CN105675867A - Fluorescence immunochromatographic assay method of thymidine kinase 1 and kit - Google Patents
Fluorescence immunochromatographic assay method of thymidine kinase 1 and kit Download PDFInfo
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- CN105675867A CN105675867A CN201511020024.4A CN201511020024A CN105675867A CN 105675867 A CN105675867 A CN 105675867A CN 201511020024 A CN201511020024 A CN 201511020024A CN 105675867 A CN105675867 A CN 105675867A
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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Abstract
The invention relates to a fluorescence immunochromatographic assay method of thymidine kinase 1 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 thymidine kinase 1 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 thymidine kinase 1 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 thymidine kinase 1 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 thymidine kinase 1 and test kit, particularly relate to fluorescence immune chromatography detection method and the detection kit thereof of thymidine kinase 1.
Background technology
Thymidine kinase 1, also known as TK1, is that cancerous tumor cell DNA synthesizes required precursor, is the specific enzymes of regulating cell synthesis, and its content and cellular abnormality propagation are closely related. Tumour is the chronic disease of a kind of cellular abnormality propagation, it is hyperplasia and the abnormal cancer cells being differentiated to form under such environmental effects of the normal cell in body, cancer cells is on the increase and is just defined solid tumor, but this process is very very long, often just can be detected by test sets such as CT after reaching certain size by a few year. At the cell carcinogenesis initial stage, TK1 just can detect that these change exactly, and then just carried out Risk-warning before forming solid tumor. The detection mode of TK1 extracts blood of human body to carry out detection and draw corresponding TK1 value, assesses the risk suffering from tumour according to TK1 value.
TK1 catalysis thymidine (TdR) is 1-phosphoric acid thymidylic acid (TMP), is the cell cycle rely on enzyme, is mainly present in tenuigenin. When a large amount of proliferative cell occurs in body, the level of TK-1 raises rapidly. Once generation canceration, with the sharply propagation of tumour cell, in body, the activity of TK-1 and content all will raise, and can exceed 20～100 times of normal level. TK1 is internationally recognized cellular abnormality proliferation marker, the propagation activity information of cancer cells in the level of thymidine kinase 1 (STK1), dynamic knowledge body in detection serum, the result for the treatment of of assessment tumour are had vital role by it, can more early indicate tumor recurrence risk. Therefore the detection of TK1 can be used for assessment surgical effect, monitoring chemotherapy effect, predicting recurrence risk, judges tumor prognosis etc. TK1 test kit many employings enzyme immunization method of current home and abroad exploitation, enzyme immunization method detection complicated operation, manual operation easily causes resultant error, and machine operation needs expensive biochemical instrument, and detection time is long, and when detecting a small amount of sample, cost is higher.
Prior art fluorescent chromatographic immune analysis method is a kind of trace analysis method, is that immunofluorescence technique and tradition immunochromatography technique combine a kind of quantitative novel detection technique of development innovation. This technology is retaining outside the advantage that colloidal gold immunochromatographimethod technological operation is easy, detection is quick, portability is strong, the accurate quantification of detected result is also achieved by fluorescent tracing enhancement techniques, convenient material is carried out qualitative or quantitative analysis, have highly sensitive, selectivity strong, need the advantages such as sample amount is few and easy and simple to handle. Its principle is a certain zone that special antibody is first fixed on nitrocellulose filter, after sample is immersed in nitrocellulose one end of this drying, due to capillary action, sample will move forward along this film, when moving to the region being fixed with antibody, in sample corresponding antigen namely with this antibody generation specific binding, adopt immune colloid gold etc. that this region can be made to show certain color, thus realize the immunodiagnosis of specificity.
Chinese patent CN101231288B discloses the test kit of a kind of fluorescence immunoassay detection by quantitative TK1 based on magnetic nanometer particles, by TK1 being fixed on the superparamagnetic nano particle fixation support surface of amino silicone alkanisation, adopt competition law, it take horseradish peroxidase as enzyme labelling, para hydroxybenzene propionic acid it is fluorogenic substrate, it is achieved the detection of TK1. But the immune response time that the method needs when detecting is longer, the first TK1 monoclonal antibody reactive with enzyme labelling of TK1 to be measured, add the magnetic microsphere generation competing reaction of TK1 bag quilt again, it is separated under magnetic field after washing, finally add fluorogenic substrate to carry out developing the color and measure, the required step of detection is longer, and process is 1.5～3h about.
