CN116068179B - Colloidal gold test strip for detecting human tissue type plasminogen activator - Google Patents
Colloidal gold test strip for detecting human tissue type plasminogen activator Download PDFInfo
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- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 title claims abstract description 44
- 238000012360 testing method Methods 0.000 title claims abstract description 19
- 101000801481 Homo sapiens Tissue-type plasminogen activator Proteins 0.000 title claims abstract description 6
- 108090000373 Tissue Plasminogen Activator Proteins 0.000 claims abstract description 42
- 238000001514 detection method Methods 0.000 claims abstract description 36
- 239000012528 membrane Substances 0.000 claims abstract description 27
- 239000002245 particle Substances 0.000 claims abstract description 20
- 239000000020 Nitrocellulose Substances 0.000 claims abstract description 18
- 229920001220 nitrocellulos Polymers 0.000 claims abstract description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 5
- 238000010521 absorption reaction Methods 0.000 claims abstract description 4
- 238000003908 quality control method Methods 0.000 claims description 17
- 241001529936 Murinae Species 0.000 claims description 4
- 102000019400 Tissue-type plasminogen activator Human genes 0.000 claims 2
- 102000003978 Tissue Plasminogen Activator Human genes 0.000 abstract description 40
- 230000002980 postoperative effect Effects 0.000 abstract description 5
- 230000028327 secretion Effects 0.000 abstract description 5
- 238000006243 chemical reaction Methods 0.000 abstract description 3
- 229960000187 tissue plasminogen activator Drugs 0.000 description 36
- 210000004379 membrane Anatomy 0.000 description 7
- 208000000592 Nasal Polyps Diseases 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 125000003275 alpha amino acid group Chemical group 0.000 description 5
- 241001416177 Vicugna pacos Species 0.000 description 4
- 239000000427 antigen Substances 0.000 description 4
- 102000036639 antigens Human genes 0.000 description 4
- 108091007433 antigens Proteins 0.000 description 4
- 239000008363 phosphate buffer Substances 0.000 description 4
- 229920000742 Cotton Polymers 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 238000009835 boiling Methods 0.000 description 3
- 238000009792 diffusion process Methods 0.000 description 3
- 239000003365 glass fiber Substances 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 208000016366 nasal cavity polyp Diseases 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 238000001356 surgical procedure Methods 0.000 description 3
- 206010061218 Inflammation Diseases 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 238000002649 immunization Methods 0.000 description 2
- 230000003053 immunization Effects 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 2
- 230000037452 priming Effects 0.000 description 2
- 238000003118 sandwich ELISA Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 206010009137 Chronic sinusitis Diseases 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 108010003723 Single-Domain Antibodies Proteins 0.000 description 1
- 239000002250 absorbent Substances 0.000 description 1
- 230000002745 absorbent Effects 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 208000027157 chronic rhinosinusitis Diseases 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000000875 corresponding effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000001804 emulsifying effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000005484 gravity Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 230000016784 immunoglobulin production Effects 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000003928 nasal cavity Anatomy 0.000 description 1
- 210000002850 nasal mucosa Anatomy 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 1
- 229940038773 trisodium citrate Drugs 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- 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
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
-
- 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
-
- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
- G01N33/587—Nanoparticles
-
- 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
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
- G01N2333/972—Plasminogen activators
- G01N2333/9726—Tissue plasminogen activator
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention relates to a test strip for detecting human tissue type plasminogen activator, which comprises a bottom plate, and a sample pad, a colloidal gold binding pad, a nitrocellulose membrane and a water absorption pad which are sequentially connected on the bottom plate, wherein a detection line is arranged on the nitrocellulose membrane; the colloidal gold binding pad contains colloidal gold particles marked by binding antibodies; the capture antibody is anchored in the detection line, and both the binding antibody and the capture antibody can specifically recognize and bind to tissue type plasminogen activator and do not interfere with each other. The colloidal gold test strip provided by the invention is convenient and quick to use, can complete chromatographic reaction within 10 minutes, can be used for qualitative detection and quantitative detection, and helps a doctor to judge the tPA condition in nasal secretion in a short time so as to determine the postoperative recurrence risk of a patient. The test strip is simple to operate and short in time, can be directly used for detection in an outpatient service, and does not need to be transferred to a clinical laboratory.
