CN103926403A - Streptococcus-A quantum dot immunochromatographic detection reagent card and preparation method thereof - Google Patents
Streptococcus-A quantum dot immunochromatographic detection reagent card and preparation method thereof Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
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Abstract
The invention discloses a streptococcus-A quantum dot immunochromatographic detection reagent card and a preparation method of the streptococcus-A quantum dot immunochromatographic detection reagent card, and aims to provide the streptococcus-A quantum dot immunochromatographic detection reagent card which is simple, high in sensitivity and reliable in detection results. The detection reagent card comprises a box body (6), wherein a piece of detection test paper is arranged in the box body (6); the detection test paper comprises a bottom plate (1); a sample pad (2), an anti-streptococcus-A quantum dot anti-body markup pad (3), a coating film (4) and a water absorption pad (5) are connected with the bottom plate (1) in sequence; a streptococcus-A quantum antibody detection area line T and a goat anti-rabbit polyclonal anti-body quality control area line C are arranged on the coating film (4); the two lines are arranged in parallel; the streptococcus-A quantum dot immunochromatographic detection reagent card and the preparation method thereof belong to the field of fluorescence immunoassay.
Description
Technical field
The invention discloses a kind of reagent card that detects, specifically, one is measured the streptococcic detection reagent card of A family in pharyngeal swab, the invention also discloses the preparation method of this detection reagent card, belongs to fluorescence immunoassay detection field.
Background technology
One of common disease substance of children's upper respiratory tract infection, in children with respiratory infection, A family streptococcus positive rate is about 23.2%.As accepted not in time anti-infective therapy, comprise the allergic disease causing after acpuei pharyngitis, purulence born of the same parents disease, TSS, aggressive fascitis, scarlet fever, pneumonia, erysipelas and the streptococcal infection of A family, as acute rheumatic fever, rheumatic heart disease, acute glomerulonephritis.General A family streptococcus authentication method mainly contains clinical experience examination, bacterial cultivation at present.
On the low side according to the streptococcic accuracy rate of clinical experience diagnosis A family, according to statistics, even if the accuracy rate of veteran diagnosis also only has 45%-75%.Bacterial cultivation is A family streptococcus diagnosis " goldstandard ", and the method is that the throat swab of taking is inoculated in and on blood agar plate, hatches 24～48h, 10%CO
2or under anaerobic condition, can increase the streptococcic separation rate of A family, finally take serological method to differentiate A family streptococcus and other β hemolytic streptococcus, particularly C family and G family, but the time needing is longer.
Summary of the invention
For above-mentioned deficiency, the object of the present invention is to provide a kind of simple to operate, diagnosis rapidly, the reliable A of inspection testing result family streptococcus quantum dot immune chromatography detects reagent card.
For solving the problems of the technologies described above, last technical scheme provided by the invention is as follows: this A family streptococcus quantum dot immune chromatography detects reagent card, comprise the box body with lid, in box body, be provided with Test paper, described Test paper comprises base plate, on base plate, be connected and have sample pad, the quantum dot-labeled pad of anti-A family's streptococcus antibody, coated film and adsorptive pads successively, described coated film is provided with an A family streptococcus antibody test district T line and a goat-anti rabbit polyclonal antibody Quality Control district C line, and two line parallels are arranged.
Above-mentioned A family streptococcus quantum dot immune chromatography detects reagent card, and the lid of described box body is provided with well and result watch window.
Above-mentioned A family streptococcus quantum dot immune chromatography detects reagent card, described coated film two ends respectively with adsorptive pads and anti-A family's streptococcus antibody mutual overlapping connection of quantum dot-labeled pad, on the quantum dot-labeled pad of anti-A family's streptococcus antibody, overlayed sample pad.
Further, above-mentioned A family streptococcus quantum dot immune chromatography detects reagent card, and described sample pad is glass fiber sample pad.
Further, above-mentioned A family streptococcus quantum dot immune chromatography detects reagent card, and described coated film is nitrocellulose filter.
