CN105403704A - Fluorescence immunoassay detection method and kit for HER-2 - Google Patents
Fluorescence immunoassay detection method and kit for HER-2 Download PDFInfo
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Abstract
The invention relates to fluorescence immunoassay detection method and kit for HER-2. The method comprises the following steps: preparation of a probe fixing pad; preparation of an immunoassay test strip; preparation of sample diluent; and inspection of the sample. HER-2 monoclonal antibody fluorescence latex particles and goat anti-rabbit IgG fluorescence latex particles are mixed and then diluted by using gold diluent, and the diluted mixture is sprayed on glass fiber so as to prepare the probe fixing pad; and a detection line is coated by an HER-2 monoclonal antibody, and a control line is coated by goat anti-rabbit IgG so as to prepare the fluorescence immunoassay test strip. The detection kit provided by the invention comprises a PVC liner plate, a sample pad, the probe fixing pad, a cellulose nitrate membrane and absorbent paper. The probe fixing pad is prepared by mixing and drying the HER-2 monoclonal antibody fluorescence latex particles and the goat anti-rabbit IgG fluorescence latex particles. Thus, the fluorescence immunoassay detection method and kit have the advantages of high sensitivity, high accuracy, simple operation and low cost.
Description
Technical field
The present invention relates to detection method and the kit of a kind of HER-2, particularly relate to fluorescence immune chromatography detection method and the detection kit thereof of HER-2.
Background technology
HER-2 gene, ErbB-2 gene, also referred to as nen, C-erbB-2, Her-2/neu gene, its protein product of expressing is human epidermal growth factor receptor 2, and HER-2 gene is by expressing the effect of HER-2 protein exhibits.HER-2 proto-oncogene is positioned at No. 17 chromosome long arm, and the people such as Shih is with 3-MECA induction mouse NIH3T3 cultured cell system the earliest, and is detected in the neuroblastoma oncocyte transited out of.The mankind, 20% ~ 30% patient with breast cancer's cancer cell has overexpression, and its reason mainly HER-2 gene magnification (95%) or transcribe increases (5%).HER-2 is a member of EGF-R ELISA (EGFR) family, when can tyrosine kinase in active cell after extracellular respective segments is combined with HER-2, thus activate other a series of intracellular messengers pipeline, final signal arrives in core, causes regulating cell to copy and the gene that breaks up is transcribed.
The general less expression of normal human cell or appropriateness express HER-2 albumen, play the effect of regulation and control normal cell growth and growth.And in tumor tissues, HER-2 gene increases in a large number, cause HER-2 protein overexpression, cause cell proliferation rapidly, grow out of control, promote breast cancer progression.Analysis of clinical shows, early-stage breast cancer patient 10 years survival rates of HER-2 gene magnification or protein overexpression are significantly lower than the low expression group of HER-2, the median survival interval of the recurrent and metastatic breast cancer patient of HER-2 gene magnification or protein overexpression is only 3 years, and the median survival interval of HER-2 negative patient is 6-7.Therefore, HER-2 process LAN is one of index pointing out Prognosis in Breast Cancer poor, and the meaning of its overexpression in breast cancer and detection method thereof are the focuses that people pay close attention to always.
At present breakthrough is achieved to the research of HER-2 gene, 1987, the people such as Slamon first reported HER-2 amplification and clinical prognosis bad between remarkable relation, its conspicuousness higher than indexs such as estrogen (ER), progestational hormone (PR), and is confirmed in research afterwards in a large number.It is found that afterwards HER-2 high expressed patient with breast cancer to tamoxifen treatment, independent hormonotherapy and endoxan, methopterin, 5-fluor-uracil combined chemotherapy produce tolerance, and to PTX, doxorubicin become more responsive.In recent years, a kind of success of the monoclonal antibodies drug development for HER-2, and obtained U.S. food and FAD license for clinical treatment three primary breast cancer, it is only for the tumour cell generation effect of HER-2 high expressed, can effectively extending the survival of patients phase and its quality of life is improved, is the extremely promising therapy of one.Therefore, patient with breast cancer, with or without HER-2 high expressed, is detected it how objective and accurately, has very important meaning to the selection of patient treatment and prognosis.
