CN116930489A - Chromatographic test strip, detection kit and method - Google Patents
Chromatographic test strip, detection kit and method Download PDFInfo
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- CN116930489A CN116930489A CN202210339918.3A CN202210339918A CN116930489A CN 116930489 A CN116930489 A CN 116930489A CN 202210339918 A CN202210339918 A CN 202210339918A CN 116930489 A CN116930489 A CN 116930489A
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- biotin
- test strip
- streptavidin
- chromatographic
- pad
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- 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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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/5436—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand physically entrapped within the solid phase
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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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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- 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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- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The application relates to a chromatographic test strip, a detection kit and a method. The chromatographic test strip comprises a bottom plate, a sample pad, a binding pad, a chromatographic membrane and a water absorption pad, wherein the sample pad, the binding pad, the chromatographic membrane and the water absorption pad are arranged on the bottom plate, a marker agent marked by a marker is arranged on the binding pad, the marker agent marked by the marker can form a compound with an analyte and a capturing agent connected with biotin and/or biotin derivatives, a T line is arranged on the chromatographic membrane, the T line is provided with poly-streptavidin, the poly-streptavidin is used for binding the compound, and the sensitivity of the chromatographic test strip is high.
Description
Technical Field
The application relates to the technical field of immunodetection, in particular to a chromatographic test strip, a detection kit and a detection method.
Background
The traditional immunochromatography test strip is a combination of immunology and thin layer chromatography technology, and the structure of the traditional immunochromatography test strip mainly comprises five parts: a sample application area (sample pad), a labeling area (conjugate pad), a display area (chromatographic membrane), a water absorption area (water absorption pad), and a bottom plate. The sample pad is mainly used for sample adding; the conjugate pad is mainly used for loading markers (such as monoclonal antibodies or secondary antibodies or antigens marked by colloidal gold) and stably releasing the markers in chromatographic detection; the chromatographic membrane has the main functions of solidifying antigen, antibody and the like and providing a place for chromatographic detection reaction, and a sample application instrument is mainly used for linearly solidifying the antigen, antibody and the like on the membrane to form a detection line (T line) and/or a quality control line (C line) in an immunochromatography experiment; the water absorption pad is a power source of the chromatographic reaction and controls the continuous flowing direction of the sample to be detected in the chromatographic reaction; the bottom plate is used for providing rigid support for the laminated structure of the immunochromatographic test strip.
Various immunological reaction forms can be applied to the immunochromatographic test strip, such as a common double-antibody sandwich method, a double-antigen sandwich method, an indirect method, a capturing method and the like. The sandwich-based chromatographic test strip utilizes a immobilized capture antibody or antigen on a detection line to capture and fix an analyte in a sample to be detected, and simultaneously, the labeled antibody or antigen is combined with the analyte, so that a sandwich structure compound with a label is formed on the detection line, and a detectable/visible signal is displayed through the label. Specifically, the chromatography test strip based on the indirect method utilizes immobilized antigen on a detection line to capture and fix an analyte (antibody) in a sample to be detected, and simultaneously, a labeled secondary antibody (anti-antibody) is combined with the analyte, so that a sandwich structure compound with a label is formed on the detection line, and a detectable/visible signal is displayed through the label; the capture method is to solidify the secondary antibody and take the antigen as a labeling end to form an immune complex form of the secondary antibody-antigen-label to be detected so as to present signals.
When using a sandwich-based chromatographic test strip, a sample to be tested (e.g., serum, urine, etc.) is added to the sample pad, and if an analyte is present in the sample to be tested, it reacts with the labeling agent in the conjugate pad to form a complex, which flows through the chromatographic membrane toward the absorbent pad by capillary action and is captured by the immobilized ligand molecules (capture site on the detection line) of the corresponding antigen or antibody on the chromatographic membrane, so that it can be seen visually or detected by a device capable of recognizing the label signal.
In addition to immunochromatography, chemiluminescence and enzyme-linked immunity can also carry multiple immunological reaction forms; in contrast to immunochromatography, solid phase forms are usually employed, for example, as magnetic beads or microwell plates; labels are typically employed such as horseradish peroxidase, alkaline phosphatase, acridinium esters, and the like.
The sensitivity of these detection methods and modes has room for improvement.
