CN105445466A - Detection method for interleukin 6 and reagent kit of detection method - Google Patents
Detection method for interleukin 6 and reagent kit of detection method Download PDFInfo
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Abstract
The invention discloses a detection method for interleukin 6 and a reagent kit of the detection method. The detection method comprises the steps that fluorescent latex particles are coupled with albumin and then coupled with an interleukin 6 monoclonal antibody, and a probe pad is obtained; a sample pad, the probe pad, a nitrocellulose membrane and absorbent paper are attached to a PVC lining plate in sequence, the interleukin 6 monoclonal antibody and rabbit IgG are wrapped on a detection line and a control line of the nitrocellulose membrane respectively, immunochromatographic test paper strips are obtained after drying and cutting are carried out, and thus the reagent kit for interleukin 6 detection is obtained. The detection method for interleukin 6 and the reagent kit of the detection method have the advantages of being high in sensitivity and accuracy, simple in operation, low in cost and the like.
Description
Technical field
The invention belongs to hormone test technical field, particularly relate to a kind of detection method and kit thereof of interleukin 6.
Background technology
Interleukin-6, is called for short interleukin 6 (IL-6), is a kind of cell factor, belongs to the one of interleukins.It produced by fibroblast, Monocytes/Macrophages, T lymphocyte, bone-marrow-derived lymphocyte, epithelial cell, horn cell and multiple oncocyte.IL-1, TNF-a, PDGF, virus infections, double-stranded RNA and cAMP etc., all can induce normal cell to produce interleukin 6.Interleukin 6 can stimulate and participates in immunoreactive cell proliferation, differentiation improve its function.Interleukin 6 (IL-6) is the most important instrumentality that liver cell produces acute phase protein, the protein of to be molecular weight under native state be 23-30KD, have glycosylation in various degree and multimerization, this inhomogeneity and vigor and tissue specificity have certain relation.Except macrophage, IL-6 can by other emiocytosises many, as fibroblast, endothelial cell etc.People IL-6 and C reactive protein (one of Acute radiation reaction), serum amylase A, fibrinogen, myosin, Copper slag connect each other.IL-6 also has wide application outward liver, and at liver, the effect of IL-6 is by being combined with the specific cell surface receptor of its high-affinity.IL-6 acceptor (IL-6R) belongs to immunoglobulin superfamily, and in IL-6R, only the part of a 80KD is required by being combined with II-6, and ligand/receptor compound and membrane glycoprotein (gp130) combine, and participates in the information transduction of growth signals.IL-6 occurs in rna transcription level to the molecular effect that Hepatocyte matter is synthesized.Sternzellen is the main source of IL-6 in liver.Bacteria lipopolysaccharide is its effective stimulant.In chronic liver disease, IL-6 level rises, and can cause a series of immunologic function disorder and immunopathogenesis reaction, as mentioned above, IL-6 can be described as hepatocyte-stimulating factor again, various kinds of cell can be promoted to breed and differentiation, and by white secretion feedback loop antiproliferative effect.
At present, the detection of domestic interleukin 6 mainly adopts import reagent and SABC reagent, kit many employings chemiluminescence method of domestic-developed and euzymelinked immunosorbent assay (ELISA), and these method detecting steps are many, and the influence factor of introduction is more, and testing result easily causes a deviation.
Fluorescent chromatographic immune analysis method is a kind of microanalysis method, according to fluorescence intensity, carries out qualitative or quantitative test to material, have high sensitivity, selectivity strong, need the advantages such as sample amount is few and easy and simple to handle.Chinese patent publication No. is the patent of invention of CN104330551A, disclose the kit that a kind of immunofluorescence based on chemoluminescence method quantitatively detects interleukin 6, anti-reagent is prepared with the interleukin 6 labelled antibody of the interleukin 6 coated antibody of marked by fluorescein isothiocyanate and alkali phosphatase enzyme mark, magnetic particle reagent is obtained with anti-fluorescein isothiocynate antibody coupling carboxyl magnetic bead, two sandwich method is in conjunction with interleukin 6, with new chemical luminous substrate ALPS for substrate, detect luminous intensity with Chemiluminescence Apparatus and carry out quantitatively.But the preparation process of this kit is more complicated, need cost higher, need the process such as water-bath, separation during detection, operation is many, length consuming time.Chinese patent publication No. is the patent of invention of CN103969438A, adopts quantitative sandwich enzyme-linked immunoassay method to detect interleukin 6.But the method detecting step have the bag of ELISA Plate by and close, the process of standard items or tested serum, monoclonal antibody detect, in testing process brooding time long, wash plate often, need support equipment and professional.
Summary of the invention
The object of this invention is to provide a kind of detection method and kit thereof of interleukin 6, have highly sensitive, accuracy is high, simple to operate, low cost and other advantages.
