CN103675264A - Colloidal gold film for detecting antibodies on sensitization nitrocellulose membrane and preparation method and application thereof - Google Patents
Colloidal gold film for detecting antibodies on sensitization nitrocellulose membrane and preparation method and application thereof Download PDFInfo
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- CN103675264A CN103675264A CN201310653323.6A CN201310653323A CN103675264A CN 103675264 A CN103675264 A CN 103675264A CN 201310653323 A CN201310653323 A CN 201310653323A CN 103675264 A CN103675264 A CN 103675264A
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Abstract
The invention discloses a colloidal gold film for detecting antibody location and pollution on a sensitization nitrocellulose membrane, and the colloidal gold film includes a colloidal-gold-probe-containing gold labeled pad, a PVC (polyvinyl chloride) rubber board, sample filter paper and a gold probe complex solution. In addition, the invention also discloses a preparation method of the colloidal gold film and its application in detection of initial-stage production of the sensitization nitrocellulose membrane. The colloidal gold film is used for detecting that in the initial-stage production of the sensitization nitrocellulose membrane if the position of a quality control antibody and the position of a capture antibody are exchanged and are mutually polluted, and the colloidal gold film has the advantages of being safe, simple, fast and accurate in operation, suitable for the production field operation, and the like.
Description
Technical field
The invention belongs to biological medicine technology field, be specifically related to a kind of collaurum film, relate in particular to a kind of collaurum film for detection of antibody on sensitization nitrocellulose filter, be applicable to detect on sensitization nitrocellulose filter Quality Control antibody and whether capture antibody position is exchanged and whether Quality Control antibody and capture antibody are polluted mutually.In addition the invention still further relates to, the preparation method and application of this collaurum film.
Background technology
It is one of key link during immuno-chromatographic test paper strip is manufactured that Quality Control antibody, capture antibody are coated with to production sensitization nitrocellulose filter on nitrocellulose filter.Current spray membrane technology is about to antibody and is coated with on nitrocellulose filter, be divided into two kinds, a kind of is contact dotter skill, in contact spot injection system, shower nozzle streaks nitrocellulose filter surface, and while pump promotes a certain amount of liquid and is discharged on nitrocellulose filter surface from shower nozzle; Another kind is specking technique, use solenoid valve to connect high precision gradient pump, specking liquid specking is formed to continuous lines to nitrocellulose filter and with crossover point closely, and specking process is connected with imaging system, can check that the quality of specking lines is with any bad part of tense marker.These two kinds of techniques all can be distinguished specking appointed area (being respectively nature controlling line region, detection line region) on nitrocellulose filter equably by Quality Control antibody and capture antibody.
Because current spray membrane technology all cannot be distinguished Quality Control antibody and capture antibody, can not determine that Quality Control antibody and capture antibody position are exchanged or detected between two kinds of antibody mutually and (for example pollute, the antibody that when preparing/using because spraying membrane antibody and capture antibody, batch mixing causes pollutes), thus cause sensitization nitrocellulose filter by the gross to be scrapped.
Therefore, need that exploitation is a kind of can be used for Quality Control antibody on fast detecting sensitization nitrocellulose filter and whether capture antibody position is exchanged and whether Quality Control antibody and capture antibody are polluted mutually new product badly.
Summary of the invention
For the deficiencies in the prior art, whether whether one of the technical problem to be solved in the present invention is to provide a kind of collaurum film for detection of antibody on sensitization nitrocellulose filter, for detection of Quality Control antibody on sensitization nitrocellulose filter and capture antibody position, mutually polluted being in harmonious proportion two kinds of antibody.
Two of the technical problem to be solved in the present invention is to provide the preparation method of this collaurum film.
Three of the technical problem to be solved in the present invention is to provide the application of this collaurum film in sensitization nitrocellulose filter detects.
For solving the problems of the technologies described above, in one aspect of the invention, a kind of collaurum film for detection of antibody on sensitization nitrocellulose filter is provided, this sensitization nitrocellulose filter is provided with the coated nature controlling line of Quality Control antibody and the detection line of coated capture antibody, and this collaurum film comprises gold mark pad and matching used PVC offset plate, filter sample paper and the Au probe redissolution liquid that contains colloidal gold probe.On PVC offset plate, be superimposed with successively filter sample paper, the gold mark pad that contains colloidal gold probe, and sensitization nitrocellulose filter to be checked; Au probe redissolution drop is added on filter sample paper.