Description
Technical Field
The invention belongs to the technical field of biomedical detection, and particularly relates to a colloidal gold test strip for detecting human tissue type plasminogen activator.
Background
Chronic sinusitis with nasal polyps is a complex and heterogeneous inflammation of the nasal mucosa characterized by the presence of nasal polyps estimated to occur in 1% to 4% of the world population. In europeans and americans, CRSwNP is characterized immunologically predominantly by Th2 inflammation; whereas in asian countries such as china and korea, at least half of CRSwNP patients exhibit Th1/Th17 inflammatory responses.
Although surgery is the first line of treatment for nasal polyps, the recurrence rate of patients one year after surgery is as high as 30%. If the postoperative recurrence condition of the patient can be judged before the operation, the doctor can be prompted, and the operation type of the patient and the postoperative medication can be selected individually. Earlier we have found that protein levels of tissue plasminogen activator (tPA) in nasal secretions are correlated with postoperative recurrence in nasal polyp patients, and that recurrence after nasal polyp surgery can be predicted.
At present, no clinical method for detecting nasal secretion tPA exists, so that the tPA kit developed by us is used for making up the defect of clinical detection, judging whether a patient recurs after operation or not, and providing personalized operation and postoperative treatment scheme for the operator.
Disclosure of Invention
In order to solve the problems, the invention provides a test strip for detecting tissue type plasminogen activator (tPA), which comprises a bottom plate, and a sample pad, a colloidal gold binding pad, a nitrocellulose membrane and a water absorption pad which are sequentially connected on the bottom plate, wherein a detection line is arranged on the nitrocellulose membrane;
the colloidal gold binding pad contains colloidal gold particles marked by binding antibodies; the capture antibody is anchored in the detection line, the binding antibody and the capture antibody can specifically recognize and bind to tissue type plasminogen activator, and the binding antibody and the capture antibody do not interfere with the site of tissue type plasminogen activator binding.
The invention uses the double-antibody sandwich strategy to prepare the colloidal gold test strip for detecting the human tissue type plasminogen activator (tPA), can rapidly detect whether the tPA is contained in a target sample in a short time, can accurately and quantitatively detect the content of the tPA, and helps doctors to rapidly judge the recurrence risk of nasal polyp patients after operation.
In a preferred embodiment, the binding antibody is a nanobody comprising three CDR regions having the sequence shown in SEQ ID NO. 6-8.
Preferably, the variable region sequence of the binding antibody is shown in SEQ ID NO. 5.
In a preferred embodiment, the capture antibody is a nanobody comprising three CDR regions having sequences as shown in SEQ ID NOS.2-4.
Preferably, the variable region sequence of the capture antibody is shown in SEQ ID NO. 1.
The tPA3 (with the sequence shown as SEQ ID NO: 1) capture antibody and the AtPA5 (with the sequence shown as SEQ ID NO: 5) as binding antibody can be used for efficiently and specifically detecting tPA in a sample. Both AtPA3 and AtPA5 are nanobodies, comprising only a heavy chain variable region (VHH) with 4 framework regions and 3 CDRs, which are stable, have good affinity, high potency, and are easy to express, which is advantageous for reducing antibody production costs.
In a preferred embodiment, a quality control line is further arranged on the nitrocellulose membrane, and the quality control line is far away from the colloidal gold binding pad than the detection line.
In a preferred embodiment, murine anti-alpaca IgG is anchored in the quality control line.
The colloidal gold test strip provided by the invention is convenient and quick to use, can complete chromatographic reaction within 10 minutes, is used for quantitative detection, and greatly shortens the detection time, thereby helping doctors judge the tPA condition in nasal secretion of patients in a short time. The test strip is simple to operate and short in time, can be directly used for detection in an outpatient service, and does not need to be transferred to a clinical laboratory.