The present invention is achieved through the following technical solutions above-mentioned purpose:
A detection method for interleukin 6, comprises the following steps:
1) preparation of probe pads: add in fluorescent latex particles solution activator reaction, centrifugal, washing after obtain activate fluorescent latex particles;
By albumin and goat anti-rabbit igg respectively with the fluorescent latex particles coupling reaction of activation, obtain albumin fluorescent latex particles and goat anti-rabbit igg fluorescent latex particles; Albumin fluorescent latex particles solution is added activator reaction, centrifugal, washing after obtain activate albumin fluorescent latex particles;
By the albumin fluorescent latex particles coupling reaction of interleukin 6 monoclonal antibody and activation, obtain interleukin 6 monoclonal antibody fluorescent latex particles;
Be mixed to get with goat anti-rabbit igg fluorescent latex particles by interleukin 6 monoclonal antibody fluorescent latex particles and mix fluorescent latex particles, use fluorescence probe microballoon diluted subsequently, evenly spray on the glass fibers, dries and obtain probe pads;
2) preparation of immuno-chromatographic test paper strip: sample pad, probe pads, nitrocellulose filter, thieving paper are attached to successively on PVC liner plate, on the detection line that interleukin 6 monoclonal antibody and rabbit igg are coated on nitrocellulose filter respectively and control line, after drying, cutting, obtain immuno-chromatographic test paper strip;
3) preparation of sample diluting liquid: sample diluting liquid is the damping fluid containing albumin, surfactant, spreading agent and antiseptic;
4) sample survey: get serum to be detected, blood plasma or whole blood sample and be added in sample diluting liquid, to be mixed evenly after be added on immuno-chromatographic test paper strip and carry out immunochromatography reaction; Under fluorescence detector, adopt the wavelength of transmitted light corresponding with fluorescent latex particles to carry out fluoroscopic examination subsequently.
Further, described step 1) in, wavelength of transmitted light be 500nm ~ 590nm with the fluorescent latex particles solution of carboxyl or amino in add activator 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and N-hydroxy thiosuccinimide, 0.5h ~ 2h is reacted under room temperature, centrifugal, the fluorescent latex particles activated is obtained after washing, wherein, the weight ratio of 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and N-hydroxy thiosuccinimide is 1:6 ~ 1:1, the weight ratio of 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and fluorescent latex particles is 1:100 ~ 5:100, preferably, described step 1) in, wavelength of transmitted light be 500nm ~ 590nm with the fluorescent latex particles solution of carboxyl or amino in add activator 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and N-hydroxy thiosuccinimide, 1h is reacted under room temperature, centrifugal, the fluorescent latex particles activated is obtained after washing, wherein, the weight ratio of 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and N-hydroxy thiosuccinimide is 1:3, the weight ratio of 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and fluorescent latex particles is 1:100.
Further, described albumin comprises any one in human serum albumins, bovine serum albumin(BSA), sheep blood serum albumin and mouse serum albumin.
Further, described step 1) in, the blending ratio of the fluorescent latex particles of albumin and activation is 0.05:100 ~ 0.2:100, and after mixing, coupling reaction 1h ~ 5h under room temperature, obtains albumin fluorescent latex particles; The blending ratio of the fluorescent latex particles of goat anti-rabbit igg and activation is 0.05:100 ~ 0.2:100, and after mixing, coupling reaction 1h ~ 5h under room temperature, obtains goat anti-rabbit igg fluorescent latex particles; The blending ratio of the albumin fluorescent latex particles of interleukin 6 monoclonal antibody and activation is 0.05:100 ~ 0.2:100, and after mixing, coupling reaction 1h ~ 5h under room temperature, obtains interleukin 6 monoclonal antibody fluorescent latex particles; Preferably, described step 1) in, the blending ratio of the fluorescent latex particles of albumin and activation is 0.12:100, and after mixing, coupling reaction 3h under room temperature, obtains albumin fluorescent latex particles; The blending ratio of the fluorescent latex particles of goat anti-rabbit igg and activation is 0.12:100, and after mixing, coupling reaction 3h under room temperature, obtains goat anti-rabbit igg fluorescent latex particles; The blending ratio of the albumin fluorescent latex particles of interleukin 6 monoclonal antibody and activation is 0.12:100, and after mixing, coupling reaction 3h under room temperature, obtains interleukin 6 monoclonal antibody fluorescent latex particles.
Further, described step 1) in, in ratio mixing interleukin 6 monoclonal antibody fluorescent latex particles and the goat anti-rabbit igg fluorescent latex particles of 2:1 ~ 6:1, obtain mixing fluorescent latex particles, fluorescent latex particles fluorescence probe microballoon diluted 2 ~ 6 times will be mixed, evenly be sprayed on the glass fibre that width is 0.7cm ~ 1.3cm with the speed of 2 μ l/cm ~ 6 μ l/cm, at 37 DEG C, dry 2h ~ 6h obtains probe pads.
Further, described step 1) in, described fluorescence probe microballoon dilution is the damping fluid comprising sucrose, bovine serum albumin(BSA) and surfactant; Described damping fluid comprises one or more in PBS damping fluid, Tris damping fluid, MES damping fluid, HEPES damping fluid, and described surfactant is polysorbas20 or Tween 80.
Further, described step 3) in, described damping fluid is one or more in PBS damping fluid, Tris damping fluid, glycine buffer, MES damping fluid, HEPES damping fluid; Described surfactant is polysorbas20, Tween 80, triton x-100 or polyglycol; Described antiseptic is Sodium azide or procline; Described spreading agent is polyvinylpyrrolidone 10K, PVP K or polyvinyl alcohol (PVA).
Further, described step 2) in, sample pad, probe pads, nitrocellulose filter, thieving paper are attached to successively on PVC liner plate, adopt and draw on film metal spraying machine detection line that interleukin 6 monoclonal antibody and rabbit igg are coated on chromatographic test paper respectively and control line, after 37 DEG C of dry 12h ~ 36h, obtain the immuno-chromatographic test paper strip of 3mm ~ 5mm width through cutting cutter cutting; Preferably, sample pad, probe pads, nitrocellulose filter, thieving paper are attached to successively on PVC liner plate, adopt and draw on film metal spraying machine detection line that interleukin 6 monoclonal antibody and rabbit igg are coated on chromatographic test paper respectively and control line, after 37 DEG C of dry 24h, obtain the immuno-chromatographic test paper strip of 4mm width through cutting cutter cutting.