As preferred technical scheme, described gold mark pad is the glass fibre of coated Au probe.The described gold mark pad that contains colloidal gold probe is rabbit antibody gold label pad or sheep anti-mouse antibody gold mark pad; The Au probe that described rabbit antibody gold label pad is rabbit polyclonal antibody mark is coated on glass fibre, and the Au probe that described sheep anti-mouse antibody gold mark pad is sheep anti mouse Anti-TNF-α body tag is coated on glass fibre.
As preferred technical scheme, described Au probe is the colloid gold particle of coupling antibody, and described colloidal gold probe adopts colloid gold particle to be combined by the effect coupling of physisorption with antibody molecule.
As preferred technical scheme, the diameter range of described colloid gold particle is 20-60nm.
As preferred technical scheme, described antibody molecule is rabbit polyclonal antibody or sheep anti mouse polyclonal antibody.Be gold mark pad and matching used PVC offset plate, filter sample paper and the Au probe redissolution liquid that described rabbit antibody colloidal gold film comprises the Au probe that is coated with rabbit polyclonal antibody mark.Described sheep anti-mouse antibody collaurum film comprises gold mark pad and matching used PVC offset plate, filter sample paper and the Au probe redissolution liquid of the Au probe of coated sheep anti mouse Anti-TNF-α body tag.
As preferred technical scheme, described Au probe redissolution liquid comprises following component (mass percent):
Triton X-100 0.05%；
NaH
2PO
4·2H
2O 0.38%；
Na
2HPO
4·12H
2O 2.95%；
PH=8.0 ± 0.1 that this Au probe redissolves liquid.
In another aspect of this invention, provide a kind of preparation method of above-mentioned collaurum film, comprise the steps:
(1) preparation of colloid gold particle;
(2) preparation of Au probe redissolution liquid;
(3) preparation of Au probe: get colloidal gold solution, with 0.1M solution of potassium carbonate, adjusting its PH is 8.0-8.4, add antibody, continue to stir 30 minutes, adding final concentration is 1% bovine serum albumin(BSA), continues to stir 30 minutes, 4 ℃ of 11000RTC rotating speeds are centrifugal, abandon supernatant, precipitation is resuspended with Au probe redissolution liquid, and 2-8 ℃ saves backup;
(4) preparation of Au probe dilution;
(5) preparation of gold mark pad solution; Measure colloidal gold probe OD530 value prepared by above-mentioned steps (3), with Au probe diluted to OD530 be 1-2;
(6) preparation of gold mark pad: the long glass fibre of 30cm is soaked in the gold mark pad solution of preparing in above-mentioned steps (5), take out after glass fibre is completely moistening, after 37 ℃ of oven dry, room temperature sealing saves backup;
(7) collaurum film redissolves liquid and is formed by filtering Au probe that gold mark pad that sample paper and PVC offset plate and step (6) make and step (2) make.
As preferred technical scheme, in step (2), described Au probe redissolution liquid comprises the component of following percentage by weight:
Triton X-100 0.05%；
NaH
2PO
4·2H
2O 0.38%
Na
2HPO
4·12H
2O 2.95%
PH=8.0 ± 0.1 that this Au probe redissolves liquid;
In step (4), described Au probe dilution comprises the component of following mass percent:
pH=8.0±0.1。
In another aspect of this invention, provide the application of a kind of above-mentioned collaurum film in sensitization nitrocellulose filter detects, a bit of sensitization nitrocellulose filter under shearing when the sensitization nitrocellulose filter detecting begins to produce for opening at sensitization nitrocellulose filter.
As preferred technical scheme, in described testing process, when starting production sensitization nitrocellulose filter, initial a bit of sensitization nitrocellulose filter is cut, use described collaurum film to detect, the sensitization nitrocellulose filter first cutting being got off sticks on PVC offset plate, again gold mark pad and filter sample paper are sticked on this PVC offset plate successively, then be cut into test strips, on the filter sample paper of test strips, drip Au probe redissolution liquid, in 3-5 minute, observe the colour developing situation of nature controlling line and detection line on sensitization nitrocellulose filter, with this, judge on sensitization nitrocellulose filter whether Quality Control antibody and capture antibody position are exchanged, whether two kinds of antibody are polluted mutually.
