Utility model content
In view of this, this utility model provides a kind of tumor markers detection kit, including box body, lid, quantum dot fluorescence detectable card；Described reagent card includes base and is placed in sample pad, quantum dot conjugate pad, coated film, adsorptive pads and the name of product identification pad being sequentially connected with on base；Described quantum dot conjugate pad contains quantum dot-labeled cancer antigen 125 monoclonal antibody, quantum dot-labeled cancer antigen 1 5-3 monoclonal antibody, quantum dot-labeled squamous cell carcinoma antigen monoclonal antibody, quantum dot-labeled people's epididymal proteins 4 monoclonal antibody；Described coated film along the direction deviating from quantum dot conjugate pad be provided with four roads detection line T with together with nature controlling line C, wherein, Article 1, detection line T is coated with immobilised cancer antigen 125 monoclonal antibody, Article 2 detection line T is coated with immobilised cancer antigen 15-3 monoclonal antibody, Article 3 detection line T is coated with immobilised anti-squamous cell carcinoma antigen monoclonal antibody, Article 4 detection line T is coated with immobilised anti-human epididymal proteins 4 monoclonal antibody, and nature controlling line C is coated with the sheep anti mouse polyclonal antibody of solidification.
Preferably, described lid is provided with well, result detection window and name of product watch window.
Preferably, described sample pad right-hand member has the sample clinch being overlapped on described quantum dot conjugate pad, described quantum dot conjugate pad right-hand member has the conjugate clinch being overlapped on described coated film, and described adsorptive pads left end has the water suction clinch being overlapped on coated film.
Preferably, described sample clinch leaves spacing with described conjugate clinch in their extension direction.
Preferably, the sample pad that described sample pad is made up of glass fibre, polyester film, cellulosic filter paper or non-woven fabrics.
Preferably, the quantum dot conjugate pad that described quantum dot conjugate pad is made up of polyester cellulose film or glass fibre membrane.
Preferably, the coated film that described coated film is made up of nitrocellulose filter.
Preferably, tumor markers detection kit farther includes one or more ingredients being selected from lower group: for firm banking, upper cover, desiccant, dropper, description, color label, sample collection tube and the sample diluting liquid of fixing described quantum dot fluorescence detectable card.
Preferably, described well is provided with the sealing aluminum membrane matched with it.
Preferably, described lid is connected by the rotating shaft being fixed on box body with box body.
The test kit that this utility model provides have employed quantum dot as fluorescent marker, utilizes the determinand in the immunological method detection sample of double-antibody sandwich.Can not only detect determinand qualitatively, it is also possible to use fluorescence immunity analyzer to be measured the quantum dot emission fluorescence intensity in detection line T region, the concentration of determinand becomes positive correlation with the transmitting fluorescence intensity of corresponding detection line T.By setting up calibration object concentration and fluorescence intensity curves, cancer antigen 125 in sample to be tested, cancer antigen 15-3, squamous cell carcinoma antigen, the content of anti-human epididymal proteins 4 can be gone out with the Fitting Calculation.It can thus be seen that compared with prior art, experimentation is simple, quick, safety, and limitation is little, applied widely, highly sensitive, particularly can detect the content of tumor markers to be checked by the quantum dot fluorescence productivity of quantum dot marked tumor mark.
Detailed description of the invention
Claim of the present utility model is described in further detail by the mode below in conjunction with specific embodiment, but it is not intended that any restriction of the present utility model, the amendment of anyone limited number of time made in this utility model right, still within right of the present utility model.