A kind of kit applying the detection method of above-mentioned interleukin 6, comprise kit box and dilution bottle, described kit box comprises PVC liner plate and is fixed on sample pad, probe pads, nitrocellulose filter and the thieving paper on described PVC liner plate, described sample pad is overlapped in described probe pads, described probe pads and described thieving paper are overlapped on the both sides of described nitrocellulose filter respectively, described nitrocellulose filter is provided with detection line and control line, in described detection line and control line, is coated with interleukin 6 monoclonal antibody and rabbit igg respectively; Sample diluting liquid has been deposited in described dilution bottle.
Further, also comprise 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 at described thieving paper place.
Compared with prior art, a kind of detection method of interleukin 6 of the present invention and the beneficial effect of kit thereof are:
1) the present invention utilizes fluorescence immune chromatography method, achieves the external qualitative and quantitative detection of interleukin 6;
2), in fluorescence immune chromatography method of the present invention, detection line region is coated with and can forms the interleukin 6 monoclonal antibody of compound with interleukin 6, and partner probe then uses the interleukin 6 monoclonal antibody of 2 kinds of fluorescent latex particles marks; Control line region is coated with rabbit igg antibody, partner probe uses 2 kinds of fluorescent latex particles of goat anti-rabbit igg antibody mark, control line and detection line independent reaction, be independent of each other and disturb, probe is being dispersed in the quality in mixing material to utilize control line signal accurately to judge, is used for correct detection line signal;
3) in the present invention, fluorescent latex particles first carries out coupling with albumin, and then carry out coupling with interleukin 6 monoclonal antibody, obtain interleukin 6 monoclonal antibody fluorescent latex particles, decrease the space resistance of double fastener core structure in testing process, increase sensitivity, the interleukin 6 of lower concentration can be detected;
4) probe is placed on the end of sample pad by the present invention, and probe pads and sample pad are made of one formula, is conducive to strengthening sample pad to the processing power of serum, blood plasma and whole blood, reduces the detection difference between them;
5) the interleukin 6 detection kit prepared of the present invention, detection sensitivity is high, can detect the interleukin 6 of 0.05ng/ml in serum sample; Detecting instrument is simple, simple to operate, without the need to professional operator; Detect fast, within 10 ~ 20 minutes, can testing result be obtained; Kit containment is convenient.
Accompanying drawing explanation
Fig. 1 is the structural representation of immuno-chromatographic test paper strip of the present invention;
Fig. 2 is the structural representation of kit of the present invention.
Embodiment
The detection method of a kind of interleukin 6 of the present invention, comprises the following steps:
1) probe pads preparation:
Be that the fluorescent latex particles solution with carboxyl or amino of 500nm ~ 590nm adds activator 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) and N-hydroxy thiosuccinimide (Sulfo-NHS) by wavelength of transmitted light, 0.5h ~ 1h is reacted under room temperature, wherein, the weight ratio of 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and N-hydroxy thiosuccinimide is 1:6 ~ 1:1, 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) is 1:100 ~ 5:100 with the weight ratio of fluorescent latex particles, centrifugal, the fluorescent latex particles activated is obtained after washing,
Should be noted that: a) wavelength of transmitted light is that 500nm ~ 590nm fluorescent latex particles is easily obtaining on the market, thus ensure accuracy of detection prerequisite under, economy and applicability higher; B) 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 limited extent that the activation rate of present latex particulate increases with EDC amount and increases, cost raises, and optimal proportion is 1:100; C) weight ratio of EDC and Sulfo-NHS is greater than 1:1, and the present latex particulate facile hydrolysis of activation, reduces activation efficiency; The weight ratio of EDC and Sulfo-NHS is less than 1:6, and the activation rate of present latex particulate reaches capacity, cost increase, and optimal proportion is 1:3; D) 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.
Mixed by the 0.05:100 ~ 0.2:100 in proportion of albumin with the fluorescent latex particles of activation, coupling reaction 1h ~ 5h under room temperature, obtains albumin fluorescent latex particles; By the fluorescent latex particles of goat anti-rabbit igg and activation in proportion 0.05:100 ~ 0.2:100 mix, coupling reaction 1h ~ 5h under room temperature, obtains goat anti-rabbit igg fluorescent latex particles; Albumin fluorescent latex particles solution is added activator reaction, centrifugal, washing after obtain activate albumin fluorescent latex particles; The blending ratio of the albumin fluorescent latex particles of interleukin 6 monoclonal antibody and activation is 0.05:100 ~ 0.2:100, and after mixing, coupling reaction 1h ~ 5h under room temperature, obtains interleukin 6 monoclonal antibody fluorescent latex particles.
Should be noted that: when a) weight ratio of antibody and fluorescent latex particles is less than 0.05:100, coupling efficiency is high, but waste present latex particulate, the non-binding antibody of major part activation point of present latex particulate; When being greater than 0.2:100, the most activation point of present latex particulate is combined, and the amount continuing to increase antibody has no significant effect coupling efficiency, waste antibody, and optimal proportion is 0.12:100; B) reaction time 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.
In ratio mixing interleukin 6 monoclonal antibody fluorescent latex particles and the goat anti-rabbit igg fluorescent latex particles of 2:1 ~ 6:1, obtain mixing fluorescent latex particles, fluorescent latex particles fluorescence probe microballoon diluted 2 ~ 6 times will be mixed, evenly be sprayed on the glass fibre that width is 0.7cm ~ 1.3cm with the speed of 2 μ l/cm ~ 6 μ l/cm, at 37 DEG C, dry 2h ~ 6h, obtains probe pads.Fluorescence probe microballoon dilution is the damping fluid containing sucrose, BSA and surfactant; Described damping fluid comprises one or more in PBS damping fluid, Tris damping fluid, MES damping fluid, HEPES damping fluid, and described surfactant is polysorbas20, Tween 80.