The HER-2 detection method of current home and abroad exploitation has SABC or FISH to detect, and these method detecting steps are many, and testing process influence factor is more, and testing result easily causes a deviation.Prior art 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 CN104195246A, disclose a kind of HER-2 kit detected based on the quick FISH of HER-2 gene, by the principle of base pair complementarity, specific DNA sequence dna and the complementation of intracellular target sequence combine, probe is with fluorescence, under suitable exciting light irradiates, hybridization probe and target dna can be detected, adopt the short data records fluorescence probe optimized, required hybridization time is shortened, the detection time of FISH can be foreshortened to 1 ~ 2h.But the method detecting step have washing, degerming, hybridize, hatch, detection etc., testing process is complicated, and loaded down with trivial details, elapsed time is long, needs professional to operate, and particularly has strict demand to testing environment, is unfavorable for large batch of pattern detection.
Chinese patent publication No. is the patent of invention of CN104122394A, disclose a kind of enzyme-catalyzed chemical luminescence method and detect HER-2 quantitative determination reagent kit, the nano-magnetic microsphere of the monoclonal antibody complex containing mouse anti human HER-2 is adopted to fix HER-2, then be combined with the anti-HER-2 monoclonal antibody containing alkali phosphatase enzyme mark, by producing signal with the reaction of enzyme-catalyzed chemical luminescence substrate.The method needs different HER-2 antibody to be combined with nanometer magnetic bead and phosphatase, and cost is higher; Operation steps has fixing, the temperature of HER-2 to bathe, go supernatant, cleaning, chemiluminescence reaction and luminometer to detect, and complicated operation, easily brings error, needs professional, and detection time is longer.
Therefore, length when how to develop detection, easy to operate, the accurate HER-2 detection method of testing result and testing product become problem demanding prompt solution.
Summary of the invention
For solving the problems of the technologies described above, the object of this invention is to provide detection method and the kit of a kind of highly sensitive, accuracy is high, simple to operate, cost is low HER-2.
The fluorescence immune chromatography detection method of a kind of HER-2 of the present invention, comprises the following steps:
Step 1) preparation of probe fixed bolster: HER-2 monoclonal antibody is obtained HER-2 monoclonal antibody fluorescent latex particles with activation albumin fluorescent latex particles coupling reaction in proportion, again described HER-2 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 fluorescence probe microballoon diluted, spray obtained probe fixed bolster on the glass fibers;
Step 2) immuno-chromatographic test paper strip preparation: sample pad, probe fixed bolster, chromatographic film, thieving paper are attached on PVC liner plate successively, described HER-2 monoclonal antibody is coated on the detection line of chromatographic film, goat anti-rabbit igg is coated on chromatographic film control line, and then obtained immuno-chromatographic test paper strip, the preferred nitrocellulose filter of described chromatographic film;
Step 3) sample diluting liquid preparation: the potpourri including bovine serum albumin(BSA), surfactant, spreading agent and antiseptic is mixed obtained sample diluting liquid with damping fluid;
Step 4) sample survey: get blood serum sample and be mixed to described sample diluting liquid, add immuno-chromatographic test paper strip and carry out immunochromatography reaction, through fluorescence detector fluoroscopic examination after chromatography reaction terminates.
The present invention further, step 1) in, the preparation of described activation albumin fluorescent latex particles first fluorescent latex particles and activator is reacted obtained to activate fluorescent latex particles, again with albumin in proportion coupling reaction obtain albumin fluorescent latex particles, described albumin is 0.05:100 ~ 0.2:100 with the ratio of activation fluorescent latex particles, and described albumin fluorescent latex particles reacts obtained described activation albumin fluorescent latex particles further with activator.