Drawings
FIG. 1 is a statistical plot of the binding values of AtPA1-7 to tPA, binding value = tPA-containing 0D 450/blank OD450;
FIG. 2 is a statistical chart of OD450 of double-antibody sandwich ELISA detection by using AtPA3 as capture antibody and AtPA1-2, 4-7 as binding antibody;
FIG. 3 is a statistical chart of OD450 of double-antibody sandwich ELISA detection by using AtPA5 as capture antibody and AtPA1-4, 6-7 as binding antibody;
FIG. 4 is a schematic diagram of a test strip according to the present invention;
FIG. 5 is a top view of a nitrocellulose membrane of a paper strip of the present invention;
wherein, 1, bottom plate, 2, sample pad, 3, colloidal gold bond pad, 4, nitrocellulose membrane, 411, detection line, 412, quality control line, 5, absorbent pad.
Detailed Description
The principles and features of the present invention are described below with examples given for the purpose of illustration only and are not intended to limit the scope of the invention.
1. Screening and preparation of antibodies
Anti-human tPA antibodies were prepared using full length tissue plasminogen activator (tPA) for immunization of alpaca. The priming was accomplished by emulsifying 250 μg antigen in admixture with 250 μl Freund's complete adjuvant and applying the mixture to an immunized alpaca. Alpaca was boosted once each after 2 weeks and 4 weeks of priming, with 1250 μg antigen and 250 μl of incomplete Freund's adjuvant. After one week of the second and third, blood was collected and assayed for antiserum titers.
The results showed that antigen di-and tri-priming antisera titers were 3.26X10, respectively 6 And 6.37X10 7 . It can be seen that antigen-induced alpaca produced high titres of antisera against the corresponding antigenic peptides.
Peripheral blood after one week of three-phase immunization was taken, peripheral Blood Mononuclear Cells (PBMC) were isolated, RNA was extracted, amplified using universal primers after reverse transcription, and cloned into phagemids, transformed into TG1 strain, and phage libraries were established for screening monoclonal antibodies. After multiple rounds of screening we obtained 7 monoclonal antibodies, atPA1-7, with high specificity and affinity. As shown in FIG. 1, the 7 monoclonal antibodies were found to be at 4X 10 -3 The binding value reaches more than 2 at the concentration of mug/mL. The antibody combinations were further studied and the results are shown in figures 2 and 3, with AtPA3 and AtPA5 forming the antibody pair and with AtPA3 as the capture antibody and AtPA5 as the binding antibody for the double antibody sandwich immunoassay.
The results of analysis of the sequences of AtPA3 and AtPA5 show that the amino acid sequence of AtPA3 is shown as SEQ ID NO. 1, which includes 3 CDR regions, and the amino acid sequences of CDR1-3 are shown as SEQ ID NO. 2-4, respectively. The amino acid sequence of AtPA5 is shown as SEQ ID NO. 5, which comprises 3 CDR regions, and the amino acid sequences of CDR1-3 are shown as SEQ ID NO. 6-8, respectively.
Further studies using the tPA mutant library found that the recognition sites for AtPA1 and 3 were located at the N-terminus of the tPA amino acid sequence, atPA2 and 7 were located in the middle of tPA, and the recognition site for AtPA4-6 was located at the C-terminus of tPA. It is speculated that AtPA3 and AtPA5 are beneficial to pairing for double-antibody sandwich detection because the recognition sites are far apart and the binding of antibodies does not affect each other. Furthermore, according to our experiments, it was shown that when AtPA3 was used as the capture antibody and AtPA5 was used as the binding antibody, the binding value reached 20 or more, which is far higher than other combinations.
2. Preparation and application methods of tPA quantitative detection test strip
the tPA test strip has a structure shown in fig. 4 and 5, and comprises a rectangular bottom plate 1, and a sample pad 2, a colloidal gold bonding pad 3, a nitrocellulose membrane 4 and a water absorption pad 5 which are sequentially overlapped on the bottom plate 1. In a preferred embodiment, the interface between the sample pad 2 and the colloidal gold conjugate pad 3 is increased to improve the sample diffusion efficiency. Due to the interface, the sample pad 2 is above, the colloidal gold binding pad 3 is below, and the diffusion of the sample from the sample pad 2 to the colloidal gold binding pad 3 is facilitated under the action of gravity.