Should be noted that: a) probe is kept in sample diluting liquid and easily reunites, used fluorescence probe microballoon diluted to spray drying on the glass fibers and made probe pads, the component of fluorescence probe microballoon dilution can make probe dispersed, avoids the reunion between probe; Probe in probe pads, when slow releasing, can slow down immune response, and make the reaction time longer, detection signal is stronger.B) metal spraying speed is crossed and probe solution can be caused slowly very few, and disperse uneven on the glass fibers, metal spraying excessive velocities can cause glass fibre Premature saturation, and probe solution penetrates on metal spraying platform, causes experimental error, amplifies batch interpolation.C) drying time of probe pads is not easily long, also needs to continue drying after making immuno-chromatographic test paper strip, and this step only needs moisture fully dry.
2) immuno-chromatographic test paper strip preparation: sample pad, probe pads, nitrocellulose filter, thieving paper are attached to successively on PVC liner plate, adopt and draw on the film metal spraying machine detection line (T line) that interleukin 6 monoclonal antibody and rabbit igg is coated on respectively chromatographic test paper and control line (C line), after 37 DEG C of dry 12h ~ 36h, obtain the immuno-chromatographic test paper strip of 3mm ~ 5mm width through cutting cutter cutting;
Should be noted that: a) traditional quantitative immune chromatographic technique adopts bag by sheep anti-mouse antibody as control line, along with the increase of interleukin 6 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 p-wire is also without reference frame.The present invention adopts rabbit igg antibody and goat anti-rabbit antibody to match as control line, and the reaction of C line and T line is independently carried out, and without cross influence, C line and T line can be avoided to contend with one other probe particulate and the repeated deviation produced; B) be less than 12h drying time, the joint efficiency of antibody on nitrocellulose filter is low, and product detect signal can change with the prolongation of holding time, causes the detectability of interleukin 6 to raise and detect unstable; Drying time is greater than 36h, the easy inactivation of antibody, causes the sensitivity decrease detected, and best drying time is 24h; C), when the width of test strips is less than 3mm, the precision of cutting machine is comparatively large on the impact of test strips, and can reduce the repeatability of testing result, when the width of test strips is greater than 5mm, can increase the cost of test card, optimized test strips width is 4mm.
3) sample diluting liquid preparation: sample diluting liquid is the damping fluid containing bovine serum albumin(BSA), surfactant, spreading agent and antiseptic; Damping fluid comprises one or more in PBS damping fluid, Tris damping fluid, glycine buffer, MES damping fluid, HEPES damping fluid, surfactant is polysorbas20, Tween 80, triton x-100 or polyglycol, antiseptic is Sodium azide or procline, and spreading agent is polyvinylpyrrolidone 10K, PVP K or polyvinyl alcohol (PVA);
Should be noted that: a) bovine serum albumin(BSA) 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 and non-specific binding of probe on film, reduce false Yangxin number; B) surfactant can improve the water wettability of detected sample and probe, makes the combination on NC film of sample and probe and is more evenly distributed, and avoids basal signal fluctuation and the uneven impact on signal of immune response; C) antiseptic 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.
4) sample survey: get serum to be detected, blood plasma or whole blood sample and add in sample diluting liquid, to be mixed evenly after drop on immunochromatography reagent card and carry out immunochromatography reaction; Under fluorescence detector, adopt the wavelength of transmitted light corresponding with fluorescent latex particles to carry out fluoroscopic examination subsequently.Mixing material penetrates in sample pad, mixing material arrives probe pads after glass fiber filter impurity and process, interleukin 6 monoclonal antibody on interleukin 6 wherein and probe is combined into compound, compound penetrates on detection line along nitrocellulose filter, the new compound of double-antibody sandwich is formed with the interleukin 6 monoclonal antibody be fixed on detection line, interleukin 6 monoclonal antibody on detection line and the interleukin 6 monoclonal antibody in probe, its epi-position on interleukin 6 is different; Remaining antigen-fluorescently-labeled antibody complex, the unconjugated interleukin 6 monoclonal antibody being marked with fluorescent latex particles and the goat anti-rabbit igg being marked with fluorescent latex particles continue to penetrate into control line, and the rabbit igg that the goat anti-rabbit igg being marked with fluorescent latex particles can be fixed on control line is combined and forms antigen-antibody complex; Under fluorescence detector during fluoroscopic examination, only signal detected on the control line, could prove that testing result is effective.
As shown in Fig. 1 to 2, it is the interleukin 6 fluorescence immune chromatography detection kit prepared according to the detection method of interleukin 6 of the present invention, comprise kit box and dilution bottle, kit box comprises PVC liner plate 1 and is fixed on sample pad 3, probe pads 4, nitrocellulose filter 2 and the thieving paper 7 on PVC liner plate.
Sample pad 3 is overlapped in probe pads 4, probe pads 4 and thieving paper 7 are overlapped on the both sides of nitrocellulose filter 2 respectively, nitrocellulose filter 2 is provided with detection line 5 and control line 6, in detection line 5 and control line 6, is coated with interleukin 6 monoclonal antibody and rabbit igg respectively.Probe pads 4 is obtained through metal spraying drying by interleukin 6 monoclonal antibody fluorescent latex particles and goat anti-rabbit igg fluorescent latex particles.Sample diluting liquid has been deposited in dilution bottle.Kit also comprises housing 9, housing 9 is provided with well 8, detection window 10, code area 11 and handle 12, well 8 is positioned at sample pad 3 place, and detection window 10 is positioned at detection line 5 and control line 6 place, code area 11 is between detection window 10 and handle 12, and handle 12 is positioned at thieving paper 7 place.
