CN105675879A - Fluorescence immunochromatographic assay method of serum amyloid protein A and kit - Google Patents
Fluorescence immunochromatographic assay method of serum amyloid protein A and kit Download PDFInfo
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Abstract
The invention relates to a fluorescence immunochromatographic method of serum amyloid protein A and a detection kit thereof. The method includes the steps of preparing a probe fixing pad, preparing an immunochromatographic test strip, preparing a sample diluting liquid and detecting a sample. The probe fixing pad is prepared by mixing serum amyloid protein A monoclonal antibody fluorescent latex particles and goat-anti-rabbit IgG fluorescent latex particles, and diluting the mixture with a gold diluent and spraying the liquid onto glass fibers. A detection line is coated with serum amyloid protein A monoclonal antibody and a control line is coated with goat-anti-rabbit IgG to prepare the immune-chromatographic test strip. The detection kit includes an immune-chromatographic detection card including a PVC lining plate, a sample pad, the probe fixing pad, a nitrocellulose membrane and a water absorbing paper. The probe fixing pad is prepared by mixing and drying the serum amyloid protein A monoclonal antibody fluorescent latex particles and the goat-anti-rabbit IgG fluorescent latex particles, so that the detection kit is high in detection sensitivity and accuracy, is simple in operation and is low in cost.
Description
Technical field
The present invention relates to the detection method of a kind of serum amyloid A protein and test kit, particularly relate to fluorescence immune chromatography detection method and the detection kit thereof of serum amyloid A protein.
Background technology
Serum amyloid A protein (serumamyloidAprotein, SAA) is the multiform state protein family of a class polygene coding, is the prerequisite material organizing amyloid A, belongs to Acute reaction protein, its relative molecular weight about 12000. In acute-phase response, such as inflammation or acute stage of infection, stimulate through IL-1, IL-6 and TNF, SAA is synthesized by the scavenger cell being activated and fibroblast in liver, in 48～72 hours, concentration raises rapidly, can be elevated to 100～1000 times of initial concentration, but its transformation period is short, only about 50 minutes, and at Recovery Phase of diseases rapid decrease. Some disease, such as virus infection, graft-rejection, coronary heart disease etc., the susceptibility of SAA, higher than c reactive protein (CRP), can be the better reference value of clinical offer, and as a new Testing index, SAA receives the concern that people get more and more.
On the one hand, the key property of SAA is to there being very big diagnostic value in acute renal allograft rejection. The degraded product of SAA can be deposited in different organs in the way of amyloid A (AA) protofibril, and in chronic inflammatory diseases, this is a kind of serious complication. Normal human serum SAA mean concentration is that measuring of 2.33mg/L, SAA concentration is more more responsive than change of serum C r for the diagnosis of renal transplantation acute rejection. Such as when getting rid of infection, the exception of SAA raises has very big value to diagnosis renal transplantation acute rejection.
On the other hand, SAA reaction is sensitive, especially can provide better discriminating to " normally " and small Acute-phase protein. After usual human body Inflammatory response is about 8h, SAA starts to raise, and exceedes with reference to scope upper limit time early than CRP, CRP healthy human body I d median and about 10 times of the gap with reference to the scope upper limit, and only has 5 times in SAA.Such as mild infection such as many virus infectiones, SAA raises more more common than CRP, and in diseases such as infectivities, the absolute rise of SAA is higher than CRP. And for example, about 2/3 flu patient SAA raises usually, but the patient identical performance CRP less than 1/2 raises, and in virus infection case, SAA and CRP concentration raises and sees adenovirus infection person. In other diseases, as do not raised in lupus erythematosus and ulcerative colitis SAA. Malignant tumor of bonal metastasis stage SAA raises and is usually confined to the higher numerical value of organ stage display than tumour. In transplant rejection, in research to a renal transplantation recipients, the inspection of the generation rejection of 97% is the rising according to SAA, in irreversible transplant rejection detects, its mean concns reaches 690 ± 29mg/L, and the related levels of reversible rejection outbreak case is 271 ± 31mg/L. In rheumatoid arthritis, tuberculosis or leprosy, the chronic rising of SAA concentration is the prerequisite of synthesis AA-starch fiber, is also used to diagnosis Secondary cases amyloidosis pathology. Therefore SAA the better reference value of clinical offer is provided.
The SAA detection method variation that current home and abroad is existing, strengthens the methods such as turbidimetry as adopted enzyme linked immunosorbent assay, radioimmunoassay, latex and carries out SAA detection. But these method detecting steps are many, it is necessary to relevant biochemical instrument detects, and cost is higher, is not suitable for general small-sized or medium-sized hospital. Relative to above-mentioned detection method, fluorescent chromatographic immune analysis method is a kind of trace analysis method, has the advantages such as highly sensitive, equipment is simple and easy to get and easy and simple to handle.
