CN105548567A - Kit for time resolution fluorescent quantitative detection on PCT - Google Patents
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- CN105548567A CN105548567A CN201610031801.3A CN201610031801A CN105548567A CN 105548567 A CN105548567 A CN 105548567A CN 201610031801 A CN201610031801 A CN 201610031801A CN 105548567 A CN105548567 A CN 105548567A
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- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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Abstract
The invention discloses a kit for time resolution fluorescent quantitative detection on PCT. The kit comprises a fluorescent microsphere antibody complex and an immune fluorescent test paper card, wherein the fluorescent microsphere antibody complex is prepared by marking rare earth fluorescent microsphere with a PCT monoclonal antibody to form a compound and adding the compound on a pipette tip, and is used as a detection antibody after freeze-drying; the immune fluorescent test paper card comprises a detection test paper card; the detection test paper card consists of a sample pad, a nitrocellulose membrane and a water absorbing pad which are adhered to a lining plate in sequence in a lap joint manner; the position of a detection line on the nitrocellulose membrane is wrapped by another PCT monoclonal antibody; the position of a quality control line is wrapped by goat anti-mouse polyclonal antibody. By adopting the kit, the fluorescence influence caused by a sample self can be avoided, the rare earth fluorescent microsphere is adopted as a marking carrier, good stability can be achieved, the microsphere can be connected with the antibody through a covalent bond, and a stable marking product can be generated. The kit is rapid, simple and convenient in detecting the sample and high in sensitivity, and full quantitative detection can be achieved.
Description
Technical field
The present invention relates to technical field of immunoassay, be specifically related to the kit that a kind of time-resolved fluorescence quantitatively detects PCT.
Background technology
Procalcitonin (PCT) is a kind of protein, and when serious bacterial, fungi, parasitic infection and pyemia and MOFE, its level in blood plasma raises.PCT have occur early, the half life period is short, under whole body and local infection, all present the features such as positively related growth by immunosuppressive effects.Based on this, the inflammatory factor (as TNF-a, IL-16, IL-6, CRP etc.) of the more most of classics of PCT has better susceptibility, specificity and real-time, has important practical value in clinical detection and monitoring.
Present stage, the clinically detection method of PCT common double antibodies sandwich immunochemiluminescence method, radioimmunoassays, euzymelinked immunosorbent assay (ELISA) (ELISA), immunochromatographic method etc.
Double antibodies sandwich immunochemiluminescence method has the advantages such as susceptibility is high, required time is short, but can not detect the PCT in normal human blood.
Radioimmunoassays carrys out by radioactive molecules being covalently cross-linking to the rear emission signal measuring radioactive molecules of PCT antibody mediated immunity reaction a kind of ultramicro-analysis method that indirect quantification detects PCT.There is the advantages such as specificity good, highly sensitive (can reach ng and pg level), easy and simple to handle, sample preparation are simple, accurate quantification, but operating personnel can touch radiomaterial in the method, may damage the health of operating personnel, and how to dispose radioactive material after having detected also be a serious problem.
The enzyme-linked immune detection method of PCT is double crush syndrome method, a pair PCT monoclonal antibody is carried out ELISA Plate solidification and enzyme labeling respectively, after antigen-antibody reaction, by the color reaction of substrate and enzyme, specific antigen can be shown whether exist, and quantitative test can be carried out according to the depth of colour generation.There is the features such as high specificity, highly sensitive, easy and simple to handle, quick, sample energy is large, not only can carry out qualitative detection and also can quantitatively detect, but consuming time longer.
Side direction immunochromatographic method the field such as to detect fast at disease quick diagnosis, Small molecular and is widely used.Its principle be by specific antigen or antibody bag by nitrocellulose filter, form detection line, the two anti-distance detection lines that are coated on formed nature controlling line in the region of 2 ~ 3 millimeters.After the target molecule in sample is combined with the detection antibody with particular marker, from the loading end of cellulose nitrate enzyme to suction side chromatography.Due to capillarity, compound will move forward along film, but when moving to detection line, the capture antibody in tested survey line catches and rests on detection line by the target molecule in sample, unnecessary detection antibody is then by anti-being caught by two of nature controlling line after detection line.Conventional label is colloid gold particle, fluorescent microsphere etc.