During use, get serum to be detected, blood plasma or whole blood sample, join in well 8 after mixing; Under fluorescence detector, adopt the wavelength of transmitted light corresponding with fluorescent latex particles to carry out fluoroscopic examination subsequently.Mixing material penetrates in sample pad 3, probe pads 4 is arrived after glass fiber filter impurity and process, interleukin 6 monoclonal antibody in interleukin 6 wherein and probe pads 4 is combined into compound, compound penetrates on detection line 5 along nitrocellulose filter 2, the new compound of double-antibody sandwich is formed with the interleukin 6 monoclonal antibody be fixed on detection line 5, interleukin 6 monoclonal antibody on detection line 5 and the interleukin 6 monoclonal antibody in probe pads 4, its epi-position on interleukin 6 is different; Remaining antigen-fluorescently-labeled antibody complex, the unconjugated interleukin 6 monoclonal antibody being marked with fluorescent latex particles 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 fluorescence detector during fluoroscopic examination, only on control line 6, signal detected, could prove that testing result is effective.
Detection fluorescence detector of the present invention, comprises excitation-detection module, pre-amplifying module, control analysis module and software systems.The wherein light emitting diode of the light source of excitation-detection module to be emission wavelength be 400nm ~ 600nm, pre-amplifying module is a pre-amplification circuit.
Embodiment 1:
The fluorescence immune chromatography detection method of a kind of interleukin 6 of the present embodiment, comprises the following steps successively:
The first step: the preparation of probe pads
Getting 200 μ l wavelength of transmitted light is after fluorescent latex particles solution (containing carboxyl) the pH6.0MES buffer solution centrifuging three times of 590nm, precipitation pH6.0MES damping fluid dilution, after adding 5mgEDC and 15mgSulfo-NHS mixing, at room temperature reaction activation 30min, after centrifuging, precipitation continues to use pH6.5MES buffer solution three times, dilute with postprecipitation pH6.5MES damping fluid, add 100 μ g bovine serum albumin(BSA)s, 3h is reacted under room temperature, the ethanolamine solutions added containing bovine serum albumin(BSA) is closed, continue reaction 1h, centrifuged deposit pH7.4PBS buffer solution four times, obtain the bovine serum albumin(BSA) precipitation and the bovine serum albumin(BSA) fluorescent latex particles that are marked with fluorescent latex particles, in like manner can obtain the goat anti-rabbit igg precipitation and the goat anti-rabbit igg fluorescent latex particles that are marked with fluorescent latex particles, precipitation is resuspended in 200 μ lpH7.4PBS damping fluids, add 0.6 μ lproclin, preserve at 4 DEG C, obtain bovine serum albumin(BSA) activation coupling thymidine kinase 1 monoclonal antibody being marked with fluorescent latex particles to be 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.
By interleukin 6 monoclonal antibody fluorescent latex particles and goat anti-rabbit igg fluorescent latex particles by volume 2:1 mix, epoxy glue lactoconium mixes with the fluorescence probe microballoon dilution whirlpool of 2 times of volumes, obtain probe solution, evenly be sprayed on the glass fibre that width is 1cm with the speed of 4 μ l/cm, at 37 DEG C, dry 4h obtains probe pads.
Second step: the preparation of immunochromatographydetection detection card
Liner plate pastes nitrocellulose filter, probe pads, sample pad and thieving paper successively, and sample pad is overlapped in probe pads, and probe pads and thieving paper are overlapped on nitrocellulose filter, and are closely connected.Respectively interleukin 6 monoclonal antibody and rabbit igg coating buffer are mixed with respectively the antibody coating buffer of 1mg/ml and 1mg/ml.Interleukin 6 monoclonal antibody coating buffer and rabbit igg coating buffer are coated on detection line corresponding on nitrocellulose filter and control line with the linear speed of 1 μ l/cm, detection line and control line interval 6mm, under humidity <30% condition after 37 DEG C of oven dry 24h, make fluorescence immune chromatography test paper plate.
With cutting cutter, the fluorescence immune chromatography test paper plate prepared longitudinally is cut into the wide fluorescence immune chromatography test paper bar of 4mm, putting it into gets stuck interiorly obtains immunochromatographydetection detection card through case pressing machine process.
3rd step: the preparation of sample diluting liquid
Get the PBS damping fluid 1L of 0.02MpH7.0, add 8ml polysorbas20,5.1g bovine serum albumin(BSA), 3g polyvinylpyrrolidone 10K and 0.19g Sodium azide, ultrasonic until solid all dissolves, mixing.
4th step: sample detection
1) linearly, detectability and precision assessment:
Adopt negative cow's serum as dilution, interleukin 6 standard items are mixed with the standard solution that concentration is 10,9,8,7,6,5,4,3,2,1 and 0ng/ml; get standard items 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, after 15 minutes, adopt fluorescence detector to detect.Result shows, linear correlation coefficient r ^2 is greater than 0.99, and detect and be limited to 0.05ng/ml, the detection coefficient of variation of each concentration is all less than 8%.
2) checking of linear dimensions:
Adopt low value human serum and high level human serum, compound concentration is the interleukin 6 human serum solution of 10,8,6,4 and 2ng/ml, and each sample repeats 4 times, and result shows, r^2 is greater than 0.99, and highest detection scope can reach 10ng/ml.
3) assessment of accuracy
Adopt Interleukin-6 Concentration be 0.1ng/ml human serum sample based on sample, add the interleukin 6 reference material human serum of same volume variable concentrations, be mixed with the interleukin 6 human serum solution that concentration is 7,5,3 and 2ng/ml; Another increment originally adds the negative human serum of same volume, carries out 4 duplicate detection analyses, and calculate recovery sample and basic sample.Result shows, the recovery is within the scope of 90%-110%.
