CN103869079B - A kind of colloid gold test paper of Quantitative detection CBP-35 - Google Patents
A kind of colloid gold test paper of Quantitative detection CBP-35 Download PDFInfo
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- CN103869079B CN103869079B CN201410080580.XA CN201410080580A CN103869079B CN 103869079 B CN103869079 B CN 103869079B CN 201410080580 A CN201410080580 A CN 201410080580A CN 103869079 B CN103869079 B CN 103869079B
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- test paper
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4724—Lectins
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/32—Cardiovascular disorders
- G01N2800/325—Heart failure or cardiac arrest, e.g. cardiomyopathy, congestive heart failure
Abstract
The invention discloses a kind of colloid gold test paper of Quantitative detection CBP-35, described colloid gold test paper comprises support pad 5, be positioned at the nitrocellulose filter 1 in described support pad, one end of described nitrocellulose filter connects adsorptive pads 4, the other end connects conjugates pad 2, described conjugates pad connects sample pad 3, the below of described sample pad the other end is provided with the first fixed part 7, top is provided with the second fixed part 6, it is characterized in that, described nitrocellulose filter 1 middle part detection line 8 is coated with Gal-3 antibody, detection line both sides are respectively equipped with control line 9, described conjugates pad 2 is coated with Gal-3 monoclonal antibody or polyclonal antibody and the control line conjugates of gold mark.
Description
Technical field
The invention belongs to clinical diagnose field, being specifically related to a kind of for quantitatively detecting the colloid gold test paper of CBP-35 (Gal-3).
Background technology
CBP-35 (Galectin-3, Gal-3) also claim galactose-binding protein-3, and molecular weight is 31kD, is one of important member of galactose agglutinin family.Member's demand fulfillment two standards of Galectin family: the affinity of the first p-galactose; Second has remarkable sequence similarity at carbohydrate recognition domain (thecarbohydraterecognitiondomain, CRD).Galectin-3 has two functional domains: c-terminus is carbohydrate recognition domain, contains 135 amino acid residues; Aminoterminal comprises 100 ~ 150 amino acid residues, is made up of 9 amino acid residue tandem sequence repeats of Pro-rich and glycocoll.Galectin-3 mainly expresses in macrophage, acidophic cell, neutrophil leucocyte and mast cell.Its function according to it in intracellular location.In akinete, be mainly present in endochylema, in proliferative cell, be then mainly present in nucleus.Galectin-3 participates in regulating Growth of Cells, anti-apoptotic, mediates cell attachment, participates in the biological function such as vascularization and inflammatory reaction.Research shows that CBP-35 is myocardial fibrosis mark, increases relevant to the generation of heart failure and mortality risk.The CBP-35 content of ventricular dysfunction patient can raise, and content is directly related in the order of severity of heart failure.The CBP-35 level of Quantitative detection patient has great clinical meaning.
Immune colloidal gold technique (Immunecolloidalgoldtechnique) is a kind of novel immunolabelling technique being applied to antigen-antibody using collaurum as tracer label thing.Collaurum be by gold chloride (HAuCl4) at reductive agent as under the effect such as white phosphorus, ascorbic acid, sodium citrate, tannic acid, polymerization becomes the gold grain of specific size, and becomes a kind of stable colloidal state due to electrostatic interaction, is called collaurum.Collaurum is electronegative under mild alkaline conditions, can be formed firmly be combined, because this combination is electrostatical binding, so do not affect the biological nature of protein with the positive charge group of protein molecule.Collaurum except with protein bound except, can also be combined with other biomacromolecules many.According to some physical behaviors of collaurum, as high electron density, grain size, shape and color reaction, add immunity and the biological characteristics of bond, thus make collaurum be widely used in the fields such as immunology, histology, pathology and cell biology.
At present, detect the current mainly Enzyme-multiplied immune technique (ELISA) of method of CBP-35 (Gal-3), there is following shortcoming in elisa technique: checkout equipment requires high, and cost is high; Disturbing factor is more, and repeatability is bad; Detection time is long.Therefore Enzyme-multiplied immune technique detects CBP-35 and is not suitable for clinical quick diagnosis.In view of there is no quantitative detecting method fast at present, the object of this invention is to provide and a kind ofly can be used for the colloid gold test paper of Quantitative detection CBP-35 and a kind of colloid gold test paper box that can be used for Quantitative detection CBP-35, described colloid gold test paper and colloid gold test paper box have the advantages such as convenient and swift, simple to operate, result is accurate.
Summary of the invention
An object of the present invention is to provide a kind of colloid gold test paper of Quantitative detection CBP-35, the colloid gold test paper of CBP-35 of the present invention has the advantages such as convenient and swift, simple to operate, result is accurate, is suitable for clinical quick diagnosis.
