CN103926404A - Group B streptococcus quantum dot immunochromatographic detection reagent card and preparation method for same - Google Patents

Group B streptococcus quantum dot immunochromatographic detection reagent card and preparation method for same Download PDF

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Publication number
CN103926404A
CN103926404A CN201410178962.6A CN201410178962A CN103926404A CN 103926404 A CN103926404 A CN 103926404A CN 201410178962 A CN201410178962 A CN 201410178962A CN 103926404 A CN103926404 A CN 103926404A
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quantum dot
streptococcus
family
antibody
line
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CN103926404B (en
Inventor
汤永平
潘秀华
张晓丽
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GUANGZHOU MICRON BIOTECHNOLOGY Co Ltd
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GUANGZHOU MICRON BIOTECHNOLOGY Co Ltd
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911Bacteria
    • G01N33/56944Streptococcus
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366Apparatus specially adapted for solid-phase testing
    • G01N33/54386Analytical elements
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/315Assays involving biological materials from specific organisms or of a specific nature from bacteria from Streptococcus (G), e.g. Enterococci

Abstract

The invention discloses a group B streptococcus quantum dot immunochromatographic detection reagent card and a preparation method for the same, and aims to provide a simple, convenient and high-sensitivity group B streptococcus quantum dot immunochromatographic detection reagent card by using which a reliable detection result can be obtained. The detection reagent card comprises a box body (1) with a box cover, wherein test paper is arranged in the box body (1), and comprises a bottom plate (1); a sample pad (2), an anti-group B streptococcus quantum dot marker pad (3), a coating membrane (4) and an absorbent pad (5) are sequentially connected with the bottom plate (1); a group B streptococcus antibody detection area T line and a goat anti-rabbit polyclonal antibody quality control area C line are arranged in parallel on the coating membrane (4). The group B streptococcus quantum dot immunochromatographic detection reagent card and the preparation method for the same belong to the field of fluorescent quantum dot labeled immunological detection.