Chinese patent CN10376455B discloses the test kit of a kind of Colloidal Gold detection by quantitative serum amyloid A protein, be combined by anti-SAA immune colloid gold and another SAA monoclonal antibody being coated on Sptting plate or many anti-SAA in testing sample, Radioactive colloidal gold is fixed on Sptting plate, measures SAA content by gold mark quantitative instrument. The method needs immune colloid gold is carried out freeze-drying preservation, redissolves before detection, and Sptting plate needs to close before reactions, then adds sample and reacts, and reaction terminates rear Sptting plate to be needed to wash and complete just can detect. The whole testing process of this method is longer, operates more, it is necessary to relevant equipment, testing cost height.
Chinese patent CN104569414A discloses the test strip of a kind of colloidal gold immunity chromatography joint-detection Procalcitonin/serum amyloid A protein (PCT/SAA), nitrocellulose filter wrap by anti-SAA monoclonal antibody, combine with the Radioactive colloidal gold of the SAA in sample He the anti-SAA monoclonal antibody being marked with pairing, coordinate immunochromatography readout instrument, detect SAA fast. The method is quick, easy, and detected result is easy to judge, is applicable to qualitative detection, accurately quantitatively still can not satisfy the demands for SAA content.
Therefore, when how to develop detection, length, easy to operate, the accurate serum amyloid A protein detection method of detected result and testing product become problem demanding prompt solution.
Summary of the invention
For solving the problems of the technologies described above, it is an object of the invention to provide a kind of highly sensitive, accuracy height, the detection method of the serum amyloid A protein that simple to operate, cost is low and test kit.
The fluorescence immune chromatography detection method of a kind of serum amyloid A protein of the present invention, comprises the following steps:
Step 1) probe fixing pad preparation: by serum amyloid A protein monoclonal antibody serum amyloid A protein monoclonal antibody fluorescent latex particles obtained with activation albumin fluorescent latex particles linked reaction in proportion, again described serum amyloid A protein monoclonal antibody fluorescent latex particles is mixed to get by a certain percentage with goat anti-rabbit igg fluorescent latex particles and mixes fluorescent latex particles, after described mixing fluorescent latex particles fluorescent probe microballoon diluted, spray the fixing pad of obtained probe on the glass fibers;
Step 2) immuno-chromatographic test paper strip preparation: sample pad, probe fixing pad, chromatographic film, thieving paper are attached on PVC liner plate successively, described serum amyloid A protein monoclonal antibody is coated on the detection line of chromatographic film, goat anti-rabbit igg is coated on chromatographic film control line, and then obtained immuno-chromatographic test paper strip, the preferred nitrocellulose filter of described chromatographic film;
Step 3) sample diluting liquid preparation: the mixture including bovine serum albumin, tensio-active agent, dispersion agent and sanitas is mixed with damping fluid obtained sample diluting liquid;
Step 4) sample survey: get serum sample and be mixed to described sample diluting liquid, add immuno-chromatographic test paper strip and carry out immunochromatography reaction, layer analysis reaction terminate after through fluorimetric detector fluoroscopic examination.
The present invention is further, step 1) in, the preparation of described activation albumin fluorescent latex particles is first by fluorescent latex particles and activator reaction obtained activation fluorescent latex particles, again with albumin in proportion linked reaction obtain albumin fluorescent latex particles, described albumin is 0.05:100～0.2:100 with the ratio of activation fluorescent latex particles, and described albumin fluorescent latex particles is described activation albumin fluorescent latex particles obtained with activator reaction further.
The present invention is further, step 1) in, the preparation of described goat anti-rabbit igg fluorescent latex particles is obtained with activation fluorescent latex particles linked reaction in proportion by goat anti-rabbit igg, and described goat anti-rabbit igg is 0.05:100～0.2:100 with the blending ratio of activation fluorescent latex particles.
The present invention is further, step 1) in, described activator comprises 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and N-hydroxy thiosuccinimide, the two weight ratio is 1:6～1:1, and the weight ratio of described 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and fluorescent latex particles is 1:100～5:100. Three can at room temperature react 0.5～1h after mixing.
Further, the kind of described albumin comprises people, ox, sheep or mouse in the present invention.
The present invention further, step 1) in, described serum amyloid A protein monoclonal antibody is 0.05:100～0.2:100 with the blending ratio of activation fluorescent latex particles, can at room temperature linked reaction 1～5h after mixing; Ratio 2:1～6:1 that described serum amyloid A protein monoclonal antibody fluorescent latex particles mixes mutually with goat anti-rabbit igg fluorescent latex particles, linked reaction 1～5h under room temperature after mixing.
The present invention is further, step 1) in, fluorescent latex particles fluorescent probe microballoon diluted 2～6 times will be mixed, evenly it will be sprayed on width as, on the glass fibre of 0.7～1.3cm, after dry 2～24h, obtained probe is fixing at 30～55 DEG C pads taking the speed of 2～6ul/cm.