Seeing Fig. 1, this utility model provides a kind of tumor markers detection kit, including box body 1, lid 2, quantum dot fluorescence detectable card 3；Described reagent card 3 includes base 36 and is placed on base 36 sample pad 31, quantum dot conjugate pad 32, coated film 33, adsorptive pads 34 and the name of product identification pad 35 being sequentially connected with；Described quantum dot conjugate pad 32 is containing quantum dot-labeled cancer antigen 125 monoclonal antibody, quantum dot-labeled cancer antigen 1 5-3 monoclonal antibody, quantum dot-labeled squamous cell carcinoma antigen monoclonal antibody, quantum dot-labeled people's epididymal proteins 4 monoclonal antibody；Described coated film 33 along the direction deviating from quantum dot conjugate pad 32 be provided with four roads detection line T with together with nature controlling line C, wherein, Article 1, detection line T is coated with immobilised cancer antigen 125 monoclonal antibody, Article 2 detection line T is coated with immobilised cancer antigen 15-3 monoclonal antibody, Article 3 detection line T is coated with immobilised anti-squamous cell carcinoma antigen monoclonal antibody, Article 4 detection line T is coated with immobilised anti-human epididymal proteins 4 monoclonal antibody, and nature controlling line C is coated with the sheep anti mouse polyclonal antibody of solidification.
Above-mentioned, after sample is added in sample pad 31, material therein is acted on by chromatography, moves to successively on quantum dot conjugate pad 32 and coated film 33.When sample moves to quantum dot conjugate pad 32, quantum dot redissolves, detection antibody therein, such as people's antibody, can combine with the coated specific antibody of quantum dot on quantum dot conjugate pad 32, then the sample containing the material combining from quantum dot conjugate pad 32 or mixing continues to move on coated film 33, and combines with coated specific antibody on coated film 33 and be trapped, so that the analyte in sample is detected.It should be understood that the coated liquid of specific antibody is coated on coated film 33, i.e. detection line T；Nature controlling line C is usually coated liquid and is coated and the sheep anti mouse polyclonal antibody on coated film 33 or goat-anti rabbit polyclonal antibody.Suitable spacing should be left between detection line T and nature controlling line C, and be set to the region being parallel to each other, both differentiations of being more convenient for, see Fig. 2.
It is understood that quantum dot conjugate pad 32 can contain quantum dot-labeled cancer antigen 125 monoclonal antibody, quantum dot-labeled cancer antigen 1 5-3 monoclonal antibody, quantum dot-labeled squamous cell carcinoma antigen monoclonal antibody, quantum dot-labeled people's epididymal proteins 4 monoclonal antibody simultaneously.Certainly, described quantum dot conjugate pad 32 can also comprise other other quantum dot-labeled tumor markers, and these tumor markers should possess following characteristics:
(1) sensitivity is high, and tumor markers is beyond expression in normal human cell, does not contains tumor markers composition in serum, once detecting tumor markers, i.e. can determine whether that measuring samples is positive, patient may be tumor patient, tumor can detected in early days, not fail to pinpoint a disease in diagnosis.
(2) high specificity, different tumors can only be expressed relevant specific antigen, can conveniently identify the non-tumor of tumor.
(3) it is present in the middle of human serum, it is easy to clinical sampling and process, it is simple to detection.
(4) serum tumor mark content and gross tumor volume size, clinical stages, are relevant, in order to judge the possible course of disease and the prognosis of predictive disease.
(5) whether the half-life is short, can reflect the dynamic change of tumor, monitoring therapeuticing effect in real time, it is judged that postoperative recur and shift.
(6) assay method precision, accuracy height, easy to operate.
In this utility model embodiment, described lid 2 is provided with well 21, result detection window 22 and name of product watch window 23.
Above-mentioned, sample is loaded by adding mouth 21, the fluorescence radiation situation of quantum dot is observed by result detection window 22, should be understood that, in embodiment, it is able to observe that the name of product identification pad 35 being positioned on adsorptive pads 34 by the name of product watch window 23 being opened on lid 2, thus the detection project to detection device that obtains carries out Accurate classification, the effect of fast recording testing result according to title.