Embodiment 2:
The fluorescence immune chromatography detection method of a kind of interleukin 6 of the present embodiment, comprises the following steps successively:
The first step: the preparation of probe pads
Get fluorescent latex particles solution that 100 μ l wavelength of transmitted light are 500nm (containing amino) with after pH6.5MES buffer solution centrifuging three times, precipitation pH6.5MES damping fluid dilution, after adding 1mgEDC and 6mgSulfo-NHS mixing, at room temperature reaction activation 1h, after centrifuging, precipitation continues to use pH6.5MES buffer solution three times, dilute with postprecipitation pH6.5MES damping fluid, add 200 μ g bovine serum albumin(BSA)s, 5h is reacted under room temperature, the ethanolamine solutions added containing bovine serum albumin(BSA) is closed, continue reaction 1h, centrifuged deposit pH7.4PBS buffer solution four times, obtain the bovine serum albumin(BSA) precipitation and the bovine serum albumin(BSA) fluorescent latex particles that are marked with fluorescent latex particles, in like manner can obtain the goat anti-rabbit igg precipitation and the goat anti-rabbit igg fluorescent latex particles that are marked with fluorescent latex particles, precipitation is resuspended in 200 μ lpH7.4PBS damping fluids, add 0.6 μ lproclin, preserve at 4 DEG C, obtain bovine serum albumin(BSA) activation coupling thymidine kinase 1 monoclonal antibody being marked with fluorescent latex particles to be 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.
By interleukin 6 monoclonal antibody fluorescent latex particles and goat anti-rabbit igg fluorescent latex particles by volume 6:1 mix, epoxy glue lactoconium mixes with the fluorescence probe microballoon dilution whirlpool of 6 times of volumes, obtain probe solution, evenly be sprayed on the glass fibre that width is 1.3cm with the speed of 6 μ l/cm, at 37 DEG C, dry 6h obtains probe pads.
Second step: the preparation of immunochromatographydetection detection card
Liner plate pastes nitrocellulose filter, probe pads, sample pad and thieving paper successively, and sample pad is overlapped in probe pads, and probe pads and thieving paper are overlapped on nitrocellulose filter, and are closely connected.Respectively interleukin 6 monoclonal antibody and rabbit igg coating buffer are mixed with respectively the antibody coating buffer of 1mg/ml and 1mg/ml.Interleukin 6 monoclonal antibody coating buffer and rabbit igg coating buffer are coated on detection line corresponding on nitrocellulose filter and control line with the linear speed of 1 μ l/cm, detection line and control line interval 6mm, under humidity <30% condition after 37 DEG C of oven dry 36h, make fluorescence immune chromatography test paper plate.
With cutting cutter, the fluorescence immune chromatography test paper plate prepared longitudinally is cut into the wide fluorescence immune chromatography test paper bar of 5mm, putting it into gets stuck interiorly obtains immunochromatographydetection detection card through case pressing machine process.
3rd step: the preparation of sample diluting liquid
Get the PBS damping fluid 1L of 0.015MpH7.0, add 10ml Tween 80,5.0g bovine serum albumin(BSA), 3g PVP K and 300 μ lprocline, ultrasonic until solid all dissolves, mixing.
4th step: sample detection
1) linearly, detectability and precision assessment:
Adopt negative cow's serum as dilution, interleukin 6 standard items are mixed with the standard solution that concentration is 10,9,8,7,6,5,4,3,2,1 and 0ng/ml; get standard items 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, after 15 minutes, adopt fluorescence detector to detect.Result shows, linear correlation coefficient r ^2 is greater than 0.99, and detect and be limited to 0.05ng/ml, the detection coefficient of variation of each concentration is all less than 8%.
2) checking of linear dimensions:
Adopt low value human serum and high level human serum, compound concentration is the interleukin 6 human serum solution of 10,8,6,4 and 2ng/ml, and each sample repeats 4 times, and result shows, r^2 is greater than 0.99, and highest detection scope can reach 10ng/ml.
3) assessment of accuracy
Adopt Interleukin-6 Concentration be 0.1ng/ml human serum sample based on sample, add the interleukin 6 reference material human serum of same volume variable concentrations, be mixed with the interleukin 6 human serum solution that concentration is 7,5,3 and 2ng/ml; Another increment originally adds the negative human serum of same volume, carries out 4 duplicate detection analyses, and calculate recovery sample and basic sample.Result shows, the recovery is within the scope of 90%-110%.
Embodiment 3:
The fluorescence immune chromatography detection method of a kind of interleukin 6 of the present embodiment, comprises the following steps successively:
The first step: the preparation of probe pads
Getting 300 μ l wavelength of transmitted light is after fluorescent latex particles solution (containing carboxyl) the pH6.0MES buffer solution centrifuging three times of 540nm, precipitation pH6.0MES damping fluid dilution, after adding 15mgEDC and 15mgSulfo-NHS mixing, at room temperature reaction activation 45min, after centrifuging, precipitation continues to use pH6.5MES buffer solution three times, dilute with postprecipitation pH6.5MES damping fluid, add 150 μ g bovine serum albumin(BSA)s, 1h is reacted under room temperature, the ethanolamine solutions added containing bovine serum albumin(BSA) is closed, continue reaction 1h, centrifuged deposit pH7.4PBS buffer solution four times, obtain the bovine serum albumin(BSA) precipitation and the bovine serum albumin(BSA) fluorescent latex particles that are marked with fluorescent latex particles, in like manner can obtain the goat anti-rabbit igg precipitation and the goat anti-rabbit igg fluorescent latex particles that are marked with fluorescent latex particles, precipitation is resuspended in 200 μ lpH7.4PBS damping fluids, add 0.6 μ lproclin, preserve at 4 DEG C, obtain bovine serum albumin(BSA) activation coupling thymidine kinase 1 monoclonal antibody being marked with fluorescent latex particles to be 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.
