity junction mixture-thing to be checked " Compound, i.e., described compound 3；

4）The second thing specificity junction mixture to be checked is coated with the immobilon-p；

5）With the liquid phase containing the compound 3, by chromatography, to flow through coated second thing to be checked on the immobilon-p special Property conjugate, formed " particulate-thing to be checked specificity junction mixture-thing-the second to be checked thing specificity junction mixture to be checked " compound, i.e., The compound 4, and be captured and be fixed on the immobilon-p；

6）With the liquid phase containing chemiluminescent substance label by chromatography flow through be trapped in it is described multiple on the immobilon-p Compound 4, by being combined with the thing specificity junction mixture to be checked, form " chemiluminescent substance-thing to be checked specificity junction mixture Specificity junction mixture-the compound of compound 4 ", i.e. compound 13, and be captured and be fixed on the immobilon-p；

6）Using centrifugal chromatography, cleaned with cleaning fluid the chemiluminescent substance label that is not associated with the immobilon-p and The chemiluminescent substance of residual；

7）The immobilon-p after cleaning is placed in the starting luminescence reagent and reacted, and quilt is detected with the luminescence detector Luminous value caused by the chemiluminescent substance decomposition on the immobilon-p is indirectly fixed to, to detect the content of the thing to be checked.

In above-mentioned paper chromatography chemical luminescence detection method, the intermediate material is anti-with specific binding capacity At least one of original, antibody, Avidin, biotin and the like.

It is described to chromatograph the reaction for flowing through the immobilon-p in centrifugation dress in above-mentioned paper chromatography chemical luminescence detection method Put and complete and using centrifugal chromatography driving liquid chromatographic flow on immobilon-p.

In above-mentioned paper chromatography chemiluminescence detection combined complete or detection method, the particle diameter of particulate selection 1nm ~ 10um, concretely 10 ~ 3000 nm, 20 ~ 2000 nm or 43 ~ 1000 nm.The particulate is can directly and/or passing through Crosslinking method is learned to form non-specific binding with protein and/or the chemiluminescent reactant and maintain stable particle, mesh Preceding conventional particulate have colloidal gold particles, colloidal selenium particles, colloid gold-magnetic particles, fluorescent microsphere, magnetic particle, gold-magnetic particles, Microgel particle, emulsion particle, plastic microsphere, microsphere silica gel, agarose beads, ps particle, silicon dioxide microsphere, polyphenyl Ethene microballoon, carboxyl microballoon, chloromethyl microballoon etc..

In above-mentioned paper chromatography chemiluminescence detection combined complete, the rotating speed of the centrifugal device uses programme-control side Formula；The rotating speed of the centrifugal device is 200 ~ 10000 revs/min, concretely 500 ~ 5000 revs/min, 800 ~ 3000 Rev/min or 800 ~ 2000 revs/min.

In above-mentioned paper chromatography chemical luminescence detection method, the rotating speed of the centrifugal chromatography uses program controlled mode； The rotating speed of the centrichromatography is 200 ~ 10000 revs/min, concretely 500 ~ 5000 revs/min, 800 ~ 3000 revs/min Clock or 800 ~ 2000 revs/min.

In above-mentioned paper chromatography chemiluminescence detection combined complete or detection method, the chemiluminescence enzyme includes horseradish mistake At least one of oxidizing ferment, alkaline phosphatase and xanthine oxidase.

In above-mentioned paper chromatography chemiluminescence detection combined complete or detection method, the immobilon-p includes nitrocellulose Film, polyvinylidene fluoride film, nylon membrane, DEAE cellulose membranes, the one or both sides of the immobilon-p carry backing；In the present invention, The polyvinylidene fluoride film abbreviation pvdf membrane；The DEAE cellulose membranes refer to diethyl amino ethyl group (DEAE) introducing cellulose Manufactured paper-like film after molecule, it is a kind of weak base type anion-exchange material；The detector is chemiluminescence detector, institute State the one or both sides that detector is located at the immobilon-p.

In above-mentioned paper chromatography chemiluminescence detection combined complete, the detection combination suit also includes the cleaning Liquid, the cleaning fluid are the normal experiment cushioning liquid containing surfactant.Currently used surfactant has TWEEN20, TritonX-100, TritonX-405 etc..

In above-mentioned paper chromatography chemical luminescence detection method, the cleaning fluid is used for the normal experiment containing surfactant Cushioning liquid.

In above-mentioned paper chromatography chemiluminescence detection combined complete, the detection combination suit also includes the second thing to be checked Specificity junction mixture, intermediate material A, intermediate material B, thing specificity junction mixture to be checked specificity junction mixture at least one Kind.

Application of the described combined complete or detection method in immune detection product development.

In above-mentioned paper chromatography chemical luminescence detection method, the chemical luminous substrate of the HRPO often with luminol and The different Shandong promise of base-N- ethyls of different luminol and its derivative species, such as different luminol, 4- amino and AHEI and ABEI, mesh Preceding conventional product has the West Pico chemiluminescence detections substrate of PIERCE companies production, West Dura chemiluminescence detections Substrate, West Femto chemiluminescence detection substrates.What the chemical luminous substrate of alkaline phosphatase was commonly used has (golden steel alkane) -1,2- Dichloroethane and its derivative, AMPPD, CDP-STAR, Lumi-Phos 530.The chemical luminous substrate of xanthine oxidase has Xanthine, myricetin, Quercetin.

In above-mentioned paper chromatography chemical luminescence detection method, non-enzyme-catalyzed chemical luminescence substrate, i.e., direct chemiluminescent substance, It is the immunoassay method with the direct labelled antigen of chemiluminescence agent or antibody.The chemiluminescent substance for being usually used in mark has acridine Ester type compound-acridinium ester (AE), are effective luminous markers, it is by starting luminescence reagent （NaOH、H₂O₂）Act on and light, mainly there is acridinium ester and a word used for translation shallow lake amide-type, tris (bipyridine) ruthenium etc..

For the present invention due to taking above technical scheme, it has advantages below：

1st, the present invention uses carrying carrier of the particulate as thing to be checked and its intermediate reaction material, and chromatographic flow is simultaneously on immobilon-p Reaction is completed, improves capture binding ability of the immobilon-p to thing to be checked and its intermediate reaction material.

2nd, the liquid phase that the present invention is driven using centrifugal device chromatographic flow on immobilon-p, can be effectively reduced to be special Property capture chemiluminescent substance and immobilon-p non-specific binding, reduce immobilon-p ambient noise interference, improve inspection Survey sensitivity.

3rd, existing flat board and magnetic microparticle separating chemiluminescence detection technique, using multi-step, too many levels drive control, It is related to detection sample, detection phase and the transposition and movement of reaction carriers and needs to carry out under temperature control.The present invention adopts Chemiluminescence detection is carried out with paper chromatography, room temperature is optimized for and the single mode of chromatographic flow is reacted, shorten detection time, letter Operation and the design of corresponding instrument and cost of manufacture are changed.

4th, operating procedure of the present invention is simple, it is easy to accomplish automation mechanized operation.The inventive method has high sensitivity, Quan Ding The characteristics of amount, automation, while have that detection is quick, uses the simple detection technique of equipment again；Not only easy to use, reduction original The waste of material, while operating efficiency is also significantly improved, applied to detection and analysis, the numerous areas of separation.

Brief description of the drawings

Fig. 1 is the schematic diagram of the paper chromatography chemiluminescence detection combined complete of the embodiment of the present invention 1.

1 thing specificity junction mixture to be checked；2 particulates；3 immobilon-ps；4 chemiluminescent reactants；5 centrifugal devices；6 luminous inspections Survey device；7 second thing specificity junction mixtures or intermediate material B to be checked.

Embodiment

The present invention is further described below in conjunction with the accompanying drawings, but the invention is not limited in following examples.

The preparation of embodiment 1, paper chromatography chemiluminescence detection combined complete of the present invention：

As shown in figure 1, paper chromatography chemiluminescence detection combined complete of the present invention includes, thing specificity junction mixture 1 to be checked, particulate 2, Immobilon-p 3, chemiluminescence enzyme or chemiluminescent substance 4, centrifugal device 5, luminescence detector 6, chemiluminescence enzyme reaction substrate or Luminescence reagent 7 is started, while also includes the second thing specificity junction mixture or intermediate material B 8 to be checked, mark adsorbed film pad 9, inhale Water film bearing 10, support egative film 11.Thing specificity junction mixture 1 and chemiluminescence enzyme or chemiluminescent substance 4 to be checked and the knot of particulate 2 Close, and be printed on mark adsorbed film pad 8.The second thing specificity junction mixture or intermediate material B to be checked is printed with immobilon-p 3 to catch Obtain detection line 8.Binding mark adsorbed film pad 9, immobilon-p 3 and absorbing membrane pad 10 successively on support egative film 11, detection examination is made Paper slip.During detection, test strip is placed on centrifugal device 5, loads on mark adsorbed film pad 8 and is treated containing thing to be checked Sample sheet, be now marked with the particulate 2 of thing specificity junction mixture 1 and chemiluminescence enzyme or chemiluminescent substance 4 to be checked with it is to be checked Thing combines, and forms thing compound to be checked, and chromatography flows through immobilon-p 3, and the thing compound to be checked formed is coated on immobilon-p 3 Acquisition Detection line 7 captured, carry out chromatography cleaning, then by chemiluminescence enzyme reaction substrate or start luminescence reagent 7 it is anti- Should, and then luminous, the chemiluminescence captured indirectly using Acquisition Detection line 8 of the detection of luminescence detector 6 on immobilon-p 3 The luminous quantity of enzyme or chemiluminescent substance 4, and then obtain the content of thing to be checked, wherein sample introduction, capture and the process of cleaning from Completed on center device 5 by centrichromatography.

The detection method and its effect of following description of test present invention, but be not limitation of the invention.In following experiments Used experimental method is conventional method unless otherwise specified.Material used, reagent etc. in following experiments, such as without spy Different explanation, is commercially obtained.

Experiment one, paper chromatography chemiluminescence detection of the present invention experiment（One）

Using colloidal gold particles as liquid phase reactor delivery vehicle, tested using methods described A.