The present invention is further, described fluorescent probe microballoon diluent is the damping fluid containing sucrose, bovine serum albumin and tensio-active agent, described damping fluid comprises one or more in PBS, Tris damping fluid, MES damping fluid, HEPES damping fluid, and described tensio-active agent is polysorbas20 or tween 80.
The present invention is further, step 3) in, described damping fluid comprises one or more in PBS, Tris damping fluid, glycine buffer, MES damping fluid, HEPES damping fluid, described tensio-active agent is polysorbas20, tween 80, triton x-100 or polyoxyethylene glycol, described sanitas is sodium azide or proclin, and described dispersion agent is polyvinylpyrrolidone 10K, PVP K or polyvinyl alcohol.
On the other hand, the fluorescence immune chromatography detection kit of a kind of serum amyloid A protein of the present invention, comprise kit box and diluent bottle, described kit box comprises, the sample pad that described immunochromatographydetection detection card comprises PVC liner plate and is fixed on PVC liner plate, the fixing pad of probe, nitrocellulose filter and thieving paper, described sample pad is overlapped on the fixing pad of probe, the fixing pad of described probe and thieving paper are overlapped on described nitrocellulose filter both sides respectively, described nitrocellulose filter is provided with detection line and control line, serum amyloid A protein monoclonal antibody it is coated with in described detection line, it is coated with goat anti-rabbit igg in described control line, described probe fixes pad for obtained by the mixing fluorescent latex particles drying of serum amyloid A protein monoclonal antibody fluorescent latex particles and goat anti-rabbit igg fluorescent latex particles, has deposited sample diluting liquid in described diluent bottle.
The present invention is further, described immunochromatographydetection detection card also comprises housing, described housing is provided with well, detection window, coding region and handle, described well is positioned at described sample pad place, described detection window is positioned at described detection line and control line place, described coding region is between detection window and handle, and described handle is positioned on described housing to be positioned at the side near thieving paper.
By such scheme, the present invention at least has the following advantages:
1. fluorescence immune chromatography method of the present invention is adopted, it is achieved external highly sensitive, the accuracy height of serum amyloid A protein, the detection by quantitative that simple to operate, cost is low.
2. fluorescence immune chromatography method of the present invention, detection line region adopts and can form the serum amyloid A protein monoclonal antibody of mixture with serum amyloid A protein, and partner probe then uses the serum amyloid A protein monoclonal antibody that fluorescent latex particles marks; Control line region is coated with rabbit igg antibody, and partner probe uses the fluorescent latex particles of goat anti-rabbit igg antibody mark. This control line and detection line independent reaction, be independent of each other and disturb. Control line signal is utilized can accurately to judge the quality that probe is dispersed in mixing liquid, for correct detection line signal.
3. fluorescence immune chromatography method of the present invention, fluorescent latex particles needs first to carry out coupling with albumin, and then carry out coupling with serum amyloid A protein monoclonal antibody, reduce the space resistance of two sandwich structure in testing process so that it is the serum amyloid A protein of lower concentration can be detected.
4. the serum amyloid A protein detection kit that prepared by the present invention, store convenient, detection sensitivity height, concentration in blood sample can be detected and it is low to moderate the serum amyloid A protein of 0.5mg/L, the detecting instrument used is simple, simple to operate, without the need to professional operator, have simultaneously and detect advantage fast, within 10～20 minutes, detected result can be obtained.
5. the serum amyloid A protein detection kit that prepared by the present invention, due in advance to the special processing of sample pad and the improvement of sample diluting liquid, applied widely, serum, blood plasma and whole blood can be detected.
Above-mentioned explanation is only the general introduction of technical solution of the present invention, in order to better understand the technique means of the present invention, and can be implemented according to the content of specification sheets, below with the better embodiment of the present invention and coordinate accompanying drawing to be described in detail as follows.
Accompanying drawing explanation
Fig. 1 is the structural representation of immuno-chromatographic test paper strip in the present invention;
Fig. 2 is the structural representation of reagent card in the present invention.
In figure, the implication of each Reference numeral is as follows.
1PVC liner plate 2 sample pad
3 probes fixing pad 4 nitrocellulose filters
5 detection line 6 control lines
7 thieving paper 8 wells
9 housings 10 detect window
11 coding region 12 handles
Embodiment
Technical scheme in the embodiment of the present invention is carried out clear, complete description below, it is clear that described embodiment is only the present invention's part embodiment, instead of whole embodiments, is not used for limiting the scope of the invention. Based on embodiments of the invention, those of ordinary skill in the art, not making other embodiments all obtained under creative work prerequisite, belong to the scope of protection of the invention.
The fluorescence immune chromatography detection method of the serum amyloid A protein of the present invention, comprises the following steps:
Step 1) the fixing pad preparation of probe: by wavelength of transmitted light be 500～590nm add activator 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) and N-hydroxy thiosuccinimide (Sulfo-NHS) room temperature with the fluorescent latex particles solution of carboxyl or amino under react 0.5～1h, wherein, N-hydroxy thiosuccinimide (Sulfo-NHS) is 1:1～6:1 with the weight ratio of 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC), 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) is 1:100～5:100 with the weight ratio of fluorescent latex particles, centrifugal, activation fluorescent latex particles is obtained after washing.