Further, described albuminous kind comprises people, ox, sheep or mouse in the present invention.
The present invention further, step 1) in, the preparation of described goat anti-rabbit igg fluorescent latex particles is obtained with activation fluorescent latex particles coupling reaction in proportion by goat anti-rabbit igg, and described goat anti-rabbit igg is 0.05:100 ~ 0.2:100 with the blending ratio of activation fluorescent latex particles.
The present invention 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.At room temperature 0.5 ~ 1h can be reacted after three's mixing.
The present invention further, step 1) in, described HER-2 monoclonal antibody is 0.05:100 ~ 0.2:100 with the blending ratio of activation fluorescent latex particles, can at room temperature coupling reaction 1 ~ 5h after mixing; Ratio 2:1 ~ 6:1 that described HER-2 monoclonal antibody fluorescent latex particles mixes mutually with goat anti-rabbit igg fluorescent latex particles, coupling reaction 1 ~ 5h under room temperature after mixing.
The present invention further, step 1) in, fluorescent latex particles fluorescence probe microballoon diluted 2 ~ 6 times will be mixed, be evenly sprayed on the glass fibre that width is 0.7 ~ 1.3cm with the speed of 2 ~ 6ul/cm, obtained probe fixed bolster after dry 2 ~ 24h at 30 ~ 55 DEG C.
The present invention further, described fluorescence probe microballoon dilution is the damping fluid containing 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.
The present invention further, step 3) in, described damping fluid comprises 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 proclin, and described spreading agent is polyvinylpyrrolidone 10K, PVP K or polyvinyl alcohol (PVA).
On the other hand, the fluorescence immune chromatography detection kit of a kind of HER-2 of the present invention, comprise kit box and dilution bottle, described kit box comprises, the sample pad that described immunochromatographydetection detection card comprises PVC liner plate and is fixed on PVC liner plate, probe fixed bolster, nitrocellulose filter and thieving paper, described sample pad is overlapped on probe fixed bolster, described probe fixed bolster and thieving paper are overlapped on described nitrocellulose filter both sides respectively, described nitrocellulose filter is provided with detection line and control line, described detection line endoperidium has HER-2 monoclonal antibody, described control line endoperidium has goat anti-rabbit igg, described probe fixed bolster is obtained by the mixing fluorescent latex particles drying of HER-2 monoclonal antibody fluorescent latex particles and goat anti-rabbit igg fluorescent latex particles, in described dilution bottle, deposited sample diluting liquid.
The present invention 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 at the side described housing being positioned at close thieving paper.
By such scheme, the present invention at least has the following advantages:
1. adopt fluorescence immune chromatography method of the present invention, realize external highly sensitive, accuracy is high, simple to operate, cost the is low quantitative detection of HER-2.
2. fluorescence immune chromatography method of the present invention, detection line region adopts and can form the HER-2 monoclonal antibody of compound, the HER-2 monoclonal antibody that partner probe then uses fluorescent latex particles to mark with HER-2; 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.Probe is dispersed in the quality in mixing material to utilize control line signal accurately to judge, 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 HER-2 monoclonal antibody, reduces the space resistance of double fastener core structure in testing process, can detect the HER-2 of lower concentration.
4. the HER-2 detection kit prepared of the present invention, storage is convenient, detection sensitivity is high, concentration in blood sample can be detected and be low to moderate the HER-2 of 10pg/ml, 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, can testing result be obtained.
5. the HER-2 detection kit prepared of the present invention, due in advance to the special processing of sample pad and the improvement of sample diluting liquid, applied widely, can detect serum, blood plasma and whole blood.
Above-mentioned explanation is only the general introduction of technical solution of the present invention, in order to better understand technological means of the present invention, and can be implemented according to the content of instructions, coordinates accompanying drawing to be described in detail as follows below with preferred embodiment of the present invention.