Description

B family streptococcus quantum dot immune chromatography detects reagent card and preparation method thereof
Technical field
The invention discloses a kind of reagent card that detects, specifically, one is measured the streptococcic detection reagent card of B family in pregnant woman's uterine neck or vagina, the invention also discloses the preparation method of this detection reagent card, belongs to fluorescence quantum point mark field of immunodetection.
Background technology
B family streptococcus (Group B Streptococcus is called for short GBS) can cause sepsis of the newborn, pneumonia, meningitis, even dead.The neonate of surviving afterwards in infection, also likely has serious nervous system sequelae, comprises hydrocephalus, dysnoesia, microcephalus, deafness etc.Meanwhile, B family streptococcus also can cause infection of pregnant women, cause premature labor, development of fetus bad (infant of low-birth weight), premature rupture of fetal membranes and late abortion.Therefore, extremely important and necessary to the streptococcic detection of pregnant woman B family.At present, the streptococcic detection of B family mainly contains following three kinds of methods:
1, traditional microbe growth detection method, the testing result of the method is more accurate, but detection time is longer, generally needs 2-3 days out results, and efficiency is lower;
2, PCR detection method, this detection method is easy fast, and sensitivity is higher, still, when detection, easily cause intersecting amplified reaction, thereby occur false positive results, so, need technical professional and special instrument and equipment, therefore, the range of application of the method is very restricted.
3, collaurum detection method, the method is simple to operate, but sensitivity is low, and particularly infective dose is few, may occur situation undetected, flase drop, brings serious harm to like this prenatal and postnatal care of fetus.
Summary of the invention
For above-mentioned deficiency, the object of the present invention is to provide a kind of detection method simple, quick, highly sensitive, the reliable B of testing result family streptococcus quantum dot immune chromatography detects reagent card.
For solving the problems of the technologies described above, the last technical scheme providing of the present invention is such: this B 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-B family's streptococcus antibody, coated film and adsorptive pads successively, described coated film is provided with a B 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.
Further, above-mentioned B family streptococcus quantum dot immune chromatography detects reagent card, and the lid of described box body is provided with well and result watch window.
Further, above-mentioned B family streptococcus quantum dot immune chromatography detects reagent card, described coated film two ends respectively with adsorptive pads and anti-B family's streptococcus antibody mutual overlapping connection of quantum dot-labeled pad, on the quantum dot-labeled pad of anti-B family's streptococcus antibody, overlayed sample pad.
Further, above-mentioned B family streptococcus quantum dot immune chromatography detects reagent card, and described sample pad is glass fiber sample pad.
Further, above-mentioned B family streptococcus quantum dot immune chromatography detects reagent card, and described coated film is nitrocellulose filter.
Further, above-mentioned B family streptococcus quantum dot immune chromatography detects reagent card, and the described quantum dot-labeled pad of anti-B family's streptococcus antibody is the polyester cellulose film of coated quantum dot-labeled B family streptococcus antibody.
After of the present invention, a technical scheme is to provide the preparation method of this B family streptococcus quantum dot immune chromatography detection reagent card, and the method comprises the steps: to be connected and to have sample pad, the quantum dot-labeled pad of anti-B family streptococcus, coated film and adsorptive pads successively on base plate successively:
Wherein:
1) preparation method of sample pad is: sample pad is cut into the size of 20mm × 300mm, is immersed in sample pad damping fluid, take out, in drying at room temperature 16-18 hour after 1 hour.
The buffer formulation of sample pad is as follows: 2%BSA, 1%PVP, 0.5%Tween are dissolved in 0.01 PBS (pH7.4) damping fluid.
2) preparation method of the quantum dot-labeled pad of anti-B family's streptococcus antibody is:
1. use the borate buffer (2.5mM borax: 10mM boric acid=4.5:5.5 (V:V)) of 10mM pH8.4 that the QD525 of water-soluble carboxyl based quantum dot 8 μ M is diluted to 1 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 above-mentioned damping fluid and revert to original volume.
2. anti-B family streptococcus antibody is added in the super filter tube of 100kDa, antibody concentration is adjusted into 4mg/mL with the borate buffer of the pH8.4 of 0.05M.
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 glycocoll to step in 3., 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. at 4 DEG C of 1800g centrifuging and taking supernatants of solution;
7. 6. in the supernatant of gained, add 100 μ L antibody to preserve liquid in step, described preservation liquid is 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. coated