Disclosure of Invention
Based on this, it is necessary to provide a chromatographic test strip having improved sensitivity.
Meanwhile, a detection reagent, a detection kit and an immune detection method capable of improving sensitivity are also provided.
The chromatographic test strip comprises a bottom plate, a sample pad, a binding pad, a chromatographic membrane and a water absorption pad, wherein the sample pad, the binding pad, the chromatographic membrane and the water absorption pad are arranged on the bottom plate, a marker marked by a marker is arranged on the binding pad, the marker marked by the marker can form a compound with an analyte and a capturing agent connected with biotin and/or biotin derivatives, a T line is arranged on the chromatographic membrane, and the T line is provided with streptavidin.
The chromatographic test strip captures the analyte in the sample to be detected by using the capture agent connected with the biotin and/or the biotin derivative, the marker marked by the marker on the binding pad can be combined with the capture agent capturing the analyte to form a compound, and the compound is enriched on a T line by using the specific combination of the biotin and/or the biotin derivative and the poly-streptavidin, so that the detection sensitivity is improved compared with the common streptavidin mode.
In one embodiment, the capture agent with attached biotin and/or a biological derivative is provided on the sample pad.
In one embodiment, the label comprises at least one of a chromophore, a digoxin-labeled probe, an electron dense substance, a metal particle, and an enzyme that produces a detectable signal.
In one embodiment, the molecular weight of the polymeric streptavidin is above 150 kDa.
In one embodiment, the molecular weight of the polymeric streptavidin is 150kDa to 1340kDa.
In one embodiment, the analyte is an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimitope, or aptamer.
In one embodiment, the analyte is one of SARS-CoV-2, N-protein of SARS-CoV-2, SARS-CoV-2 antibody, C-reactive protein, serum amyloid A, procalcitonin, interleukin 6, urinary microalbumin, beta 2-microglobulin, retinol binding protein, cystatin C, human neutrophil gelatinase-associated lipocalin, D-dimer, troponin I, troponin T, N terminal brain natriuretic peptide, N-terminal brain natriuretic peptide precursor, brain natriuretic peptide, creatine kinase isozymes or myoglobin.
In one embodiment, the chromatographic test strip is based on a sandwich method.
A detection reagent comprising a solid support and a polymeric streptavidin coated on the solid support.
In one embodiment, the molecular weight of the polymeric streptavidin is above 150 kDa.
In one embodiment, the solid support comprises at least one of a tube, a plate, a membrane, and a microsphere.
In one embodiment, the molecular weight of the polymeric streptavidin is 150kDa to 1340kDa.
A detection kit comprising the above-described detection reagent;
in one embodiment, the detection kit further comprises a capture agent to which biotin and/or a biotin derivative is attached; and/or the detection kit further comprises a labeling agent labeled by a labeling substance.
In one embodiment, the analyte of the detection kit is an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimitope, or aptamer.
In one embodiment, the label comprises at least one of a chromophore, a digoxin-labeled probe, an electron dense substance, a metal particle, and an enzyme that produces a detectable signal.
A chromatography kit comprising:
a capture agent to which biotin and/or a biotin derivative is attached; and
The chromatographic test strip.
An immunoassay method using a complex of a label and a capture agent to which biotin and/or a biotin derivative is attached, the complex being bound to a streptavidin-coated solid support.
In one embodiment, the molecular weight of the polymeric streptavidin is above 150 kDa.
In one embodiment, the molecular weight of the polymeric streptavidin is 150kDa to 1340kDa.
In one embodiment, the solid support comprises at least one of a tube, a plate, a membrane, and a microsphere.
In one embodiment, the marking agent is a marking agent that is labeled with a marker.
Drawings
FIG. 1 is a schematic diagram of a chromatographic test strip according to an embodiment;
FIG. 2 is a SEC spectrum of example 1;
FIG. 3 is a standard curve of the test strips in Table 1 in example 2.
Reference numerals;
10. a chromatographic test strip; 110. a bottom plate; 120. a sample pad; 130. a bonding pad; 140. a chromatographic membrane; 150. a water absorbing pad; 141. a T line; 142. and C line.