By interleukin 6 monoclonal antibody fluorescent latex particles and goat anti-rabbit igg fluorescent latex particles by volume 4:1 mix, epoxy glue lactoconium mixes with the fluorescence probe microballoon dilution whirlpool of 4 times of volumes, obtain probe solution, evenly be sprayed on the glass fibre that width is 0.7cm with the speed of 2 μ l/cm, at 37 DEG C, dry 2h obtains probe pads.
Second step: the preparation of immunochromatographydetection detection card
Liner plate pastes nitrocellulose filter, probe pads, sample pad and thieving paper successively, and sample pad is overlapped in probe pads, and probe pads and thieving paper are overlapped on nitrocellulose filter, and are closely connected.Respectively interleukin 6 monoclonal antibody and rabbit igg coating buffer are mixed with respectively the antibody coating buffer of 1mg/ml and 1mg/ml.Interleukin 6 monoclonal antibody coating buffer and rabbit igg coating buffer are coated on detection line corresponding on nitrocellulose filter and control line with the linear speed of 1 μ l/cm, detection line and control line interval 6mm, under humidity <30% condition after 37 DEG C of oven dry 12h, make fluorescence immune chromatography test paper plate.
With cutting cutter, the fluorescence immune chromatography test paper plate prepared longitudinally is cut into the wide fluorescence immune chromatography test paper bar of 3mm, putting it into gets stuck interiorly obtains immunochromatographydetection detection card through case pressing machine process.
3rd step: the preparation of sample diluting liquid
Get the PBS damping fluid 1L of 0.01MpH7.2, add 5ml polysorbas20,4.9g bovine serum albumin(BSA), 3.1g polyvinyl alcohol (PVA) and 0.2g Sodium azide, ultrasonic until solid all dissolves, mixing.
4th step: sample detection
1) linearly, detectability and precision assessment:
Adopt negative cow's serum as dilution, interleukin 6 standard items are mixed with the standard solution that concentration is 10,9,8,7,6,5,4,3,2,1 and 0ng/ml; get standard items 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, after 15 minutes, adopt fluorescence detector to detect.Result shows, linear correlation coefficient r ^2 is greater than 0.99, and detect and be limited to 0.05ng/ml, the detection coefficient of variation of each concentration is all less than 8%.
2) checking of linear dimensions:
Adopt low value human serum and high level human serum, compound concentration is the interleukin 6 human serum solution of 10,8,6,4 and 2ng/ml, and each sample repeats 4 times, and result shows, r^2 is greater than 0.99, and highest detection scope can reach 10ng/ml.
3) assessment of accuracy
Adopt Interleukin-6 Concentration be 0.1ng/ml human serum sample based on sample, add the interleukin 6 reference material human serum of same volume variable concentrations, be mixed with the interleukin 6 human serum solution that concentration is 7,5,3 and 2ng/ml; Another increment originally adds the negative human serum of same volume, carries out 4 duplicate detection analyses, and calculate recovery sample and basic sample.Result shows, the recovery is within the scope of 90%-110%.
Above-described is only some embodiments of the present invention.For the person of ordinary skill of the art, without departing from the concept of the premise of the invention, can also make some distortion and improvement, these all belong to protection scope of the present invention.
Claims (10)
1. a detection method for interleukin 6, is characterized in that: comprise the following steps:
1) preparation of probe pads: add in fluorescent latex particles solution activator reaction, centrifugal, washing after obtain activate fluorescent latex particles;
By albumin and goat anti-rabbit igg respectively with the fluorescent latex particles coupling reaction of activation, obtain albumin fluorescent latex particles and goat anti-rabbit igg fluorescent latex particles; Albumin fluorescent latex particles solution is added activator reaction, centrifugal, washing after obtain activate albumin fluorescent latex particles;
By the albumin fluorescent latex particles coupling reaction of interleukin 6 monoclonal antibody and activation, obtain interleukin 6 monoclonal antibody fluorescent latex particles;
Be mixed to get with goat anti-rabbit igg fluorescent latex particles by interleukin 6 monoclonal antibody fluorescent latex particles and mix fluorescent latex particles, use fluorescence probe microballoon diluted subsequently, evenly spray on the glass fibers, dries and obtain probe pads;
2) preparation of immuno-chromatographic test paper strip: sample pad, probe pads, nitrocellulose filter, thieving paper are attached to successively on PVC liner plate, on the detection line that interleukin 6 monoclonal antibody and rabbit igg are coated on nitrocellulose filter respectively and control line, after drying, cutting, obtain immuno-chromatographic test paper strip;
3) preparation of sample diluting liquid: sample diluting liquid is the damping fluid containing albumin, surfactant, spreading agent and antiseptic;
4) sample survey: get serum to be detected, blood plasma or whole blood sample and be added in sample diluting liquid, to be mixed evenly after be added on immuno-chromatographic test paper strip and carry out immunochromatography reaction; Under fluorescence detector, adopt the wavelength of transmitted light corresponding with fluorescent latex particles to carry out fluoroscopic examination subsequently.