Sample pad 2 was a glass fiber membrane treated with phosphate buffer, which was phosphate buffer containing 1-5% bsa and surfactant.
The colloidal gold conjugate pad 3 is a glass fiber membrane coated with colloidal gold particles labeled with an antibody AtPA5. The colloidal gold binding pad 3 contains colloidal gold particles, and the antibodies AtPA5 are bound on the colloidal gold particles. Samples dropped in sample pad 2 if tPA is present, the tPA diffuses into the colloidal gold conjugate pad 3 and then the colloidal gold particles can be coupled by AtPA5. tPA with colloidal gold particles continues to diffuse toward nitrocellulose membrane 4 and aggregate on nitrocellulose membrane 4.
The method for labeling the AtPA5 antibody by the colloidal gold particles is as follows:
1) Preparing colloidal gold: diluting 1% chloroauric acid solution into 0.01% with double distilled water, boiling, adding trisodium citrate solution, continuously boiling until the liquid is bright red, stopping heating, and supplementing water lost due to boiling to obtain colloidal gold;
2) Adding an AtPA5 antibody into the colloidal gold, uniformly mixing, standing, centrifuging, taking the precipitate, and washing twice to obtain the colloidal gold particles marked with the AtPA5. The colloidal gold particles labeled with AtPA5 were sprayed on a glass fiber film to prepare a colloidal gold conjugate pad 3.
The nitrocellulose membrane 4 is provided with a detection line 411. When the coated antibody AtPA3 is anchored on the detection line 411 and tPA with colloidal gold particles diffuses on the nitrocellulose membrane 4, the tPA with colloidal gold particles is combined with the coated antibody AtPA3 anchored in the detection line 411 and accumulated on the detection line 411 when encountering the detection line 411, and the more the tPA with colloidal gold particles is accumulated on the detection line, the darker the color development.
And a quality control line 412 is further arranged on the nitrocellulose membrane 4, and the quality control line 412 is far away from the colloidal gold bonding pad 3 than the detection line 411. A murine anti-alpaca IgG is anchored to the control line 412. When the colloidal gold particles diffuse to the quality control line 412, they are collected on the quality control line 412. Therefore, during the diffusion process, the colloidal gold particles first encounter the detection line 411, the colloidal gold particles combined with tPA are collected on the detection line 411, the colloidal gold particles not combined with tPA continue to diffuse forward, and when encountering the quality control line 412, the colloidal gold particles are collected on the quality control line 412. Since colloidal gold particles are much more abundant than tPA, the quality control line 412 will develop regardless of whether tPA is present in the sample.
The preparation method of the nitrocellulose membrane 4 is as follows:
1) Blocking the nitrocellulose membrane in blocking solution of 0.01M phosphate buffer (pH 7.0) containing 1% BSA and 0.1% Tween-20 for 60 min;
2) And respectively adding the capture antibody AtPA3 and the mouse anti-alpaca IgG into spot membrane diluent to obtain AtPA3 spot membrane liquid and mouse anti-alpaca IgG antibody spot membrane liquid, and respectively spraying the AtPA3 spot membrane liquid and the mouse anti-alpaca IgG spot membrane liquid on a detection line and a quality control line which are 5mm apart according to the amount of 2 mu L/cm. Here, the spot film dilution was a 0.01M phosphate buffer (pH 7.4) containing 0.15M sodium chloride, 10mM ethylenediamine tetraacetic acid, 1g/L sodium azide and 25g/L methanol. The concentration of the AtPA3 spot membrane solution is 1.25 mug/mL, and the concentration of the murine anti-alpaca IgG spot membrane solution is 1.5 mug/L.