Detailed Description
The present application will be described more fully hereinafter in order to facilitate an understanding of the application, which may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
It is noted that the term "and/or" includes any and all combinations of one or more of the associated listed items.
The term "antibody" refers to a protein or polypeptide of an immunoglobulin molecule that specifically binds to a corresponding antigen. Antibodies may be polyclonal or monoclonal, multi-chain or single-chain or intact immunoglobulins, derived from natural sources or recombinant sources. For example, naturally occurring IgG antibodies are tetramers comprising at least two heavy (H) chains and two light (L) chains connected to each other by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains (CH 1, CH2 and CH 3). Each light chain is composed of a light chain variable region (VL) and a light chain constant region. The light chain constant region is composed of one domain (CL). VH and VL regions can be further subdivided into regions of hypervariability, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FR). Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody may mediate the binding of an immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q). "antibodies" include, but are not limited to, monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, and anti-idiotype (anti-Id) antibodies. Antibodies can be of any isotype/class (e.g., igG, igE, igM, igD, igA and IgY) or subclass (e.g., igG1, igG2, igG3, igG4, igA1, and IgA 2).
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the description of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.
Referring to fig. 1, an embodiment of the present application provides a chromatographic test strip 10 for detecting an analyte in a sample, where the chromatographic test strip 10 includes a base plate 110, a sample pad 120, a binding pad 130, a chromatographic membrane 140 and a water absorbing pad 150 disposed on the base plate 110. The conjugate pad 130 is provided with a labeling agent labeled with a labeling substance, the labeling agent labeled with a labeling substance can form a complex with an analyte and a capturing agent to which biotin and/or a biotin derivative is attached, the chromatographic carrier 140 is provided with a T line 141 and a C line 142, and the T line 141 is provided with a streptavidin.
Biotin (B) is widely distributed in animal and plant tissues and is often extracted from egg yolk and liver tissues with high content. Molecular weight of biotin 244.31Da. The biotin molecule has two cyclic structures, wherein the I ring is an imidazolone ring and is a main part combined with avidin; the ring II is thiophene ring, C2 has a valeric acid side chain, and its terminal carboxyl is the only structure for combining antibody and other biomacromolecule, after chemical modification, biotin can be made into derivative-activated biotin with various active groups. Activated biotin can be coupled to almost all known biomacromolecules, including proteins, nucleic acids, polysaccharides, lipids, etc., mediated by protein cross-linking agents.
Streptavidin (SA) is a protein secreted by streptomyces and has a molecular weight of 50 kDa-70 kDa. Streptavidin does not contain cysteine residues, carbohydrate side chains or related cofactors. Streptavidin is a homotetramer consisting of 4 identical peptide chains, each consisting of 159 amino acids, each capable of binding one biotin. That is, one streptavidin molecule can bind to 4 biotin molecules. Streptavidin has an affinity constant (K) for biotin of about 10 -15 mol/L。
Taking conventional immunochromatography as an example, when a labeled complex (i.e., a complex formed by a labeling agent labeled with a label and an analyte, the same applies hereinafter) reacts with an antigen or antibody immobilized on a T-line, a large amount of labeled complex cannot bind to the antigen or antibody on the T-line due to a short reaction time, so that the sensitivity of the test paper is lost. In contrast, in the case of using an avidin-biotin amplification system, for example, a label-streptavidin-biotin is attached to an antigen or antibody, and after the antigen or antibody to which the label-streptavidin-biotin is attached forms a complex with an analyte and another antigen or antibody immobilized on a T line, detection is performed by naked eyes or by detecting the amount of the label, but the antigen or antibody to which the label-streptavidin-biotin is attached is also affected by steric hindrance, and thus the improvement of sensitivity is not good. For example, streptavidin may be immobilized on the T-line to capture the biotin-antibody-antigen-antibody-label complex, but it has been found that the binding efficiency of biotin to streptavidin in this mode is affected and does not exert a true amplification effect.