As preferred technical scheme, on described sensitization nitrocellulose filter, Quality Control antibody is goat-anti rabbit polyclonal antibody, capture antibody is mouse monoclonal antibody, and the collaurum film that detects use is respectively the rabbit antibody colloidal gold film of Au probe and the sheep anti-mouse antibody collaurum film of the Au probe that contains sheep anti mouse Anti-TNF-α body tag that contains rabbit polyclonal antibody mark.
As preferred technical scheme, the result determination methods that described sensitization nitrocellulose filter detects is as follows:
The present invention adopts physisorption colloid gold particle labelling method, prepare respectively rabbit antibody colloidal gold film and sheep anti-mouse antibody collaurum film, wherein rabbit antibody colloidal gold film is for detection of Quality Control antibody on sensitization nitrocellulose filter, and sheep anti-mouse antibody collaurum film is for detection of capture antibody on sensitization nitrocellulose filter.
The principle that collaurum film of the present invention detects antibody on sensitization nitrocellulose filter is that 20-60nm colloid gold particle and antibody molecule are combined by the effect coupling of physisorption as rabbit polyclonal antibody/sheep anti mouse polyclonal antibody, when this bond combination specifically can occur when Quality Control antibody (goat-anti rabbit polyclonal antibody) and capture antibody (mouse monoclonal antibody) on by sensitization nitrocellulose filter, form macroscopic red lines, thereby can judge rapidly and accurately on sensitization nitrocellulose filter Quality Control antibody and whether capture antibody position is exchanged and whether two kinds of antibody are polluted mutually.
Compared to the prior art, collaurum film of the present invention has following beneficial effect: the present invention is for detection of the sensitization nitrocellulose filter of the initial period of producing, can detect and judge on sensitization nitrocellulose filter Quality Control antibody and whether capture antibody position is exchanged, whether two kinds of antibody are polluted mutually in production scene fast and easy, avoid in time because of Quality Control antibody and capture antibody position is exchanged or the contaminated time sensitization nitrocellulose filter product rejection by the gross causing of two kinds of antibody and the waste of time/manpowers, there is good economic benefit.Collaurum film of the present invention, has the advantages such as handling safety, easy, quick, accurate, applicable production scene operation.
Accompanying drawing explanation
Fig. 1 is the assembling schematic diagram of test strips in the embodiment of the present invention 2;
Fig. 2 is rabbit antibody colloidal gold film testing result schematic diagram in the embodiment of the present invention 2.Wherein, Fig. 2 A1 is qualified, and Fig. 2 B1 is defective, and Fig. 2 C1 is defective.
Fig. 3 is sheep anti-mouse antibody collaurum film testing result schematic diagram in the embodiment of the present invention 2.Wherein, Fig. 3 A2 is qualified, and Fig. 3 B2 is defective, and Fig. 3 C2 is defective.
Wherein, description of reference numerals is as follows:
1 is filter sample paper, and 2 is the gold mark pad that contains colloidal gold probe, and 3 is detection line, and 4 is nature controlling line, and 5 is sensitization nitrocellulose filter to be checked, and 6 is PVC offset plate, and 7 is Au probe redissolution liquid.
Embodiment
Below in conjunction with drawings and Examples, the present invention is further detailed explanation.
As shown in Figure 1, a kind of collaurum film for detection of antibody on sensitization nitrocellulose filter the present invention relates to is comprised of the gold mark pad 2 that contains colloidal gold probe, filter sample paper 1, PVC offset plate 6 and Au probe redissolution liquid 7, superimposed filter sample paper 1 successively on PVC offset plate 6, the gold mark pad 2 that contains colloidal gold probe, and sensitization nitrocellulose filter 5 to be checked; Au probe redissolves liquid 7 droppings on filter sample paper 1; Sensitization nitrocellulose filter 5 to be checked is provided with the coated nature controlling line 4 of Quality Control antibody and the detection line 3 of coated capture antibody.The gold mark pad 2 that contains colloidal gold probe can be rabbit antibody gold label pad or sheep anti-mouse antibody gold mark pad.The Au probe that described rabbit antibody gold label pad is rabbit polyclonal antibody mark is coated on glass fibre, and the Au probe that described sheep anti-mouse antibody gold mark pad is sheep anti mouse Anti-TNF-α body tag is coated on glass fibre.Colloid gold particle diameter in the gold mark pad 2 that contains colloidal gold probe is at 20-60nm.
