CN102135536B - Colloidal gold immune chromatographic test paper box of silver staining enhancement technology and preparation method thereof - Google Patents
Colloidal gold immune chromatographic test paper box of silver staining enhancement technology and preparation method thereof Download PDFInfo
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- CN102135536B CN102135536B CN 201010565942 CN201010565942A CN102135536B CN 102135536 B CN102135536 B CN 102135536B CN 201010565942 CN201010565942 CN 201010565942 CN 201010565942 A CN201010565942 A CN 201010565942A CN 102135536 B CN102135536 B CN 102135536B
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- test paper
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- colloidal gold
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Abstract
The invention relates to a colloidal gold immune chromatographic test paper box of a silver staining enhancement technology. The test paper box is characterized by mainly comprising a colloidal gold immune chromatographic test paper strip, a silver salt-coated bottom membrane and a top membrane which contains a reducing agent for silver salt reduction reaction. The test paper box has the advantages of easiness for operation, judgment and storage, high sensitivity, high stability, quick detection, clear result, no need of any instrument or equipment and the like, is particularly suitable for field test of clinical and sudden events and is suitable for large-scale application of epidemiological survey.
Description
Technical field
The invention provides a kind of silver and dye enhancing technology colloidal gold immune chromatography test box, be used for improving the detection performance of colloidal gold immuno-chromatography test paper strip, the invention also discloses preparation method and the application in test strips detects of above-mentioned paper box, belong to field of biological detection.
Background technology
Colloidal gold probe is a new technology that grows up the seventies in 20th century.Noticeable especially is to be the collaurum quick diagnosis technology of solid phase carrier with the film, and this technology is a novel vitro diagnostic techniques that grows up on enzyme linked immunosorbent assay, latex agglutination test, monoclonal antibody technique, colloid gold immune technology and new material technology basis the eighties in 20th century.This technology more and more is subject to people's attention in recent years, and its technical development is rapid, has particularly obtained widespread use in physianthropy, the veterinary inspection at biomedical sector.
Colloidal gold immunochromatographimethod test-strips binding immunoassay collaurum is made with atopic and the chromatographic technique of corresponding antigen (antibody).A colloidal gold immunochromatographimethod test-strips is made up of sample application zone, reaction zone and adsorption zone three parts usually.Sample application zone contains the immune colloid gold particle, by glass fibre the immune colloid gold particle is adsorbed on this district usually; Reaction zone then sprays or puts and adds two response lines, and one is detection line, and one is nature controlling line.Detection line is in order to detect the reactivity of the envelope antigen (antigen that antibody or antibody carry) of (antigen) material and immune colloid gold particle herein; Nature controlling line is then in order to detect activity and the degree of the coating protein matter on the immune colloid gold particle, and after the immune colloid gold particle was discharged by sample application zone, through reaction zone, part proceeded to adsorption zone, finishes chromatography, thereby reaches the purpose of fast detecting.
It is reductive agent and silver salt solution that silver dyes enhancing technology principal ingredient.Under the effect of reductive agent, silver ion is core with the nanogold particle easily, and the surface reduction that accumulates in nanogold particle becomes silver atoms, like this, the particle of nanoscale size just can become millimetre-sized particle by gathering and the reduction of silver ion, and therefore, the signal of nanogold particle obtains amplifying.
Do not see that by retrieval having Ben Yin to dye the enhancing technology is applied to colloidal gold immuno-chromatography test paper strip, detect the disclosed bibliographical information of performance to strengthen it.
Summary of the invention
The objective of the invention is to disclose a kind of silver and dye enhancing technology colloidal gold immune chromatography test box, be fit to field quick detection, have high specificity, highly sensitive, characteristics easy and simple to handle.
The present invention also provides the preparation method of above-mentioned immunity-chromatography test carton, is applicable to batch process.
Silver of the present invention dyes enhancing technology colloidal gold immune chromatography test box,Comprise: a colloidal gold immuno-chromatography test paper strip; Two counterdies wherein contain silver salt solution; Two teleblems wherein contain the reductive agent that participates in the silver salt reduction reaction; A plastic dropper and a plastic tweezer.