Detection line (T line): rule on nitrocellulose membrane, carry out the mark of C line and T line with marking pen in one end of film, the distance of C line and T line is 0.5cm, the distance of T line and nitrocellulose filter lower edge is 1cm, B family streptococcus antibody dilution is coated with to 1.2mg/mL with the PBS (pH7.4) of 0.01M, line concentration is 1 μ L/cm, speed 100mm/s.
Nature controlling line (C line): with the PBS (pH7.4) of 0.01M, goat-anti rabbit polyclonal antibody is diluted to 1.5mg/mL and is coated with, line concentration is 1 μ L/cm, speed 100mm/s.
2. dry
Put into 37 DEG C of baking ovens, dry 2~5 hours.
Further, above-mentioned B family streptococcus quantum dot immune chromatography detects the preparation method of reagent card, the borate buffer of the step pH8.4 that 1. described quantum dot damping fluid is 0.05M.
Further, above-mentioned B family streptococcus quantum dot immune chromatography detects the preparation method of reagent card, and the formula of described sample pad damping fluid is the 0.01M PBS damping fluid containing 2wt%BSA, 1w%PVP, 0.5w%Tween.
Further, above-mentioned B family streptococcus quantum dot immune chromatography detects the preparation method of reagent card, and described preservation liquid is for containing 1w%BSA, 20w% sucrose, 10w% trehalose, the 0.05MTris-HCl damping fluid of 0.1w% Tween-20 and 0.1w% polyvinylpyrrolidone.
Compared with prior art, B provided by the present invention family streptococcus quantum dot immune chromatography detects reagent card tool and has the following advantages:
1, compared with traditional microbe growth detection method: microbe growth needs within 2-3 days, to go out result, and the present invention only needs 10-15min, has greatly shortened detection time, has improved detection efficiency;
2, compared with PCR detection method: do not need large-scale instrument and equipment, without technical skill personnel operation, saved cost;
3, compared with colloidal gold method: improved sensitivity and recall rate, the sensitivity of gold-immunochromatographyreagent reagent for assay is 10 5cFU/mL, the sensitivity that the present invention detects reagent is 10 3cFU/mL, is obviously better than colloidal gold method.
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-B family's streptococcus antibody, coated film 4, adsorptive pads 5, the streptococcus antibody test district T of B 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 B provided by the invention family streptococcus quantum dot immune chromatography detects reagent card, consults Fig. 1 to Fig. 4, comprises the box body 6 with lid, and in order to facilitate application of sample and to observe testing result, the lid 61 of described box body 6 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, base plate 1 is PVC base plate, on base plate 1, be connected and have sample pad 2, the quantum dot-labeled pad 3 of anti-B family's streptococcus antibody, coated film 4 and adsorptive pads 5 successively, on coated film 4, be provided with a B 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 of anti-B family's streptococcus antibody is coated quantum dot-labeled B family streptococcus antibody 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-B family's streptococcus antibody mutual overlapping connection of quantum dot-labeled pad 3, on the quantum dot-labeled pad 3 of anti-B family's streptococcus antibody, overlayed sample pad 2.
Sample pad 2 covers the quantum dot-labeled pad of anti-B family's streptococcus antibody 1mm left and right, the quantum dot-labeled pad 3 of anti-B family's streptococcus antibody covers the about 1mm of coated film 4, the streptococcus antibody test district T of B family linear distance coated film 3 left end 7mm, goat-anti rabbit polyclonal antibody Quality Control district C linear distance coated film 4 right-hand member 8mm, C, T spacing 5mm, adsorptive pads is pressed in coated film 2mm place.
This B family streptococcus quantum dot immune chromatography that the present invention also provides detects the preparation method of reagent card:
Wherein:
1) preparation method of sample pad is: sample pad is cut into the size of 20mm × 300mm, is immersed in sample pad damping fluid
In, within 1 hour, take out afterwards, in drying at room temperature 16-18 hour.
The buffer formulation of sample pad is as follows: 2%BSA, 1%PVP, 0.5%Tween are dissolved in 0.01 PBS (pH7.4) damping fluid.
2) preparation method of the quantum dot-labeled pad of anti-B family's streptococcus antibody is:
1. use the borate buffer (2.5mM borax: 10mM boric acid=4.5:5.5 (V:V)) of 10mM pH8.4 that the QD525 of water-soluble carboxyl based quantum dot 8 μ M is diluted to 1 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 above-mentioned damping fluid and revert to original volume.
2. anti-B family streptococcus antibody is added in the super filter tube of 100kDa, antibody concentration is adjusted into 4mg/mL with the borate buffer of the pH8.4 of 0.05M.
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 glycocoll to step in 3., 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. at 4 DEG C of 1800g centrifuging and taking supernatants of solution;
7. 6. in the supernatant of gained, add 100 μ L antibody to preserve liquid in step, described preservation liquid is 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. coated
