C reactive protein, serum amyloid A protein immunochromatography quantify combined detection test paperTechnical field
The utility model belongs to field of immunodetection, and in particular to a kind of high sensitivity C reactive protein/serum amyloid sampleAlbumin A immunochromatography quantifies combined detection test paper.
Background technique
C reactive protein (CRP) is a kind of very sensitive inflammation and tissue damage marker, various acute or chronicWhens infection, tissue damage, myocardial infarction etc., just there are the trend that is doubled and redoubled, even as high as 2000 in CRP a few hours after disease generationTimes, and the growth of more other Phasic proteins is fast and significant, the degree of raised amplitude and infection is positively correlated, and can be used as infectionEarly diagnosis index also can be used for the clinical monitoring course of disease.CRP usually increases after bacterium infection, and does not increase after virus infectionOr slightly increase, therefore, clinically using CRP as discriminating bacteria or an important indicator of virus infection.Healthy Human Serum is denseIt spends very low (≤8mg/L), the long-term low concentration of CRP increases the presence that (< 10mg/L) prompt has atherosclerotic inflammation, thereforeIt can be used as the important indicator of evaluation coronary heart disease risk.
Serum amyloid A protein (SAA) is a kind of small molecule apolipoprotein, relative molecular weight 12000Da, main sourceIn liver, also there is expression in cell outside liver, is equally a kind of Acute reaction protein.SAA is feminine gender under normal operation,But it can increase 1000 times or more when inflammation occurs, and be more easily detected than high quick CRP.Detect SAA content in serumHelp to diagnose non-specific inflammation, monitoring course inflammatory activity and outcome.
Since CRP lacks sensibility to virus infection, and SAA can be increased in bacterium and virus infection early stage.The twoThe diagnosis efficiency of virus infection early stage can be improved in joint-detection, and is virus and the identification of bacterium infection and the selection of therapeutic schemeUseful reference information is provided.In addition, there are correlation, prisons for the severity of the ratio of CRP and SAA and infant infectious diseasesIt surveys CRP/SAA odds ratio and individually detects CRP or SAA with bigger application value.
As inflammatory reaction effect played in the development process of coronary heart disease is further elucidated with, SAA and high quick CRP areIt is acute stage Phasic proteins, is all sensitive Inflammatory parameter, reflects the mark of Stability of Atherosclerotic Plaques.BloodThe severity of CRP/SAA concentration level and many cardiovascular diseases is in certain positive correlation in liquid, and the combined detection canFor the auxiliary diagnosis of cardiovascular disease, Treatment monitoring and prognosis evaluation.
The product of CRP/SAA joint-detection is considerably less currently on the market, clinically carries out CRP and SAA concentration to patientDetection can only be detected individually substantially.Time, manpower and cost are consumed, direct judgement of the doctor to the state of an illness is not easy to.CauseThis, establishes quick, the convenient, highly sensitive C reactive protein/serum amyloid A protein combined detection reagent of one kind and answers clinicIt is very urgent with diagnosing.
Biotin-avidin system (biotinavidin system, BAS) is a kind of widely applied life of medical domainObject reacts amplification system.A large number of studies show that BAS can almost be studied in conjunction with successfully various markers with current.By the systemApplied to immunohistochemistry, enzyme linked immunological, fluorescence immunoassay in the detection techniques such as radio-immunity, improves the above technical side in which can dramaticallySensibility, specificity and the stability of method facilitate clinical quick diagnosis, and are conducive to carry out extensive epidemiological survey, atFor the powerful of research immune response.It is polluted since BAS detection system economy is quick, and without radiogen, is not required to complicated instrumentDevice has a possibility that great potential and application.
Biotin-avidin system testing principle: biotin can become after chemical modification with various active groupDerivative -- activated biotin, activated biotin can be under the mediations of protein cross agent, with known almost all creaturesMacromolecular coupling, including protein, nucleic acid, polysaccharide, lipid etc..Avidin is the sugared egg of a kind of alkalinity extracted in ovalbuminIt is white, very high affinity is known as to biology, affinity costant (K) is 10mol/L, and the good specificity of the two combination stability is strong.ThisSample, the avidin molecule for combining fluorescent material is generated with the biotin molecule for being combined with specific antibody reacts, and plays moreGrade amplification, emits specific fluorescent signal and the excitation to fluorescent material, reaches detection unknown antigen (or antibody) pointThe purpose of son.Streptavidin is and a kind of affine protein for being known as similar biological properties, Streptavidin and AvidinIn the same molecule every peptide chain can in conjunction with a biotin, because nearly all substance for label can with Avidin orStreptavidin combines.Developing anti-Miao Le pipe hormone test examination fast diagnosis reagent using biotin-avidin system there is no reportRoad.