Accompanying drawing explanation
Fig. 1 is the structural representation of immuno-chromatographic test paper strip in the present invention;
Fig. 2 is the structural representation of reagent card in the present invention.
In figure, the implication of each Reference numeral is as follows.
1PVC liner plate 2 sample pad
3 probe fixed bolster 4 nitrocellulose filters
5 detection line 6 control lines
7 thieving paper 8 wells
9 housing 10 detection windows
11 coding region 12 handles
Embodiment
Carry out clear, complete description to the technical scheme in the embodiment of the present invention below, obviously, 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 the every other embodiment obtained under creative work prerequisite, belong to the scope of protection of the invention.
The fluorescence immune chromatography detection method of HER-2 of the present invention, comprises the following steps:
Step 1) preparation of probe fixed bolster: be react 0.5 ~ 1h under the fluorescent latex particles solution with carboxyl or amino of 500 ~ 590nm adds activator 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) and N-hydroxy thiosuccinimide (Sulfo-NHS) room temperature by wavelength of transmitted light, 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, obtain after washing activating fluorescent latex particles.
Should be noted that the above-mentioned wavelength of transmitted light of (1) the present invention is that 500 ~ 590nm fluorescent latex is easily obtaining on the market, thus ensure accuracy of detection prerequisite under, economy and applicability 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 limited extent that the activation rate of present latex particulate increases with EDC amount and increases, cost raises, and optimal proportion is 1:100; (3) the weight ratio of EDC and Sulfo-NHS is less than 1:1, and the present latex particulate facile hydrolysis of activation, reduces activation efficiency; The weight ratio of EDC and Sulfo-NHS is greater than 6:1, and the activation rate of present latex particulate reaches capacity, cost increase, and optimal proportion is 1:3; (4) soak time is less than 0.5h, and the efficiency of activation is not high, waste starting material; Soak time is greater than 2h, and the speed of activation is less than the speed of activating substance hydrolysis, reduces activation efficiency, optimum activating time 1h.
In proportion 0.05:100 ~ the 0.2:100 of albumin with activation fluorescent latex particles mixes by the present invention, and coupling 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, coupling reaction 2 ~ 5h under room temperature, obtains goat anti-rabbit igg fluorescent latex particles.The blending ratio of the albumin fluorescent latex particles of HER-2 monoclonal antibody and activation is 0.05:100 ~ 0.2:100, coupling reaction 1 ~ 5h under room temperature after mixing.
When should be noted that the weight ratio of (1) HER-2 monoclonal 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, and when being greater than 0.2:100, the most activation point of present latex particulate is combined, the amount continuing to increase HER-2 monoclonal antibody has no significant effect coupling efficiency, wastage of material, optimal proportion is 0.12:100; (2) the 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.
The present invention mixes HER-2 monoclonal antibody fluorescent latex particles and goat anti-rabbit igg fluorescent latex particles in the ratio 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 0.7 ~ 1.3cm with the speed of 2 ~ 6ul/cm, dry 2 ~ 24h at 30 ~ 55 DEG C.Fluorescence probe microballoon dilution is the damping fluid containing 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, Tween 80.
Should be noted that (1) probe is kept in sample diluting liquid easily to reunite, used fluorescence probe microballoon diluted to spray drying on the glass fibers and made probe fixed bolster, the component of fluorescence probe microballoon dilution can make probe dispersed, avoids the reunion between probe; Probe on probe fixed bolster, when slow releasing, can slow down immune response, and make the reaction time longer, detection signal is stronger; (2) 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; (3) the drying time of probe fixed bolster is not easily long, also needs to continue drying 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 fixed bolster, chromatographic film, thieving paper are attached on PVC liner plate successively, adopt and draw on the film metal spraying machine detection line (T line) that HER-2 monoclonal antibody and rabbit igg is coated on respectively chromatographic test paper and control line (C line), the immuno-chromatographic test paper strip of 3 ~ 5mm width is obtained through cutting cutter cutting, the preferred cellulose nitrate of above-mentioned chromatographic film after 37 DEG C of drying 12 ~ 36h.