Technical scheme of the present invention is:
A kind of colloid gold test paper of Quantitative detection CBP-35, comprise support pad 5, be positioned at the nitrocellulose filter 1 in described support pad, one end of described nitrocellulose filter connects adsorptive pads 4, the other end connects conjugates pad 2, described conjugates pad connects sample pad 3, the below of described sample pad the other end is provided with the first fixed part 7, top is provided with the second fixed part 6, it is characterized in that, described nitrocellulose filter 1 middle part detection line 8 is coated with Gal-3 antibody, detection line both sides are respectively equipped with control line 9, described conjugates pad 2 is coated with Gal-3 monoclonal antibody or polyclonal antibody and the control line conjugates of gold mark.
Preferably, described control line 9 is coated with anti-DNP antibody, and described control line conjugates is the collaurum of DNP-BSA mark.BSA is bovine serum albumin(BSA).
Preferably, described first fixed part 7 is double faced adhesive tape, and the second fixed part 6 is one side glue.
Preferably, described two control lines 9 and detection line 8 are for be arrangeding in parallel.
Another object of the present invention there is provided a kind of colloid gold test paper box of Quantitative detection CBP-35, and the basis of the colloid gold test paper of quantitative detection CBP-35 of the present invention also comprises a shell 10.
The preferred plastic casing of described shell.
Described shell wraps up described test paper, exposes detection line 8 on sample pad 3, nitrocellulose filter 1 and two control lines 9 and adsorptive pads 4.
Paper box of the present invention has the advantage being convenient for carrying and storing.
Another object of the present invention is to provide colloid gold test paper of the present invention and is detecting the application in CBP-35.
Another object of the present invention is to provide a kind of method of Quantitative detection CBP-35, comprises and uses colloid gold test paper described in claim 1 to carry out the step of Quantitative detection CBP-35.
The colloid gold test paper of Quantitative detection CBP-35 of the present invention or the principle of work of paper box are: adopt two-way effluent immune response principle, be prepared from by double antibody sandwich method.Gal-3 antigen during inspection in sample first with the anti-Gal-3 antibody conjugates generation immune response on coupling pad, formed immune complex.Thereafter immune complex is along with sample chromatographic flow on NC Nitroncellulose film, when immune complex chromatography is to detection zone (Gal-3 detection line) on NC Nitroncellulose film, reacts with the anti-GAL-3 antibody be coated in advance on NC Nitroncellulose film thus be fixed on the detection line of NC Nitroncellulose film.GAL-3 in blood is more, and the compound on detection line is more, and the optical density value on band is higher.Simultaneously, in testing process, control line conjugates (i.e. the collaurum of DNP-BSA mark), also can with sample chromatography on NC Nitroncellulose film, when chromatography is to the control line of NC Nitroncellulose film, DNP-BSA collaurum can react with the anti-DNP antibody be coated in advance on NC Nitroncellulose film thus be fixed on check plot (control line).Because collaurum is red, the DNP-BSA colloid gold immune compound be thus fixed in the anti-DNP on NC Nitroncellulose film control line will show redness.When not containing GAL-3 in sample, then only show two control lines.When sample is containing GAL-3, detection line will show clearly.
After reaction terminates, utilize multifunction immunity detector (auspicious Lay bioengineering (Shenzhen) company limited) optical density of control line and detection line to be analyzed, and by analyze the result obtained and carry out computing, thus obtain relative optical density number (RI).Then detector calculates according to the concentration of the typical curve be set in advance in detector to GAL-3 and shows result, represents in units of ng/mL.
Accompanying drawing explanation
Fig. 1 is the structural representation of a kind of preferred implementation of the colloid gold test paper of Quantitative detection CBP-35 of the present invention.
Wherein, 1: nitrocellulose filter, 2: conjugates pad, 3: sample pad, 4: adsorptive pads, 5: support pad, 6: one side glue, 7: double faced adhesive tape, 8: detection line, 9: control line.
Fig. 2 is the diagram of a kind of preferred implementation of the colloid gold test paper box of Quantitative detection CBP-35 of the present invention.
Wherein, 3: sample pad, 8: detection line, 9: control line, 4: adsorptive pads, 10: shell.
Embodiment
Below in conjunction with accompanying drawing, the colloid gold test paper of Quantitative detection CBP-35 of the present invention or paper box are described in detail, can not be interpreted as it is limitation of the present invention.The material used in following examples and reagent unless otherwise indicated, are common commercially available.