The colloidal gold immunity chromatography of PCT is surperficial to colloid gold particle by PCT antibody labeling by Electrostatic Absorption, judges testing result by the gathering colour developing of observing colloid gold grain on detection line.There is the advantages such as quick, easy, directly perceived, but also exist to exist simultaneously rely on naked eyes identification, sensitivity low, cannot accurate quantification, mark by Electrostatic Absorption, the shortcomings such as marked product is unstable.
The immunofluorescence chromatographic technique of PCT is the reactive group (-the COOH ,-NH that PCT antibody are covalently bonded in fluorescent microsphere surface
2) on, whether produce fluorescence by exciting rear detection line and judge testing result, continued the advantage of colloidal gold immunochromatographimethod method, have simultaneously high sensitive, completely quantitatively, the stable advantage of label.Increasingly extensive application is obtained in medical science, the animal and plant quarantine, each field of food safety supervision.
Many compounds in biofluid and serum and albumen inherently can fluoresce, and therefore using traditional chromophore and then carry out the sensitivity of fluoroscopic examination will degradation.Major part background fluorescence signal exists in short-term, is therefore combined with time-resolved fluorescence technology by the label of long Decay, prompt fluorescence interference just can be made to reduce to and minimize.
Summary of the invention
In order to solve the above-mentioned defect that existing several detection PCT technology exists, the invention provides the kit that a kind of time-resolved fluorescence quantitatively detects PCT.
Time-resolved fluorescence provided by the invention quantitatively detects the kit of PCT, comprises fluorescent microsphere antibody complex and immunofluorescence test card, wherein,
Fluorescent microsphere antibody complex: be PCT monoclonal antibody is marked on the complex that rare-earth fluorescent microballoon formed, adds to this complex in rifle head and freeze-drying forms, as detection antibody;
Immunofluorescence test card comprises Test paper card, and Test paper card is overlapped successively by sample pad, nitrocellulose filter and adsorptive pads and is affixed on liner plate and forms; Wherein, the detection line position on nitrocellulose filter is coated with another PCT monoclonal antibody, and nature controlling line position is coated with sheep anti-Mouse and resists more.
PCT monoclonal antibody is that the hybridoma that the bone-marrow-derived lymphocyte that obtained by PCT mice immunized with antigen and murine myeloma cell merge produces, and therefore sheep anti-Mouse is many anti-ly can be combined with PCT monoclonal antibody.
Time resolved fluoro-immunoassay (Timed-resolvedfluoroimmunoassay, TRFIA) be a kind of sensitive high-end quantitative immunological detection technique, come labelled antigen or antibody using rare-earth fluorescent microballoon (La rear earth ion) as label, because La rear earth ion Stokes displacement is large, avoid exciting light and radiative overlapping two and affect result, and La rear earth ion fluorescence lifetime is long, can by extending the interference eliminating detection material background fluorescence detection time, improve sensitivity, the fluorescence intensity of rare earth element is high in addition, the sensitivity of detection can be improved equally.Conventional rare-earth fluorescent microballoon is EU fluorescent microsphere.
Preferably, above-mentioned time-resolved fluorescence quantitatively detects in the kit of PCT, described complex adopts carboxylated rare-earth fluorescent microballoon as carrier, by carbodiimide and hydroxy thiosuccinimide by activated carboxylic, and-the NH in the carboxyl of activation and PCT monoclonal antibody
2covalent bond, the fluorescent microsphere complex of formation.