First, experiment material

Anti-human myoglobins polyclonal antibody（Genagates companies of the U.S.）, anti-human myoglobins monoclonal antibody（The U.S. Genagates companies）, HRPO（HRP, SIGMA product）, spectrophotometer（The limited public affairs of Shanghai mountain valley with clumps of trees and bamboo China tech equipment Department, 752 ultraviolet-uisible spectrophotometers）, human muscle hemoglobin（Sigma-Aldrich products）, BioFlow die instrument（The U.S. IMAGENE companies）, Index cutting machines（A-point companies of the U.S.）, DBF-900 sealing machines（Wenzhou south of the River packing factory）, ACBO dehumidifiers（Jiangsu wuxi Ao Bo dehumidifiers company）, desk centrifuge（Eppendoff companies of the U.S.）, bovine serum albumin In vain（Abbreviation BSA, SIGMA product）, nitrocellulose diaphragm（AE 99, provided by Genagates companies of the U.S.）, multi-polyester fibre Plain film (Reemay 2033, U.S.'s Alstrom Products) is tieed up, absorb water paper membrane pad（Grade 470, the production of S＆S companies of the U.S. Product）, gold chloride（SIGMA products）, chemiluminescence detector（Promega, Glomax Multi JR Detection System）, West Pico luminescence reagents（Thermo scientific）.

It is as follows that above-mentioned material corresponds to Patent Application Request content, and the anti-human myoglobins polyclonal antibody of this experiment is second to treat Thing specificity junction mixture is examined, anti-human myoglobins monoclonal antibody is thing specificity junction mixture to be checked, and HRPO is change Chemiluminescence enzyme, the human muscle hemoglobin for learning luminescence-producing reaction thing are thing to be checked, and desk centrifuge is centrifugal device, nitrocellulose filter Piece is immobilon-p, and gold chloride is the material for preparing particulate, and chemiluminescence detector is luminescence detector, and West Pico chemistry is sent out Light detection substrate is the chemiluminescence enzyme reaction substrate of chemiluminescent reactant.

2nd, experimental method

The preparation of human muscle hemoglobin solution：The human muscle hemoglobin solution of concentration known is taken, uses sample dilution buffer（1%BSA, 100mM glycine, 50mM PBS, 150mM NaCl, pH7.4）Dilution configuration 0.1,1.0,10,100,500,1000ng/ml Serial human muscle hemoglobin solution.

Colloidal gold particles mark：10ml pure water is taken, heating stirring, it is molten that the gold chlorides of 500 μ l 10% are added when boiling water Liquid, heating are boiled 5 minutes, add the citric acid three sodium solutions of 500 μ l 12%, are kept this solution stirring boiling 10 minutes, are dropped naturally Warm to room temperature, produce particle diameter 50nm colloidal gold particles solution.Colloidal gold solution volume 10ml is taken, pH is adjusted extremely with 10% potassium carbonate 8.3, the anti-human μ g of myoglobins monoclonal antibody 100, the μ g of HRPO 100 are rapidly added, to each 10 μ g/ml final concentrations, Beaker mixing is rocked, room temperature is placed 30 minutes, is rapidly added 10% bovine serum albumin solution 100ul, makes final concentration of 1%, together When shake beaker, room temperature is placed 30 minutes, and 12000rpm is centrifuged 20 minutes, carefully suctions out supernatant；Add 5ml 50mM phosphorus Hydrochlorate (PBS) buffer solution, pH7.4, precipitation is suspended, 12000rpm is centrifuged 20 minutes, suctions out supernatant, precipitation is dissolved in 1.0ml contains in 1% bovine serum albumin(BSA) and the phosphate buffer of 3% sucrose, and 4 DEG C are kept in dark place.

Colloidal gold particles mark absorption film preparation：Preparation contains 0.5%PVA（That is polyvinyl alcohol）, 50mM PBS liquid, 0.5% BSA, 0.88% NaCl, pH 7.4 multi-polyester cellulose membrane pretreatment fluid, pending multi-polyester cellulose membrane is put in pre- place Manage in liquid, soaking at room temperature 1 hour, take the film out, put and seal after 37 DEG C of dryings standby, can also be used directly as dispersion membrane.Take Colloidal gold particles labelled antibody solution, with colloid buffer solution（1%BSA, 3% sucrose, 50mM PBS, pH7.4）It is diluted to OD530 For 30, start die instrument, load antibody, open pressurized nitrogen, take multi-polyester cellulose membrane, start die, set die condition For：The airbrush translational speed 30mm/ seconds, the μ l/cm of liquid fltting speed 5.0, the film after printing is put into drying box, 37 DEG C dry Dry 6 hours, it is subsequently placed in the hermetic bag containing drier and preserves use.

It is prepared by polyclonal antibody die：Anti-human myoglobins Anti-TNF-α liquid solution is taken, with 50mM phosphate buffers (pH 7.4) is diluted to 1mg/ml concentration.Start die instrument, load antibody, take the PVC pieces for posting nitrocellulose filter（That is polychlorostyrene Piece of vinyl）, start die, set die condition as：Airbrush translational speed 30mm/ seconds, the μ l/cm of liquid fltting speed 1.5.Will print The film made is put into 37 DEG C of drying boxes, dries 6 hours, then film is placed in the drying receptacle containing drier and preserves use.

Semi-finished product assemble method：Starting dehumidifier makes the humidity in operating room be reduced to less than 25%, is printed in polyclonal antibody Water suction paper membrane pad and colloidal gold particles mark adsorbed film are pasted in film both ends respectively, then with Pressure sensitive adhesive tape sealing label surface.Put stickup Good detection lug is on cutting machine, being cut into 3.5mm test strips.Test strips are put into the aluminium amber hermetic bag of drier, sealed Sealed on mouth machine, labelling.

Experimental group：The test strips of above-mentioned preparation are taken, with the side of colloidal gold particles mark adsorbed film upward, are placed in centrifuge In rotor, the human muscle hemoglobin solution 80ul for the various concentrations that preparation is added dropwise on adsorbed film is marked to colloidal gold particles, stands 2 points Clock, with 1000 revs/min centrifuge 1 minute, then to colloidal gold particles mark adsorbed film on be added dropwise pH7.4 0.05% Tween-20, 50mM PBSs 80ul, 2000 revs/min of centrifugations are cleaned for 30 seconds, and cleaning twice, takes out test strips, cuts polyclonal antibody print Film detection line, is positioned in cuvette, adds West Pico chemiluminescence detection substrates, 5 minutes is stood, in chemiluminescence 6 seconds luminous quantities are read on detector, experiment in triplicate, results averaged, then draws concentration-luminosity curve, and calculate Coefficient correlation.

Control group：The test strips of above-mentioned preparation are taken, are placed in desktop, is marked to colloidal gold particles and is added dropwise what is prepared on adsorbed film The human muscle hemoglobin solution 80ul of various concentrations, stand, treat the red colloid gold particulate mark on colloidal gold particles mark adsorbed film Remember thing all chromatography flow into nitrocellulose filter, then to colloidal gold particles mark adsorbed film on be added dropwise pH7.4 0.05% tween- 20th, 50mM PBSs 80ul, stand, liquid all flows into nitrocellulose filter, and cleaning twice, is removed test strips, cut Polyclonal antibody die detection line, is positioned in cuvette, adds West Pico chemiluminescence detection substrates, stands 5 points Clock, reads 6 seconds luminous quantities on chemiluminescence detector, and experiment in triplicate, results averaged, then draws concentration-hair Light curve, and calculate coefficient correlation.

3rd, experimental result

The present invention is tested using colloidal gold particles as liquid phase reactor delivery vehicle using methods described A, as a result, the present invention is real Test group test strips single detection process time average out to 4.6 minutes, control test group（Do not do centrifugal treating）Test strips single is examined Survey processing time average out to 47 minutes；Experimental group detection data of the present invention are in the dependency relation of good concentration-luminous value, related Coefficient r is 0.995, and control group detects data in below 100ng/ml, the substantially correlation without concentration-luminous value, mainly by It can not be effectively cleaned in non-specific binding of the chemiluminescence enzyme on immobilon-p, cause background too high relevant, phase relation Number r is 0.776, P ＜ 0.05, and result of the present invention is significantly better than the testing result for not doing centrifugal treating, illustrates that the technology of the present invention carries The high linear and accuracy of prior art detection.Experimental result is as shown in table 1.

The paper chromatography chemical luminescence detection method A experimental results of the present invention of table 1（Unit：Luminous value）

Experiment two, paper chromatography chemiluminescence detection of the present invention experiment（Two）

Using colloidal gold particles as liquid phase reactor delivery vehicle, tested using methods described B.

First, experiment material

Alkaline phosphatase（ALP, Roche Holding Ag's product）, sheep anti-mouse igg polyclonal antibody（Genagates companies of the U.S.）, APLS Luminescence reagent（Zhengzhou English promise biology）, it is other with experiment one.

Above-mentioned material corresponds to that Patent Application Request content is as follows, and this experiment alkaline phosphatase is the change of chemiluminescent reactant Learn Luminescence Enzyme, the specificity junction mixture that sheep anti-mouse igg polyclonal antibody is thing specificity junction mixture to be checked, APLS luminescence reagents are The chemiluminescence enzyme reaction substrate of chemiluminescent reactant, it is other with experiment one.

2nd, experimental method

The preparation of human muscle hemoglobin solution：With experiment one.

The alkali phosphatase enzyme mark of sheep anti-mouse igg polyclonal antibody：Using improvement Over-voltage protection, l mg alkalescence phosphorus is taken Sour enzyme is dissolved in the M pH 8.0 of 200uL 0.3 NaHCO₃In buffer solution, add the M of l ml 0.06 sodium periodate, at room temperature lucifuge Gently stir 0.5 hour, add 1 ml 0.16M ethylene glycol, gently stir at room temperature 1 hour, oxidation is terminated, in 0.01 M pH 9.5 carbon 4 DEG C of dialysed overnights in phthalate buffer, the carbonate buffer solution l ml containing 0.5 mg antibody being added, room temperature lucifuge is gently stirred 3 hours, Add 5 mg NaHB₄, 4 DEG C overnight, in 0.01 M, the PBSs of pH 7.2 4 DEG C dialyse 24 hours, 4 DEG C of 4000rpm from The heart 30 divides, and removes precipitation, adds isometric glycerine, -20 DEG C of preservations.

Colloidal gold particles mark：Colloidal gold solution volume 10ml is taken, pH to 8.3 is adjusted with 10% potassium carbonate, is rapidly added anti-human The μ g of myoglobins monoclonal antibody 100, to 10 μ g/ml final concentrations, rock beaker mixing.Other same experiments one.