Further, above-mentioned A family streptococcus quantum dot immune chromatography detects reagent card, and the described quantum dot-labeled pad of anti-A family's streptococcus antibody is the polyester cellulose film containing quantum dot labelled antibody.
After of the present invention, a technical scheme is to provide the preparation method of this test card, and the method comprises the steps: to be connected and to have sample pad, the quantum dot-labeled pad of anti-A family's streptococcus antibody, coated film and adsorptive pads successively on base plate successively:
Wherein:
1) preparation method of sample pad is:
Sample pad is soaked 20～30 minutes with sample pad damping fluid, dry;
2) preparation method of the quantum dot-labeled pad of anti-A family's streptococcus antibody is:
1. with quantum dot damping fluid, water-soluble carboxyl based quantum dot QD525 is diluted to 1 μ M;
2. get the quantum dot of 1mL1 μ M, add 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide hydrochloric acid solution of 30 μ L, room temperature reaction 1～2 hour, with the super filter tube ultrafiltration of 30kDa, remove unreacted 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide hydrochloric acid, draw remaining liq in super filter tube, add the borate buffer of 10mM to revert to volume to 1mL;
3. anti-A family streptococcus antibody is added in the super filter tube of 100kDa, adjusting antibody content with borate buffer is 4mg/mL, by the amount of 200 μ g/1mL, by step B) in antibody join steps A) in quantum dot solution in, room temperature reaction 1～2 hour;
4. by the amount of 10 μ L/1mL, to step C) in add glycocoll, seal the not activated carboxyl site of coupling antibody, reaction 30～60min;
5. by step D) in the super filter tube that joins of the quantum dot solution of coupling antibody, add Tris-HCl solution displacement quantum dot damping fluid, repeat 3～5 times, draw remaining liq in super filter tube, adding Tris-HCl is 100 μ L to final volume, centrifuging, get supernatant, and add antibody to preserve liquid;
6. the solution of above-mentioned gained is drawn to film instrument with metal spraying and be sprayed onto on polyester cellulose film, discharge rate is 1～5 μ L/cm, dry;
3) preparation method of coated film is:
1. by 0.02M phosphate buffer dilution 0.5～2mg/mL for goat anti-rabbit antibody, add 3% methyl alcohol, be Quality Control district working fluid;
2. will resist A family streptococcus antibody 0.02M phosphate buffer to be diluted to 0.8～1.5mg/mL, add 3% methyl alcohol, be detection zone working fluid;
3. by step 1., step 2.) solution of gained, draw film instrument with metal spraying and be coated with on nitrocellulose membrane with 0.8-1.2 μ L/cm, detection zone and Quality Control Interval Distance are 0.4-0.7cm, are positioned at nitrocellulose filter centre position, nitrocellulose membrane is put into 37 DEG C of baking ovens, dry.
Further, above-mentioned A family streptococcus quantum dot immune chromatography detects the preparation method of reagent card, the step borate buffer solution that 1. described quantum dot damping fluid is pH8.4.
Further, above-mentioned A family streptococcus quantum dot immune chromatography detects the preparation method of reagent card, and described sample pad damping fluid is the 0.01M phosphate buffer containing 0.5～5wt%BSA, 0.5～5wt% polyvinylpyrrolidone, 0.5～5wt% polyglycol, 0.1～5wt% Tween-20, bent La Tong-100 of 1～5wt%.
Further, above-mentioned A family streptococcus quantum dot immune chromatography detects the preparation method of reagent card, and described antibody is preserved liquid for containing 1wt%BSA, 20wt% sucrose, 10wt% trehalose, 0.1wt% Tween-20, the 0.05M Tris-HCl damping fluid of 0.1wt% polyvinylpyrrolidone.
Compared with prior art, A provided by the present invention family streptococcus quantum dot immune chromatography detects reagent card tool and has the following advantages:
Technical scheme provided by the invention, utilize the specific binding reaction of two kinds of antibody, form special " sandwich " structure, i.e. " antibody-Ag-Ab " immune complex, binding immunoassay chromatographic technique, application fluorescence immune chromatography method detects A family streptococcal antigen, and matches with fluorescent reagent card chart scanner, increases substantially the streptococcic recall rate of A family.