[embodiment 1] prepares the colloid gold test paper of Quantitative detection CBP-35
Colloid gold test paper structure as shown in Figure 1, comprise support pad 5, be positioned at the nitrocellulose filter 1 in described support pad, stacked connection adsorptive pads 4 above one end of described nitrocellulose filter, stacked connection conjugates pad 2 above the other end, stacked connection sample pad 3 above described conjugates pad one end away from nitrocellulose filter 1, the bonding double faced adhesive tape 7 in below of described sample pad the other end, the bonding one side glue 6 in top, it is characterized in that, described nitrocellulose filter 1 middle part detection line 8 is coated with Gal-3 antibody, detection line both sides are respectively equipped with control line 9, described conjugates pad 2 is coated with Gal-3 monoclonal antibody or polyclonal antibody and the control line conjugates of gold mark.
Described control line 9 is coated with anti-DNP antibody, and described control line conjugates is the collaurum of DNP-BSA mark.
Wherein, nitrocellulose filter 1 for fixing coated antibody, is also immunoreactive nidus in colloid gold test paper simultaneously; Detection line 8 uses the damping fluid such as 1*PBS, methyl alcohol to be diluted to the concentration of 0.5mg/ml Gal-3 antibody, gets 1ul/cm and line on described nitrocellulose filter 1, and 37 DEG C of dried overnight and 60 DEG C of bakings 3 days, to obtain final product; Control line 9 uses the damping fluid such as 1*PBS, methyl alcohol to be diluted to the concentration of 0.5mg/ml anti-DNP antibody, gets 1ul/cm and line on described nitrocellulose filter 1, and 37 DEG C of dried overnight and 60 DEG C of bakings 3 days, to obtain final product.
Wherein, the starting material of described conjugates pad 2 are glass fiber filter, glass fiber filter for the preparation of conjugates pad is put into pre-Block buffer and (comprises the casein of 0.1-1%, the PEG of the bovine serum albumin(BSA) of 0.5-5%, the boric acid of 0.005-0.5%, 0.01-0.2%) middle immersion taking-up after 5 minutes, after 37 DEG C of dryings, the Gal-3 monoclonal antibody marked by gold with Biodot coating instrument or polyclonal antibody and control line conjugates are coated on conjugates pad 2, coating weight is 1ul/mm, drying, to obtain final product.Preferably, the pre-Block buffer of described conjugates pad 2 comprises the casein of 0.8%, the bovine serum albumin(BSA) of 4%, the boric acid of 0.007%, the PEG of 0.1%.
Wherein, described sample pad 3 can play preliminary filtering function by liquid towards sample.After sample pad is soaked 5 minutes with 141 confining liquids (comprising: the casein of the Tween20 of the Tris of 0.1-1%, 0.1-1%, 0.1-1%, the mouse anti-human RBC of 0.05-0.5%), 37 DEG C of dryings of spending the night, to obtain final product.Preferably, described 141 confining liquids comprise: the Tris of 0.3%, the casein of 0.8%, the mouse anti-human RBC of 0.3%.
Above-mentioned each building block is pasted onto in support pad 5 by structure shown in Fig. 1, obtains the colloid gold test paper of Quantitative detection CBP-35.
[embodiment 2] is with colloid gold test paper Quantitative detection CBP-35 of the present invention.
Get the colloid gold test paper of the preferred a kind of Quantitative detection CBP-35 of the present invention, as shown in Figure 1, described colloid gold test paper comprises support pad 5, be positioned at the nitrocellulose filter 1 in described support pad, stacked connection adsorptive pads 4 above one end of described nitrocellulose filter, stacked connection conjugates pad 2 above the other end, stacked connection sample pad 3 above described conjugates pad one end away from nitrocellulose filter 1, the bonding double faced adhesive tape 7 in below of described sample pad the other end, the bonding one side glue 6 in top, it is characterized in that, described nitrocellulose filter 1 middle part detection line 8 is coated with Gal-3 antibody, detection line both sides are respectively equipped with control line 9, described conjugates pad 2 is coated with Gal-3 monoclonal antibody or polyclonal antibody and the control line conjugates of gold mark.Preferably, described control line 9 is coated with anti-DNP antibody, and described control line conjugates is the collaurum of DNP-BSA mark.Detect according to the following steps: the patients serum 1) extracted, blood plasma or whole blood wait for that sample originally, as Cord blood sample need return to room temperature.2) testing sample is added on sample pad 3, reaction.3) interpretation, is placed in multifunction immunity detector by described colloid gold test paper, result of determination in SSJ-2 or MINI (auspicious Lay bioengineering (Shenzhen) company limited).
[embodiment 3] is with colloid gold test paper box Quantitative detection CBP-35 of the present invention.