The chromatographic test strip 10 of the present application is characterized in that a labeling agent for binding to a capture agent for capturing an analyte is labeled with a labeling agent by attaching biotin and/or a biotin derivative to the capture agent for capturing an analyte, and the labeling agent labeled with the labeling agent is provided on the binding pad 130, so that a complex formed by the labeling agent, the analyte and the capture agent is formed on the binding pad 130, and then moves to the T line 141 under the continued action of capillary force and binds to the poly-streptomycin provided on the T line 141, thereby quantitatively and/or qualitatively judging the analyte by the labeling agent enriched on the T line 141. The chromatographic test strip utilizes the poly SA envelope to increase the molecular weight of the streptavidin, so that the binding sites of SA and biotin are fully exposed, more complexes can be bound to the streptavidin, the real amplification effect is achieved, and the detection sensitivity is improved.
Specifically, the sample pad 120 is used for sample application. The material of the sample pad 120 is not particularly limited. In some embodiments, sample pad 120 is a filtration medium. The filter media may filter particles in the sample, adjust the pH of the sample, and bind components of the sample that interfere with subsequent chromatographic reactions. In an alternative specific example, the sample pad 120 is made of cellulose, fiberglass, or rayon.
Specifically, a capture agent having biotin and/or a biotin derivative attached thereto is used to capture an analyte, and specifically binds to the poly-streptomycin on the T-line 141 through biotin and/or a bio derivative thereon after forming a complex with the analyte. The biotin derivative herein is a derivative having a function equivalent to biotin, and specifically binds to streptavidin. Alternatively, the biotin derivative is selected from D-biotin, activated biotin, ethylenediamine biotin, cadaverine biotin or desthiobiotin.
In some embodiments, a capture agent with biotin and/or a biological derivative attached thereto is disposed on the sample pad 120. It will be appreciated that in other embodiments, capture agents with biotin and/or biological derivatives attached thereto may not be disposed on the sample pad 120. For example, in some embodiments, the capture agent to which biotin and/or a biotin derivative is attached is disposed independently outside of the chromatographic test strip. In use, a sample to be tested is mixed with a capture agent having biotin and/or a biological derivative attached thereto and applied to the sample pad 120.
In some embodiments, the sample pad 120 may also be optionally coated with a blocking agent. The blocking agent is used for removing endogenous interference and can be combined with substances with interference in a sample to be detected. Blocking agents commercially available in the art, such as those effective against HAMA and rheumatoid factors, can generally be selected.
Specifically, the conjugate pad 130 is used to load the label agent labeled with the label and to enable stable release of the label agent labeled with the label in chromatographic detection. In some embodiments, the bonding pad 130 is made of glass fiber, polyester fiber, or rayon. It is understood that the material of the bonding pad 130 is not particularly limited.
Herein, a label refers to a substance capable of providing a detected signal. In some embodiments, the label is selected from at least one of a chromophore, a digoxin-labeled probe, an electron dense substance, a metal particle, and an enzyme that produces a detectable signal. Optionally, the chromophore is selected from one or more of fluorescence, quantum dots, fluorescent microspheres, luminescent compounds, and dyes. In some embodimentsThe chromophore is a luminescent compound. Such as acridinium esters, acridinium ester derivatives, adamantane, luminol, isoluminol, and the like. Optionally, the acridinium ester derivative is selected from at least one of acridinium ester sulfonamide, acridinium ester tosylamide, acridinium ester para-methylsulfonamide and acridinium ester trifluoromethylsulfonamide. The electron dense material is a radioactive molecule. For example 32 P， 35 S or 125 I. In some embodiments, the enzyme that produces the detectable signal is selected from one of horseradish peroxidase, alkaline phosphatase, beta-galactosidase, and glucose-6-phosphate dehydrogenase. In some embodiments, the metal particles are colloidal metals, such as colloidal gold, colloidal silver, colloidal selenium, and the like. It will be appreciated that the markers are not limited to the above, but may be other substances having a color that can be directly observed by the naked eye and detected by means of an instrument.
In some embodiments, the label is directly attached to the labeling agent.