Colloidal gold particles mark absorption film preparation：With experiment one.

It is prepared by polyclonal antibody die：With experiment one.

Semi-finished product assemble method：With experiment one.

Experimental group：The test strips of above-mentioned preparation are taken, with the side of colloidal gold particles mark adsorbed film upward, are placed in centrifuge In rotor, the human muscle hemoglobin solution 80ul for the various concentrations that preparation is added dropwise on adsorbed film is marked to colloidal gold particles, stands 2 points Clock, centrifuged 1 minute with 1000 revs/min, mark dropwise addition alkali phosphatase enzyme mark sheep anti-mouse igg on adsorbed film more to colloidal gold particles Clonal antibody solution 80ul, 2 minutes are stood, centrifuged 1 minute with 1000 revs/min, then marked to colloidal gold particles and dripped on adsorbed film Add pH7.4 0.05% Tween-20,50mM PBSs 80ul, 2000 revs/min of centrifugations are cleaned for 30 seconds, and cleaning twice, is taken out Test strips, polyclonal antibody die detection line is cut, is positioned in cuvette, add APLS chemiluminescence detection substrates, it is quiet Put 1 minute, 6 seconds luminous quantities are read on chemiluminescence detector, experiment in triplicate, results averaged, is then drawn dense Degree-luminosity curve, and calculate coefficient correlation.

Control group：The test strips of above-mentioned preparation are taken, are placed in desktop, is marked to colloidal gold particles and is added dropwise what is prepared on adsorbed film The human muscle hemoglobin solution 80ul of various concentrations, stand, treat the red colloid gold particulate mark on colloidal gold particles mark adsorbed film Remember that all chromatography flows into nitrocellulose filter to thing, marks to colloidal gold particles and alkali phosphatase enzyme mark sheep anti mouse is added dropwise on adsorbed film IgG Anti-TNF-α liquid solution 80ul, stand, liquid all flows into nitrocellulose filter, then marks adsorbed film to colloidal gold particles Upper dropwise addition pH7.4 0.05% Tween-20,50mM PBS 80ul, stand, liquid all flows into nitrocellulose filter, clearly Wash twice, remove test strips, cut polyclonal antibody die detection line, be positioned in cuvette, add APLS chemiluminescences Detection substrate, 1 minute is stood, 6 seconds luminous quantities is read on chemiluminescence detector, tested in triplicate, results averaged, Then concentration-luminosity curve is drawn, and calculates coefficient correlation.

3rd, experimental result

This experiment is tested using colloidal gold particles as liquid phase reactor delivery vehicle using methods described B, as a result, the present invention is real Test group test strips single detection process time average out to 7.8 minutes, control test group（Do not do centrifugal treating）Test strips single is examined Survey processing time average out to 62 minutes；Experimental group detection data of the present invention are in the dependency relation of good concentration-luminous value, related Coefficient r is 0.994, and control group detects data in below 100ng/ml, the substantially correlation without concentration-luminous value, mainly by It can not be effectively cleaned in non-specific binding of the chemiluminescence enzyme on immobilon-p, cause background too high relevant, phase relation Number r is 0.807, P ＜ 0.05, and result of the present invention is significantly better than the testing result for not doing centrifugal treating, illustrates that the technology of the present invention carries The high linear and accuracy of prior art detection.Experimental result is as shown in table 2.

The paper chromatography chemical luminescence detection method B experimental results of the present invention of table 2（Unit：Luminous value）

Experiment three, paper chromatography chemiluminescence detection of the present invention experiment（Three）

Using colloidal gold particles as liquid phase reactor delivery vehicle, tested using methods described C.

First, experiment material

NHS activated biotins（SIGMA,）, Avidin（SIGMA）, it is other with experiment one.

Above-mentioned material corresponds to that Patent Application Request content is as follows, and this bioorganism element is intermediate material A, Avidin is middle Substance B, it is other with experiment one.

2nd, experimental method

The preparation of human muscle hemoglobin solution：With experiment one.

Anti-human myoglobins polyclonal antibody biotin labeling：Anti-human myoglobins polyclonal antibody is taken in 0.1M pH9.5 Dialysed overnight in sodium carbonate buffer, adjust final concentration of 2mg/ml.NHS activated biotins 20mg is taken to be dissolved in 1ml dimethyl methyls Acid amides, 50ul is taken, be added in above-mentioned solution, reacted at room temperature 4 hours.Reaction solution is dialyzed overnight in PBS, -20 DEG C Preserve.

Colloidal gold particles mark：With experiment one.

Colloidal gold particles mark absorption film preparation：Colloidal gold particles labelled antibody solution is taken, with colloid buffer solution（1%BSA, 3% sucrose, 50mM PBS, pH7.4）It is 30 to be diluted to OD530, adds the anti-human myoglobins polyclonal antibody 10 of biotin labeling μ g/ml, mix.Other same experiments one.

It is prepared by Avidin die：Avidin solution is taken, it is dense to be diluted to 1mg/ml with 50mM phosphate buffers (pH 7.4) Degree.Start die instrument, load avidin solution, it is other with experiment one.

Semi-finished product assemble method：Starting dehumidifier makes the humidity in operating room be reduced to less than 25%, in Avidin die two Water suction paper membrane pad and colloidal gold particles mark adsorbed film are pasted in end respectively, other with experiment one.

Experimental group：The test strips of above-mentioned preparation are taken, with the side of colloidal gold particles mark adsorbed film upward, are placed in centrifuge In rotor, the human muscle hemoglobin solution 80ul for the various concentrations that preparation is added dropwise on adsorbed film is marked to colloidal gold particles, stands 2 points Clock, with 1000 revs/min centrifuge 1 minute, then to colloidal gold particles mark adsorbed film on be added dropwise pH7.4 0.05% Tween-20, 50mM PBSs 80ul, 2000 revs/min of centrifugations are cleaned for 30 seconds, and cleaning twice, takes out test strips, cuts the inspection of Avidin die Survey line, it is positioned in cuvette, adds West Pico chemiluminescence detection substrates, 5 minutes is stood, in chemiluminescence detection 6 seconds luminous quantities are read on instrument, experiment in triplicate, results averaged, then draws concentration-luminosity curve, and calculate correlation Coefficient.

Control group：The test strips of above-mentioned preparation are taken, are placed in desktop, is marked to colloidal gold particles and is added dropwise what is prepared on adsorbed film The human muscle hemoglobin solution 80ul of various concentrations, stand, treat the red colloid gold particulate mark on colloidal gold particles mark adsorbed film Remember thing all chromatography flow into nitrocellulose filter, then to colloidal gold particles mark adsorbed film on be added dropwise pH7.4 0.05% tween- 20th, 50mM PBSs 80ul, stand, liquid all flows into nitrocellulose filter, and cleaning twice, is removed test strips, cut Avidin die detection line, is positioned in cuvette, adds West Pico chemiluminescence detection substrates, stands 5 minutes, 6 seconds luminous quantities are read on chemiluminescence detector, experiment in triplicate, results averaged, then draws concentration-luminous song Line, and calculate coefficient correlation.

3rd, experimental result

This experiment is tested using colloidal gold particles as liquid phase reactor delivery vehicle using methods described C, as a result, the present invention is real Test group test strips single detection process time average out to 4.6 minutes, control test group（Do not do centrifugal treating）Test strips single is examined Survey processing time average out to 42 minutes；Experimental group detection data of the present invention are in the dependency relation of good concentration-luminous value, 500ng/ml has just reached reaction platform, correlation coefficient r 0.987, and control group detects data in below 100ng/ml, does not have substantially There is the correlation of concentration-luminous value, can not be by effectively mainly due to non-specific binding of the chemiluminescence enzyme on immobilon-p Cleaning, causes background too high relevant, correlation coefficient r 0.866, P ＜ 0.05, result of the present invention, which is significantly better than, does not do centrifugal treating Testing result, illustrate the technology of the present invention improve prior art detection linear and accuracy.Meanwhile add intermediate material production Signal amplification has been given birth to, has improved the response intensity of each concentration.Experimental result is as shown in table 3.

The paper chromatography chemical luminescence detection method C experimental results of the present invention of table 3（Unit：Luminous value）

Experiment four, paper chromatography chemiluminescence detection of the present invention experiment（Four）

Using colloidal gold particles as liquid phase reactor delivery vehicle, tested using methods described D.

First, experiment material

NHS activated biotins（SIGMA,）, Avidin（SIGMA）, alkaline phosphatase（ALP, Roche Holding Ag's product）, sheep anti mouse IgG polyclonal antibodies（Genagates companies of the U.S.）, APLS luminescence reagents（Zhengzhou English promise biology）, it is other with experiment one.

Above-mentioned material corresponds to that Patent Application Request content is as follows, and this experiment alkaline phosphatase is the change of chemiluminescent reactant Learn Luminescence Enzyme, the specificity junction mixture that sheep anti-mouse igg polyclonal antibody is thing specificity junction mixture to be checked, biotin is intermediate Matter A, Avidin are the chemiluminescence enzyme reaction substrate that intermediate material B, APLS luminescence reagent are chemiluminescent reactant, Qi Tatong Experiment one.

2nd, experimental method

The preparation of human muscle hemoglobin solution：With experiment one.

Sheep anti-mouse igg polyclonal antibody alkali phosphatase enzyme mark：With experiment two.

Anti-human myoglobins polyclonal antibody biotin labeling：With experiment three.

Colloidal gold particles mark：With experiment two.

Colloidal gold particles mark absorption film preparation：With experiment three.

It is prepared by Avidin die：With experiment three.

Semi-finished product assemble method：With experiment three.

Experimental group：The test strips of above-mentioned preparation are taken, with the side of colloidal gold particles mark adsorbed film upward, are placed in centrifuge In rotor, the human muscle hemoglobin solution 80ul for the various concentrations that preparation is added dropwise on adsorbed film is marked to colloidal gold particles, stands 2 points Clock, centrifuged 1 minute with 1000 revs/min, mark dropwise addition alkali phosphatase enzyme mark sheep anti-mouse igg on adsorbed film more to colloidal gold particles Clonal antibody solution 80ul, 2 minutes are stood, centrifuged 1 minute with 1000 revs/min, then marked to colloidal gold particles and dripped on adsorbed film Add pH7.4 0.05% Tween-20,50mM PBSs 80ul, 2000 revs/min of centrifugations are cleaned for 30 seconds, and cleaning twice, is taken out Test strips, Avidin die detection line is cut, is positioned in cuvette, add APLS chemiluminescence detection substrates, stand 1 point Clock, reads 6 seconds luminous quantities on chemiluminescence detector, and experiment in triplicate, results averaged, then draws concentration-hair Light curve, and calculate coefficient correlation.