Should be noted that the above-mentioned wavelength of transmitted light of (1) the present invention is that 500～590nm fluorescent latex is easily obtaining on the market, thus under the prerequisite ensureing accuracy of detection, economy and suitability are higher; (2) when the weight ratio of EDC and fluorescent latex particles is less than 1:100, the activation rate of present latex particulate is very low, and on coupling microballoon, the combination of antibody is little, and sensitivity during detection is low; When the weight ratio of EDC and fluorescent latex particles is greater than 5:100, the amplitude that the activation rate of present latex particulate increases and increase with EDC amount is limited, and cost raises, and optimum proportion is 1:100; (3) the weight ratio of EDC and Sulfo-NHS is less than 1:1, and the present latex particulate facile hydrolysis of activation, reduces activation efficiency; The weight ratio of EDC and Sulfo-NHS is greater than 6:1, and the activation rate of present latex particulate reaches capacity, cost increase, and optimum proportion is 1:3; (4) soak time is less than 0.5h, and the efficiency of activation is not high, waste starting material; Soak time is greater than 2h, and the speed of activation is less than the speed of activating substance hydrolysis, reduces activation efficiency, optimum activating time 1h.
0.05:100～the 0.2:100 in proportion of albumin with activation fluorescent latex particles is mixed by the present invention, and linked reaction 2～5h under room temperature, obtains albumin fluorescent latex particles. By goat anti-rabbit igg with activation fluorescent latex particles in proportion 0.05:100～0.2:100 mix, linked reaction 2～5h under room temperature, obtains goat anti-rabbit igg fluorescent latex particles. The blending ratio of the albumin fluorescent latex particles of serum amyloid A protein monoclonal antibody and activation is 0.05:100～0.2:100, linked reaction 1～5h under room temperature after mixing.
When should be noted that the weight ratio of (1) serum amyloid A protein monoclonal antibody and fluorescent latex particles is less than 0.05:100, coupling efficiency height, but waste present latex particulate, the major part activation non-binding antibody of point of present latex particulate, and when being greater than 0.2:100, the activation point of the major part of present latex particulate is combined, coupling efficiency is had no significant effect by the amount continuing to increase serum amyloid A protein monoclonal antibody, wastage of material, optimum proportion is 0.12:100;(2) the reaction times is relevant with Conjugate ratio, and the time is too short is less than 1 hour, then antibody and present latex particulate in conjunction with less, uneconomical, overlong time is greater than 5 hours, then Conjugate ratio increases limited, loses time, optimum reacting time 3h.
The present invention is in the ratio pooled serum amyloid A monoclonal antibody fluorescent latex particles of 2:1～6:1 and goat anti-rabbit igg fluorescent latex particles, obtain mixing fluorescent latex particles, fluorescent latex particles fluorescent probe microballoon diluted 2～6 times will be mixed, width evenly it is sprayed on as on the glass fibre of 0.7～1.3cm, dry 2～24h at 30～55 DEG C taking the speed of 2～6ul/cm. Fluorescent probe microballoon diluent is the damping fluid containing sucrose, bovine serum albumin and tensio-active agent, described damping fluid comprises one or more in PBS, Tris damping fluid, MES damping fluid, HEPES damping fluid, and described tensio-active agent is polysorbas20, tween 80.
Should be noted that (1) probe is kept in sample diluting liquid easily to reunite, it is sprayed drying on the glass fibers by fluorescent probe microballoon diluted and makes the fixing pad of probe, the component of fluorescent probe microballoon diluent so that probe evenly disperses, can avoid the reunion between probe; Probe fixes the probe on pad when slow releasing, it is possible to slow down immune response, makes the reaction times longer, and detection signal is stronger; (2) spray gold speed is crossed and probe solution can be caused slowly very few, disperses uneven on the glass fibers, sprays gold excessive velocities and can cause glass fibre Premature saturation, and probe solution penetrates on spray gold platform, causes experimental error, and it is poor to amplify in criticizing; (3) to fix time of drying of pad not easily excessively long for probe, also needs to continue dry after making immuno-chromatographic test paper strip, and this step only needs moisture fully dry.
Step 2) immuno-chromatographic test paper strip preparation: sample pad, probe fixing pad, chromatographic film, thieving paper are attached on PVC liner plate successively, adopt on stroke detection line (T line) that serum amyloid A protein monoclonal antibody and rabbit igg are coated on chromatographic test paper by film spray gold machine respectively and control line (C line), the immuno-chromatographic test paper strip of 3～5mm width is obtained through slitting shear machine cutting, the preferred nitrocellulose of above-mentioned chromatographic film after 37 DEG C of drying 12～36h.