Should be noted that (1) traditional quantitative immune chromatographic technique adopts bag by sheep anti-mouse antibody as control line, along with the increase of HER-2 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.Goat anti-rabbit igg antibody of the present invention adopts rabbit igg antibody and goat-anti chicken antibody to match as control line, and the reaction of C line and T line is independently carried out, and without cross influence, thus C line and T line can be avoided to contend with one other probe particulate and the repeated deviation that produces; (2) be less than 12h drying time, the joint efficiency of antibody on nitrocellulose filter is low, product detect signal can change with the prolongation of holding time, the detectability of HER-2 is caused 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; (3) when the width of immuno-chromatographic test paper strip is less than 3mm, the precision of cutting machine is comparatively large on the impact of immuno-chromatographic test paper strip, can reduce the repeatability of testing result, when the width of immuno-chromatographic test paper strip is greater than 5mm, can increase the cost of test card, optimized immuno-chromatographic test paper strip is 4mm.
Step 3) sample diluting liquid preparation: sample diluting liquid is the damping fluid containing bovine serum albumin(BSA), surfactant and antiseptic.This 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 proclin, and described spreading agent is polyvinylpyrrolidone 10K, PVP K or polyvinyl alcohol (PVA).
Above-mentioned proclin antiseptic, its active component is MIT (MCI) and CMIT (CMCI) mainly, is high-performance bio antiseptic of new generation.The growth of these two kinds of composition T suppression cell and impel apoptotic principle identical.Active component with cell membrane contact a few minutes after, can to penetrate into immediately in film and the activity of T suppression cell endoenzyme.Target enzyme is in the center that cellular metabolism KREBS circulates, four diverse locations of ProClin antiseptic effect KREBS circulation (tricarboxylic acid cycle): pyruvic dehydrogenase, a-ketoglutaric acid dehydrogenasa, succinate dehydrogenase and nadh dehydrogenase.Thus Cell growth inhibition metabolism, macromolecular synthesis, cause intracellular energy level to decline rapidly.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 be suitable for of ProClin antiseptic widely.Secondly, ProClin antiseptic has many targets action site, therefore greatly reduces the drug resistance of the microorganism because of variation generation.Along with KREBS circulation is blocked, the ability of the bioactivators such as cell produce power and synzyme declines rapidly, but its using dosage keeps lower level to person poultry harmless, can effectively control microbial growth in external diagnosis reagent.Thus there is broad spectrum antibacterial, speed of action be 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(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; (2) 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; (3) 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.
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 on immunochromatography immunochromatographydetection detection 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.As depicted in figs. 1 and 2, mixing material penetrates in sample pad 2, mixing material arrives tat probe fixed bolster 3 after glass fiber filter impurity and process, HER-2 monoclonal antibody on HER-2 wherein and probe is combined into compound, compound to penetrate on detection line 5 along nitrocellulose filter 4, the new compound of double-antibody sandwich is formed with the HER-2 monoclonal antibody be fixed on detection line 5, HER-2 monoclonal antibody on detection line 5 and the HER-2 monoclonal antibody in probe, its epi-position on HER-2 is different.Remaining antigen-fluorescently-labeled antibody complex, unconjugated be marked with fluorescent latex particles HER-2 monoclonal antibody precipitation and HER-2 monoclonal antibody fluorescent latex particles antibody and the goat anti-rabbit igg that is marked with fluorescent latex particles continue to penetrate into control line 6, 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.