Utility model content
The purpose of this utility model is to provide C reactive protein, serum amyloid A protein immunochromatography quantitatively combines inspectionPaper slip is tested, double antibody sandwich method, fluorescence immunoassay lateral chromatography and biotin-Streptavidin amplification system is based on, providesA kind of C reactive protein of high sensitivity/serum amyloid A protein fast quantification joint-detection.When using the utility model, mentionHigh detection sensitivity and detection stability, reduce non-specific binding, joint-detection increases substantially the accuracy of detectionAnd convenience, detection time are not more than 15 minutes, substantially increase diagnosis efficiency.
The solution that the utility model solves its technical problem is: layer is immunized in C reactive protein, serum amyloid A proteinAnalyse quantitative combined detection test paper comprising get stuck, get stuck in be provided with bottom plate, sample pad, glimmering is successively arranged on the bottom plateSignal object bonding pad, nitrocellulose filter and water absorption pad, the sample pad are located at bottom plate one end, and the water absorption pad is located at bottomThe plate other end, the fluorescent marker bonding pad and sample pad one end are overlapping, the fluorescent marker bonding pad and water absorption padIt overlaps respectively and forms detection zone on the surface of nitrocellulose filter after being pressed in the both ends of nitrocellulose filter, detection zone is equipped with firstDetection line, the second detection line and nature controlling line, first detection line is fixed with the monoclonal antibody of identification C reactive protein, describedSecond detection line is fixed with the monoclonal antibody of identification serum amyloid A protein, and the nature controlling line is fixed with more grams of sheep anti-mouse iggGrand antibody, the C reactive protein monoclonal antibody of biotin labeling is fixed on fluorescent marker bonding pad, and to be fixed with identification C- anti-Answer the monoclonal antibody of another epitope of albumen, the serum amyloid A protein monoclonal antibody of biotin labeling and strepto- parentWith the fluorescin of element label.
As a further improvement of the above technical scheme, the fluorescent marker bonding pad includes the first fluorescence mark of stackingNote object bonding pad and the second fluorescent marker bonding pad, the first fluorescent marker bonding pad are fixed with the knowledge of biotin labelingThe monoclonal antibody of another epitope of other C reactive protein and the identification serum amyloid A protein of biotin labeling another tableThe monoclonal antibody of position, the second fluorescent marker bonding pad are fixed with the fluorescin of marked by streptavidin, and described theFor one end pad of one fluorescent marker bonding pad in the lower section of sample pad, the second fluorescent marker bonding pad is overlapping to be pressed in nitric acidOne end of cellulose membrane.
As a further improvement of the above technical scheme, the fluorescin is green fluorescent protein, in phycobniliproteinIt is a kind of.
As a further improvement of the above technical scheme, first detection line is located in detection zone close to fluorescent markerOne end of bonding pad, the nature controlling line are located at one end in detection zone close to water absorption pad, first detection line and nature controlling line phaseEvery distance be 4-8mm, the second detection line is between the first detection line and nature controlling line.
As a further improvement of the above technical scheme, the sample pad is after surfactant buffer immersion treatmentDry glass fibre component.
As a further improvement of the above technical scheme, the bottom plate is polystyrene component or polyethylene component.
As a further improvement of the above technical scheme, described to get stuck including plastics upper casing and plastics lower casing, the plasticsUpper casing is formed after fastening on plastics lower casing to get stuck.
As a further improvement of the above technical scheme, the position in the shell surface that gets stuck corresponding to nitrocellulose membrane is equipped withObservation window, the position that sample pad is corresponded in the shell surface that gets stuck are equipped with well, and the observation window is slot, the wellFor slotted eye.
The utility model compared with prior art, has the advantages that
(1) using fluorescin as marker, this marker has good stability the utility model, is conducive to improve detection surelyIt is qualitative.