Should be noted that (1) traditional qualitative immunochromatography technique adopts bag by sheep anti-mouse antibody as control line, along with the increase of serum amyloid A protein in serum or some against murine source antibody blocking agent, signal on control line decreases, its signal value just can not be used for calculating, and the accuracy of the signal of testing wire is also without reference frame. Goat anti-rabbit igg antibody of the present invention adopts rabbit igg antibody and sheep anti-chicken antibody to match as control line, and the reaction of C line and T line independently carries out, without cross influence, it is thus possible to avoid the repeated deviation that C line and T line are competed probe particulate each other and produced; (2) it is less than 12h time of drying, low in conjunction with efficiency on nitrocellulose filter of antibody, product detect signal can change with the prolongation of shelf time, the detectability of serum amyloid A protein is caused to raise and detection instability, time of drying is greater than 36h, the easy inactivation of antibody, causes the sensitivity decrease of detection, and best time of drying is 24h; (3) when the width of immuno-chromatographic test paper strip is less than 3mm, the precision of cutting machine is relatively big on the impact of immuno-chromatographic test paper strip, can reduce the repeatability of detected result, when the width of immuno-chromatographic test paper strip is greater than 5mm, increasing the cost of test card, the immuno-chromatographic test paper strip of optimization is 4mm.
Step 3) sample diluting liquid preparation: sample diluting liquid is the damping fluid containing bovine serum albumin, tensio-active agent and sanitas. This damping fluid comprises one or more in PBS, Tris damping fluid, glycine buffer, MES damping fluid, HEPES damping fluid, tensio-active agent is polysorbas20, tween 80, triton x-100 or polyoxyethylene glycol, sanitas is sodium azide or proclin, and described dispersion agent is polyvinylpyrrolidone 10K, PVP K or polyvinyl alcohol.
Above-mentioned proclin sanitas, its activeconstituents is MIT (MCI) and CMIT (CMCI) mainly, is a new generation's high-performance bio sanitas. The growth of these two kinds of composition T suppression cell and impel the principle of apoptosis identical. Activeconstituents with cell membrane contact several minutes after, can penetrate in film immediately and the activity of T suppression cell endoenzyme. Target enzyme is in the central position that cellular metabolism KREBS circulates, four different positionss of ProClin sanitas effect KREBS circulation (tricarboxylic acid cycle): pyruvic oxidase, a-ketoglutaric acid desaturase, succinodehydrogenase and nadh dehydrogenase. Thus the synthesis of cell growth inhibiting metabolism, macromole, cause the horizontal rapid decrease of intracellular energy. Due to the collapse of energy system, cell can not synthesize the compound required for daily metabolism. All bacteriums and fungi at least have part KREBS circulation, so the scope being suitable for of ProClin sanitas is widely. Secondly, ProClin sanitas has many mark target action sites, therefore greatly reduces the resistance of the microorganism because of variation generation. Along with KREBS circulation is blocked, the ability rapid decrease of the biologically active substances such as cell generate energy and synthetic enzyme, but its using dosage keeps lower level to person poultry harmless, it is possible to effectively control microbial growth in external diagnosis reagent. Thus there is broad spectrum antibacterial, speed of action is fast, using dosage is few, PH (PH2～8.5) applied widely, chemical stability is good, toxicity is lower, with water arbitrarily than the feature such as mixing.
Should be noted that (1) bovine serum albumin has the effect of flow sealing, can be combined by the foreign protein in human serum or blood plasma, close the blank site on nitrocellulose filter, reduce the residual of probe on film and non-specific binding, reduce false Yangxin number; (2) tensio-active agent can improve the wetting ability of detected sample and probe so that combination on NC film of sample and probe and being more evenly distributed, and avoids basal signal fluctuation and the uneven impact on signal of immune response; (3) sanitas postpones or suppresses microbial growth, avoids the corruption of sample diluting liquid; D) damping fluid is used for the reaction environment of immunity moderation reaction, and the difference of the difference and serum plasma that reduce different people serum is on the impact of reaction system.
Step 4) sample survey: take out a certain amount of serum or blood plasma adds in sample diluting liquid, to be mixed evenly after drop to immunochromatography immunochromatographydetection detection card carry out immunochromatography reaction, adopt the wavelength of transmitted light corresponding with fluorescent latex particles to carry out fluoroscopic examination subsequently under fluorimetric detector. as depicted in figs. 1 and 2, mixing liquid penetrates in sample pad 2, mixing liquid fixes pad 3 to tat probe after glass fibre impurity screening and process, serum amyloid A protein monoclonal antibody on serum amyloid A protein wherein and probe is combined into mixture, mixture penetrates on detection line 5 along nitrocellulose filter 4, the new mixture of double-antibody sandwich is formed with the serum amyloid A protein monoclonal antibody being fixed on detection line 5, serum amyloid A protein monoclonal antibody on detection line 5 and the serum amyloid A protein monoclonal antibody in probe, its table position on serum amyloid A protein is different.The antibody complex of remaining antigen-fluorescent mark, the serum amyloid A protein monoclonal antibody precipitation being marked with fluorescent latex particles not combined and serum amyloid A protein monoclonal antibody fluorescent latex particles antibody and the goat anti-rabbit igg being marked with fluorescent latex particles continue to penetrate into control line 6, and the rabbit igg that the goat anti-rabbit igg being marked with fluorescent latex particles can be fixed on control line 6 is combined and forms antigen-antibody complex. Under fluorimetric detector during fluoroscopic examination, only on control line 6, signal detected, could prove that detected result is effective.