In qualitative detection, the cotton piece was placed in the nasal cavities on both sides of the patient for 5 minutes, after which the cotton piece was taken out and placed in a 5mL EP tube, and 2mL of sterile physiological saline was added thereto and left for 5 minutes. The cotton piece was removed with forceps and placed in a 10mL syringe and the liquid was squeezed into another EP tube. The EP tube was centrifuged at 1500g for 10 minutes, after which the supernatant was transferred to another EP tube for detecting the level of tPA.
Adding 50 μl of nasal secretion supernatant into 50 μl of sample pretreatment solution, stirring, mixing, dripping into sample adding region of sample pad of test strip, and performing chromatography at 20-30deg.C for 5min. And determining whether the sample contains tPA or not by observing the detection line and the quality control line. Namely, when both the detection line and the quality control line develop, tPA is contained in the sample; when the detection line does not develop color, and the quality control line develops color, the sample does not contain tPA.
For quantitative detection, a color value reading system is required to be prepared. And placing the chromatographic reaction test strip under a scanning device of a color value reading system for scanning, and processing and judging the scanned image through the color value reading system to obtain the concentration of tPA.
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.
Claims (5)
1. The test strip for detecting human tissue type plasminogen activator is characterized by comprising a base plate (1), and a sample pad (2), a colloidal gold binding pad (3), a nitrocellulose membrane (4) and a water absorption pad (5) which are sequentially connected on the base plate (1), wherein a detection line (41) is arranged on the nitrocellulose membrane (4);
the colloidal gold binding pad (3) contains colloidal gold particles marked by binding antibodies; a capture antibody is anchored in the detection line (41), the binding antibody and the capture antibody can specifically recognize and bind to tissue type plasminogen activator, and the binding antibody and the capture antibody do not interfere with the site of tissue type plasminogen activator binding;
the binding antibody is a nano antibody and comprises three CDR regions, and the sequence of the binding antibody is shown as SEQ ID NO. 6-8;
the capture antibody is a nano antibody and comprises three CDR regions, and the sequence of the capture antibody is shown as SEQ ID NO. 2-4.
2. The test strip of claim 1, wherein the binding antibody has a sequence as shown in SEQ ID NO. 5.
3. The test strip of claim 1, wherein the capture antibody has a sequence as shown in SEQ ID NO. 1.
4. A test strip according to any one of claims 1-3, characterized in that a quality control line (42) is further provided on the nitrocellulose membrane (4), the quality control line (42) being further away from the colloidal gold binding pad (3) than the detection line (41).
5. The strip of claim 4, wherein murine anti-alpaca IgG is anchored in the control line (42).
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| CN113156121A (en) * | 2021-04-16 | 2021-07-23 | 苏州东尼生物技术有限公司 | Colloidal gold test strip for detecting human thyroglobulin |
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| WO2003062824A1 (en) * | 2002-01-23 | 2003-07-31 | Boditech Inc. | Lateral flow quantitative assay method and strip and laser-induced fluoerescence detection device therefor |
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|---|---|---|---|---|
| EP0190711A2 (en) * | 1985-02-07 | 1986-08-13 | Kowa Company, Ltd. | Monoclonal antibodies to tissue plasminogen activator derived from human normal cells |
| US5102787A (en) * | 1987-07-10 | 1992-04-07 | Miho Sasamata | Method, device and kit for measurement of tissue plasminogen activator activity |
| EP0339302A1 (en) * | 1988-04-04 | 1989-11-02 | Teijin Limited | Reagent system for immunologically assaying complex of human plasminogen activator inhibitor and human tissue plasminogen activator, and assay kit therefor |
| JP2015068764A (en) * | 2013-09-30 | 2015-04-13 | 凸版印刷株式会社 | Method for measuring density of analysis object and test strip for lateral flow |
| CN112881714A (en) * | 2021-02-01 | 2021-06-01 | 德林医疗科技(武汉)有限公司 | Colloidal gold test strip for detecting human parathyroid hormone |
| CN113156121A (en) * | 2021-04-16 | 2021-07-23 | 苏州东尼生物技术有限公司 | Colloidal gold test strip for detecting human thyroglobulin |
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| CN116068179A (en) | 2023-05-05 |
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