(2) the utility model improves detection sensitivity, reduces by utilizing " Streptavidin-biotin amplification system "Non-specific binding is conducive to improve kit performance.
(3) C reactive protein is carried out using the utility model, the detection time of serum amyloid A protein is not more than 15 pointsClock, it is 0.5mg/L~200.0mg/L that C reactive protein, which detects the range of linearity, and the serum amyloid A protein detection range of linearity is3.0mg/L~200.0mg/L greatly improves detection efficiency.
(4) the utility model can carry out interpretation to result by fluorescence immunity analyzer, it can be achieved that automation, is reduced subjectiveThe influence of factor provides convenient, quick, reliable diagnostic result.
(5) the utility model is easy to make, small in size, easy to carry and cost is relatively low, overcomes enzyme linked immunosorbent assayAnd the shortcomings that chemoluminescence method price is high, time-consuming and high operation requirements, realize single part quantitative detection.
(6) the utility model can be mass, and be suitable for clinical quick diagnosis and live quick diagnosis；It is easy to save, hasConducive to grass-roots unit's popularization.
(7) the utility model uses associated detection technique, and two detections are integrated into same reagent strip and are carried out, on the one handOperation is simplified, detection time is shortened, keeps detection more rapidly convenient；On the other hand, by combining two joint detection results,The accuracy for improving clinical diagnosis makes doctor have more reliability to the prediction of conditions of patients, diagnosis and monitoring.
Detailed description of the invention
It, below will be to required in embodiment description in order to illustrate more clearly of the technical scheme in the embodiment of the utility modelAttached drawing to be used is briefly described.Obviously, described attached drawing is a part of the embodiment of the utility model, rather than completePortion's embodiment, those skilled in the art without creative efforts, can also be obtained according to these attached drawings itHis design scheme and attached drawing.
Fig. 1 is the structural schematic diagram of the utility model embodiment one；
Fig. 2 is the structural schematic diagram of the utility model embodiment two；
Fig. 3 is the structural schematic diagram of the utility model embodiment three；
Fig. 4 is the structural schematic diagram of the utility model embodiment four.
Specific embodiment
It is carried out below with reference to technical effect of the embodiment and attached drawing to the design of the utility model, specific structure and generationIt clearly and completely describes, to be completely understood by the purpose of this utility model, feature and effect.Obviously, described embodimentIt is a part of the embodiment of the utility model, rather than whole embodiments, it is based on the embodiments of the present invention, the skill of this fieldArt personnel other embodiments obtained without creative efforts belong to the model of the utility model protectionIt encloses.In addition, all connection/connection relationships being previously mentioned in text, not singly refer to that component directly connects, and referring to can be according to specific realitySituation is applied, by adding or reducing couple auxiliary, to form more preferably coupling structure.
Referring to Figures 1 and 2, C reactive protein, serum amyloid A protein immunochromatography quantify combined detection test paper,Including getting stuck 6, gets stuck and be provided with bottom plate 5 in 6, sample pad 1, fluorescent marker bonding pad 2, nitre are successively arranged on the bottom plate 5Acid cellulose film 3 and water absorption pad 4, the sample pad 1 are located at 5 one end of bottom plate, and the water absorption pad 4 is located at 5 other end of bottom plate, instituteThe fluorescent marker bonding pad 2 stated and 1 one end of sample pad are overlapping, and the fluorescent marker bonding pad 2 overlaps respectively with water absorption pad 4It is pressed in behind the both ends of nitrocellulose filter 3 and forms detection zone on the surface of nitrocellulose filter 3, the nitric acid in the detection zone is fineIt ties up and is fixed with the first detection line T1 (0.5~2.0mg/ of concentration that the monoclonal antibody of identification C reactive protein is constituted on plain film 3ML), the second detection line T2 (0.5~2.0mg/mL of concentration) and sheep that the monoclonal antibody of serum amyloid A protein is constituted are identifiedThe nature controlling line C (0.5~2.0mg/mL of concentration) that anti-mouse IgG polyclonal antibody is constituted, it is fixed on the fluorescent marker bonding pad 2There are monoclonal antibody (0.3~1.5mg/mL of concentration), the biology of identification another epitope of C reactive protein of biotin labelingThe monoclonal antibody (0.3~1.5mg/mL of concentration) of identification another epitope of serum amyloid A protein of element label and strepto- parentWith element label (or Avidin) fluorescin (use excitation wavelength 530nm, wavelength of transmitted light 570nm, concentration 0.1~2.0mg/mL)。