The colloidal gold immuno-chromatography test paper strip of immunity-chromatography test carton of the present invention can be all colloidal gold immuno-chromatography test paper strips, its structure is that sample pad, pad, nitrocellulose filter, absorbent filter are by being fixed in successively from top to bottom on the PVC lamina membranacea, wherein pad is coated with the colloid gold label thing, and whether association colloid gold label is foundation for its result's be judged to be detection line on the nitrocellulose filter and nature controlling line.
The counterdie of immunity-chromatography test carton of the present invention is that the plain film of untreated glass fibre was soaked in liquor argenti nitratis ophthalmicus 1 minute, takes out the dry processing of back lucifuge, and dried counterdie is cut into suitable size sealing and keeps in Dark Place.
The teleblem of epidemic disease chromatographic test paper box of the present invention is that the plain film of untreated glass fibre was soaked 1 minute in p-dihydroxy-benzene solution, takes out the dry processing of back lucifuge, and dried teleblem is cut into suitable size sealing and keeps in Dark Place.
Silver of the present invention dyes the preparation method of enhancing technology colloidal gold immune chromatography test box,Comprise following key step:
1) preparation of immuno-chromatographic test paper strip
Sample pad, pad, nitrocellulose filter, absorbent filter are prepared immuno-chromatographic test paper strip by being fixed in successively on the PVC lamina membranacea from top to bottom, and wherein pad is coated with the colloid gold label thing, the tested survey line of bag and nature controlling line on the nitrocellulose filter.
2) preparation of counterdie and teleblem
Counterdie was soaked in liquor argenti nitratis ophthalmicus 1 minute, and 60 ℃ of lucifuge dryings are loaded on the counterdie of handling well in the opaque sealing bag and preserve.
Teleblem was soaked 1 minute in p-dihydroxy-benzene solution, and 60 ℃ of lucifuge dryings are loaded on the teleblem of handling well in the opaque sealing bag and preserve.
3) silver dyes the assembling that strengthens the colloidal gold immune chromatography test box
To the opaque sealing bag of counterdie be housed; The opaque sealing bag of counterdie is housed; Immuno-chromatographic test paper strip; Application of sample dropper and tweezers are loaded in the paper box.
Good effect of the present invention is:Utilize colloidal gold immunochromatographimethod technology and silver-colored dyeing technique to prepare the immunity-chromatography test carton, both strengthened the detection sensitivity of colloidal gold immuno-chromatography test paper strip, also kept the characteristics of its high specificity.Immunity-chromatography test carton of the present invention wherein silver dyes the enhancing technology applicable to all colloidal gold immuno-chromatography test paper strips, immunity-chromatography test carton of the present invention is suitable for the scene of clinical and accident and detects, be fit to epidemiology survey, have simple to operate, highly sensitive, good stability, detection is quick, the result is clear, is easy to judge and preserve, and need not advantage such as any instrument and equipment, the scene that is particularly suitable for clinical and accident is detected, and is fit to the epidemiology survey large-scale application.
Description of drawings
Fig. 1 is the testing result figure of colloid gold test paper box;
Wherein, 1,1ng/mL toxin sample detection result; 2,100 pg/mL toxin sample detection results; 3, negative findings.
Embodiment
The following example is intended to further illustrate, rather than restriction the present invention.It will be appreciated by those skilled in the art that, under the prerequisite that does not deviate from the spirit and principles in the present invention, all will fall in the claim scope that awaits the reply of the present invention any parallel change of the present invention and change.
Embodiment 1
The preparation of colloidal gold probe
1, the preparation of colloid gold particle
Get one of the 250mL triangular flask of silication, add 100mL deionized water and 1mL 1% gold chloride, ebuillition of heated; Stir the accurate 1.5mL1% of adding trisodium citrate down, continue to boil 15min, the cooling back returns to the colloid gold particle that original volume can obtain particle diameter 25 nm with deionized water.By regulating the colloid gold particle that the amount that adds 1～2mL1% trisodium citrate can be prepared particle diameter 15～40nm.