As shown in Fig. 1 to 2, the present invention also provides the fluorescence immune chromatography detection kit of a kind of serum amyloid A protein based on aforesaid method, comprise kit box and diluent bottle, the sample pad 2 that kit box comprises PVC liner plate 1 and is fixed on PVC liner plate, pad 3 fixed by probe, nitrocellulose filter 4 and thieving paper 7, sample pad is overlapped on the fixing pad of probe, pad 3 fixed by probe and thieving paper 7 is overlapped on the both sides of nitrocellulose filter respectively, nitrocellulose filter is provided with detection line 5 and control line 6, serum amyloid A protein monoclonal antibody and rabbit igg it is coated with respectively in detection line 5 and control line 6, pad 3 fixed by probe is that the mixing fluorescent latex particles by serum amyloid A protein monoclonal antibody and goat anti-rabbit igg is dry obtained through spraying gold, has deposited sample diluting liquid in diluent bottle. immunochromatographydetection detection card also comprises housing 9, housing 9 is provided with well 8, detection window 10, coding region 11 and handle 12, well 8 is positioned at sample pad 2 place, detection window 10 is positioned at detection line 5 and control line 6 place, coding region 11 is between detection window 10 and handle 12, and handle 12 is positioned on housing to be positioned at the side near thieving paper 7.
When detection kit of the present invention uses, get a certain amount of serum or plasma sample joins in sample diluting liquid, join after mixing in well 8. the wavelength of transmitted light corresponding with fluorescent latex particles is adopted to carry out fluoroscopic examination subsequently under fluorimetric detector. mixing liquid penetrates in sample pad 2, to the fixing pad 3 of tat probe after glass fibre impurity screening and process, serum amyloid A protein monoclonal antibody on serum amyloid A protein wherein and probe is combined into mixture, mixture penetrates on detection line 5 along nitrocellulose filter 4, the new mixture of double-antibody sandwich is formed with the serum amyloid A protein monoclonal antibody being fixed on detection line 5, serum amyloid A protein monoclonal antibody on detection line 5 and the serum amyloid A protein monoclonal antibody in probe, its table position on serum amyloid A protein is different. the antibody complex of remaining antigen-fluorescent mark, the serum amyloid A protein monoclonal antibody precipitation being marked with fluorescent latex particles not combined and serum amyloid A protein monoclonal antibody fluorescent latex particles antibody and the goat anti-rabbit igg being marked with fluorescent latex particles continue to penetrate into control line 6, and the rabbit igg that the goat anti-rabbit igg being marked with fluorescent latex particles can be fixed on control line 6 is combined and forms antigen-antibody complex. under fluorimetric detector during fluoroscopic examination, only on control line 6, signal detected, could prove that detected result is effective.
The detection fluorimetric detector of above-mentioned the present invention, comprises excitation-detection module, pre-amplifying module, control analysis module and software system. Wherein the light source of excitation-detection module is emission wavelength is the photodiode of 400～600nm, and pre-amplifying module is a pre-amplification circuit.
The fluorescence immune chromatography detection of " embodiment " serum amyloid A protein
The first step: the preparation of pad fixed by probe
1) after getting fluorescent latex particles solution (containing carboxyl) the pH6.0MES damping fluid washing centrifugation three times that 200 μ l wavelength of transmitted light are 570nm, precipitation is diluted with pH6.0MES damping fluid, add 5mgEDC and 15mgSulfo-NHS mix even after, at room temperature reaction activation 30min, after centrifugation, precipitation continuation pH6.5MES damping fluid washs three times, dilute with postprecipitation pH6.5MES damping fluid, add 100 μ g bovine serum albumins, 3h is reacted under room temperature, add the ethanolamine solutions containing bovine serum albumin to close, continue reaction 1h, centrifuged deposit pH7.4PBS damping fluid washs four times, obtain being marked with bovine serum albumin precipitation and the bovine serum albumin fluorescent latex particles of fluorescent latex particles, goat anti-rabbit igg precipitation and the goat anti-rabbit igg fluorescent latex particles of fluorescent latex particles can be obtained being marked with reason, precipitation is resuspended in 200 μ lpH7.4PBS damping fluids, add 0.6 μ lproclin, preserve at 4 DEG C, the bovine serum albumin activation conjugated sera amyloid A monoclonal antibody being marked with fluorescent latex particles obtains being marked with the serum amyloid A protein monoclonal antibody precipitation of fluorescent latex particles in the same way, precipitation is resuspended in 200 μ lpH7.4PBS damping fluids, add 0.6 μ lproclin, preserve at 4 DEG C.