Preferably, above-mentioned time-resolved fluorescence quantitatively detects in the kit of PCT, and the concrete preparation method of described complex is:
(1) get rare-earth fluorescent microballoon, centrifuging and taking precipitates, with the MES buffer solution of pH6.0;
(2) activate: the carbodiimide and the hydroxy thiosuccinimide that add the volumetric molar concentrations such as equal-volume in microballoon after washing, activated carboxylic reaction is carried out in room temperature concussion;
(3) labelled antibody: the microballoon centrifuging and taking precipitation after step (2) activation, with adding appropriate PCT monoclonal antibody after PB buffer solution, room temperature concussion makes carboxyl and-NH
2covalent bond, realize antibody labeling;
(4) the microballoon centrifuging and taking through antibody labeling is precipitated, add azanol damping fluid cancellation reaction;
(5) through the microballoon centrifuging and taking precipitation of cancellation reaction, add confining liquid and close, finally add storage liquid and preserve;
Described confining liquid is 10mMPB solution, wherein containing 0.5wt% casein;
Described storage liquid is 10mMPB solution, wherein containing 0.5wt%BSA, 0.5% (v/v) Tween-20,0.02wt% sodium azide.
In the present invention, the pH value of PB damping fluid used is 7.5, and concentration is 10mM.For conventional reagent, directly can buy commodity or prepare voluntarily.Described azanol damping fluid refers to the aqueous solution of azanol, and wherein conventional azanol volumetric molar concentration is 50mM.
Preferably, above-mentioned time-resolved fluorescence quantitatively detects in the kit of PCT, and described sample pad is the glass fibre membrane through the 0.01MPB buffer blind containing 3wt%BSA.
Preferably, above-mentioned time-resolved fluorescence quantitatively detects in the kit of PCT, and the distance between described detection line and nature controlling line is 4mm.
More preferably, the bee-line of detection line and sample pad end is 7mm, and the bee-line of nature controlling line distance adsorptive pads is 11mm.
Preferably, above-mentioned time-resolved fluorescence quantitatively detects in the kit of PCT, immunofluorescence test card fastens on plastic bottom card by Test paper, face, test paper surface card compresses and forms, and face is stuck in position reserved well and the view window respectively of counter sample pad and nitrocellulose filter.
Preferably, above-mentioned time-resolved fluorescence quantitatively detects in the kit of PCT, also comprises sample buffer, and it is the PB solution containing 0.25wt% casein and 40mMEDTA.Sample buffer is used for chromatography samples.
Preferably, above arbitrary described time-resolved fluorescence quantitatively detects in the kit of PCT, also comprises dilution, and diluent ingredient is the PB solution containing 6wt%BSA and 5mM glucose.Dilution is used for dilute sample, prepares in addition, use convenient in kit after adding dilution without the need to operating personnel.
Time-resolved fluorescence provided by the invention quantitatively detects the kit of PCT, and Cleaning Principle is as follows:
After the sample diluted is mixed with fluoroscopic examination antibody, join the adding mouth of test card one end, moved forward by capillary action, after reaction a period of time, utilize Fluorescence Scanner, the viewing mask of scanning test card, the fluorescence intensity that seizure nature controlling line and detection line are excited, if containing corresponding PCT antigen in sample, be coated on the PCT monoclonal antibody on detection line and the antigen of PCT monoclonal antibody all in sample in fluorescent microsphere antibody complex is combined, form immune complex, PCT monoclonal antibody in unnecessary unconjugated fluorescent microsphere antibody complex will move to the many anti-bindings of sheep anti-Mouse of nature controlling line and nature controlling line bag quilt because of capillary action, such detection line and nature controlling line all can produce fluorescence, if there is no corresponding antigens in measuring samples, PCT monoclonal antibody in fluorescent microsphere antibody complex can not be combined with being coated on another PCT monoclonal antibody on detection line, fluorescent microsphere antibody complex can not enrichment, then detection line can not produce fluorescence, and PCT monoclonal antibody in fluorescent microsphere antibody complex only with the many anti-bindings of sheep anti-Mouse of nature controlling line bag quilt, nature controlling line there will be fluorescence.