After sample to be tested is added in sample pad 1, is acted on and being moved forward by chromatography, C reactive protein, serum in sampleAmyloid A respectively corresponds and the C reactive protein antibody (Mab-CRP* on fluorescent marker bonding pad 2 in conjunction with fluorescinFluoro), compound CRP-is formed in conjunction with serum amyloid A protein antibody (Mab-SAA*Fluoro) reaction of fluorescinMab-CRP*Fluoro, SAA-Mab-SAA*Fluoro, reaction compound is continued to move along through pernitric acid under chromatography effectOn tunica fibrosa when coated CRP antibody (T1), SAA antibody (T2), compound is reacted respectively by the coated CRP antibody of correspondence, SAAAntibody capture forms compound (Mab-CRP-CRP-Mab-CRP*Fluoro, Mab-SAA-SAA-Mab-SAA*Fluoro)(detection line) reads the reaction signal of detection line by fluorescence immunity analyzer, under the action of excitation light source, fluorescent material hairThe fluorescence signal of specific wavelength is penetrated, fluorescence immunity analyzer captures fluorescence signal, the standard curve for converting and setting by signalIt is automatically converted to quantitative value, calculates the concentration of C reactive protein in sample, serum amyloid A protein, obtains C- reaction eggWhite, serum amyloid A protein testing result.
As a further improvement of the above technical scheme, referring to Fig. 3 and Fig. 4, the fluorescent marker bonding pad 2 includes layerFolded the first fluorescent marker bonding pad 20 and the second fluorescent marker bonding pad 21, the first fluorescent marker bonding pad 20It is fixed with the monoclonal antibody of identification another epitope of C reactive protein of biotin labeling and the identification blood of biotin labelingThe monoclonal antibody of another epitope of clear amyloid A, it is affine that the second fluorescent marker bonding pad 21 is fixed with strepto-The fluorescin of element label.One end pad of the first fluorescent marker bonding pad 20 is glimmering in the lower section of sample pad 1, described secondThe overlapping one end for being pressed in nitrocellulose filter 3 of signal object bonding pad 21.The layering of fluorescent marker bonding pad 2 is arranged, and is convenient forFluorescent marker bonding pad 2 is mounted between sample pad 1 and nitrocellulose filter 3.
As a further improvement of the above technical scheme, the fluorescin is green fluorescent protein, in phycobniliproteinIt is a kind of.
As a further improvement of the above technical scheme, the detection line T is located in detection zone close to fluorescent markerOne end of bonding pad 2, the nature controlling line be located at detection go in close to one end of water absorption pad 4, first detection line T1 and Quality ControlLine C separated by a distance is 4-8mm, and the second detection line T2 is between the first detection line T1 and nature controlling line C.
As a further improvement of the above technical scheme, the sample pad 1 is through surfactant buffer immersion treatmentDry glass fibre component afterwards.Sample pad is made of glass fibre, through surfactant buffer immersion treatment, is made after dryWith.The cutting width of the sample pad 1 is 2.5~5.0mm.
As a further improvement of the above technical scheme, the bottom plate 5 is polystyrene component or polyethylene component.
As a further improvement of the above technical scheme, it is described get stuck 6 include plastics upper casing 60 and plastics lower casing 61, it is describedPlastics upper casing 60 is formed after fastening on plastics lower casing 61 gets stuck 6.
As a further improvement of the above technical scheme, the position in 6 shell surfaces that get stuck corresponding to nitrocellulose membrane 3 is setThere is observation window 8, be equipped with well 7 corresponding to the position of sample pad 1 in 6 shell surfaces that get stuck, the observation window 8 is slot, instituteStating well 7 is slotted eye.
Be above the better embodiment of the utility model is illustrated, but the utility model create and it is unlimitedIn the embodiment, those skilled in the art can also make various without departing from the spirit of the present inventionEquivalent modifications or replacement, these equivalent variation or replacement are all included in the scope defined by the claims of the present application.