2, colloid gold label mouse-anti abrin MONOCLONAL ANTIBODIES SPECIFIC FOR
Get one of the 250mL triangular flask of silication, add 100mL particle diameter 25 nm or 30nm collaurum; Add an amount of 0.2mol/L K
2CO
3Transferring pH is 8.0～9.0, most preferably 8.7, and dropwise adding monoclonal antibody to antibody final concentration is 1～1.5mg/mL, most preferably 1.1mg/mL places 15～25min on the room temperature shaking table; Add 10% BSA to final concentration be 5%, prevent antibody protein and collaurum polymerization and precipitation.
3, the preparation of gold mark pad
The plain film of glass fibre is cut into 0.8 * 30cm/ bar in Biohazard Safety Equipment, stand-by after 4 ℃ of vacuum are drained.The colloidal gold probe that purifying is good is uniformly coated on the plain film of glass fibre by the amount of 4mL/ bar, and aeration-drying is spent the night in Biohazard Safety Equipment, preserves standby under the drying condition.
Detect the immuno-chromatographic test paper strip assembling of abrin
1, the preparation of detection line and nature controlling line on the nitrocellulose filter
The anti-abrin polyclonal antibody of the rabbit that purifying is good and sheep anti-mouse igg polyclonal antibody PBS(0.01mol/mL. pH7.5) to be diluted to final concentration respectively be 1mg/mL and 1.5mg/mL to solution.The abrin polyclonal antibody solution that dilution is the good BIODOT that packs into draws film machine shower nozzle 2, be fixed on the position of nitrocellulose filter lower limb 1.1cm, the sheep anti-mouse igg polyclonal antibody that dilution the is good BIODOT that packs into draws film machine shower nozzle 1, is fixed on the position of nitrocellulose filter lower limb 1.6cm.Parameter is 1.0 μ L/cm and is sprayed on the nitrocellulose membrane.
2, the assembling of immuno-chromatographic test paper strip
The assemble method of immune chromatography test paper: every operation all carries out under free from dust, aseptic, no oarse-grained situation, get size and be the PVC base plate of 30cm * 80mm, nitrocellulose filter (30cm * 25mm) be assembled on the viscosity PVC base plate with coated antibody, require its lower limb must align density bullet line on the mould and careful floating face.With adsorptive pads (30cm * 35mm) be assembled on the adhesive base near the mould coboundary, and carefully floating.To be coated with the plain film of golden labeling antibody glass fibre (30cm * 8mm) be assembled on the adhesive base near the scale lower limb, and carefully floating.With sample pad (30cm * 25mm) be assembled on the adhesive base near the mould lower limb, and carefully floating.Be cut into the wide test paper of 4.5mm with cutting cutter, in the assembly section test strips that cuts be sub-packed in the sealing bag of suitable size, can adorn 1-2 test strips for every bag, also the test strips that cuts can be loaded on immunochromatography with in the plastic clip.
The assembling of the immunity-chromatography test carton of abrin
1, the processing of counterdie
The plain film of untreated glass fibre was soaked in liquor argenti nitratis ophthalmicus 1 minute, take out the dry processing of back lucifuge, dried counterdie is cut into suitable size: be that the width of naked counterdie should be identical with the test strips width as test strips, length covers detection line with can be good the time and nature controlling line is advisable; Be loaded in the plastic clip as test strips, the size of counterdie and big or small identical the getting final product of the display window as a result of plastic clip, the counterdie that cuts is sub-packed in the sealing bag of suitable size, 2 every bag.
2, the processing of teleblem
The plain film of untreated glass fibre was soaked 1 minute in p-dihydroxy-benzene solution, take out the dry processing of back lucifuge, dried teleblem is cut into identical size with counterdie and gets final product, and the teleblem that cuts is sub-packed in the sealing bag of suitable size, 2 every bag.
3, the assembling of the immunity-chromatography test carton of abrin
1 bag of the test strips that branch is installed or 1 of 1 bag of 1 bag of 1 of plastic clip, counterdie, teleblem, plastic dropper that test strips is housed and 1 of plastic tweezer are loaded in the plastic packaging bag and get final product after the sealing.