Compared with existing detection technique, the kit that time-resolved fluorescence of the present invention quantitatively detects PCT has following beneficial effect:
(1) fluorescence of sample itself can be avoided to affect.
(2) labeled vector is stablized.
(3) microballoon is connected with antibody by covalent bond by mark, and marked product is stablized.
(4) detect fast and convenient, susceptibility is high.
(5) can entirely quantitatively detect.
Accompanying drawing explanation
Fig. 1 is the reagent cartridge configuration composition schematic diagram that time-resolved fluorescence provided by the invention quantitatively detects PCT.
Fig. 2 is the chromatography curve of variable concentrations PCT on immunofluorescence test card.
Fig. 3 is the PCT typical curve using kit of the present invention to draw.
Fig. 4: use kit of the present invention and ELISA method to detect the correlativity of the accuracy of blood sample.
Embodiment
In order to make those skilled in the art person understand the present invention program better, below in conjunction with preferred embodiment, the present invention is described in further detail.
Embodiment 1 time-resolved fluorescence quantitatively detects the kit preparation of PCT
(1) preparation of fluorescent microsphere antibody complex
1. get 0.5mL rare-earth fluorescent microballoon (lanthanide series) (purchased from Shanghai Wei Ke power, article No.: DF020EU), centrifuging and taking precipitates, and adds the MES buffer solution twice of pH6.0.MES buffer formulation: the Brij35 weighing 9.76gMES (2-(N-morpholine) ethyl sulfonic acid), 29.22gNaCl, 5ml10%, is dissolved in 1L distilled water, regulates pH=6.0 with 3mol/LNaOH.
2. activate: the hydroxy thiosuccinimide (Sulfo-NHS) adding the carbodiimide (EDAC) of the 50mM of 114 μ L and the 50mM of 114 μ L in microballoon after washing respectively, room temperature concussion 1h.
3. labelled antibody: precipitation got by centrifugal microballoon, with PB buffer solution twice.
4. add the PCT monoclonal antibody A of appropriate amount, room temperature concussion 2h, makes carboxyl and-NH
2covalent bond, realize antibody labeling.
5. precipitation got by centrifugal microballoon, adds the azanol/10mMpH7.5PB damping fluid cancellation reaction of 50mM, room temperature concussion 5min; Then centrifugal microballoon, adds the thorough cancellation reaction of azanol damping fluid of 50mM, room temperature concussion 30min
6. centrifugal microballoon, adds confining liquid (0.5% casein, 10mMPB), room temperature concussion 2h.
7. add 400 μ L storage liquid (0.5wt%BSA, 0.5% (v/v) Tween-20,0.02wt% sodium azide, 10mMPB) and be stored in 4 DEG C.
(2) preparation of fluorescent microsphere antibody complex:
By the fluorescent microsphere specking being marked with PCT monoclonal antibody for preparing in rifle head, each rifle head 2.5 μ L, after-80 DEG C of refrigerator freezings spend the night, is fluorescent microsphere antibody complex after vacuum freezedrying 2h, can be used for assembling kit.
(3) preparation of coated film:
1. quilt is wrapped
Open the diaphragm of PVC base plate intermediate width 25mm, nitrocellulose filter is pasted on this, namely can be used for the bag quilt of T line and C line antibody.
The position of adjustment line is T line-spacing NC film lower edge (sample pad end) 7mm, C line-spacing NC film lower edge (adsorptive pads end) 11mm, T line and C distance between centers of tracks is 4mm.
Detection line (T line): with the PBS (pH7.4 comprises 3% methyl alcohol) of 0.01M, anti-PCT antibody (mouse source) is diluted to 1mg/mL and carries out bag quilt, line concentration is 1 μ L/cm, speed 100mm/s.
Nature controlling line (C line): with the PBS (pH7.4 comprises 3% methyl alcohol) of 0.01M, sheep anti-Mouse polyclonal antibody is diluted to 1mg/mL and carries out bag quilt, line concentration is 1 μ L/cm, speed 100mm/s.