Chinese patent CN101275954B discloses a kind of early warning chip utilizing immunochromatographic method simple determination TK1, by being coated with the fibre strip layer analysis solid phase carrier NC film of primary antibodie TK1 monoclonal antibody, filter sample paper, water adsorption glass fibrous paper, the polyester film of two anti-TK1 monoclonal antibody binding substancess that is marked with Radioactive colloidal gold be pasted onto PVC plastic tablet and form, the method detection is simple, but can not accurate quantitative TK1, clear and definite data can not be provided to TK1 in cancer treatment procedure monitoring.
Therefore, when how to develop detection, length, easy to operate, the accurate TK1 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 thymidine kinase 1 that simple to operate, cost is low and test kit.
The fluorescence immune chromatography detection method of a kind of thymidine kinase 1 of the present invention, comprises the following steps:
Step 1) probe fixing pad preparation: by thymidine kinase 1 monoclonal antibody thymidine kinase 1 monoclonal antibody fluorescent latex particles obtained with activation albumin fluorescent latex particles linked reaction in proportion, again described thymidine kinase 1 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 thymidine kinase 1 monoclonal antibody is coated on the detection line of chromatographic film, described 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 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 weight ratio of activation fluorescent latex particles, and described albumin fluorescent latex particles is described activation albumin fluorescent latex particles obtained with activator reaction further.
Further, the kind of described albumin comprises people, ox, sheep or mouse in the present invention.
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 mixed weight 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.
The present invention further, step 1) in, the mixed weight of described thymidine kinase 1 monoclonal antibody and activation fluorescent latex particles, can at room temperature linked reaction 1～5h after mixing than for 0.05:100～0.2:100; Ratio 2:1～6:1 that described thymidine kinase 1 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 thymidine kinase 1 of the present invention, comprise kit box and diluent bottle, described kit box comprises immunochromatographydetection detection card, 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, thymidine kinase 1 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 thymidine kinase 1 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 thymidine kinase 1, 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 thymidine kinase 1 monoclonal antibody of mixture with thymidine kinase 1, and partner probe then uses thymidine kinase 1 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 thymidine kinase 1 monoclonal antibody, reduce the space resistance of two sandwich structure in testing process so that it is the thymidine kinase 1 of lower concentration can be detected.
4. thymidine kinase 1 detection kit that prepared by the present invention, store convenient, detection sensitivity height, the thymidine kinase 1 that concentration in blood sample is low to moderate 15pg/ml can be detected, 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. thymidine kinase 1 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 immunochromatographydetection detection 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 thymidine kinase 1 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.
It should be noted that (1) the above-mentioned wavelength of transmitted light of 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 weight ratio 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 weight ratio 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. Thymidine kinase 1 monoclonal antibody is 0.05:100～0.2:100 with the mixed weight ratio of the albumin fluorescent latex particles of activation, linked reaction 1～5h under room temperature after mixing.
When should be noted that the weight ratio of (1) thymidine kinase 1 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 thymidine kinase 1 monoclonal antibody, wastage of material, optimum weight ratio 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 mixing thymidine kinase 1 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 thymidine kinase 1 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 thymidine kinase in serum 1 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 thymidine kinase 1 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, thymidine kinase 1 wherein is combined into mixture with thymidine kinase 1 monoclonal antibody on probe, mixture penetrates on detection line 5 along nitrocellulose filter 4, the new mixture of double-antibody sandwich is formed with thymidine kinase 1 monoclonal antibody being fixed on detection line 5, thymidine kinase 1 monoclonal antibody on detection line 5 and thymidine kinase 1 monoclonal antibody in probe, its table position on thymidine kinase 1 is different. The antibody complex of remaining antigen-fluorescent mark, the thymidine kinase 1 monoclonal antibody precipitation being marked with fluorescent latex particles not combined and thymidine kinase 1 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 thymidine kinase 1 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, thymidine kinase 1 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 thymidine kinase 1 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, thymidine kinase 1 wherein is combined into mixture with thymidine kinase 1 monoclonal antibody on probe, mixture penetrates on detection line 5 along nitrocellulose filter 4, the new mixture of double-antibody sandwich is formed with thymidine kinase 1 monoclonal antibody being fixed on detection line 5, thymidine kinase 1 monoclonal antibody on detection line 5 and thymidine kinase 1 monoclonal antibody in probe, its table position on thymidine kinase 1 is different. The antibody complex of remaining antigen-fluorescent mark, the thymidine kinase 1 monoclonal antibody precipitation being marked with fluorescent latex particles not combined and thymidine kinase 1 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 " thymidine kinase 1
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 650nm, 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, bovine serum albumin activation coupling thymidine kinase 1 monoclonal antibody being marked with fluorescent latex particles obtains being marked with the thymidine kinase 1 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 thymidine kinase 1 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 thymidine kinase 1 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 thymidine kinase 1 monoclonal antibody that fluorescent latex particles marks. Thymidine kinase 1 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, thymidine kinase 1 standard substance are mixed with the standard solution that concentration is 2000,1800,1600,1400,1200,1000,800,600,400,200 and 0pg/ml; 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 15pg/ml, 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 thymidine kinase 1 human serum solution of 2000,1600,1200,800 and 400pg/ml, and each sample repeats 4 times, and result shows, r^2 is greater than 0.99, and highest detection scope can reach 2000pg/ml.