This method is simple to operate, and diagnosis is rapid, and high specificity is highly sensitive, easily stores, and operating personnel that need not be special, only need simple instrument and equipment, can obtain a result.Be conducive to like this medical personnel and catch golden hour, avoid antibiotic abuse, reduce A family streptococcus allergic reaction odds simultaneously.
Brief description of the drawings
Fig. 1 is detection reagent card structural representation provided by the invention;
Fig. 2 is the lid vertical view of detection reagent card provided by the invention;
Fig. 3 is Test paper structural representation provided by the invention;
Fig. 4 is Test paper vertical view provided by the invention;
Fig. 5 is test card result of determination provided by the invention schematic diagram when negative;
Fig. 6 is test card result of determination provided by the invention schematic diagram when positive.
Wherein: base plate 1, sample pad 2, the quantum dot-labeled pad 3 of anti-A family's streptococcus antibody, coated film 4, adsorptive pads 5, the streptococcus antibody test district T of A family line, goat-anti rabbit polyclonal antibody Quality Control district C line, box body 6, lid 61, well 71, result watch window 72.
Embodiment
Mode below in conjunction with specific embodiment is described in further detail claim of the present invention, but do not form any limitation of the invention, the amendment of anyone limited number of time making within the scope of the claims in the present invention, still within claim scope of the present invention.
Embodiment 1
A kind of A provided by the invention family streptococcus quantum dot immune chromatography detects reagent card, consult Fig. 1 to Fig. 4, described detection reagent card comprises the box body 6 with lid 61, and in order to facilitate application of sample and to observe testing result, described lid 61 is provided with well 71 and result watch window 72.
In box body 6, be provided with Test paper, described Test paper comprises base plate 1, described base plate 1 is PVC plate, on base plate 1, be connected and have sample pad 2, the quantum dot-labeled pad 3 of anti-A family's streptococcus antibody, coated film 4 and adsorptive pads 5 successively, on coated film 4, be provided with an A family streptococcus antibody test district T line and a goat-anti rabbit polyclonal antibody Quality Control district C line, two line parallels are arranged.In order to reach testing result fast and accurately, described sample pad 2 preferred glass fibers sample pad; The described preferred nitrocellulose filter of coated film 4; The described quantum dot-labeled pad 3 of anti-A family's streptococcus antibody is polyester cellulose film.
In particular, described coated film 4 overlays in base plate 1 middle part, the two ends of coated film 4 respectively with adsorptive pads 5 and anti-A family's streptococcus antibody mutual overlapping connection of quantum dot-labeled pad 3, on the quantum dot-labeled pad 3 of anti-A family's streptococcus antibody, overlayed sample pad 2.
Sample pad 2 is pressed in 1/3～1/4 left and right of the quantum dot-labeled pad 3 of anti-A family's streptococcus antibody, the quantum dot-labeled pad 3 of anti-A family's streptococcus antibody is pressed in the 1mm place of coated film, the streptococcus antibody test district T of A family linear distance coated film 4 left end 10mm, goat-anti rabbit polyclonal antibody Quality Control district C linear distance coated film 4 right-hand member 10mm, C, T distance between centers of tracks 4～7mm (preferably 5mm), adsorptive pads is pressed in coated film 1～2.5mm (preferably 2mm) and locates.
This A family streptococcus quantum dot immune chromatography that the present invention also provides detects the preparation method of reagent card:
1) preparation method of sample pad is:
After sample pad (pH7.4) is soaked completely with the 0.01M phosphate buffer (0.01M sodium hydrogen phosphate: 0.01M sodium dihydrogen phosphate=81:19 (V:V)) containing 0.5～5wt%BSA, 0.5～5wt% polyvinylpyrrolidone, 0.5～5wt% polyglycol, 0.1～5wt% Tween-20, bent La Tong-100 of 1～5wt%, 37 DEG C of oven dry or dry under the environment of relative humidity≤35%.