In this utility model embodiment, described sample pad 31 right-hand member has the sample clinch being overlapped on described quantum dot conjugate pad 32, described quantum dot conjugate pad 32 right-hand member has the conjugate clinch being overlapped on described coated film 33, and described adsorptive pads 34 left end has the water suction clinch being overlapped on coated film 33.
Above-mentioned, " overlap joint " refers to, the head and the tail of two adjacent assemblies partly overlap, and form the attachment structure that fluid sample can be allowed to move at overlapping.For example, it is possible to the head and the tail part at two adjacent assemblies forms overlay structure more closely by pressing operation so that sample can be moved to downstream components by this overlay structure from the afterbody of upstream component.Upstream component can overlap the top of downstream components, it is also possible to overlaps lower section.When detection, can be by the sample of liquid condition (such as blood, blood plasma and other body fluid diluents etc.) it is loaded onto in sample pad 31, the sample of this liquid condition can pass through water sorption, move along sample pad 31 to the quantum dot conjugate pad 32 overlapped therewith, move to the coated film 33 overlapped therewith further along quantum pad conjugate pad 32 after touching quantum dot conjugate pad 32, move to the adsorptive pads 34 overlapped therewith further along coated film 33 after touching coated film 33.
Sample clinch described in this utility model embodiment leaves spacing with described conjugate clinch in their extension direction.
Above-mentioned, sample is commonly referred to chromatography effect by sample pad 31 to the movement of adsorptive pads 34, it can thus be appreciated that, further, described sample clinch leaves spacing with described conjugate clinch in their extension direction, so that after quantum labelling, specific antibody can leave sufficient binding time with the determined antigen in sample on quantum dot conjugate pad 32, as testing sample exists specific antigen to be detected, then can form antigen-antibody quantum dot complex at the conjugate pad containing quantum dot-labeled rear specific antibody；Described conjugate clinch leaves spacing with described water suction clinch in their extension direction, so that above-mentioned antigen-antibody quantum dot complex forms antibody-antigen-antibody quantum dot complex on coated film 33, i.e. there is the specific binding of double antibodies sandwich in reserved sufficient binding time.
In this utility model embodiment, the sample pad 31 that described sample pad 31 is made up of glass fibre, polyester film, cellulosic filter paper or non-woven fabrics.
Above-mentioned, that sample pad 31 can be made up of polyester film, cellulosic filter paper or non-woven fabrics sample pad, preferably glass fiber sample pad；Glass fibre is good relative to other materials dehydration property, be not adhered sample.
In this utility model embodiment, the quantum dot conjugate pad 32 that described quantum dot conjugate pad 32 is made up of polyester cellulose film or glass fibre membrane.
Above-mentioned, quantum dot conjugate pad 3 can be made up of glass fibre membrane, is preferably made up of polyester cellulose film, and polyester cellulose film is better than, with the affinity of gold colloidal, the conjugate pad that other materials is made, and prevents gold colloidal from occurring to assemble or form precipitation.
In this utility model embodiment, the coated film 33 that described coated film 33 is made up of nitrocellulose filter.
Above-mentioned, that the preferred nitrocellulose filter of coated film 33 is made coated film 33, the proteopexy ability of nitrocellulose filter is better than the coated film 33 that other materials is made, it is possible to preferably solidification is coated on protein thereon.
In this utility model embodiment, tumor markers detection kit farther includes one or more ingredients being selected from lower group: for firm banking, upper cover, desiccant, dropper, description, color label, sample collection tube and the sample diluting liquid of fixing described quantum dot fluorescence detectable card 3.
Above-mentioned, composition described above part is when tumor markers detection kit uses or the ordinary articles in the time of preservation, it is simple to operation, can be combined into the detection suit being easy to clinical manipulation with detection kit.
In this utility model embodiment, described well 21 is provided with the sealing aluminum membrane matched with it.
Above-mentioned, lid 2 also includes the sealing aluminum membrane matched with well 21, and before being used for using, protection quantum dot luciferase assay reagent card 3 is not contaminated, and sealing aluminum membrane can exist various ways.