Control group：The test strips of above-mentioned preparation are taken, are placed in desktop, is marked to colloidal gold particles and is added dropwise what is prepared on adsorbed film The human muscle hemoglobin solution 80ul of various concentrations, stand, treat the red colloid gold particulate mark on colloidal gold particles mark adsorbed film Remember that all chromatography flows into nitrocellulose filter to thing, marks to colloidal gold particles and alkali phosphatase enzyme mark sheep anti mouse is added dropwise on adsorbed film IgG Anti-TNF-α liquid solution 80ul, stand, liquid all flows into nitrocellulose filter, then marks adsorbed film to colloidal gold particles Upper dropwise addition pH7.4 0.05% Tween-20,50mM PBS 80ul, stand, liquid all flows into nitrocellulose filter, clearly Wash twice, remove test strips, cut Avidin die detection line, be positioned in cuvette, add APLS chemiluminescence detections Substrate, 1 minute is stood, 6 seconds luminous quantities are read on chemiluminescence detector, tested in triplicate, results averaged, then Concentration-luminosity curve is drawn, and calculates coefficient correlation.

3rd, experimental result

This experiment is tested using colloidal gold particles as liquid phase reactor delivery vehicle using methods described D, as a result, the present invention is real Test group test strips single detection process time average out to 7.9 minutes, control test group（Do not do centrifugal treating）Test strips single is examined Survey processing time average out to 58 minutes；Experimental group detection data of the present invention are in the dependency relation of good concentration-luminous value, related Coefficient r is 0.987, and control group detects data in below 100ng/ml, the substantially correlation without concentration-luminous value, mainly by It can not be effectively cleaned in non-specific binding of the chemiluminescence enzyme on immobilon-p, cause background too high relevant, phase relation Number r is 0.841, P ＜ 0.05, and result of the present invention is significantly better than the testing result for not doing centrifugal treating, illustrates that the technology of the present invention carries The high linear and accuracy of prior art detection.Meanwhile add intermediate material and generate signal amplification, improve each dense The response intensity of degree.Experimental result is as shown in table 4.

The paper chromatography chemical luminescence detection method D experimental results of the present invention of table 4（Unit：Luminous value）

Experiment five, paper chromatography chemiluminescence detection of the present invention experiment（Five）

Using fluorescent microsphere as liquid phase reactor delivery vehicle, tested using methods described E.

First, experiment material

Fluorescent microsphere（The biology of Shanghai outstanding person one）, trehalose（SIGMA）, nitrocellulose filter（Millipore companies）, EDC （PIERCE companies）, NHS（PIERCE companies）, NHS activation acridinium esters（Acridinium NHS ester, Shanghai are prosperous happy raw Thing）, start luminescence reagent（26.8 mM H₂O₂The ul of solution 50 and containing the ul of 0.2 M NaOH solution 300, used time mixing）, its It is the same as experiment one.

It is as follows that above-mentioned material corresponds to Patent Application Request content, and this experimental fluorescence microballoon is particulate, and NHS activation acridinium esters are The chemiluminescent substance of chemiluminescent reactant, the starting luminescence reagent that luminescence reagent is chemiluminescent reactant is started, it is other With experiment one.

2nd, experimental method

The preparation of human muscle hemoglobin solution：With experiment one.

Anti-human myoglobins monoclonal antibody acridinium ester label：The anti-human myoglobins monoclonal antibodies of 5mg are taken, add the concentration to be 0.2M sodium bicarbonate aqueous solution（pH8.3）2ml dissolves.Resisted with the mg/ml NHS of 8ul concentration 20 acridinium esters activated with 1 mg The ratio hybrid reaction of human muscle hemoglobin polyclonal antibody, 4 DEG C, concussion reaction 4 hours.Stayed overnight again with pH7.4 PBS Dialysis, removes free acridinium ester, 4 DEG C save backup.

Fluorescent microsphere marks：0.5ml microballoons are taken, using pH7.2 0.1M phosphate buffers eccentric cleaning 4 times, 13000rpm is centrifuged, and is redissolved with pH7.2 0.1M phosphate buffers to 1ml, is added the 200 anti-human flesh red eggs of μ g acridinium ester labels White monoclonal antibody, mix, add 250ul 40mg/ml EDC solution, add 250ul 40mg/ml NHS solution, mix It is even, react at room temperature 60 minutes, add 20mg bovine serum albumin(BSA), mix, react at room temperature 60 minutes.Supernatant is abandoned in centrifugation suction, is made After 0.05M Tris pH7.6 eccentric cleanings 4 times, with 0.5% trehalose, 1%BSA, 0.05M Tris pH7.6 redissolve to 10ml, 4 DEG C be kept in dark place it is stand-by.

Fluorescent microsphere mark absorption film preparation：Fluorescent microsphere labelled antibody solution is taken, with pH7.2 0.1M phosphoric acid buffers Liquid dilutes 2 times, other same experiments one.

It is prepared by polyclonal antibody die：With experiment one.

Semi-finished product assemble method：With experiment one.

Experimental group：The test strips of above-mentioned preparation are taken, with the side of fluorescent microsphere mark adsorbed film upward, are placed in centrifugal basket In son, the human muscle hemoglobin solution 80ul for the various concentrations that preparation is added dropwise on adsorbed film is marked to fluorescent microsphere, stands 2 minutes, Centrifuged 1 minute with 1000 revs/min, then 0.05% Tween-20,50mM PBS to dropwise addition pH7.4 on fluorescent microsphere mark adsorbed film Buffer solution 80ul, 2000 revs/min of centrifugations are cleaned for 30 seconds, and cleaning twice, takes out test strips, cuts the detection of polyclonal antibody die Line, it is positioned in cuvette, is placed on chemiluminescence detector, adds and start luminescence reagent, time delay is 0.30 second, The luminous quantity time of integration is 10 seconds, and experiment in triplicate, results averaged, then draws concentration-luminosity curve, and calculate phase Relation number.

Control group：The test strips of above-mentioned preparation are taken, are placed in desktop, marks to be added dropwise on adsorbed film to fluorescent microsphere and prepares not With the human muscle hemoglobin solution 80ul of concentration, stand, treat that the liquid on fluorescent microsphere mark adsorbed film all chromatographs and flow into nitric acid Cellulose membrane, then 0.05% Tween-20,50mM PBS 80ul to dropwise addition pH7.4 on fluorescent microsphere mark adsorbed film, Stand, liquid all flows into nitrocellulose filter, and cleaning twice, removes test strips, cuts polyclonal antibody die detection line, put It is placed in cuvette, is placed on chemiluminescence detector, add and start luminescence reagent, time delay is 0.30 second, luminous quantity The time of integration is 10 seconds, and experiment in triplicate, results averaged, then draws concentration-luminosity curve, and calculate phase relation Number.

3rd, experimental result

This experiment is tested using fluorescent microsphere as liquid phase reactor delivery vehicle using methods described E, as a result, present invention experiment Group test strips single detection process time average out to 4.7 minutes, control test group（Do not do centrifugal treating）Test strips single detects Processing time average out to 48 minutes；Experimental group detection data of the present invention are in the dependency relation of good concentration-luminous value, phase relation Number r are 0.981, and control group detects data in below 100ng/ml, the substantially correlation without concentration-luminous value, mainly due to Non-specific binding of the chemiluminescent substance on immobilon-p can not be effectively cleaned, and cause background too high relevant, phase relation Number r is 0.908, P ＜ 0.05, and result of the present invention is significantly better than the testing result for not doing centrifugal treating, illustrates that the technology of the present invention carries The high linear and accuracy of prior art detection.Experimental result is as shown in table 5.

The paper chromatography chemical luminescence detection method E experimental results of the present invention of table 5（Unit：Luminous value）

Experiment six, paper chromatography chemiluminescence detection of the present invention experiment（Six）

Using fluorescent microsphere as liquid phase reactor delivery vehicle, tested using methods described F.

First, experiment material

Sheep anti-mouse igg polyclonal antibody（Genagates companies of the U.S.）, it is other with experiment five.

It is as follows that above-mentioned material corresponds to Patent Application Request content, and this experiment sheep anti-mouse igg polyclonal antibody is that thing to be checked is special The specificity junction mixture of specific binding agent, it is other with experiment five.

2nd, experimental method

The preparation of human muscle hemoglobin solution：With experiment one.

Sheep anti-mouse igg polyclonal antibody acridinium ester label：The anti-human myoglobins polyclonal antibodies of 5mg are taken, add the concentration to be 0.2M sodium bicarbonate aqueous solution（pH8.3）2 ml dissolve.The acridinium ester and 1 mg activated with the mg/ml NHS of 8ul concentration 20 The ratio hybrid reaction of anti-human myoglobins polyclonal antibody, 4 DEG C, concussion reaction 4 hours.PH7.4 PBS salting liquid mistakes are used again Night dialyses, and removes free acridinium ester, 4 DEG C save backup.

Fluorescent microsphere marks：0.5ml microballoons are taken, using pH7.2 0.1M phosphate buffers eccentric cleaning 4 times, 13000rpm is centrifuged, and is redissolved with pH7.2 0.1M phosphate buffers to 1ml, is added the anti-human myoglobins monoclonals of 200 μ g and is resisted Body, mix, it is other with experiment five.

Fluorescent microsphere mark absorption film preparation：With experiment five.

It is prepared by polyclonal antibody die：With experiment five.

Semi-finished product assemble method：With experiment five.

Experimental group：The test strips of above-mentioned preparation are taken, with the side of fluorescent microsphere mark adsorbed film upward, are placed in centrifugal basket In son, the human muscle hemoglobin solution 80ul for the various concentrations that preparation is added dropwise on adsorbed film is marked to fluorescent microsphere, stands 2 minutes, Centrifuged 1 minute with 1000 revs/min, marked to fluorescent microsphere and acridinium ester label sheep anti-mouse igg polyclonal antibody is added dropwise on adsorbed film Solution 80ul, 2 minutes are stood, centrifuged 1 minute with 1000 revs/min, then marked to fluorescent microsphere and be added dropwise pH7.4's on adsorbed film 0.05% Tween-20,50mM PBSs 80ul, 2000 revs/min of centrifugations are cleaned for 30 seconds, and cleaning twice, is taken out test strips, cut Polyclonal antibody die detection line is taken, is positioned in cuvette, is placed on chemiluminescence detector, adds and starts luminous examination Agent, time delay are 0.30 second, and the luminous quantity time of integration is 10 seconds, and experiment in triplicate, results averaged, is then drawn Concentration-luminosity curve, and calculate coefficient correlation.