2. dry
Put into 37 DEG C of baking ovens, dry 30-40min.
(4) preparation of sample pad:
Glass fibre membrane is cut to 31mm × 100mm, with rifle head, 1.91mL confining liquid (0.01MPB damping fluid, comprises 3wt%BSA) is spread evenly across glass fibre membrane surface, puts 37 DEG C of oven overnight dryings.
(5) assembling of Test paper card and shearing
Pasting nitrocellulose filter (NC film, be coated with PCT monoclonal antibody detection zone and sheep anti-Mouse quality control region) PVC base plate (wide 77mm) up and down paste adsorptive pads and processed good sample pad respectively in two ends, adsorptive pads is in narrower one end, width is 25mm, is cross-linked 2mm with coated film; Sample pad is in wider one end, and width is 31mm, is cross-linked 2mm with coated film.
Assembling completes, and cutting cutter is cut into 5mm width, is Test paper card.Concrete assembling mode as shown in Figure 1.
(6) preparation of immunofluorescence test card
A Test paper is fastened on plastic bottom card, and test card surface face card compresses, and face is stuck in the sample pad of corresponding Test paper card and position reserved well and the view window respectively of NC film.Be immunofluorescence test card.
(7) preparation of kit
By immunofluorescence test card, containing the rifle head of freeze-drying fluorescent microsphere antibody complex, containing sample diluting liquid (6%BSA, 5mM glucose, PB) cryopreservation tube, two bag 1g drying agent put into aluminium foil bag jointly, 4 DEG C of preservations after vacuum seal, have namely prepared the immunofluorescent reagent box detecting PCT.
Embodiment 2 uses kit of the present invention to detect PCT standard items
Standard items are carried out 10 times of dilutions with sample diluting liquid, make the sample of 100000ng/L, 10000ng/L, 1000ng/L, 100ng/L, 10ng/L by serial dilution PCT standard items (our company).Be arranged on pipettor with the rifle head containing freeze-drying fluorescent microsphere compound and get 75 μ L sample solutions, join in the sample buffer (0.25% casein, 40mMEDTA, PB) of 300 μ L, get 75 μ L points after mixing and be added in the well of test card.Room temperature leaves standstill 20min, test card is put into reading in Fluorescence Scanner.Be measured value with detection line fluorescent value divided by nature controlling line fluorescent value.Each sample concentration carries out measurement three times, after averaging, maps to sample concentration with measured value.As shown in Figure 2, peak shape is very sensitive for the peak shape figure of detection line and nature controlling line.The linear result of standard items is as shown in Figure 3, linear also very good.
The detection of PCT in the clinical blood sample of embodiment 3
Gather the positive and the negative sample of hospital PCT, detect by this kit (method is the same) and EISA two kinds of methods, calculate recall rate respectively.Result is as shown in table 1, and time-resolved fluorescence of the present invention quantitatively detects the method for recall rate apparently higher than ELISA of the kit of PCT.
The detection of PCT in table 1, clinical blood sample
| Negative sample number | Positive sample number | Negative recall rate | Positive rate | |
| ELISA | 128 | 356 | 95％ | 97％ |
| Kit of the present invention | 128 | 356 | 97％ | 99％ |
Blood sample is carried out to the experiment of accuracy correlativity simultaneously, the testing result of testing result to ELISA quantitatively detecting the kit of PCT with time-resolved fluorescence is mapped, as shown in Figure 4, the distribution of point is mainly distributed near miter angle straight line, matching obtains correlation curve, the related coefficient obtaining two kinds of methods is 0.9946, and the testing result of visible two kinds of methods is very consistent.
The explanation of above embodiment just understands core concept of the present invention for helping.It should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention, can also carry out some improvement and modification to the present invention, these improve and modify and also fall in the protection domain of the claims in the present invention.