As shown in Fig. 1 to 2, the present invention is based on the fluorescence immune chromatography detection kit that said method also provides a kind of HER-2, comprise kit box and dilution bottle, kit box comprises PVC liner plate 1 and is fixed on the sample pad 2 on PVC liner plate, probe fixed bolster 3, nitrocellulose filter 4 and thieving paper 7, sample pad is overlapped on probe fixed bolster, probe fixed bolster 3 and thieving paper 7 are overlapped on the both sides of nitrocellulose filter respectively, nitrocellulose filter is provided with detection line 5 and control line 6, HER-2 monoclonal antibody and rabbit igg is coated with respectively in detection line 5 and control line 6, probe fixed bolster 3 is obtained through metal spraying drying by the mixing fluorescent latex particles of HER-2 monoclonal antibody and goat anti-rabbit igg, has deposited sample diluting liquid in dilution bottle.Immunochromatographydetection detection card 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 2 place, 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 side housing being positioned at close 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 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 2, to tat probe fixed bolster 3 after glass fiber filter impurity and process, HER-2 monoclonal antibody on HER-2 wherein and probe is combined into compound, compound penetrates on detection line 5 along nitrocellulose filter 4, the new compound of double-antibody sandwich is formed with the HER-2 monoclonal antibody be fixed on detection line 5, HER-2 monoclonal antibody on detection line 5 and the HER-2 monoclonal antibody in probe, its epi-position on HER-2 is different.Remaining antigen-fluorescently-labeled antibody complex, unconjugated be marked with fluorescent latex particles HER-2 monoclonal antibody precipitation and HER-2 monoclonal antibody fluorescent latex particles antibody and the goat anti-rabbit igg that is marked with fluorescent latex particles continue to penetrate into control line 6, 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.
The detection fluorescence detector of the invention described above, 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 400 ~ 600nm, pre-amplifying module is a pre-amplification circuit.
The fluorescence immune chromatography of " embodiment " HER-2 detects
The first step: the preparation of probe fixed bolster
1) 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 mouse serum albumins, 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 mouse serum albumin precipitation and the mouse serum albumin 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 the mouse serum albumin activation coupling HER-2 monoclonal antibody being marked with fluorescent latex particles to be marked with the HER-2 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 HER-2 monoclonal antibody fluorescent latex particles and goat anti-rabbit igg fluorescent latex particles by volume 6:1 mix, the epoxy glue lactoconium be mixed to get is mixed 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 4ul/cm, dry 4h at 37 DEG C.
Second step: the preparation of immunochromatographydetection detection card
Liner plate is pasted nitrocellulose filter 4, probe fixed bolster 3, sample pad 2 and thieving paper 7 successively, and sample pad 2 is overlapped on probe fixed bolster 3, and probe fixed bolster 3 and thieving paper 7 are overlapped on nitrocellulose filter 4, and are closely connected.The antibody coating buffer that another strain of different epi-position HER-2 monoclonal antibody (coated antibody) and goat anti-rabbit igg coating buffer are mixed with 1mg/ml and 1mg/ml is respectively positioned at respectively by from the HER-2 monoclonal antibody that fluorescent latex particles marks.HER-2 monoclonal antibody coating buffer and rabbit igg coating buffer are coated on detection line 5 corresponding on nitrocellulose filter 4 and control line 6 with the linear speed of 1 μ l/cm, detection line 5 and control line 6 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) and 0.19g Sodium azide, ultrasonic until solid all dissolves mixes.
4th step: pattern detection
1) linearly, detectability and precision assessment:
Adopt negative cow's serum as dilution, HER-2 standard items are mixed with the standard solution that concentration is 2000,1800,1600,1400,1200,1000,800,600,400,200 and 0pg/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 8, 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 10pg/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 HER-2 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 HER-2 concentration be 40pg/ml human serum sample based on sample, add the HER-2 reference material human serum of same volume variable concentrations, be mixed with the HER-2 human serum solution that concentration is 1400,1000,600 and 500pg/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 in 95% ~ 105% scope.
The above is only the preferred embodiment of the present invention; be 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 make some improvement and modification, these improve and modification also should be considered as protection of the present invention.