2) by serum amyloid A protein monoclonal antibody fluorescent latex particles and goat anti-rabbit igg fluorescent latex particles by volume 6:1 mix, the epoxy glue lactoconium being mixed to get is mixed with the fluorescent probe microballoon diluent whirlpool of 2 times of volumes, obtain probe solution, width evenly it is sprayed on as on the glass fibre of 1cm, dry 4h at 37 DEG C taking the speed of 4ul/cm.
2nd step: the preparation of immunochromatographydetection detection card
Pasting nitrocellulose filter 4, the fixing pad 3 of probe, sample pad 2 and thieving paper 7 on liner plate successively, sample pad 2 is overlapped on the fixing pad 3 of probe, and the fixing pad 3 of probe and thieving paper 7 are overlapped on nitrocellulose filter 4, and are closely connected. Another strain serum amyloid A protein monoclonal antibody (coated antibody) of different table position will be positioned at and goat anti-rabbit igg coating buffer is mixed with the antibody coating buffer of 1mg/ml and 1mg/ml respectively respectively from the serum amyloid A protein monoclonal antibody that fluorescent latex particles marks. Serum amyloid A protein monoclonal antibody coating buffer and rabbit igg coating buffer are coated on nitrocellulose filter 4 corresponding detection line 5 with the line speed of 1 μ l/cm and on control line 6, detection line 5 and control line 6 interval 6mm, <under 30% condition after 37 DEG C of oven dry 24h, fluorescence immune chromatography test paper plate is made in humidity.
With slitting shear machine, the fluorescence immune chromatography test paper plate prepared longitudinally is cut into the wide fluorescence immune chromatography test paper bar of 4mm, puts it into interior process through case pressing machine of getting stuck and obtain immunochromatographydetection detection card.
3rd step: the preparation of sample diluting liquid
Get the PBS 1L of 0.02MpH7.0, add 8ml polysorbas20,5.1g bovine serum albumin and 0.19g sodium azide, ultrasonic until solid all dissolves mixes.
4th step: pattern detection
1) linear, detectability and precision assessment:
Adopt negative bovine serum as diluent, serum amyloid A protein standard substance are mixed with the standard solution that concentration is 200,180,160,140,120,100,80,60,40,20 and 0mg/Ll; get standard substance 100 μ l and add 100 μ l sample diluting liquids; get 100 μ l after blowing and beating 15 times and join in well 8, after 15 minutes, adopt fluorimetric detector to detect.Result shows, linear correlation coefficient r ^2 is greater than 0.99, and detection is limited to 0.5mg/L, and the detection variation coefficient of each concentration is all less than 8%.
2) checking of linear dimensions:
Adopting low value human serum and high level human serum, compound concentration is the serum amyloid A protein human serum solution of 200,100,120,80 and 40mg/L, and each sample repeats 4 times, and result shows, r^2 is greater than 0.99, and highest detection scope can reach 200mg/L.
3) assessment of accuracy:
Serum amyloid A protein concentration is adopted to be sample based on the human serum sample of 2mg/L, add the serum amyloid A protein standard substance human serum of same volume different concns, it is mixed with the serum amyloid A protein human serum solution that concentration is 140,80,14 and 3mg/L. Another part of sample adds the negative human serum of same volume, recovery sample and basis sample is carried out 4 duplicate detection analyses, and calculates. Result shows, the rate of recovery is in 95%～105% scope.
The above is only the preferred embodiment of the present invention; it is not limited to the present invention; should be understood that; for those skilled in the art; under the prerequisite not departing from the technology of the present invention principle; can also making some improvement and modification, these improve the protection that also should be considered as the present invention with modification.
Claims (10)
1. the fluorescence immune chromatography detection method of a serum amyloid A protein, it is characterised in that, comprise the following steps:
Step 1) probe fixing pad preparation: by serum amyloid A protein monoclonal antibody serum amyloid A protein monoclonal antibody fluorescent latex particles obtained with activation albumin fluorescent latex particles linked reaction in proportion, again described serum amyloid A protein monoclonal antibody fluorescent latex particles is mixed to get by a certain percentage with goat anti-rabbit igg fluorescent latex particles and mixes fluorescent latex particles, after described mixing fluorescent latex particles fluorescent probe microballoon diluted, spray the fixing pad of obtained probe on the glass fibers;
Step 2) immuno-chromatographic test paper strip preparation: sample pad, probe fixing pad, chromatographic film, thieving paper are attached on PVC liner plate successively, described serum amyloid A protein monoclonal antibody is coated on the detection line of chromatographic film, goat anti-rabbit igg is coated on chromatographic film control line, and then obtained immuno-chromatographic test paper strip;
Step 3) sample diluting liquid preparation: the mixture including bovine serum albumin, tensio-active agent, dispersion agent and sanitas is mixed with damping fluid obtained sample diluting liquid;
Step 4) sample survey: get serum, blood plasma or whole blood sample and be mixed to described sample diluting liquid, add immuno-chromatographic test paper strip and carry out immunochromatography reaction, layer analysis reaction terminate after through fluorimetric detector fluoroscopic examination.