Sample pad damping fluid is preferably and in 0.01M phosphate buffer, dissolves bent La Tong-100 of 0.5wt%BSA, 0.5wt% polyvinylpyrrolidone, 0.5wt% polyglycol, 0.25wt% Tween-20 and 1wt%.
2) preparation method of the quantum dot-labeled pad of anti-A family's streptococcus antibody is:
1. the borate buffer (2.5mM borax: 10mM boric acid=4.5:5.5 (V:V)) of using 10mM pH8.4, is diluted to 1 μ M by the QD525 of water-soluble carboxyl based quantum dot 8 μ M.
2. get the quantum dot of 1mL1 μ M, add 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide hydrochloric acid solution of 30 μ L10mg/mL, room temperature reaction 1～2 hour.With the super filter tube ultrafiltration of 30kDa, remove unreacted 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide hydrochloric acid, draw remaining liq in super filter tube, add the borate buffer of 10mM to revert to volume to 1mL.
3. anti-A family streptococcus antibody is added in the super filter tube of 100kDa, adjusting antibody content with 10mM borate buffer is 4mg/mL.
3. by the amount of 200 μ g/1mL, the antibody that 2. step is prepared joins step 1. in quantum dot solution, room temperature reaction 1～2 hour.
4. by the amount of 10 μ L/1mL, add the glycocoll of 10mg/mL in 3. to step, seal the not activated carboxyl site of coupling antibody, reaction 30～60min.
5. the quantum dot solution of coupling antibody is joined in the super filter tube of 30kDa, add pH9.0 concentration 0.05M Tris-HCl solution substitutional solution buffer system, repeat 3～5 times, recovering final volume is 100 μ L.
6. by step in 5. to be dissolved in 4 DEG C of 1800g of liquid centrifugal, get supernatant.
7. 6. in the supernatant of gained, add 100 μ L antibody to preserve liquid in step, described body is preserved liquid for containing 1wt%BSA, 20wt% sucrose, 10wt% trehalose, the 0.05M Tris-HCl damping fluid of 0.1wt% Tween-20 and 0.1wt% polyvinylpyrrolidone.
8. the solution of above-mentioned gained is drawn to film instrument with metal spraying and be sprayed onto on polyester cellulose film, discharge rate is 1～5 μ L/cm (preferably 2 μ L/cm), puts into 37 DEG C of baking ovens, dry 2～5 hours.
3) preparation method of coated film is:
1. goat anti-rabbit antibody is diluted to 1mg/mL with 0.02M phosphate buffer, adds 3% methyl alcohol, be Quality Control district working fluid.
2. will resist A family streptococcus antibody 2 use 0.02M phosphate buffers to be diluted to 1.2mg/mL, add 3% methyl alcohol, be detection zone working fluid.
3. by 1., the 2. solution of gained, draw film instrument with metal spraying and be coated with on nitrocellulose membrane with 1 μ L/cm.Detection zone and Quality Control Interval Distance are 0.4～0.7cm (preferably 0.5cm), are positioned at nitrocellulose filter centre position.
4. by step 3. nitrocellulose membrane put into 37 DEG C of baking ovens, dried overnight.
4) assembling:
1. the stickup of nitrocellulose filter: open gently the diaphragm of NC film location for paste on PVC plate, NC film is evenly advanced from left to right, it is firmly sticked on PVC plate.
2. the stickup of adsorptive pads: PVC plate is laid on work top, opens gently the diaphragm of absorption pad location for paste on PVC plate, absorption pad is adhered thereto; evenly, slight tumbling-type advances; to add strong adhesive power, and prevent that bubble, absorption pad cover 1mm place on NC film.
The stickup of the quantum dot-labeled pad of ③Kang A family streptococcus antibody: quantum dot-labeled pad is cut out as 5mm × 300mm.Open gently the diaphragm of the quantum dot-labeled pad of NC film upper limb location for paste, by adhered thereto quantum dot-labeled collaurum pad, the same absorption pad of method.The quantum dot-labeled pad 3 of anti-A family's streptococcus antibody covers 1mm place on NC film.