Claims (10)
1. a fluorescence immune chromatography detection method of HER-2, is characterized in that, comprise the following steps:
Step 1) preparation of probe fixed bolster: HER-2 monoclonal antibody is obtained HER-2 monoclonal antibody fluorescent latex particles with activation albumin fluorescent latex particles coupling reaction in proportion, again described HER-2 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 fluorescence probe microballoon diluted, spray obtained probe fixed bolster on the glass fibers;
Step 2) immuno-chromatographic test paper strip preparation: sample pad, probe fixed bolster, chromatographic film, thieving paper are attached on PVC liner plate successively, described HER-2 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 potpourri including bovine serum albumin(BSA), surfactant, spreading agent and antiseptic is mixed obtained sample diluting liquid with damping fluid;
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, through fluorescence detector fluoroscopic examination after chromatography reaction terminates.
2. the fluorescence immune chromatography detection method of HER-2 according to claim 1, it is characterized in that: step 1) in, the preparation of described activation albumin fluorescent latex particles first fluorescent latex particles and activator is reacted obtained to activate fluorescent latex particles, again with albumin in proportion coupling reaction obtain albumin fluorescent latex particles, described albumin is 0.05:100 ~ 0.2:100 with the ratio of activation fluorescent latex particles, described albumin fluorescent latex particles reacts obtained described activation albumin fluorescent latex particles further with activator, wherein, described albuminous kind comprises people, ox, sheep or mouse.
3. the fluorescence immune chromatography detection method of HER-2 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 coupling reaction in proportion by goat anti-rabbit igg, and described goat anti-rabbit igg is 0.05:100 ~ 0.2:100 with the blending ratio of activation fluorescent latex particles.
4. the fluorescence immune chromatography detection method of HER-2 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 HER-2 according to claim 1, it is characterized in that: step 1) in, described HER-2 monoclonal antibody is 0.05:100 ~ 0.2:100 with the blending ratio of activation albumin fluorescent latex particles, described goat anti-rabbit igg is 0.05:100 ~ 0.2:100 with the blending ratio of activation fluorescent latex particles, ratio 2:1 ~ 6:1 that described HER-2 monoclonal antibody fluorescent latex particles mixes mutually with goat anti-rabbit igg fluorescent latex particles.
6. the fluorescence immune chromatography detection method of HER-2 according to claim 1, it is characterized in that: step 1) in, fluorescent latex particles fluorescence probe microballoon diluted 2 ~ 6 times will be mixed, evenly be sprayed on the glass fibre that width is 0.7 ~ 1.3cm with the speed of 2 ~ 6ul/cm, obtained probe fixed bolster after dry 2 ~ 24h at 30 ~ 55 DEG C.
7. the fluorescence immune chromatography detection method of HER-2 according to claim 1, it is characterized in that: described fluorescence probe microballoon dilution is the damping fluid containing 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.
8. the fluorescence immune chromatography detection method of HER-2 according to claim 1, it is characterized in that: step 3) in, described damping fluid comprises 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 proclin, and described spreading agent is polyvinylpyrrolidone 10K, PVP K or polyvinyl alcohol (PVA).
9. the fluorescence immune chromatography detection kit of a HER-2, comprise kit box and dilution bottle, described kit box comprises immunochromatographydetection detection card, described immunochromatographydetection detection card comprises PVC liner plate and is fixed on sample pad, probe fixed bolster, nitrocellulose filter and the thieving paper on PVC liner plate, it is characterized in that:
Described sample pad is overlapped on probe fixed bolster, described probe fixed bolster and thieving paper are overlapped on described nitrocellulose filter both sides respectively, described nitrocellulose filter is provided with detection line and control line, described detection line endoperidium has HER-2 monoclonal antibody, and described control line endoperidium has goat anti-rabbit igg;
The mixing fluorescent latex particles drying that described probe fixed bolster is made up of HER-2 monoclonal antibody fluorescent latex particles and goat anti-rabbit igg fluorescent latex particles is obtained.
10. the fluorescence immune chromatography detection kit of HER-2 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 at the side described housing being positioned at close thieving paper.
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