2. the fluorescence immune chromatography detection method of serum amyloid A protein according to claim 1, it is characterized in that: step 1) in, the preparation of described activation albumin fluorescent latex particles is first by fluorescent latex particles and activator reaction obtained activation fluorescent latex particles, again with albumin in proportion linked reaction obtain albumin fluorescent latex particles, described albumin is 0.05:100～0.2:100 with the ratio of activation fluorescent latex particles, described albumin fluorescent latex particles is described activation albumin fluorescent latex particles obtained with activator reaction further, wherein, the kind of described albumin comprises people, ox, sheep or mouse.
3. the fluorescence immune chromatography detection method of serum amyloid A protein according to claim 2, it is characterized in that: step 1) in, the preparation of described goat anti-rabbit igg fluorescent latex particles is obtained with activation fluorescent latex particles linked reaction in proportion by goat anti-rabbit igg, and described goat anti-rabbit igg is 0.05:100～0.2:100 with the blending ratio of activation fluorescent latex particles.
4. the fluorescence immune chromatography detection method of serum amyloid A protein according to claim 2, it is characterized in that: step 1) in, described activator comprises 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and N-hydroxy thiosuccinimide, the two weight ratio is 1:6～1:1, and the weight ratio of described 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and fluorescent latex particles is 1:100～5:100.
5. the fluorescence immune chromatography detection method of serum amyloid A protein according to claim 1, it is characterized in that: step 1) in, described serum amyloid A protein monoclonal antibody is 0.05:100～0.2:100 with the blending ratio of activation albumin fluorescent latex particles, described goat anti-rabbit igg is 0.05:100～0.2:100 with the blending ratio of activation fluorescent latex particles, ratio 2:1～6:1 that described serum amyloid A protein monoclonal antibody fluorescent latex particles mixes mutually with goat anti-rabbit igg fluorescent latex particles.
6. the fluorescence immune chromatography detection method of serum amyloid A protein according to claim 1, it is characterized in that: step 1) in, fluorescent latex particles fluorescent probe microballoon diluted 2～6 times will be mixed, width evenly it is sprayed on as, on the glass fibre of 0.7～1.3cm, after dry 2～24h, obtained probe is fixing at 30～55 DEG C pads taking the speed of 2～6ul/cm.
7. the fluorescence immune chromatography detection method of serum amyloid A protein according to claim 1, it is characterized in that: described fluorescent probe microballoon diluent is the damping fluid containing sucrose, bovine serum albumin and tensio-active agent, described damping fluid comprises one or more in PBS, Tris damping fluid, MES damping fluid, HEPES damping fluid, and described tensio-active agent is polysorbas20 or tween 80.
8. the fluorescence immune chromatography detection method of serum amyloid A protein according to claim 1, it is characterized in that: step 3) in, described damping fluid comprises one or more in PBS, Tris damping fluid, glycine buffer, MES damping fluid, HEPES damping fluid, described tensio-active agent is polysorbas20, tween 80, triton x-100 or polyoxyethylene glycol, described sanitas is sodium azide or proclin, and described dispersion agent is polyvinylpyrrolidone 10K, PVP K or polyvinyl alcohol.
9. the fluorescence immune chromatography detection kit of a serum amyloid A protein, comprise kit box and diluent bottle, described kit box comprises immunochromatographydetection detection card, described immunochromatographydetection detection card comprises PVC liner plate and the sample pad being fixed on PVC liner plate, the fixing pad of probe, nitrocellulose filter and thieving paper, it is characterised in that:
Described sample pad is overlapped on the fixing pad of probe, the fixing pad of described probe and thieving paper are overlapped on described nitrocellulose filter both sides respectively, described nitrocellulose filter is provided with detection line and control line, it is coated with serum amyloid A protein monoclonal antibody in described detection line, in described control line, it is coated with goat anti-rabbit igg; Described probe fixes that the mixing fluorescent latex particles that be made up of serum amyloid A protein monoclonal antibody fluorescent latex particles and goat anti-rabbit igg fluorescent latex particles of pad is dry to be obtained.
10. the fluorescence immune chromatography detection kit of serum amyloid A protein according to claim 9, it is characterized in that: described immunochromatographydetection detection card also comprises housing, described housing is provided with well, detection window, coding region and handle, described well is positioned at described sample pad place, described detection window is positioned at described detection line and control line place, described coding region is between detection window and handle, and described handle is positioned on described housing to be positioned at the side near thieving paper.
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