Detection line (T line): rule on nitrocellulose membrane, carry out the mark of C line and T line with marking pen in one end of film, the distance of C line and T line is 0.5cm, the distance of T line and nitrocellulose filter lower edge is 1cm, B family streptococcus antibody dilution is coated with to 1.2mg/mL with the PBS (pH7.4) of 0.01M, line concentration is 1 μ L/cm, speed 100mm/s.
Nature controlling line (C line): with the PBS (pH7.4) of 0.01M, goat-anti rabbit polyclonal antibody is diluted to 1.5mg/mL and is coated with, line concentration is 1 μ L/cm, speed 100mm/s.
2. dry
Put into 37 DEG C of baking ovens, dry 2~5 hours.
The assemble method of B family streptococcus quantum dot immune chromatography detection reagent card is as follows:
1. the stickup of absorption pad: base plate is laid on work top; Open gently the diaphragm of absorption pad location for paste on base plate, absorption pad is adhered thereto, and even, slight tumbling-type advances, and to add strong adhesive power, and prevents that bubble, absorption pad cover 1mm on nitrocellulose filter.
The quantum dot-labeled pad of ②Kang B family streptococcus antibody: will resist the quantum dot-labeled pad of B family streptococcus antibody cut out for 10mm ×
300mm; open gently the diaphragm of the anti-B of the nitrocellulose filter upper limb quantum dot-labeled pad of family's streptococcus antibody location for paste; to resist the B family quantum dot-labeled pad of streptococcus antibody adhered thereto, the same absorption pad of method, the quantum dot-labeled pad of anti-B family's streptococcus antibody covers 1mm on nitrocellulose filter.
3. the stickup of sample pad: open gently thin plate diaphragm topmost, sample pad is adhered to the anti-B quantum dot-labeled pad of family's streptococcus antibody top, the same absorption pad of method.Sample pad covers 2mm on the quantum dot-labeled pad of anti-B family's streptococcus antibody.
3. test strips cutting: the base plate pasting is cut into the wide test strips of 4.0mm.
5. be installed and enter bag: each test strips being 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 B family streptococcus quantum dot immune chromatography detects the concrete using method of reagent card:
When use, remove external packing, take out reagent card, the B family streptococcus of getting 150 μ L left and right extractings adds in sample well, because capillarity sample will move to adsorptive pads end along reagent strip, in the time of sample Zhong You B family streptococcus, B family streptococcus in sample is after extract extracting, can with the quantum dot-labeled probe generation of anti-B family's streptococcus polyclonal antibody specific bond, then continuing mobile antibody on being coated on nitrocellulose filter is combined, thereby identified by the streptococcic polyclonal antibody of anti-B family, there is the specific bond of double antibodies sandwich, with hand-held ultraviolet light irradiation watch window, there is quantum dot fluorescence band in detection line, thereby by detecting the B family streptococcus in sample, carry out the streptococcic early screening of pregnant woman B family.
Result judgement: with hand-held ultraviolet light irradiation watch window, in the time that nature controlling line and detection line have quantum dot fluorescence to occur, in interpret sample, the streptococcic content of B family exceedes 10 3cFU/mL, positive; When only having nature controlling line to have quantum dot fluorescence, detection line occurs without quantum dot fluorescence, in interpret sample, the streptococcic content of B family is lower than 10 3cFU/mL, negative, consult Fig. 5, Fig. 6; There is not quantum dot fluorescence in nature controlling line, represents that operation is wrong or testing result is invalid, needs revision test.
For better explanation beneficial effect of the present invention, provide below adopt detection reagent provided by the invention with traditional microbe growth detection method, PCR detection method, the result contrast experiment of collaurum method in the time detecting B family streptococcus.
Test example 1
Tradition microbe growth concrete steps are as follows: vagina cotton swab is inoculated on Sheep Blood agar plate, after 14-18h cultivates, getting β hemolysin with oese drips in circular projection, tiny suspicious be streptococcic single colony edge, again flat board is hatched, respectively 30,45,60min checks haemolysis situation of change.There is the collaborative positive reaction that produces " fan-shaped " enhancing haemolysis district at the colony edge that drips part hemolysin, can be accredited as B group streptococcus.There is not strengthening the negative reaction of haemolysis, be judged to non-B group streptococcus.When each inspection, all test in contrast with known positive strain.
The concrete detection method of PCR method: use fluorescent quantitative PCR technique to detect B family streptococcus, taking the scpB gene of the C5a peptase of encoding during as target gene sensitivity as 99.6%, specificity is as 100%, sensitivity can reach 10 2cFU/mL.
Colloid gold reagent bar using method is substantially similar to method provided by the invention, but sensitivity is lower.
Using method Equipment needed thereby Detection time Sensitivity
Traditional microbe growth Selective medium 24-48 hour 10 6CFU/mL
PCR method PCR instrument 6-8 hour 10 2CFU/mL
Colloid gold reagent bar Nothing 10-20 minute 10 5CFU/mL
Method provided by the invention Hand-held ultraviolet lamp 10-20 minute 10 3CFU/mL