In this utility model embodiment, described lid 2 is connected by the rotating shaft being fixed on box body 1 with box body 1.
Above-mentioned, lid 2 can be connected with box body 1 by the vertical rotating shaft being fixed on box body 1, lid 2 can slide relative to box body 1 level, open and closed box 1, accordingly, lid 2 can also be connected with box body 1 by the horizontal rotating shaft being fixed on box body 1, and lid 2 upwards can open relative to box body 1, opens and closed box 1.
It is as follows that this utility model additionally provides quantum dot conjugate pad (32) the preparation technology program described in above-mentioned tumor markers detection kit:
(1) weigh carbodiimide 10mg and add purified water to 1mL, standby as EDC solution；Weigh glycine 75mg and add purified water to 1mL, mix standby.
(2) in microcentrifugal tube, add 10mL carboxyl water-soluble quantum dot (8 μm ol/mL), be subsequently adding above-mentioned 7.5 μ L EDC solution, 400ul 0.01M PBS solution, oscillating reactions 20min under room temperature, become quantum dot working solution.By needing the amount of antibody 1mg of activation, adding in the microcentrifugal tube of 1000 μ L quantum dot working solutions, shaken at room temperature reacts 2 hours.To add the glycine solution of excess in microcentrifugal tube after completion of the reaction, and be placed in low-temperature and high-speed centrifuge 4 DEG C, 12000r/min is centrifuged 20min.Each precipitation adds appropriate 0.01M PBS respectively so that it is dissolve.Dissolve rearmounted 4 DEG C to save backup.
(3) preparing some film machine 0.01M PBS flushing pipe, emptying flushing pipe, an adjustment point film machine hydrojet membrane type device parameter make pump head hydrojet scope be 30 μ L/cm ± 3%.Take in the quantum dot-labeled cancer antigen 125 monoclonal antibody after above-mentioned dissolving, quantum dot-labeled cancer antigen 1 5-3 monoclonal antibody, quantum dot-labeled squamous cell carcinoma antigen monoclonal antibody, quantum dot-labeled people's epididymal proteins 4 monoclonal antibody one test tube of addition, add 0.01MPBS solution and be settled to consumption required for hydrojet, after mixing in addition point film machine needle cylinder, standby.
(4) pump head position is set, by film machine continuous uniform hydrojet on quantum dot conjugate pad, monitors each glass fibre hydrojet situation, the uneven section of labelling in real time.Quantum dot conjugate pad complete for hydrojet is put drying room 37 DEG C be dried 2 hours.In valve bag, room temperature preservation is standby.
The specifically used method of quantum dot fluorescence detectable card in the tumor markers detection kit that this utility model provides:
Outer package is removed during use, take out tumor markers detection kit, by well, vertically instill about 100 microliters of sample, testing result is observed after 15 minutes, seeing 3 to Fig. 6, in Fig. 3, four detection line T and nature controlling line C all observe fluorescence radiation phenomenon, then containing above-mentioned four kinds of tumor markerses in sample under the irradiation of uviol lamp.In Fig. 4, Article 1 detection line T and Article 4 detection line T and nature controlling line C all observes fluorescence radiation phenomenon, then containing cancer antigen 125 and people's epididymal proteins 4 both tumor markers in sample under the irradiation of uviol lamp.In Fig. 5, Article 4 detection line T and nature controlling line C all observes fluorescence radiation phenomenon under the irradiation of uviol lamp, then contain only tumor marker people's epididymal proteins 4 in sample.In Fig. 6, four detection line T and nature controlling line C all do not observe fluorescence radiation phenomenon, and therefore testing result is invalid.
Further detection line T being observed, result is read in the survey that quantum dot fluorescence detectable is placed under dry type fluorescence immunity analyzer of fluorescence radiation phenomenon, then can carry out quantitative analysis.