The test example
Mensuration and the practice of abrin immune chromatography test paper performance
1, test result
(1) sample preparation
Tracer liquid breast sample goods: look sample concentration and viscosity 0.01mol/L PBS(pH7.5) solution is done suitable dilution, and gained solution namely can be used as detection liquid.
Detect blood sample: 1 volume blood sample adds the 0.01mol/L PBS(pH7.5 of 1 volume) solution, static 10 minutes of mixing is got supernatant and namely be can be used as detection liquid after the centrifugal treating.
Detect foodstuff samples: food is cooked the 0.01mol/L PBS(pH7.5 that adds appropriate amount after the pulverization process) the solution mixing, static 30min under the room temperature gets supernatant and namely can be used as detection liquid after the centrifugal treating.
(2) pattern detection
Get a toxin sample drop to be checked and be added in the sample application zone of the test strips for preparing, sample begins to spread at nitrocellulose filter, treat that gold mark pad discharges fully after, T line and C line clearly appear on the nitrocellulose filter, this reaction approximately needs 5-10 minute.After question response finishes, with with tweezers counterdie being put on the nitrocellulose filter, make it be close to nitrocellulose filter and cover the T line and the C line, again teleblem is covered on the counterdie, water droplet is added on makes enough moistening getting final product of teleblem and counterdie on the teleblem, leave standstill reaction 5-10 minute or counterdie edge and black precipitate occurs and both can, take and observe counterdie and teleblem away T line and C line, when not containing abrin in the sample, the T line is colourless and the C line has color when detecting with this paper box, sees Fig. 1.
2, detect the specific mensuration of abrin immunity-chromatography test carton
When the detection abrin immune chromatography test paper of application preparation detects the abrus agglutinin nearer with the character of abrin molecular structure, ricin (WA), ricinus agglutinin, the result is all negative, show and they between do not have cross reaction, the test strips specificity is good.
3, detect abrin immunity-chromatography test carton susceptibility
Detect abrin immunity-chromatography test carton with the present invention, when not using silver-colored dyeing technique, it is unintelligible that test strips detected colour developing when abrin concentration was lower than 12.5ng/mL in the sample; The detection line colour developing is clear but more of light color than nature controlling line when the toxin sample concentration is between 12.5～100ng/mL; When the color of toxin sample concentration detection line when 100ng/mL is above darker than nature controlling line color.Testing result is still positive when the silver-colored dyeing technique toxin sample of application is 100pg/mL.
4, detect abrin immunity-chromatography test carton repeatability
Get same sample and carry out revision test five times, testing result is consistent, illustrates that this method has repeatability.
5, detect abrin immunity-chromatography test carton stability
(1) same batch immune chromatography test paper all carries out the detection of test strips every day during 37 ℃ of failure tests, and the intensity of test strips color decreases in the time of the 12nd day.Illustrate that test strips can preserve 11 days at 37 ℃.
(2) same batch immune chromatography test paper in 4 ℃ preserve 6 months during, the develop the color color of band of the result of Ce Dinging is all very clear weekly, color intensity decreases in the time of six month; Illustrate that test strips can preserve 5 months at 4 ℃, sensitivity descends to some extent after the 6th month.
(3) during 11 months, respectively every two weeks one detects same batch immune chromatography test paper in-20 ℃ of preservations, and color intensity decreases in the time of 10th month; Illustrate that test strips can preserve 10 months at-20 ℃, sensitivity descends to some extent after the 11st month.
Embodiment 2
The ricin (WA) immuno-chromatographic test paper strip
Ricin (WA) immuno-chromatographic test paper strip wherein glue gold pad is coated with colloid gold label mouse-anti ricin (WA) monoclonal antibody, and detection line is coated with rabbit antiricin polyclonal antibody on the nitrocellulose filter, is coated with sheep anti-mouse igg on the nature controlling line.