Get the colloid gold test paper box of the preferred a kind of Quantitative detection CBP-35 of the present invention, as depicted in figs. 1 and 2, described colloid gold test paper comprises support pad 5, be positioned at the nitrocellulose filter 1 in described support pad, one end of described nitrocellulose filter connects adsorptive pads 4, the other end connects conjugates pad 2, described conjugates pad connects sample pad 3, the bonding double faced adhesive tape 7 in below of described sample pad the other end, the bonding one side glue 6 in top, it is characterized in that, described nitrocellulose filter 1 middle part detection line 8 is coated with Gal-3 antibody, detection line both sides are respectively equipped with control line 9, described conjugates pad 2 is coated with Gal-3 monoclonal antibody or polyclonal antibody and the control line conjugates of gold mark.Preferably, described control line 9 is coated with anti-DNP antibody, and described control line conjugates is the collaurum of DNP-BSA mark.Described colloid gold test paper also comprises a shell 10, and described shell wraps up described test paper, exposes detection line 8 on sample pad 3, nitrocellulose filter 1 and two control lines 9 and adsorptive pads 4.Detect according to the following steps: the patients serum 1) extracted, blood plasma or whole blood wait for that sample originally, as Cord blood sample need return to room temperature.2) testing sample is added on sample pad 3, reaction.3) interpretation, is placed in multifunction immunity detector by described colloid gold test paper, result of determination in SSJ-2 or MINI (auspicious Lay bioengineering (Shenzhen) company limited).
Claims (5)
1. the colloid gold test paper of a Quantitative detection CBP-35, comprise support pad (5), be positioned at the nitrocellulose filter (1) in described support pad, one end of described nitrocellulose filter connects adsorptive pads (4), the other end connects conjugates pad (2), described conjugates pad connects sample pad (3), the below of described sample pad the other end is provided with the first fixed part (7), top is provided with the second fixed part (6), it is characterized in that, described nitrocellulose filter (1) middle part detection line (8) is coated with Gal-3 antibody, detection line both sides are respectively equipped with control line (9), described conjugates pad (2) is coated with Gal-3 monoclonal antibody or polyclonal antibody and the control line conjugates of gold mark.
2. colloid gold test paper as claimed in claim 1, it is characterized in that, described control line (9) is coated with anti-DNP antibody, and described control line conjugates is the collaurum of DNP-BSA mark.
3. colloid gold test paper as claimed in claim 1, it is characterized in that, described first fixed part (7) is double faced adhesive tape, and the second fixed part (6) is one side glue.
4. the colloid gold test paper according to any one of claim 1-3, it is characterized in that, described colloid gold test paper also comprises a shell (10) outward, described shell wraps up outside described colloid gold test paper, exposes detection line (8) on sample pad (3), nitrocellulose filter (1) and two control lines (9) and adsorptive pads (4).
5. colloid gold test paper as claimed in claim 4, is characterized in that, described two control lines (9) and detection line (8) are for be arrangeding in parallel.
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| CN106290891B (en) * | 2016-08-10 | 2019-02-12 | 王清 | The detection method and test strip of Galectin-3 |
| CN107328926A (en) * | 2017-06-29 | 2017-11-07 | 海格德生物科技（深圳）有限公司 | Reagent card, preparation method and the detection method of the multinomial heart failure mark of quick detection |
| CN107907679A (en) * | 2017-11-06 | 2018-04-13 | 南京诺唯赞医疗科技有限公司 | A kind of immuno-chromatographic test paper strip and preparation method thereof and application |
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| CN112526123B (en) * | 2020-11-18 | 2022-05-24 | 厦门同仁心生物技术有限公司 | Quality control method for degree of polymerization of DNP-BSA (DNP-bovine serum albumin) |
| CN113311173B (en) * | 2021-07-28 | 2022-08-26 | 南京申基医药科技有限公司 | Combined detection test strip for detecting ST2 and Galectin-3 antibodies in blood sample for improving detection accuracy, preparation method and kit |
| CN115746134A (en) * | 2021-10-15 | 2023-03-07 | 深圳市睿盟创新生物科技有限公司 | Galectin-3 immunoassay |
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| KR20080063326A (en) * | 2005-10-21 | 2008-07-03 | 가부시키가이샤환케루 | Atopic dermatitis marker and technique of using the same |
| CN201083739Y (en) * | 2007-07-03 | 2008-07-09 | 丘福隆 | Sidestream chromatography test device |
| CN101839908B (en) * | 2010-05-28 | 2014-08-20 | 李金波 | Quantitative detection device and detection method of biological fluid samples |
| CN203732547U (en) * | 2014-03-06 | 2014-07-23 | 瑞莱生物工程（深圳）有限公司 | Colloidal gold test paper for rapid and quantitative detection of -3(Gal-3) |
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