The preparation of embodiment 1 collaurum film
A. the preparation of colloidal gold solution: get 0.01%HAuCl4 aqueous solution 100ml, add 1% sodium citrate aqueous solution 0.63ml after boiling, continue heating 20 minutes, the cooling rear 2-8 ℃ of room temperature saves backup;
B. the preparation of Au probe redissolution liquid:
Described Au probe redissolution liquid comprises the component of following percentage by weight:
Triton X-100 0.05%；
NaH
2pO
42H
2o(bis-hypophosphite monohydrate sodium dihydrogens) 0.38%
Na
2hPO
412H
2o(disodium hydrogen phosphate dodecahydrate) 2.95%
PH=8.0 ± 0.1 that this Au probe redissolves liquid.
Preparation 100ml Au probe redissolution liquid: take 0.38g NaH
2pO
42H
2o, 2.95g Na
2hPO
412H
2o and 0.05gTriton X-100, join in 60ml deionized water successively, slowly stirs until dissolving is completely adjusted its PH to 8.0 ± 0.1 with 0.1N NaOH and 0.1N HCl, is then settled to 100ml.
C. the preparation of rabbit antibody-gold probe:
1) rabbit polyclonal antibody optimum mark amount determines
1.1) get 10ml, with 0.1M solution of potassium carbonate, adjust PH to 8.0-8.4, stir packing 10 pipes after 20 minutes, every pipe 1ml.
1.2) rabbit polyclonal antibody (purchased from Dako) is done to serial dilution with 100mM PH8.2 ± 0.2 borate buffer solution, antibody final concentration is respectively 20-100 μ g/ml, and concentration gradient is 10 μ g/ml, gets respectively 1ml, adds in above-mentioned colloidal gold solution, mixes.Control tube only adds 1ml 100mM PH8.2 ± 0.2 borate buffer solution.
1.3) after 10min, in above-mentioned each pipe, add 0.1ml 10%NaCl solution, mix rear standing 2h, observations.
1.4) result is observed, and control tube and rabbit polyclonal antibody quantity not sufficient, with each pipe of stable colloid gold, all present the coagulation phenomenon by red stain indigo plant, adds rabbit polyclonal antibody amount to meet or exceed each quantitative pipe of minimum steady and still keeps red.Using and stablize the red constant minimum rabbit polyclonal antibody consumption of 1ml colloidal gold solution as the minimum mark consumption of rabbit polyclonal antibody, increasing on this basis 10-20% is the optimum mark amount of rabbit polyclonal antibody.
2) preparation of rabbit antibody-gold probe: get 100ml colloidal gold solution, with 0.1M solution of potassium carbonate, adjust PH to 8.0-8.4, add rabbit polyclonal antibody, stirring at room 30min, adding final concentration is 1% bovine serum albumin(BSA), continues to stir 30 minutes, 4 ℃ of 11000RTC rotating speeds are centrifugal, abandon supernatant, precipitation is resuspended with Au probe redissolution liquid, and 2-8 ℃ saves backup.
D. the preparation of sheep anti-mouse antibody Au probe:
1) sheep anti mouse polyclonal antibody optimum mark amount determines
1.1) get 10ml, with 0.1M solution of potassium carbonate, adjust PH to 8.0-8.4, stir packing 10 pipes after 20 minutes, every pipe 1ml.
1.2) sheep anti mouse polyclonal antibody (purchased from Xia Menbosheng Bioisystech Co., Ltd) is done to serial dilution with 100mM PH8.2 ± 0.2 borate buffer solution, antibody final concentration is respectively 20-100 μ g/ml, concentration gradient is 10 μ g/ml, get respectively 1ml, add in above-mentioned colloidal gold solution, mix.Control tube only adds 1ml 100mM PH8.2 ± 0.2 borate buffer solution.
1.3) after 10min, in above-mentioned each pipe, add 0.1ml 10% NaCl solution, mix rear standing 2h, observations.
1.4) result is observed, and control tube and sheep anti mouse polyclonal antibody quantity not sufficient, with each pipe of stable colloid gold, all present the coagulation phenomenon by red stain indigo plant, adds the sheep anti mouse Anti-TNF-α scale of construction to meet or exceed each quantitative pipe of minimum steady and still keeps red.Using and stablize the red constant minimum sheep anti mouse Anti-TNF-α scale of construction of 1ml colloidal gold solution as the minimum mark consumption of sheep anti mouse polyclonal antibody, increasing on this basis 10-20% is the optimum mark amount of sheep anti mouse polyclonal antibody.