Mensuration and the practice of ricin (WA) immune chromatography test paper performance
(1) sample preparation
Tracer liquid breast sample goods: look sample concentration and viscosity 0.01mol/L PBS(pH7.5) solution is done suitable dilution, and gained solution namely can be used as detection liquid.
Detect blood sample: 1 volume blood sample adds the 0.01mol/L PBS(pH7.5 of 1 volume) solution, static 10 minutes of mixing is got supernatant and namely be can be used as detection liquid after the centrifugal treating.
Detect foodstuff samples: food is cooked the 0.01mol/L PBS(pH7.5 that adds appropriate amount after the pulverization process) the solution mixing, static 30min under the room temperature gets supernatant and namely can be used as detection liquid after the centrifugal treating.
(2) pattern detection
Get a toxin sample drop to be checked and be added in the sample application zone of test strips, sample begins to spread at nitrocellulose filter, treat that gold mark pad discharges fully after, T line and C line clearly appear on the nitrocellulose filter, this reaction approximately needs 5-10 minute.After question response finishes, with with tweezers counterdie being put on the nitrocellulose filter, make it be close to nitrocellulose filter and cover the T line and the C line, again teleblem is covered on the counterdie, water droplet is added on makes enough moistening getting final product of teleblem and counterdie on the teleblem, left standstill reaction 5-10 minute or the counterdie edge occur black precipitate both can, take and observe counterdie and teleblem away T line and C line, when not containing abrin in the sample, the T line is colourless and the C line has color when detecting with this paper box.
Detect ricin (WA) immunity-chromatography test carton susceptibility
Detect ricin (WA) immunity-chromatography test carton with the present invention, when not using silver-colored dyeing technique, the lowest detection of ricin (WA) is limited to 50ng/m in the sample, and testing result is still positive when using silver-colored dyeing technique toxin sample and be 500pg/mL.
Embodiment 3
The CDV immuno-chromatographic test paper strip
Canine distemper immuno-chromatographic test paper strip wherein glue gold pad is coated with colloid gold label mouse-anti CDV monoclonal antibody, and detection line is coated with rabbit canine parvovirus prevention polyclonal antibody on the nitrocellulose filter, is coated with sheep anti-mouse igg on the nature controlling line.
Mensuration and the practice of CDV immune chromatography test paper performance
1, pattern detection
Get a sample drop to be checked and be added in the sample application zone of test strips, sample begins to spread at nitrocellulose filter, treat that gold mark pad discharges fully after, T line and C line clearly appear on the nitrocellulose filter, this reaction approximately needs 5-10 minute.After question response finishes, with with tweezers counterdie being put on the nitrocellulose filter, make it be close to nitrocellulose filter and cover the T line and the C line, again teleblem is covered on the counterdie, water droplet is added on makes enough moistening getting final product of teleblem and counterdie on the teleblem, left standstill reaction 5-10 minute or the counterdie edge occur black precipitate both can, take and observe counterdie and teleblem away T line and C line, when not containing abrin in the sample, the T line is colourless and the C line has color when detecting with this paper box.
2, detect CDV immunity-chromatography test carton susceptibility
Detect CDV immunity-chromatography test carton with the present invention, when not using silver-colored dyeing technique, the lowest detection of CDV is limited to 10
4TCID
50, be 10 when using silver-colored dyeing technique sample virus concentration
3TCID
50The time testing result still positive.
Claims (1)
1. a silver dyes enhancing technology colloidal gold immune chromatography test box, it is characterized in that mainly by colloidal gold immuno-chromatography test paper strip; The teleblem that is coated with the counterdie of silver nitrate and contains p-dihydroxy-benzene constitutes.
2, the described silver of claim 1 dyes enhancing technology colloidal gold immune chromatography test box, it is characterized in that:
The colloidal gold immuno-chromatography test paper strip structure is that sample pad, pad, nitrocellulose filter, absorbent filter are by being fixed in successively from top to bottom on the PVC lamina membranacea, wherein pad is coated with the colloid gold label thing, and whether association colloid gold label is foundation for its result's be judged to be detection line on the nitrocellulose filter and nature controlling line;
Described bag is silver salt soluble in water by the silver nitrate of counterdie;
Described bag is reduced to silver-colored compound by the p-dihydroxy-benzene of teleblem for wrapping by the silver salt in counterdie.