4. the stickup of sample pad: dried sample pad is cut out as after 18mm × 300mm, open gently thin plate diaphragm topmost, sample pad is adhered to the anti-A quantum dot-labeled pad of family's streptococcus antibody 3 tops, the same absorption pad of method.Sample pad covers 2mm place on the quantum dot-labeled pad of anti-A family's streptococcus antibody.
The reinforcing of the quantum dot-labeled pad of ⑤Kang A family streptococcus antibody: will be connected adhesive tape and cut out as 12mm × 300mm, and paste sample pad place, top is apart from test strips sample pad end 15mm.
6. slitting be installed: the PVC plate pasting is cut into the wide test strips of 3.0mm (2.5～4.5mm).Each test strips is packed in plastic clip, each reagent card is placed in to aluminum foil bag, and add 1g drying agent 1 to wrap, heat sealing.
This A family streptococcus quantum dot immune chromatography detects the concrete using method of reagent card:
After sampling, in extraction tube, add each 3～4 of lysate A (1M sodium nitrite) and lysate B (2M acetic acid), put into extruding after cotton swab and several times, leave standstill 3～5min.
When detection, consult Fig. 5 and Fig. 6, sample lysate is splashed in the well of this detection reagent to 3～4, because detecting sample, capillarity will move to adsorptive pads end along Test paper, A family streptococcus in sample is after lysate cracking, can with anti-A family streptococcus polyclonal antibody fluorescence labeling probe generation specific bond, then continuing mobile antibody on being coated on nitrocellulose filter is combined, thereby identified by the streptococcic polyclonal antibody of anti-A family, there is the specific bond of double-antibody sandwich, with hand-held ultraviolet light irradiation watch window, there is quantum dot fluorescence band in detection zone, thereby carry out the qualification of respiratory tract infection pathogeny by the A family streptococcus detecting in sample.In testing process, the fluorescence probe that is stranded in detection zone when up liquid level will show corresponding fluorescence.Setting A family streptococcus boundary value is 10
3cFU/mL, in the time that Quality Control district and detection zone have fluorescence to occur, in interpret sample, the streptococcic content of A family exceedes 10
3cFU/mL, positive; When detection zone occurs without fluorescence when only having Quality Control district to have fluorescence, in interpret sample, do not contain A family streptococcus or content lower than 10
3cFU/mL, negative.
In the time moving to goat-anti rabbit polyclonal antibody Quality Control district, no matter in sample, A family streptococcus has or not, and the goat anti-rabbit igg that fluorescence probe all can be coated on nitrocellulose filter is combined delay, makes Quality Control district show fluorescence.Therefore Quality Control district produces and represents that operation is wrong or testing result is invalid without fluorescence, needs revision test.
For better explanation beneficial effect of the present invention, provide a result contrast experiment who adopts detection reagent card provided by the invention and traditional microbe growth detection method, clinical experience to screen below.
Test example 1
Claims (10)
1. an A family streptococcus quantum dot immune chromatography detects reagent card, comprise the box body (6) with lid, box body is provided with Test paper in (6), described Test paper comprises base plate (1), it is characterized in that, on base plate (1), be connected and have sample pad (2), the anti-A quantum dot-labeled pad of family's streptococcus antibody (3), coated film (4) and adsorptive pads (5) successively, described coated film (4) is provided with an A family streptococcus antibody test district T line and a goat-anti rabbit polyclonal antibody Quality Control district C line, and two line parallels are arranged.
2. A according to claim 1 family streptococcus quantum dot immune chromatography detects reagent card, it is characterized in that, the lid (61) of described box body (6) is provided with well (71) and result detection window (72).
3. A according to claim 1 family streptococcus quantum dot immune chromatography detects reagent card, it is characterized in that, described coated film (4) two ends respectively with adsorptive pads (5) and mutual overlapping connection of the anti-A quantum dot-labeled pad of family's streptococcus antibody (3), on the anti-A quantum dot-labeled pad of family's streptococcus antibody (3), overlayed sample pad (2).
4. A according to claim 1 family streptococcus quantum dot immune chromatography detects reagent card, it is characterized in that, described sample pad (2) is glass fiber sample pad.