2) preparation of sheep anti-mouse antibody Au probe: get 100ml colloidal gold solution, with 0.1M solution of potassium carbonate, adjust PH to 8.0-8.4, add sheep anti mouse polyclonal antibody, stirring at room 30min, adding final concentration is 1% bovine serum albumin(BSA), continues to stir 30 minutes, 4 ℃ of 11000RTC rotating speeds are centrifugal, abandon supernatant, precipitation is resuspended with Au probe redissolution liquid, and 2-8 ℃ saves backup.
E. the preparation of Au probe dilution
Described Au probe dilution comprises following component (mass percent):
pH=8.0±0.1。
Preparation 100ml Au probe dilution: take 0.38g NaH
2pO
42H
2o, 2.95g Na
2hPO
412H
2o, 1gNaCl, 2g sucrose, 0.5g PEG20000 and 1.0g Triton X-100, join successively in 60ml deionized water, add until completely dissolved 1g BSA, slowly stir until dissolve completely, with 0.1N NaOH and 0.1N HCl, adjust its PH to 8.0 ± 0.1, be then settled to 100ml.
F. the preparation of rabbit antibody gold label pad solution: be 1-2 by Au probe diluted to OD530 concentration by rabbit antibody-gold probe.
G. the preparation of rabbit antibody gold label pad: glass fibre is soaked in rabbit antibody gold label pad solution, take out after glass fibre fully infiltrates, 37 ℃ are dried 6 hours, and sealing saves backup.
H. the preparation of sheep anti-mouse antibody gold mark pad solution: be 1-2 by Au probe diluted to OD530 concentration by sheep anti-mouse antibody Au probe.
I. the preparation of the golden mark pad of sheep anti-mouse antibody: glass fibre is soaked in the Au probe solution having diluted, take out after glass fibre fully infiltrates, 37 ℃ are dried 6 hours, and sealing saves backup.
J. the preparation of collaurum film, redissolves liquid and PVC offset plate and filter sample paper by the above-mentioned gold mark pad making and Au probe and is formed, and PVC offset plate and to filter sample paper be ripe commodity can be purchased from market.
When starting production sensitization nitrocellulose filter, initial sensitization nitrocellulose filter 5 is cut a bit of, stick on PVC offset plate 6, then paste successively rabbit antibody gold label pad 2(or sheep anti-mouse antibody gold mark pad) and filter sample paper 1(and see Fig. 1), then cutting is into about the wide test strips of 4mm, 200 μ l Au probes are redissolved to liquid 7 droppings on the filter sample paper 1 of test strips, in 3-5 minute, observe the colour developing situation of nature controlling line 4 and detection line 3 on sensitization nitrocellulose filter 5, with this, judge on sensitization nitrocellulose filter whether Quality Control antibody and capture antibody position are exchanged, whether two kinds of antibody are contaminated.
1. the preparation detecting
Sheep anti-mouse antibody gold mark pad (pressing embodiment 1 preparation)
Rabbit antibody gold label pad (pressing embodiment 1 preparation)
Au probe redissolution liquid (pressing embodiment 1 preparation)
PVC offset plate
Filter sample paper
2. detecting step
A. on sensitization nitrocellulose filter, nature controlling line detects: when starting production sensitization nitrocellulose filter, initial sensitization nitrocellulose filter is cut a bit of, stick on PVC offset plate, shear one section of PVC lamina membranacea of having pasted sensitization nitrocellulose filter, then paste successively rabbit antibody gold label pad and filter sample paper, cutting is into about the wide test strips of 4mm again, 200 μ l colloidal gold probes redissolution drops are added on the filter sample paper of test strips, in 3-5 minute, observe the colour developing situation of nature controlling line and detection line on sensitization nitrocellulose filter, with this, judge on sensitization nitrocellulose filter whether Quality Control antibody and capture antibody position are exchanged, whether two kinds of antibody are contaminated.
B. on sensitization nitrocellulose filter, detection line detects: when starting production sensitization nitrocellulose filter, in detecting step A, another section pasted and on the PVC offset plate of sensitization nitrocellulose filter, pasted successively sheep anti-mouse antibody gold mark pad and filter sample paper, then cutting is into about the wide test strips of 4mm, on the filter sample paper of test strips, drip 200 μ l Au probes redissolution liquid, in 3-5 minute, observe the colour developing situation of nature controlling line and detection line on sensitization nitrocellulose filter, with this, judge on sensitization nitrocellulose filter whether Quality Control antibody and capture antibody position are exchanged, whether two kinds of antibody are contaminated.