Control group：The test strips of above-mentioned preparation are taken, are placed in desktop, marks to be added dropwise on adsorbed film to fluorescent microsphere and prepares not With the human muscle hemoglobin solution 80ul of concentration, stand, treat that the liquid on fluorescent microsphere mark adsorbed film all chromatographs and flow into nitric acid Cellulose membrane, dropwise addition acridinium ester label sheep anti-mouse igg Anti-TNF-α liquid solution 80ul on adsorbed film is marked to fluorescent microsphere, it is quiet Put, liquid all flow into nitrocellulose filters, then to fluorescent microsphere mark adsorbed film on be added dropwise pH7.4 0.05% polysorbas20, 50mM PBS 80ul, stand, liquid all flows into nitrocellulose filter, and cleaning twice, removes test strips, cuts more grams Grand antibody die detection line, is positioned in cuvette, is placed on chemiluminescence detector, adds and starts luminescence reagent, delay Time is 0.30 second, and the luminous quantity time of integration is 10 seconds, and experiment in triplicate, results averaged, then draws concentration-hair Light curve, and calculate coefficient correlation.

3rd, experimental result

This experiment is tested using colloidal gold particles as liquid phase reactor delivery vehicle using methods described F, as a result, the present invention is real Test group test strips single detection process time average out to 7.8 minutes, control test group（Do not do centrifugal treating）Test strips single is examined Survey processing time average out to 59 minutes；Experimental group detection data of the present invention are in the dependency relation of good concentration-luminous value, related Coefficient r is 0.990, and control group detects data in below 100ng/ml, the substantially correlation without concentration-luminous value, mainly by It can not be effectively cleaned in non-specific binding of the chemiluminescence enzyme on immobilon-p, cause background too high relevant, phase relation Number r is 0.912, P ＜ 0.05, and result of the present invention is significantly better than the testing result for not doing centrifugal treating, illustrates that the technology of the present invention carries The high linear and accuracy of prior art detection.Experimental result is as shown in table 6.

The paper chromatography chemical luminescence detection method F experimental results of the present invention of table 6（Unit：Luminous value）

Experiment seven, influence of the centrifugal speed of the present invention to testing result

First, experiment material

With experiment one.

2nd, experimental method

Control group is not set, experimental group human muscle hemoglobin solution sample introduction uses 500,1000,2000,3000,4000,5000 revs/min Centrifugation, 2000 revs/min of centrifugations of cleaning are other with experiment one.

3rd, experimental result

This experiment is tested using colloidal gold particles as liquid phase reactor delivery vehicle using methods described A, observes different centrifugation speed Spend the influence to testing result.Experiment have detected the human muscle hemoglobin sample of various concentrations, and experimental result is as shown in table 7.By table 7 Understand, detection accuracy is relevant with centrifugal speed, and 500,1000,2000 revs/min of centrifugal speed obtains satisfactory detection As a result.3000th, the testing result that 4000,5000 revs/min of centrifugal speed obtains, luminous value significantly decrease, influence to detect Sensitivity and accuracy.Illustrate that the optimal centrifugal speed of present invention progress myoglobins detection should be below 2000 revs/min.

Influence of the centrifugal speed of table 7 to testing result（Unit：Luminous value）

Experiment eight, influence of the diameter of particle of the present invention to testing result

First, experiment material

Polystyrene microsphere（Particle diameter 45,201,376,600,1000nm, carboxylated, upper great waves space are international）, it is other with experiment one.

Above-mentioned material corresponds to that Patent Application Request content is as follows, and this experiment polystyrene microsphere is particulate, other with experiment One.

2nd, experimental method

The preparation of human muscle hemoglobin solution：With experiment one.

Polystyrene microsphere marks：Take 2ml acetate buffers, 0.01M, pH5.0,2mg antibody, the 5% of 0.2ml W/v microballoons and 20mg EDC, mix, placed 2 hours on impeller oscillator at room temperature, 3000g is centrifuged 15 minutes, is abandoned Supernatant, 4ml 50mM PBS, pH7.4 buffer solution is added to be suspended, repeated washing once, adds 2ml 50mM PBSs, 4 DEG C of guarantors Deposit standby.

Polystyrene microsphere mark absorption film preparation：Polystyrene microsphere labelled antibody solution is taken, with 50mM PBS, PH7.4 buffer solutions are diluted to 1% w/v microballoons, start die instrument, load antibody, open pressurized nitrogen, take poly ester fiber element Film, start die, set die condition as：Airbrush translational speed 30mm/ seconds, the μ l/cm of liquid fltting speed 5.0, after printing Film be put into drying box, 37 DEG C of dryings 6 hours are subsequently placed in preserve in the hermetic bag containing drier and used.

It is prepared by polyclonal antibody die：With experiment one.

Semi-finished product assemble method：With experiment one.

Experimental group：The test strips of above-mentioned preparation are taken, with the side of polystyrene microsphere mark adsorbed film upward, are placed in centrifugation In machine rotor, to polystyrene microsphere mark adsorbed film on be added dropwise preparation various concentrations human muscle hemoglobin solution 80ul, it is quiet Put 2 minutes, centrifuged 1 minute with 1000 revs/min, then 0.05% tween to dropwise addition pH7.4 on polystyrene microsphere mark adsorbed film 20th, 50mM PBSs 80ul, 2000 revs/min of centrifugations are cleaned for 30 seconds, and cleaning twice, takes out test strips, cuts Anti-TNF-α Body die detection line, is positioned in cuvette, adds West Pico chemiluminescence detection substrates, 5 minutes is stood, in chemistry 6 seconds luminous quantities are read on luminometer, experiment in triplicate, results averaged, then draws concentration-luminosity curve, and Calculate coefficient correlation.

3rd, experimental result

The present invention is tested using polystyrene microsphere as liquid phase reactor delivery vehicle using methods described A, observes different-grain diameter Influence to experimental result.As a result, particle diameter of the present invention be 45,201,376,600,1000nm detection data are in well dense The dependency relation of degree-luminous value, correlation coefficient r are all higher than 0.99, illustrate that the particulate of different-grain diameter is applied to the technology of the present invention.It is real It is as shown in table 8 to test result.

Influence of 8 diameter of particle of the present invention of table to testing result（Unit：Luminous value）

Experiment nine, the comparative experiments of linear detection range of the present invention and existing detection technique

First, experiment material

Fluorescent microsphere（The biology of Shanghai outstanding person one）, trehalose（SIGMA）, nitrocellulose filter（Millipore companies）, EDC （PIERCE companies）, NHS（PIERCE companies）, magnetic particle（Zhengzhou English promise biology）, quantitative fluorescence analysis instrument（Shanghai woman biology Company, HG-98）, the quantitative chromatographic analysis instrument of collaurum（Norway's Skannex products）, it is other with experiment one.

Above-mentioned material corresponds to that Patent Application Request content is as follows, and this experimental fluorescence microballoon is particulate, other with experiment one.

2nd, experimental method

1st, the preparation of human muscle hemoglobin solution：The human muscle hemoglobin solution of concentration known is taken, uses sample dilution buffer（1%BSA, 100mM glycine, 50mM PBS, 150mM NaCl, pH7.4）Dilution configuration 1000ng/ml human muscle hemoglobin solution.

2nd, existing colloidal gold method immunoassay technology group：

Colloidal gold particles mark：With experiment two.

Colloidal gold particles mark absorption film preparation：With experiment one.

It is prepared by polyclonal antibody die：With experiment one.

Semi-finished product assemble method：With experiment one.

The test strips of above-mentioned preparation are taken, are placed in desktop, is marked to colloidal gold particles and the different dense of preparation is added dropwise on adsorbed film The human muscle hemoglobin solution 80ul of degree, 20 minutes are stood, be positioned on the quantitative chromatographic analysis instrument of collaurum and read polyclonal antibody The digital picture of trace band, carry out image procossing and obtain corresponding chroma value.Test in triplicate, results averaged. Control experiment is using the Liquid sample introduction for not containing myoglobins.Calculate group containing human muscle hemoglobin and the ratio without human muscle hemoglobin group Value.

3rd, existing chemiluminescence detection technology group：It is polyclonal with the anti-human myoglobins of 1mg/ml using conventional labels method Magnetic particle is marked antibody, the amount ratio of anti-human myoglobins polyclonal antibody and magnetic particle（w/w）For 3:1；Using NHS The anti-human myoglobins monoclonal antibody of horseradish peroxidase-labeled of activation.EP is taken to manage, often pipe is added with anti-human myoglobins The μ l of magnetic particle 100 of polyclonal antibody mark, then it is separately added into each 100 μ l of human muscle hemoglobin solution that concentration is 1 μ g/ml, knot Close reaction and incubation is shaked at 37 DEG C 60 minutes, with magnetic separator adsorbing separation magnetic particle, abandon supernatant, add the μ l of PBS 200 cleanings Three times, with magnetic separator adsorbing separation magnetic particle, supernatant is abandoned, adds the anti-human myoglobins monoclonal of horseradish peroxidase-labeled The μ l of antibody 200, association reaction are shaked at 37 DEG C and incubated with the corresponding reaction time, micro- with magnetic separator adsorbing separation magnetic Grain, supernatant is abandoned, add the μ l of PBS 200 cleanings three times, with magnetic separator adsorbing separation magnetic particle, abandon supernatant, transfer magnetic particle is extremely In cuvette, chemiluminescence detector is put, adds 100 μ l luminous substrate working solutions, when reaction is carried out 5 minutes, record is luminous Amount 6 seconds.Control experiment is using the Liquid sample introduction for not containing myoglobins.Calculate group containing human muscle hemoglobin and be free of people's flesh red eggs The ratio organized in vain.

4th, existing fluorescence immunoassay detection technique group：

Fluorescent microsphere marks：Take 0.5ml microballoons, using pH7.2 0.1M phosphate buffers eccentric cleaning 4 times, 13000rpm from The heart, redissolved with pH7.2 0.1M phosphate buffers to 1ml, add the anti-human myoglobins monoclonal antibodies of 200 μ g, mixed, its It is the same as experiment five.