2. the detection method of a kind of interleukin 6 according to claim 1, it is characterized in that: described step 1) in, wavelength of transmitted light be 500nm ~ 590nm with the fluorescent latex particles solution of carboxyl or amino in add activator 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and N-hydroxy thiosuccinimide, 0.5h ~ 2h is reacted under room temperature, centrifugal, the fluorescent latex particles activated is obtained after washing, wherein, the weight ratio of 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and N-hydroxy thiosuccinimide is 1:6 ~ 1:1, the weight ratio of 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and fluorescent latex particles is 1:100 ~ 5:100, preferably, described step 1) in, wavelength of transmitted light be 500nm ~ 590nm with the fluorescent latex particles solution of carboxyl or amino in add activator 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and N-hydroxy thiosuccinimide, 1h is reacted under room temperature, centrifugal, the fluorescent latex particles activated is obtained after washing, wherein, the weight ratio of 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and N-hydroxy thiosuccinimide is 1:3, the weight ratio of 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and fluorescent latex particles is 1:100.
3. the detection method of a kind of interleukin 6 according to claim 1, is characterized in that: described albumin comprise in human serum albumins, bovine serum albumin(BSA), sheep blood serum albumin and mouse serum albumin any one.
4. the detection method of a kind of interleukin 6 according to claim 1, it is characterized in that: described step 1) in, the blending ratio of the fluorescent latex particles of albumin and activation is 0.05:100 ~ 0.2:100, and after mixing, coupling reaction 1h ~ 5h under room temperature, obtains albumin fluorescent latex particles; The blending ratio of the fluorescent latex particles of goat anti-rabbit igg and activation is 0.05:100 ~ 0.2:100, and after mixing, coupling reaction 1h ~ 5h under room temperature, obtains goat anti-rabbit igg fluorescent latex particles; The blending ratio of the albumin fluorescent latex particles of interleukin 6 monoclonal antibody and activation is 0.05:100 ~ 0.2:100, and after mixing, coupling reaction 1h ~ 5h under room temperature, obtains interleukin 6 monoclonal antibody fluorescent latex particles; Preferably, described step 1) in, the blending ratio of the fluorescent latex particles of albumin and activation is 0.12:100, and after mixing, coupling reaction 3h under room temperature, obtains albumin fluorescent latex particles; The blending ratio of the fluorescent latex particles of goat anti-rabbit igg and activation is 0.12:100, and after mixing, coupling reaction 3h under room temperature, obtains goat anti-rabbit igg fluorescent latex particles; The blending ratio of the albumin fluorescent latex particles of interleukin 6 monoclonal antibody and activation is 0.12:100, and after mixing, coupling reaction 3h under room temperature, obtains interleukin 6 monoclonal antibody fluorescent latex particles.
5. the detection method of a kind of interleukin 6 according to claim 1, it is characterized in that: described step 1) in, in ratio mixing interleukin 6 monoclonal antibody fluorescent latex particles and the goat anti-rabbit igg fluorescent latex particles of 2:1 ~ 6:1, obtain mixing fluorescent latex particles, fluorescent latex particles fluorescence probe microballoon diluted 2 ~ 6 times will be mixed, evenly be sprayed on the glass fibre that width is 0.7cm ~ 1.3cm with the speed of 2 μ l/cm ~ 6 μ l/cm, at 37 DEG C, dry 2h ~ 6h obtains probe pads.
6. the detection method of a kind of interleukin 6 according to claim 1, is characterized in that: described step 1) in, described fluorescence probe microballoon dilution is the damping fluid comprising sucrose, bovine serum albumin(BSA) and surfactant; Described damping fluid comprises one or more in PBS damping fluid, Tris damping fluid, MES damping fluid, HEPES damping fluid, and described surfactant is polysorbas20 or Tween 80.
7. the detection method of a kind of interleukin 6 according to claim 1, is characterized in that: described step 3) in, described damping fluid is one or more in PBS damping fluid, Tris damping fluid, glycine buffer, MES damping fluid, HEPES damping fluid; Described surfactant is polysorbas20, Tween 80, triton x-100 or polyglycol; Described antiseptic is Sodium azide or procline; Described spreading agent is polyvinylpyrrolidone 10K, PVP K or polyvinyl alcohol (PVA).
8. the detection method of a kind of interleukin 6 according to claim 1, it is characterized in that: described step 2) in, sample pad, probe pads, nitrocellulose filter, thieving paper are attached to successively on PVC liner plate, adopt and draw on film metal spraying machine detection line that interleukin 6 monoclonal antibody and rabbit igg are coated on chromatographic test paper respectively and control line, after 37 DEG C of dry 12h ~ 36h, obtain the immuno-chromatographic test paper strip of 3mm ~ 5mm width through cutting cutter cutting; Preferably, sample pad, probe pads, nitrocellulose filter, thieving paper are attached to successively on PVC liner plate, adopt and draw on film metal spraying machine detection line that interleukin 6 monoclonal antibody and rabbit igg are coated on chromatographic test paper respectively and control line, after 37 DEG C of dry 24h, obtain the immuno-chromatographic test paper strip of 4mm width through cutting cutter cutting.
9. the kit of the detection method of the interleukin 6 of an application rights requirement described in 1, it is characterized in that: comprise kit box and dilution bottle, described kit box comprises PVC liner plate and is fixed on the sample pad on described PVC liner plate, probe pads, nitrocellulose filter and thieving paper, described sample pad is overlapped in described probe pads, described probe pads and described thieving paper are overlapped on the both sides of described nitrocellulose filter respectively, described nitrocellulose filter is provided with detection line and control line, interleukin 6 monoclonal antibody and rabbit igg is coated with respectively in described detection line and control line, sample diluting liquid has been deposited in described dilution bottle.
10. kit according to claim 9, it is characterized in that: also comprise 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 at described thieving paper place.
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