3 testing results and judgement
By checking in test strips nature controlling line and the whether aobvious red lines of detection line on sensitization nitrocellulose filter, result of determination.Testing result is shown in Fig. 2 and Fig. 3.
Result determination methods is as following table 1:
Shown in Fig. 2, Fig. 3 and table 1, this interpretation is as follows:
As shown in Figure 2, use rabbit antibody colloidal gold film to detect sensitization nitrocellulose filter, if nature controlling line colour developing, detection line does not develop the color simultaneously, shows sensitization nitrocellulose filter qualified (seeing Fig. 2 A1); If nature controlling line and detection line develop the color simultaneously, show that capture antibody is polluted by Quality Control antibody, sensitization nitrocellulose filter defective (seeing Fig. 2 B1); If nature controlling line does not develop the color, detection line colour developing, shows that Quality Control antibody specking is on detection line, sensitization nitrocellulose filter defective (seeing Fig. 2 C1).
As shown in Figure 3, use sheep anti-mouse antibody collaurum to detect sensitization nitrocellulose filter, if detection line colour developing, nature controlling line does not develop the color simultaneously, shows sensitization nitrocellulose filter qualified (seeing Fig. 3 A2); If nature controlling line and detection line develop the color simultaneously, show that the Quality Control antibody antibody that is hunted down pollutes, sensitization nitrocellulose filter defective (seeing Fig. 3 B2); If detection line does not develop the color, nature controlling line colour developing, shows that capture antibody specking is on nature controlling line, sensitization nitrocellulose filter defective (seeing Fig. 3 C2).
Claims (12)
1. the collaurum film for detection of antibody on sensitization nitrocellulose filter, this sensitization nitrocellulose filter is provided with the coated nature controlling line of Quality Control antibody and the detection line of coated capture antibody, it is characterized in that, this collaurum film comprises the gold mark pad that contains colloidal gold probe, PVC offset plate, filter sample paper and Au probe redissolution liquid; On PVC offset plate, be superimposed with successively filter sample paper, the gold mark pad that contains colloidal gold probe, and sensitization nitrocellulose filter to be checked; Au probe redissolution drop is added on filter sample paper.
2. collaurum film as claimed in claim 1, is characterized in that, described gold mark pad is the glass fibre of coated Au probe; The described gold mark pad that contains colloidal gold probe is rabbit antibody gold label pad or sheep anti-mouse antibody gold mark pad; The Au probe that described rabbit antibody gold label pad is rabbit polyclonal antibody mark is coated on glass fibre, and the Au probe that described sheep anti-mouse antibody gold mark pad is sheep anti mouse Anti-TNF-α body tag is coated on glass fibre.
3. collaurum film as claimed in claim 1, is characterized in that, described colloidal gold probe adopts colloid gold particle to be combined by the effect coupling of physisorption with antibody molecule.
4. collaurum film as claimed in claim 3, is characterized in that, described colloid gold particle diameter is at 20-60nm.
5. collaurum film as claimed in claim 3, is characterized in that, described antibody molecule is rabbit polyclonal antibody or sheep anti mouse polyclonal antibody.
6. collaurum film as claimed in claim 1, is characterized in that, described Au probe redissolution liquid comprises the component of following percentage by weight:
Triton X-100 0.05%；
NaH
2PO
4·2H
2O 0.38%；
Na
2HPO
4·12H
2O 2.95%；
PH=8.0 ± 0.1 that this Au probe redissolves liquid.
7. a preparation method for the collaurum film as described in claim 1-6 any one, is characterized in that, comprises the steps:
(1) preparation of colloid gold particle;
(2) preparation of Au probe redissolution liquid;
(3) preparation of Au probe: get colloidal gold solution, with 0.1M solution of potassium carbonate, adjusting its PH is 8.0-8.4, add antibody, continue to stir 30 minutes, adding final concentration is 1% bovine serum albumin(BSA), continues to stir 30 minutes, 4 ℃ of 11000RTC rotating speeds are centrifugal, abandon supernatant, precipitation is resuspended with Au probe redissolution liquid, and 2-8 ℃ saves backup;
(4) preparation of Au probe dilution;
(5) preparation of gold mark pad solution; Measure colloidal gold probe OD530 value prepared by above-mentioned steps (3), with Au probe diluted to OD530 be 1-2;
(6) preparation of gold mark pad: glass fibre is soaked in the gold mark pad solution of preparing in above-mentioned steps (5), take out after glass fibre is completely moistening, after 37 ℃ of oven dry, room temperature sealing saves backup;
(7) collaurum film redissolves liquid and is formed by filtering Au probe that gold mark pad that sample paper and PVC offset plate and step (6) make and step (2) make.