Fluorescent microsphere mark absorption film preparation：With experiment five.

It is prepared by polyclonal antibody die：With experiment one.

Semi-finished product assemble method：With experiment one.

The test strips of above-mentioned preparation are taken, are placed in desktop, the various concentrations that preparation is added dropwise on adsorbed film are marked to fluorescent microsphere Human muscle hemoglobin solution 80ul, stand 20 minutes, be positioned on quantitative fluorescence analysis instrument reading polyclonal antibody trace band Fluorescent value.Test in triplicate, results averaged.Control experiment is using the Liquid sample introduction for not containing myoglobins.Calculate Group containing human muscle hemoglobin and the ratio without human muscle hemoglobin group.

5th, detection technique group of the present invention：

Colloidal gold particles mark：With experiment one.

Colloidal gold particles mark absorption film preparation：With experiment one.

It is prepared by polyclonal antibody die：With experiment one.

Semi-finished product assemble method：With experiment one.

The test strips of above-mentioned preparation are taken, with the side of colloidal gold particles mark adsorbed film upward, are placed in centrifuge rotor, The human muscle hemoglobin solution 80ul of the various concentrations of preparation is added dropwise on to colloidal gold particles mark adsorbed film, stands 2 minutes, with 1000 revs/min centrifuge 1 minute, then 0.05% Tween-20,50mM PBS to dropwise addition pH7.4 on colloidal gold particles mark adsorbed film Buffer solution 80ul, 2000 revs/min of centrifugations are cleaned for 30 seconds, and cleaning twice, takes out test strips, cuts the detection of polyclonal antibody die Line, it is positioned in cuvette, adds West Pico chemiluminescence detection substrates, 5 minutes is stood, in chemiluminescence detector 6 seconds luminous quantities of upper reading, test in triplicate, results averaged.Control experiment is entered using the liquid for not containing myoglobins Sample.Calculate group containing human muscle hemoglobin and the ratio without human muscle hemoglobin group.

3rd, experimental result

The present invention is tested using colloidal gold particles as liquid phase reactor delivery vehicle using methods described A, and other groups using existing Conventional method.As a result human muscle hemoglobin 1000ng groups and without human muscle hemoglobin group ratio be respectively 37 times of collaurum, it is glimmering Light is immune 74 times, 185 times of chemiluminescence, 437 times of the present invention, it is linear to illustrate that the technology of the present invention significantly improves existing detection technique Detection interval, improve sensitivity and the accuracy of detection.Experimental result is as shown in table 9.

The comparative experiments of 9 linear detection range of the present invention of table and existing detection technique

**p<0.01

Experiment ten, the of the invention and comparison of existing chemiluminescence detection technology testing result

First, experiment material

Magnetic particle（Zhengzhou English promise biology）, it is other with experiment one.

2nd, experimental method

Make standard curve：Take concentration known human muscle hemoglobin solution 3.125,6.25,12.5,25,50,100ng/ml people's flesh Hemoglobin solution, of the invention and existing chemiluminescence detection technology is respectively adopted and detects and draws standard curve.With concentration known Human muscle hemoglobin 10ng/ml as sample to be checked.

Existing chemiluminescence detection technology group：With experiment nine, reference standard curve calculates sample myoglobin concentration to be checked.

Detection technique group of the present invention：With experiment one, reference standard curve calculates sample myoglobin concentration to be checked.

3rd, experimental result

The concrete outcome for repeating experiment three times is as shown in table 10.Existing chemiluminescence detection technology measurement result shows sample to be checked The content of human muscle hemoglobin is 9.76ng/ml, and measurement result of the present invention shows that the content of sample human muscle hemoglobin to be checked is 10.43ng/ml, two kinds of experimental method acquired results are basically identical, no difference of science of statistics（P>0.05）, but the completion of the present invention is real Testing the time is significantly shorter than current art.

Table 10 is of the invention with the comparison of existing chemiluminescence detection technology testing result（Unit：ng/ml）

	Repeat 1	Repeat 2	Repeat 3	Average value
					Current art	9.67	9.09	10.53	9.76
The present invention	9.88	10.54	10.89	10.43

Test 11, the of the invention and comparison of existing Immunofluorescence test technology for detection result

First, experiment material

Fluorescent microsphere（Fluorescein used is europium compound, and Shanghai outstanding person one is biological）, trehalose（SIGMA）, EDC（PIERCE is public Department）, NHS（PIERCE companies）, sheep anti-mouse igg polyclonal antibody（Genagates companies of the U.S.）, quantitative fluorescence analysis instrument（On Extra large woman biotech firm, HG-98）, it is other with experiment one.

2nd, experimental method

1st, standard curve is made：Take concentration known human muscle hemoglobin solution 3.125,6.25,12.5,25,50,100ng/ml people Myoglobin solution, of the invention and existing fluorescence immunoassay detection technique is respectively adopted and detects and draws standard curve.With known dense The human muscle hemoglobin 10ng/ml of degree is as sample to be checked.

2nd, existing fluorescence immunoassay detection technique group：

Fluorescent microsphere marks：Take 0.5ml microballoons, using pH7.2 0.1M phosphate buffers eccentric cleaning 4 times, 13000rpm from The heart, redissolved with pH7.2 0.1M phosphate buffers to 1ml, add the anti-human myoglobins monoclonal antibodies of 200 μ g, mixed, its It is the same as experiment five.

Fluorescent microsphere mark absorption film preparation：With experiment five.

It is prepared by polyclonal antibody die：According to the antibody die method of experiment one sheep anti mouse is printed in the distal end of immobilon-p IgG polyclonal antibodies, as nature controlling line（C lines）, anti-human myoglobins polyclonal antibody is printed in the proximal part of immobilon-p, as Detection line（T lines）, it is other with experiment one.

Semi-finished product assemble method：Fluorescent labeled antibody adsorbed film is pasted on tip side, and absorbing membrane pad is pasted on C line sides, other With experiment one.

The test strips of above-mentioned preparation are taken, are placed in desktop, the various concentrations that preparation is added dropwise on adsorbed film are marked to fluorescent microsphere Human muscle hemoglobin solution 80ul, stand 20 minutes, be placed on quantitative fluorescence analysis instrument read polyclonal antibody printed film on Detection line T and nature controlling line C fluorescent value, and T/C ratios are calculated, standard curve is drawn, calculates measuring samples myoglobin concentration.

3rd, detection technique group of the present invention：

Fluorescent microsphere marks：Take 0.5ml microballoons, using pH7.2 0.1M phosphate buffers eccentric cleaning 4 times, 13000rpm from The heart, redissolved with pH7.2 0.1M phosphate buffers to 1ml, add the anti-human myoglobins monoclonal antibodies of 200 μ g, horseradish peroxide Change the μ g of enzyme 200, mix, it is other with experiment five.

Fluorescent microsphere mark absorption film preparation：With experiment five.

It is prepared by polyclonal antibody die：With experiment one.

Semi-finished product assemble method：With experiment one.

The test strips of above-mentioned preparation are taken, with the side of fluorescent microsphere labelled antibody adsorbed film upward, are placed in centrifuge rotor It is interior, to fluorescent microsphere labelled antibody adsorbed film on be added dropwise preparation various concentrations human muscle hemoglobin solution and testing sample it is each 80ul, 2 minutes are stood, 1000 revs/min centrifuge 1 minute, then to being added dropwise pH7.4's on fluorescent microsphere labelled antibody adsorbed film 0.05% Tween-20,50mM PBSs 80ul, 2000 revs/min of centrifugations are cleaned for 30 seconds, and repeated washing once, takes out test paper Bar, polyclonal antibody die detection line is cut, is positioned in cuvette, add West Pico chemiluminescence detection substrates, it is quiet Put 5 minutes, 6 seconds luminous quantities are read on chemiluminescence detector, test in triplicate, results averaged, reference standard is bent Line computation sample myoglobin concentration to be checked.

3rd, experimental result

For the technology of the present invention through operating as above, standard curve is linearly good, and correlation coefficient r value is 0.995, has carried out sample survey immediately Fixed, the ng/ml of empirical average 10.36, detection error meet the requirements within 10% three times.Determined using prior art, after point sample 20 minutes of existing Product checking are stood, standard curve is linearly good, and correlation coefficient r value is 0.989, immediately according to 20 minutes Condition has carried out sample measure, and average value is 9.13ng/ml three times, and detection error meets the requirements within 10%.Repeat three times The concrete outcome of experiment is as shown in table 11.Compared with the prior art, the present invention significantly shorten detection time.

The Analysis of test results of the present invention of table 11 and Immunofluorescence test technology（Unit：ng/ml）

	Repeat 1	Repeat 2	Repeat 3	Average value
					The present invention	10.73	10.53	9.81	10.36
Prior art	10.26	8.29	8.86	9.13

Test 12, the of the invention and comparison of existing enzyme linked immunosorbent detection technology for detection result

First, experiment material

Fluorescent microsphere（Fluorescein used is europium compound, and Shanghai outstanding person one is biological）, trehalose（SIGMA）, EDC（PIERCE is public Department）, NHS（PIERCE companies）, enzyme-linked immunosorbent assay instrument（Bio-Rad, Model 550）, horseradish peroxidase-labeled goat-anti Mouse IgG polyclonal antibodies（Genagates companies of the U.S. provide）, it is other with experiment one.

2nd, experimental method

The preparation of human muscle hemoglobin solution：Take the human muscle hemoglobin solution of concentration known, with PBS solution configuration 3.125,6.25, 12.5th, 25,50,100 ng/ml serial human muscle hemoglobin solution, is respectively adopted of the invention and existing enzyme linked immunosorbent detection technology Detect and draw standard curve.Sample to be checked is used as using the human muscle hemoglobin 10ng/ml of concentration known.