3, the described silver of claim 1 dyes the preparation method of enhancing technology colloidal gold immune chromatography test box, comprises following key step:
1) preparation of immuno-chromatographic test paper strip
Sample pad, pad, nitrocellulose filter, absorbent filter are prepared immuno-chromatographic test paper strip by being fixed in successively on the PVC lamina membranacea from top to bottom, and wherein pad is coated with the colloid gold label thing, the tested survey line of bag and nature controlling line on the nitrocellulose filter;
2) preparation of counterdie and teleblem
Counterdie was soaked in silver salt solution 1 minute, and 60 ℃ of lucifuge dryings are loaded on the counterdie of handling well in the opaque sealing bag and preserve;
Teleblem was soaked in reductive agent 1 minute, and 60 ℃ of lucifuge dryings are loaded on the teleblem of handling well in the opaque sealing bag and preserve;
3) silver dyes the assembling that strengthens the colloidal gold immune chromatography test box
To the opaque sealing bag of counterdie be housed; The opaque sealing bag of counterdie is housed; Immuno-chromatographic test paper strip; Application of sample dropper and tweezers are loaded in the paper box.
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| CN 201010565942 CN102135536B (en) | 2011-01-29 | 2011-01-29 | Colloidal gold immune chromatographic test paper box of silver staining enhancement technology and preparation method thereof |
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Families Citing this family (9)
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| CN103529217A (en) * | 2013-10-16 | 2014-01-22 | 北京华卫博沃生物科技有限公司 | Method for detecting poliovirus, quantum dot-labeled immunochromatographic test paper and preparation method thereof |
| CN103543267A (en) * | 2013-10-16 | 2014-01-29 | 北京华卫博沃生物科技有限公司 | Method for detecting hepatitis A virus (HAV), quantum dot labelled immunochromatographic test strip and preparation method thereof |
| CN103529215A (en) * | 2013-10-16 | 2014-01-22 | 北京华卫博沃生物科技有限公司 | Method for detecting measles virus, quantum-dot labeled immunochromatography test paper and preparation method thereof |
| CN103529203A (en) * | 2013-10-16 | 2014-01-22 | 北京华卫博沃生物科技有限公司 | Method for detecting mycobacterium tuberculosis, quantum-dot labeled immunochromatography test paper and preparation method thereof |
| CN104407144A (en) * | 2013-11-21 | 2015-03-11 | 王海艳 | Colloidal gold for goatpox virus colloidal gold detection reagent strips, colloidal-gold antibody and preparation method thereof |
| CN104198703B (en) * | 2014-08-18 | 2015-12-30 | 湖北工业大学 | People's mycoplasma pneumoniae gold label silver stain immunochromatographytest test kit and its preparation method and application |
| CN109342715A (en) * | 2018-10-09 | 2019-02-15 | 浙江度安生物科技有限公司 | A kind of enhanced sensitivity reagent enhancing colloid gold chromatographic test paper signal strength and the photosensitivity-enhancing method using enhanced sensitivity reagent |
| CN111505279B (en) * | 2020-04-17 | 2023-06-27 | 南昌大学 | Method for silver growth enhanced ultrasensitive detection of rotavirus |
| CN112858675B (en) * | 2021-01-05 | 2022-03-29 | 杭州奥泰生物技术股份有限公司 | Novel coronavirus antigen and antibody combined intelligent detection device |
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| CN101074959A (en) * | 2007-06-13 | 2007-11-21 | 中国人民解放军军事医学科学院军事兽医研究所 | Gold and silver combined inspection test paper for feline and canidae animals important pestilence pathogen and its production |
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|---|---|---|---|---|
| CN101074959A (en) * | 2007-06-13 | 2007-11-21 | 中国人民解放军军事医学科学院军事兽医研究所 | Gold and silver combined inspection test paper for feline and canidae animals important pestilence pathogen and its production |
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