8. preparation method as claimed in claim 7, is characterized in that,
In step (2), described Au probe redissolution liquid comprises the component of following percentage by weight:
Triton X-100 0.05%；
NaH
2PO
4·2H
2O 0.38%；
Na
2HPO
4·12H
2O 2.95%；
PH=8.0 ± 0.1 that this Au probe redissolves liquid.
In step (4), described Au probe dilution comprises the component of following mass percent:
pH=8.0±0.1。
9. the application of the collaurum film as described in claim 1-6 any one in sensitization nitrocellulose filter detects, it is characterized in that a bit of sensitization nitrocellulose filter under shearing when the sensitization nitrocellulose filter detecting begins to produce for opening at sensitization nitrocellulose filter.
10. the application of collaurum film as claimed in claim 9 in sensitization nitrocellulose filter detects, it is characterized in that, described testing process is when starting production sensitization nitrocellulose filter, initial a bit of sensitization nitrocellulose filter is cut, right to use requires the collaurum film described in 1-6 any one to detect, the sensitization nitrocellulose filter first cutting being got off sticks on PVC offset plate, again gold mark pad and filter sample paper are sticked on this PVC offset plate successively, then be cut into test strips, on the filter sample paper of test strips, drip Au probe redissolution liquid, in 3-5 minute, observe the colour developing situation of nature controlling line and detection line on sensitization nitrocellulose filter, with this, judge on sensitization nitrocellulose filter whether Quality Control antibody and capture antibody position are exchanged, whether two kinds of antibody are polluted mutually.
11. application as claimed in claim 9, it is characterized in that, on described sensitization nitrocellulose filter, Quality Control antibody is goat-anti rabbit polyclonal antibody, capture antibody is mouse monoclonal antibody, and the collaurum film that detects use is the sheep anti-mouse antibody collaurum film of the rabbit antibody colloidal gold film of the Au probe that contains rabbit polyclonal antibody mark or the Au probe that contains sheep anti mouse Anti-TNF-α body tag.
12. application as claimed in claim 9, is characterized in that, the result determination methods that described sensitization nitrocellulose filter detects is as follows:
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310653323.6A CN103675264B (en) | 2013-12-05 | 2013-12-05 | A kind of colloidal gold film for detecting antibody on sensitization nitrocellulose filter and its preparation method and application |
Applications Claiming Priority (1)
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| CN102288751A (en) * | 2011-04-25 | 2011-12-21 | 福建省淡水水产研究所 | Colloidal gold immunochromatography testing strip for rapid detection of slow Edwardsiella tarda |
| WO2011159537A2 (en) * | 2010-06-15 | 2011-12-22 | The Regents Of The University Of California | Method and device for analyte detection |
| WO2012105721A1 (en) * | 2011-02-05 | 2012-08-09 | 野地 澄晴 | Three-dimensional paper micro-diagnosis chip |
| KR20130015899A (en) * | 2011-08-05 | 2013-02-14 | 경북대학교 산학협력단 | Strip for the detection of chlorpyrifos and immunochromatographic assay using thereof |
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| WO2011159537A2 (en) * | 2010-06-15 | 2011-12-22 | The Regents Of The University Of California | Method and device for analyte detection |
| WO2012105721A1 (en) * | 2011-02-05 | 2012-08-09 | 野地 澄晴 | Three-dimensional paper micro-diagnosis chip |
| CN102288751A (en) * | 2011-04-25 | 2011-12-21 | 福建省淡水水产研究所 | Colloidal gold immunochromatography testing strip for rapid detection of slow Edwardsiella tarda |
| KR20130015899A (en) * | 2011-08-05 | 2013-02-14 | 경북대학교 산학협력단 | Strip for the detection of chlorpyrifos and immunochromatographic assay using thereof |
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