1st, existing enzyme linked immunosorbent detection technology groups

Using 96 hole elisa plates, the anti-human μ l of myoglobins polyclonal antibody 100 of often pipe addition, 4 DEG C are coated with overnight, clean three times, Human muscle hemoglobin solution or the μ l of measuring samples 100 are separately added into again, and association reaction incubates 120 minutes at 37 DEG C, and cleaning three times, adds Enter the anti-human μ l of myoglobins monoclonal antibody 100, association reaction incubates 60 minutes at 37 DEG C, and cleaning three times, abandons supernatant, adds peppery The μ l of root peroxidase labelling sheep anti-mouse igg polyclonal antibody 100, association reaction are incubated 60 minutes at 37 DEG C, and cleaning three times, is abandoned Supernatant, add 100 μ l nitrite ions（Formula：0.1M citric acids 2.43ml, 0.2M disodium hydrogen phosphate 2.57ml, o-phenylenediamine 5mg, The μ l of hydrogen peroxide 5）, lucifuge 5 minutes, add 2M sulfuric acid terminating reactions.Reading OD490 light absorption values on enzyme-linked immunosorbent assay instrument are put, Reference standard curve, calculate human muscle hemoglobin content in sample to be checked.

2nd, of the present invention group

Fluorescent microsphere marks：With experiment 11.

Fluorescent microsphere mark absorption film preparation：With experiment five.

It is prepared by polyclonal antibody die：With experiment one.

Semi-finished product assemble method：With experiment one.

The test strips of above-mentioned preparation are taken, with the side of fluorescent microsphere labelled antibody adsorbed film upward, are placed in centrifuge rotor It is interior, to fluorescent microsphere labelled antibody adsorbed film on be added dropwise preparation various concentrations human muscle hemoglobin solution and testing sample it is each 80ul, 2 minutes are stood, 1000 revs/min centrifuge 1 minute, then to being added dropwise pH7.4's on fluorescent microsphere labelled antibody adsorbed film 0.05% Tween-20,50mM PBSs 80ul, 2000 revs/min of centrifugations are cleaned for 30 seconds, and repeated washing once, takes out test paper Bar, polyclonal antibody die detection line is cut, is positioned in cuvette, add West Pico chemiluminescence detection substrates, it is quiet Put 5 minutes, 6 seconds luminous quantities are read on chemiluminescence detector, test in triplicate, results averaged, reference standard is bent Line computation sample myoglobin concentration to be checked.

3rd, experimental result

Concrete outcome is as shown in table 12.Existing enzyme linked immunosorbent detection technology measurement result shows containing for sample human muscle hemoglobin to be checked Measure and show that the content of sample human muscle hemoglobin to be checked is 10.34ng/ml for 9.83ng/ml, measurement result of the present invention, two kinds are tested Method acquired results are basically identical, no difference of science of statistics（P>0.05）, but the present invention completion experimental period be significantly shorter than it is existing Technology.

Table 12 is of the invention with the comparison of existing enzyme linked immunosorbent detection technology for detection result（Unit：ng/ml）

	Repeat 1	Repeat 2	Repeat 3	Average value
					Current art	11.09	9.18	9.22	9.83
The present invention	10.78	8.99	11.24	10.34

Test 13, the of the invention and comparison of the testing result of existing dot immune gold filtration assay

First, experiment material

The quantitative chromatographic analysis instrument of collaurum（Norway's Skannex products）, it is other with experiment one.

2nd, experimental method

The preparation of human muscle hemoglobin solution：Take the human muscle hemoglobin solution of concentration known, with PBS solution configuration 3.125,6.25, 12.5th, 25,50,100 ng/ml serial human muscle hemoglobin solution, is respectively adopted of the invention and existing enzyme linked immunosorbent detection technology Detect and draw standard curve.Sample to be checked is used as using the human muscle hemoglobin 10ng/ml of concentration known.

2nd, existing colloid gold immune detection technique group：

Colloidal gold particles mark：With experiment two.

Colloidal gold particles mark absorption film preparation：With experiment one.

It is prepared by polyclonal antibody die：According to the antibody die method of experiment one sheep anti mouse is printed in the distal end of immobilon-p IgG polyclonal antibodies, as nature controlling line（C lines）, anti-human myoglobins polyclonal antibody is printed in the proximal part of immobilon-p, as Detection line（T lines）, it is other with experiment one.

Semi-finished product assemble method：Colloidal gold particles labelled antibody adsorbed film is pasted on tip side, and absorbing membrane pad is pasted on C lines Side, it is other with experiment one.

Semi-finished product assemble method：With experiment one.

The test strips of above-mentioned preparation are taken, are placed in desktop, is marked to colloidal gold particles and the different dense of preparation is added dropwise on adsorbed film The human muscle hemoglobin solution 80ul of degree, 20 minutes are stood, be placed on the quantitative chromatographic analysis instrument of collaurum and read polyclonal antibody Detection line T and nature controlling line C pattern colour angle value on printed film, and T/C ratios are calculated, standard curve is drawn, calculates measuring samples Myoglobin concentration.

3rd, colloidal gold particles immunoassay technology group of the present invention：

Colloidal gold particles mark：With experiment one.

Colloidal gold particles mark absorption film preparation：With experiment one.

It is prepared by polyclonal antibody die：With experiment one.

Semi-finished product assemble method：With experiment one.

The test strips of above-mentioned preparation are taken, with the side of colloidal gold particles mark adsorbed film upward, are placed in centrifuge rotor, The human muscle hemoglobin solution 80ul of the various concentrations of preparation is added dropwise on to colloidal gold particles mark adsorbed film, stands 2 minutes, with 1000 revs/min centrifuge 1 minute, then 0.05% Tween-20,50mM PBS to dropwise addition pH7.4 on colloidal gold particles mark adsorbed film Buffer solution 80ul, 2000 revs/min of centrifugations are cleaned for 30 seconds, and cleaning twice, takes out test strips, cuts the detection of polyclonal antibody die Line, it is positioned in cuvette, adds West Pico chemiluminescence detection substrates, 5 minutes is stood, in chemiluminescence detector 6 seconds luminous quantities of upper reading, in triplicate, results averaged, it is dense that reference standard curve calculates sample myoglobins to be checked for experiment Degree.

3rd, experimental result

Concrete outcome is as shown in table 13.Existing colloid gold immune detection technique measurement result shows sample human muscle hemoglobin to be checked Content is 9.63ng/ml, and measurement result of the present invention shows that the content of sample human muscle hemoglobin to be checked is 9.81ng/ml, two kinds of realities Proved recipe method acquired results are basically identical, no difference of science of statistics（P>0.05）, but the completion experimental period of the present invention is significantly shorter than now Row technology.

Table 13 is of the invention with the comparison of existing colloid gold immune detection technique testing result（Unit：ng/ml）

	Repeat 1	Repeat 2	Repeat 3	Average value
					Current art	9.29	10.69	8.91	9.63
The present invention	8.12	11.22	10.08	9.81

Test 14, influence of the cleaning step of the present invention to testing result

First, experiment material

With experiment one

2nd, experimental method

Sample not contain human muscle hemoglobin does not clean as sample to be checked, control, stands chromatography；Experiment is using 40,80, 120th, 160ul is cleaned, other with experiment one.

3rd, experimental result

Experimental result is as shown in table 14.Two test results compare, and cleaning can reduce non-spy of the chemiluminescence enzyme on immobilon-p The opposite sex combines, and the effect of centrichromatography cleaning is especially pronounced, and 160ul cleanings, which have been down to immobilon-p background, can meet to test It is required that light levels.It is necessary to illustrate to test after sample introduction with the step of cleaning, and eccentric cleaning is required.

Influence of 14 cleaning step of the present invention of table to testing result（Unit：Luminous value）

	Centrifugation	Do not centrifuge
			0	2135438	2891573
40	1234571	2354216
			80	376525	1921574
120	63527	1251679
			160	12761	1022138

Claims (13)

1. a kind of paper chromatography chemiluminescence detection combined complete, is characterised by, the detection combination suit is special containing thing to be checked Property conjugate, particulate, immobilon-p, chemiluminescent reactant, centrifugal device and luminescence detector, wherein described chemiluminescence Reactant includes at least one in chemiluminescence enzyme, chemiluminescent substance, chemiluminescence enzyme reaction substrate and starting luminescence reagent Kind；The particulate is that can be formed directly and/or by chemical crosslinking mode and protein and/or the chemiluminescent reactant Non-specific binding simultaneously maintains stable particle；The immobilon-p is with the film quality thing with protein non-specific binding characteristic Matter；The centrifugal device is the structure that can centrifuge driving liquid phase chromatographic flow on the immobilon-p；The thing to be checked is special Property conjugate be at least one of antigen with specific binding capacity, antibody, Avidin, biotin and the like.

2. the detection method that detection combination described in a kind of claim 1 is set with, it is characterised in that：The detection method is as follows （A）-（F）In it is any：

（A）Comprise the following steps：

1）The particulate is marked with the thing specificity junction mixture to be checked and the chemiluminescence enzyme simultaneously；

2）Reacted with the particulate after the sample containing thing to be checked and the mark, the thing to be checked by with after the mark Particulate on the thing specificity junction mixture to be checked combine, and then form " chemiluminescence enzyme-particulate-thing to be checked specificity knot Compound-thing to be checked " compound, i.e. compound 1；

3）Coating can be treated by forming the second of specific binding with the thing to be checked on compound 1 on the immobilon-p Examine thing specificity junction mixture；

4）With the liquid phase containing the compound 1, by chromatography, to flow through coated second thing to be checked on the immobilon-p special Property conjugate, form " chemiluminescence enzyme-particulate-thing to be checked specificity junction mixture-thing-the second to be checked thing to be checked specific binding Thing " compound, i.e. compound 2, and be captured and be fixed on the immobilon-p；

5）Using centrifugal chromatography, cleaned with cleaning fluid described in the compound 1 being not associated with the immobilon-p and residual Chemiluminescence enzyme；

6）The immobilon-p after cleaning is placed in the chemiluminescence enzyme reaction substrate liquid and reacted, and with the luminous detection Chemiluminescence enzyme reaction substrate described in the chemiluminescence enzymatic that device detection is indirectly fixed on the immobilon-p produces Luminous value, to detect the content of the thing to be checked；

（B）Comprise the following steps：

1）The particulate described in the thing specific binding substance markers to be checked；

2）The specificity junction mixture of the thing specificity junction mixture to be checked is marked with the chemiluminescence enzyme, forms " chemiluminescence The specificity junction mixture of enzyme-thing to be checked specificity junction mixture ", i.e. chemiluminescence enzyme marker；

3）Reacted with the particulate after the sample containing thing to be checked and the mark, the thing to be checked by with after the mark Particulate on the thing specificity junction mixture to be checked combine, and then form " particulate-thing to be checked specificity junction mixture-thing to be checked " Compound, i.e. compound 3；