Claims (10)

1. a B 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-B 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 a B 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. B 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 watch window (72).
3. B 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-B quantum dot-labeled pad of family's streptococcus antibody (3), on the anti-B quantum dot-labeled pad of family's streptococcus antibody (3), overlayed sample pad (2).
4. B 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. B 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. B 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-B family's streptococcus antibody is coated quantum dot-labeled B family streptococcus antibody polyester cellulose film.
7. preparation B 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-B quantum dot-labeled pad of family streptococcus (3), coated film (4) and adsorptive pads (5) successively on base plate (1):
Wherein:
1) preparation method of sample pad is: sample pad is cut into the size of 20mm × 300mm, is immersed in sample pad damping fluid, take out, in drying at room temperature 16-18 hour after 1 hour.
The buffer formulation of sample pad is as follows: 2%BSA, 1%PVP, 0.5%Tween are dissolved in 0.01 PBS (pH7.4) damping fluid.
2) preparation method of the quantum dot-labeled pad of anti-B family's streptococcus antibody is:
1. use the borate buffer (2.5mM borax: 10mM boric acid=4.5:5.5 (V:V)) of 10mM pH8.4 that the QD525 of water-soluble carboxyl based quantum dot 8 μ M is diluted to 1 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 above-mentioned damping fluid and revert to original volume.
2. anti-B family streptococcus antibody is added in the super filter tube of 100kDa, antibody concentration is adjusted into 4mg/mL with the borate buffer of the pH8.4 of 0.05M.
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 glycocoll to step in 3., 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. at 4 DEG C of 1800g centrifuging and taking supernatants of solution;
7. 6. in the supernatant of gained, add 100 μ L antibody to preserve liquid in step, described preservation liquid is 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. coated
Detection line (T line): rule on nitrocellulose membrane, carry out the mark of C line and T line with marking pen in one end of film, the distance of C line and T line is 0.5cm, the distance of T line and nitrocellulose filter lower edge is 1cm, B family streptococcus antibody dilution is coated with to 1.2mg/mL with the PBS (pH7.4) of 0.01M, line concentration is 1 μ L/cm, speed 100mm/s.
Nature controlling line (C line): with the PBS (pH7.4) of 0.01M, goat-anti rabbit polyclonal antibody is diluted to 1.5mg/mL and is coated with, line concentration is 1 μ L/cm, speed 100mm/s.
2. dry
Put into 37 DEG C of baking ovens, dry 2~5 hours.
8. B according to claim 7 family streptococcus quantum dot immune chromatography detects the preparation method of reagent card, it is characterized in that the borate buffer of the step pH8.4 that 1. described quantum dot damping fluid is 0.05M.
9. B according to claim 7 family streptococcus quantum dot immune chromatography detects the preparation method of reagent card, it is characterized in that, the formula of described sample pad damping fluid is the 0.01M PBS damping fluid containing 2wt%BSA, 1w%PVP, 0.5w%Tween.
10. B according to claim 7 family streptococcus quantum dot immune chromatography detects the preparation method of reagent card, it is characterized in that, described preservation liquid is for containing 1w%BSA, 20w% sucrose, 10w% trehalose, the 0.05M Tris-HCl damping fluid of 0.1w% Tween-20 and 0.1w% polyvinylpyrrolidone.
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CN105223353A (en) * 2014-08-18 2016-01-06 董俊 Moraxelle catarrhalis quantum dot immune chromatography test card and its preparation method and application
CN105606808A (en) * 2016-03-30 2016-05-25 珠海华澳生物科技有限公司 Group A/B streptococcus colloidal gold immunochromatograohic assay detection device and detection method thereof
CN106932574A (en) * 2017-03-04 2017-07-07 天津市宝坻区人民医院 The detection method of Type B streptococcal infection
CN107132353A (en) * 2017-05-16 2017-09-05 张子林 A kind of streptococcic detection kit of B races and preparation method thereof
CN108535478A (en) * 2018-03-01 2018-09-14 珠海美华医疗科技有限公司 A kind of streptococcic POCT fluorescence quantitative detecting methods of B races and detection kit
CN108931653A (en) * 2018-08-31 2018-12-04 广州华澳生物科技有限公司 A kind of α 1- antitrypsin immunochromatographiassay assay reagent card and preparation method thereof

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CN105223353A (en) * 2014-08-18 2016-01-06 董俊 Moraxelle catarrhalis quantum dot immune chromatography test card and its preparation method and application
CN105606808A (en) * 2016-03-30 2016-05-25 珠海华澳生物科技有限公司 Group A/B streptococcus colloidal gold immunochromatograohic assay detection device and detection method thereof
CN106932574A (en) * 2017-03-04 2017-07-07 天津市宝坻区人民医院 The detection method of Type B streptococcal infection
CN107132353A (en) * 2017-05-16 2017-09-05 张子林 A kind of streptococcic detection kit of B races and preparation method thereof
CN108535478A (en) * 2018-03-01 2018-09-14 珠海美华医疗科技有限公司 A kind of streptococcic POCT fluorescence quantitative detecting methods of B races and detection kit
CN108931653A (en) * 2018-08-31 2018-12-04 广州华澳生物科技有限公司 A kind of α 1- antitrypsin immunochromatographiassay assay reagent card and preparation method thereof

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