5. A according to claim 1 family streptococcus quantum dot immune chromatography detects reagent card, it is characterized in that, described coated film (4) is nitrocellulose filter.
6. A according to claim 1 family streptococcus quantum dot immune chromatography detects reagent card, it is characterized in that, the described quantum dot-labeled pad of anti-A family's streptococcus antibody is coated quantum dot-labeled A family streptococcus antibody polyester cellulose film.
7. preparation A claimed in claim 1 family streptococcus quantum dot immune chromatography detects reagent card, it is characterized in that, comprise the steps: to be successively connected and to have sample pad (2), the anti-A quantum dot-labeled pad of family's streptococcus antibody (3), coated film (4) and adsorptive pads (5) successively on base plate (1):
Wherein:
1) preparation method of sample pad is:
Sample pad is soaked 20～30 minutes with sample pad damping fluid, dry.
2) preparation method of the quantum dot-labeled pad of anti-A family's streptococcus antibody is:
1. with quantum dot damping fluid, water-soluble carboxyl based quantum dot QD525 is diluted to 1 μ M;
2. get the quantum dot of 1mL1 μ M, add 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide hydrochloric acid solution of 30 μ L, room temperature reaction 1～2 hour, with the super filter tube ultrafiltration of 30kDa, remove unreacted 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide hydrochloric acid, draw remaining liq in super filter tube, add the borate buffer of 10mM to revert to volume to 1mL;
3. anti-A family streptococcus antibody is added in the super filter tube of 100kDa, adjusting antibody content with borate buffer is 4mg/mL, by the amount of 200 μ g/1mL, by step B) in antibody join steps A) in quantum dot solution in, room temperature reaction 1～2 hour;
4. by the amount of 10 μ L/1mL, to step C) in add glycocoll, seal the not activated carboxyl site of coupling antibody, reaction 30～60min;
5. by step D) in the super filter tube that joins of the quantum dot solution of coupling antibody, add Tris-HCl solution displacement quantum dot damping fluid, repeat 3～5 times, draw remaining liq in super filter tube, adding Tris-HCl is 100 μ L to final volume, centrifuging, get supernatant, and add antibody to preserve liquid;
6. the solution of above-mentioned gained is drawn to film instrument with metal spraying and be sprayed onto on polyester cellulose film, discharge rate is 1～5 μ L/cm, dry.
3) preparation method of coated film is:
1. by 0.02M phosphate buffer dilution 0.5～2mg/mL for goat anti-rabbit antibody, add 3% methyl alcohol, be Quality Control district working fluid;
2. will resist A family streptococcus antibody 0.02M phosphate buffer to be diluted to 0.8～1.5mg/mL, add 3% methyl alcohol, be detection zone working fluid;
3. by step 1., the 2. solution of gained of step, draw film instrument with metal spraying and be coated with on nitrocellulose membrane with 0.8-1.2 μ L/cm, detection zone and Quality Control Interval Distance are 0.4-0.7cm, are positioned at nitrocellulose filter centre position, nitrocellulose membrane is put into 37 DEG C of baking ovens, dry.
8. A according to claim 7 family streptococcus quantum dot immune chromatography detects the preparation method of reagent card, it is characterized in that step 2) borate buffer solution that is pH8.4 of the quantum dot damping fluid described in 1..
9. A according to claim 7 family streptococcus quantum dot immune chromatography detects the preparation method of reagent card, it is characterized in that, described sample pad damping fluid is the 0.01M phosphate buffer containing 0.5～5wt%BSA, 0.5～5wt% polyvinylpyrrolidone, 0.5～5wt% polyglycol, 0.1～5wt% Tween-20, bent La Tong-100 of 1～5wt%.
10. A according to claim 7 family streptococcus quantum dot immune chromatography detects the preparation method of reagent card, it is characterized in that, described antibody is preserved liquid for containing 1wt%BSA, 20wt% sucrose, 10wt% trehalose, 0.1wt% Tween-20, the 0.05M Tris-HCl damping fluid of 0.1wt% polyvinylpyrrolidone.
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