4）The described second thing specificity junction mixture to be checked is coated with the immobilon-p；

5）With the liquid phase containing the compound 3, by chromatography, to flow through coated second thing to be checked on the immobilon-p special Property conjugate, formed " particulate-thing to be checked specificity junction mixture-thing-the second to be checked thing specificity junction mixture to be checked " compound, i.e., Compound 4, and be captured and be fixed on the immobilon-p；

6）With the liquid phase containing the chemiluminescence enzyme marker by chromatography flow through be trapped in it is compound on the immobilon-p Thing 4, by being combined with the thing specificity junction mixture to be checked, formed " the chemiluminescence enzyme marker-compound of compound 4 ", i.e., Compound 5, and be captured and be fixed on the immobilon-p；

6）Using centrifugal chromatography, the chemiluminescence enzyme marker that is not associated with the immobilon-p and residual is cleaned with cleaning fluid The chemiluminescence enzyme stayed；

7）The immobilon-p after cleaning is placed in the chemiluminescence enzyme reaction substrate liquid and reacted, and with the luminous detection The chemiluminescence enzyme reaction substrate described in the chemiluminescence enzymatic that device detection is indirectly fixed on the immobilon-p Caused luminous value, to detect the content of the thing to be checked；

（C）Comprise the following steps：

1）The particulate is marked with the thing specificity junction mixture to be checked and the chemiluminescence enzyme simultaneously；

2）Reacted with the particulate after the sample containing thing to be checked and the mark, the thing to be checked by with after the mark Particulate on the thing specificity junction mixture to be checked combine, the thing to be checked by with the thing specificity junction mixture knot to be checked Close, and then form " chemiluminescence enzyme-particulate-thing to be checked specificity junction mixture-thing to be checked " compound, i.e., described compound 1；

3）The second thing specificity junction mixture to be checked is marked with intermediate material A；

4）Coating can form the intermediate material B of specific binding with the intermediate material A on the immobilon-p；

5）The the second thing specificity junction mixture to be checked marked with the liquid phase containing the compound 1 with the intermediate material A is carried out Reaction, formed " chemiluminescence enzyme-particulate-thing to be checked specificity junction mixture-thing-the second to be checked thing specificity junction mixture to be checked-in Between substance A " compound, i.e. compound 6；

6）The coated intermediate material B on the immobilon-p is flowed through by chromatography with the liquid phase containing the compound 6, formed " chemiluminescence enzyme-particulate-thing to be checked specificity junction mixture-thing-the second to be checked thing specificity junction mixture-intermediate material A- to be checked Intermediate material B " compounds, i.e. compound 7, and be captured and be fixed on the immobilon-p；

7）Using centrifugal chromatography, cleaned with cleaning fluid described in the compound 6 being not associated with the immobilon-p and residual Chemiluminescence enzyme；

8）The immobilon-p after cleaning is placed in the chemiluminescence enzyme reaction substrate liquid and reacted, and with the luminous detection Chemiluminescence enzyme reaction substrate described in the chemiluminescence enzymatic that device detection is indirectly fixed on the immobilon-p produces Luminous value, to detect the content of the thing to be checked；

（D）Comprise the following steps：

1）The particulate described in the thing specific binding substance markers to be checked；

2）The specificity junction mixture of the thing specificity junction mixture to be checked is marked with the chemiluminescence enzyme, forms " chemiluminescence The specificity junction mixture of enzyme-thing to be checked specificity junction mixture ", i.e., described chemiluminescence enzyme marker；

3）Reacted with the particulate after the sample containing thing to be checked and the mark, the thing to be checked by with after the mark Particulate on the thing specificity junction mixture to be checked combine, and then form " particulate-thing to be checked specificity junction mixture-thing to be checked " Compound, i.e., described compound 3；

4）The second thing specificity junction mixture to be checked is marked with the intermediate material A；

5）Coating can form the intermediate material B of specific binding with the intermediate material A on the immobilon-p；

6）Reacted with the second thing specificity junction mixture to be checked of the liquid phase containing the compound 3 and intermediate material A marks, Formed " particulate-thing to be checked specificity junction mixture-thing-the second to be checked thing specificity junction mixture to be checked-intermediate material A " compounds, That is compound 8；

7）The coated intermediate material B on the immobilon-p is flowed through by chromatography with the liquid phase containing the compound 8, formed " particulate-thing to be checked specificity junction mixture-thing-the second to be checked thing specificity junction mixture to be checked-intermediate material A- intermediate materials B " is multiple Compound, i.e. compound 9, and be captured and be fixed on the immobilon-p；

8）With the liquid phase containing the chemiluminescence enzyme marker by chromatography flow through be trapped in it is compound on the immobilon-p Thing 9, by being combined with thing specificity junction mixture to be checked, form " the specificity knot of chemiluminescence enzyme-thing to be checked specificity junction mixture Compound-the compound of compound 9 ", i.e. compound 10, and be captured and be fixed on the immobilon-p；

9）Using centrifugal chromatography, the chemiluminescence enzyme marker that is not associated with the immobilon-p and residual is cleaned with cleaning fluid The chemiluminescence enzyme stayed；

10）The immobilon-p after cleaning is placed in chemiluminescence enzyme reaction substrate liquid and reacted, and with the luminescence detector Detect and lighted caused by chemiluminescence enzyme reaction substrate described in the chemiluminescence enzymatic being indirectly fixed on the immobilon-p Value, to detect the content of the thing to be checked；

（E）Comprise the following steps：

1）The thing specificity junction mixture to be checked and chemiluminescent substance mark are connected on the particulate；

2）Reacted with the particulate after the sample containing thing to be checked and the mark, the thing to be checked by with after the mark Particulate on the thing specificity junction mixture to be checked combine, and then form " chemiluminescent substance-particulate-thing to be checked specificity Conjugate-thing to be checked " compound, i.e. compound 11；

3）The second thing specificity junction mixture to be checked is coated with the immobilon-p；

4）The coated second thing spy to be checked on the immobilon-p is flowed through by chromatography with the liquid phase containing the compound 11 Specific binding agent, form " chemiluminescent substance-particulate-thing to be checked specificity junction mixture-thing-the second to be checked thing specificity to be checked Conjugate " compound, i.e. compound 12, and be captured and be fixed on the immobilon-p；

5）Using centrifugal chromatography, described in cleaning fluid cleans the compound 11 being not associated with the immobilon-p and residual Chemiluminescent substance；

6）The immobilon-p after cleaning is placed in the starting luminescence reagent solution and reacted, and is examined with the luminescence detector Luminous value caused by the chemiluminescent substance decomposition being indirectly fixed on the immobilon-p is surveyed, to detect the thing to be checked Content；

（F）Comprise the following steps：

1）The particulate described in the thing specific binding substance markers to be checked；

2）The specificity junction mixture of the thing specificity junction mixture to be checked is marked with the chemiluminescent substance, forms " chemistry hair The specificity junction mixture of stimulative substance-thing to be checked specificity junction mixture ", i.e. chemiluminescent substance label；

3）Reacted with the particulate after the sample containing thing to be checked and the mark, the thing to be checked by with after the mark Particulate on the thing specificity junction mixture to be checked combine, and then form " particulate-thing to be checked specificity junction mixture-thing to be checked " Compound, i.e., described compound 3；

4）The second thing specificity junction mixture to be checked is coated with the immobilon-p；

5）With the liquid phase containing the compound 3, by chromatography, to flow through coated second thing to be checked on the immobilon-p special Property conjugate, formed " particulate-thing to be checked specificity junction mixture-thing-the second to be checked thing specificity junction mixture to be checked " compound, i.e., The compound 4, and be captured and be fixed on the immobilon-p；

6）With the liquid phase containing chemiluminescent substance label by chromatography flow through be trapped in it is described multiple on the immobilon-p Compound 4, by being combined with the thing specificity junction mixture to be checked, form " chemiluminescent substance-thing to be checked specificity junction mixture Specificity junction mixture-the compound of compound 4 ", i.e. compound 13, and be captured and be fixed on the immobilon-p；

6）Using centrifugal chromatography, cleaned with cleaning fluid the chemiluminescent substance label that is not associated with the immobilon-p and The chemiluminescent substance of residual；

7）The immobilon-p after cleaning is placed in the starting luminescence reagent and reacted, and quilt is detected with the luminescence detector Luminous value caused by the chemiluminescent substance decomposition on the immobilon-p is indirectly fixed to, to detect the content of the thing to be checked.

3. detection method according to claim 2, it is characterised in that：The intermediate material is with specific binding capacity Antigen, antibody, Avidin, at least one of biotin and the like.

4. detection method according to claim 2, it is characterised in that：The reaction that the chromatography flows through the immobilon-p exists Completed on centrifugal device and using centrifugal chromatography driving liquid chromatographic flow on immobilon-p.

5. the detection method described in combined complete according to claim 1 or claim 2, it is characterised in that：It is described micro- The particle diameter selection 1nm-10um of grain.

6. detection combination suit according to claim 1, it is characterised in that：The rotating speed of the centrifugal device uses program control Mode processed；The rotating speed of the centrifugal device is 200 ~ 10000 revs/min.

7. detection method according to claim 2, it is characterised in that：The rotating speed of the centrifugal chromatography uses programme-control Mode；The rotating speed of the centrichromatography is 200 ~ 10000 revs/min.

8. the detection method described in combined complete according to claim 1 or claim 2, it is characterised in that：Describedization Learning Luminescence Enzyme includes at least one of HRPO, alkaline phosphatase and xanthine oxidase.

9. the detection method described in combined complete according to claim 1 or claim 2 or 4, it is characterised in that：It is described Immobilon-p includes nitrocellulose filter, polyvinylidene fluoride film, nylon membrane, DEAE cellulose membranes；The detector is chemiluminescence Detector.

10. detection combination suit according to claim 1, it is characterised in that：The detection combination suit is also comprising Cleaning fluid is stated, the cleaning fluid is the normal experiment cushioning liquid containing surfactant.

11. detection method according to claim 2, it is characterised in that：The cleaning fluid is to contain the normal of surfactant Advise experiment cushioning liquid.

12. detection combination suit according to claim 1, it is characterised in that：Detection combination suit also includes the Two thing specificity junction mixtures to be checked, intermediate material A, intermediate material B, thing specificity junction mixture to be checked specificity junction mixture in It is at least one.

13. the answering in immune detection product development of the combined complete or detection method any one of claim 1-12 With.