Claims (9)
1. time-resolved fluorescence quantitatively detects a kit of PCT, comprises fluorescent microsphere antibody complex and immunofluorescence test card, it is characterized in that,
Fluorescent microsphere antibody complex: be PCT monoclonal antibody is marked on the complex that rare-earth fluorescent microballoon formed, adds to this complex in rifle head and freeze-drying forms, as detection antibody;
Immunofluorescence test card comprises Test paper card, and Test paper card is overlapped successively by sample pad, nitrocellulose filter and adsorptive pads and is affixed on liner plate and forms; Wherein, the detection line position on nitrocellulose filter is coated with another PCT monoclonal antibody, and nature controlling line position is coated with sheep anti-Mouse and resists more.
2. time-resolved fluorescence according to claim 1 quantitatively detects the kit of PCT, it is characterized in that, described complex adopts carboxylated rare-earth fluorescent microballoon as carrier, by carbodiimide and hydroxy thiosuccinimide by activated carboxylic, and-the NH in the carboxyl of activation and PCT monoclonal antibody
2covalent bond, the fluorescent microsphere complex of formation.
3. time-resolved fluorescence according to claim 2 quantitatively detects the kit of PCT, it is characterized in that, the concrete preparation method of described complex is:
(1) get rare-earth fluorescent microballoon, centrifuging and taking precipitates, with the MES buffer solution of pH6.0;
(2) activate: the carbodiimide and the hydroxy thiosuccinimide that add the volumetric molar concentrations such as equal-volume in microballoon after washing, activated carboxylic reaction is carried out in room temperature concussion;
(3) labelled antibody: the microballoon centrifuging and taking precipitation after step (2) activation, with adding appropriate PCT monoclonal antibody after PB buffer solution, room temperature concussion makes carboxyl and-NH
2covalent bond, realize antibody labeling;
(4) the microballoon centrifuging and taking through antibody labeling is precipitated, add azanol damping fluid cancellation reaction;
(5) through the microballoon centrifuging and taking precipitation of cancellation reaction, add confining liquid and close, finally add storage liquid and preserve;
Described confining liquid is 10mMPB solution, wherein containing 0.5wt% casein;
Described storage liquid is 10mMPB solution, wherein containing 0.5wt%BSA, 0.5% (v/v) Tween-20,0.02wt% sodium azide.
4. time-resolved fluorescence according to claim 1 quantitatively detects the kit of PCT, it is characterized in that, described sample pad is the glass fibre membrane through the 0.01MPB buffer blind containing 3wt%BSA.
5. time-resolved fluorescence according to claim 1 quantitatively detects the kit of PCT, it is characterized in that, the distance between described detection line and nature controlling line is 4mm.
6. time-resolved fluorescence according to claim 5 quantitatively detects the kit of PCT, it is characterized in that, the bee-line of detection line and sample pad end is 7mm, and the bee-line of nature controlling line distance adsorptive pads is 11mm.
7. time-resolved fluorescence according to claim 1 quantitatively detects the kit of PCT, it is characterized in that, immunofluorescence test card fastens on plastic bottom card by Test paper, face, test paper surface card compresses and forms, and face is stuck in position reserved well and the view window respectively of counter sample pad and nitrocellulose filter.
8. time-resolved fluorescence according to claim 1 quantitatively detects the kit of PCT, it is characterized in that, also comprises sample buffer, and it is the PB solution containing 0.25wt% casein and 40mMEDTA.
9. quantitatively detect the kit of PCT according to the arbitrary described time-resolved fluorescence of claim 1 ~ 8, it is characterized in that, also comprise dilution, diluent ingredient is the PB solution containing 6wt%BSA and 5mM glucose.
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| CN108089013A (en) * | 2018-01-05 | 2018-05-29 | 潍坊科瑞斯生物科技有限公司 | The preparation method of fluorometric reagent in a kind of dog Procalcitonin detection kit and the detection kit |
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| CN110488024B (en) * | 2019-09-23 | 2022-07-12 | 北京纳诺金生物科技有限公司 | Method for detecting multiple substances to be detected by adopting fluorescence technology and fluorescence detection product |
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