3) assessment of accuracy:
Adopt thymidine kinase 1 concentration to be sample based on the human serum sample of 40pg/ml, add the thymidine kinase 1 standard substance human serum of same volume different concns, be mixed with the thymidine kinase 1 human serum solution that concentration is 1400,1000,600 and 500pg/ml. 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 thymidine kinase 1, it is characterised in that, comprise the following steps:
Step 1) probe fixing pad preparation: by thymidine kinase 1 monoclonal antibody thymidine kinase 1 monoclonal antibody fluorescent latex particles obtained with activation albumin fluorescent latex particles linked reaction in proportion, again described thymidine kinase 1 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 thymidine kinase 1 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 thymidine kinase 1 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 weight 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 thymidine kinase 1 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 mixed weight ratio of activation fluorescent latex particles.
4. the fluorescence immune chromatography detection method of thymidine kinase 1 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 thymidine kinase 1 according to claim 1, it is characterized in that: step 1) in, described thymidine kinase 1 monoclonal antibody is 0.05:100～0.2:100, ratio 2:1～6:1 that described thymidine kinase 1 monoclonal antibody fluorescent latex particles mixes mutually with goat anti-rabbit igg fluorescent latex particles with the mixed weight ratio of activation fluorescent latex particles.
6. the fluorescence immune chromatography detection method of thymidine kinase 1 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 thymidine kinase 1 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 thymidine kinase 1 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 thymidine kinase 1, 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 thymidine kinase 1 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 thymidine kinase 1 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 thymidine kinase 1 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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| CN112462069A (en) * | 2020-11-09 | 2021-03-09 | 海卫特(广州)医疗科技有限公司 | Fluorescence immunochromatography kit for detecting canine pancreatitis and preparation method thereof |
| CN112639475A (en) * | 2018-08-31 | 2021-04-09 | 豪夫迈·罗氏有限公司 | Thymidine kinase (TK-1) in prognostic index of DLBCL |
| CN114107339A (en) * | 2021-12-09 | 2022-03-01 | 菏泽学院 | Primer, gene, fusion protein, polyclonal antibody of phytoplasma thymidine kinase gene of Paulownia arbusculari and its application |
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| CN102161716A (en) * | 2010-12-30 | 2011-08-24 | 北京九强生物技术股份有限公司 | Method and reagent for latex sensitization |
| WO2013126517A1 (en) * | 2012-02-21 | 2013-08-29 | Northwestern University | Anti-mullerian hormone detection in whole blood |
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| CN101275954A (en) * | 2008-05-09 | 2008-10-01 | 丁克祥 | Breast cancer early warning chip for easily rapid measuring human I type thymidine kinase gene protein |
| CN102161716A (en) * | 2010-12-30 | 2011-08-24 | 北京九强生物技术股份有限公司 | Method and reagent for latex sensitization |
| WO2013126517A1 (en) * | 2012-02-21 | 2013-08-29 | Northwestern University | Anti-mullerian hormone detection in whole blood |
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| CN114107339A (en) * | 2021-12-09 | 2022-03-01 | 菏泽学院 | Primer, gene, fusion protein, polyclonal antibody of phytoplasma thymidine kinase gene of Paulownia arbusculari and its application |
| CN114107339B (en) * | 2021-12-09 | 2024-06-07 | 菏泽学院 | Primers, genes, fusion proteins, polyclonal antibodies and applications of thymidine kinase gene of Paulownia witches-broom phytoplasma |
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Application publication date: 20160615 |