In other embodiments, the label is indirectly linked to the labeling agent through a bridge. Optionally, the bridge is selected from one or more of a protein, a protein complex, or a bifunctional cross-linking agent. In one embodiment, the protein used as the bridge comprises bovine serum albumin, ovalbumin, keyhole limpet hemocyanin, immunoglobulin, thyroglobulin, or polylysine. The protein complex as the bridge contains at least two of bovine serum albumin, ovalbumin, keyhole limpet hemocyanin, immunoglobulin, thyroglobulin, and polylysine. In an alternative embodiment, the bridge is bovine serum albumin. Bovine serum albumin itself has a certain effect of reducing nonspecific signals, and can increase the signal-to-noise ratio of experiments. Optionally, the bifunctional crosslinking reagent is selected from any of 4- (4-N-maleimidophenyl) butanoic acid hydrazide (4- [ 4-N-maleimidopynyl ] butyric acid hydrazide hydrochloride, MPBH), 1- [2- [2- (2-Aminoethoxy) ethoxy ] ethyl ] maleimide hydrochloride (1- [2- [2- (2-amino-methoxy) method ] ethyl ] maleimide hydrochloride, MPEG 2A), N- [ beta-maleimidopropionic acid ] hydrazide, trifluoroacetate (N- [ beta-maleimidopropionic acid ] hydrazine, trifluoroacetic acid salt, BMPH), N- [ epsilon-maleimidohexanoic acid) hydrazide, trifluoroacetate (N- [ epsilon-Maleimidocaproic acid) hydrazine, trifluoroacetic acid salt, EMCH), N- [ kappa-maleimido undecanoic acid ] hydrazide, trifluoroacetate (N- [ kappa-maleimidoundecanoic acid ] hydrazine, trifluoroacetic acid salt, KMUH). It will be appreciated that in some embodiments, the bridge has the effect of amplifying the signal. For example, the bridge may be coupled to more labels to amplify the signal.
Specifically, the analyte is a target detected by the chromatographic strip 10 described above. An analyte is a substance that is capable of specifically binding to both a capture agent and a labeling agent. The analyte is presented in the form of a binding pair with a capture agent and a labeling agent. Such as receptor/ligand pairs, antibodies/antigens, natural or synthetic receptor/ligand pairs, hapten/antibody pairs, antigen/antibody pairs, epitope/antibody pairs, mimotope/antibody pairs, aptamer/target molecule pairs, hybridization partners, and intercalator/target molecule pairs. For example, in some embodiments, the analyte is an oligonucleotide sequence, an aptamer ligand, an antibody, an antigen, a ligand, a receptor, a hapten, an epitope, or a mimotope, and the corresponding capture and labeling agents comprise complementary oligonucleotide sequences, aptamer ligands, aptamers, antigens, antibodies, receptors, ligands, or antibodies, respectively. In an alternative specific example, the analyte is an antigen and the capture and labelling agents are antibodies that specifically bind to the antigen. Of course, in sandwich-based immunoassays, the binding site of the capture agent to the analyte is different from the binding site of the label agent and the analyte. Alternatively, the analyte is SARS-CoV-2, N protein of SARS-CoV-2, SARS-CoV-2 antibody, C-reactive protein, serum amyloid A, procalcitonin, interleukin 6, urinary microalbumin, beta 2-microglobulin, retinol binding protein, cystatin C, human neutrophil gelatinase-associated lipocalin, D-dimer, troponin I (e.g., cardiac troponin I), troponin T, N terminal brain natriuretic peptide, N-terminal brain natriuretic peptide precursor, brain natriuretic peptide, creatine kinase isozymes or myoglobin.
Specifically, the chromatographic carrier 140 mainly serves to immobilize the streptavidin. In immunochromatography experiments, a detection line (T line 141) is formed by linear solidification of a poly-streptavidin on a membrane mainly by a contact or non-contact spotter, and in some embodiments, a quality control line (C line 142) is provided on the chromatographic membrane, and in some embodiments, a quality control line (C line 142) is formed by linear solidification of an antigen or antibody on the chromatographic membrane.
In some embodiments, the chromatographic membrane 140 is a nitrocellulose membrane.
The poly-streptavidin is used to bind the complex of analyte with capture and labelling agents. In this context, poly-streptavidin refers to a substance polymerized from at least two streptavidin. In some embodiments, the molecular weight of the polymeric streptavidin is above 150 kDa. Further, the molecular weight of the poly-streptavidin is 150 kDa-1340 kDa. It should be noted that the term "above" when referring to a numerical range includes the present number.
In some embodiments, the polymeric streptavidin is polymerized by condensation of amino and carboxyl groups of the streptavidin to form an amide bond. Optionally, an activator for activating the carboxyl group is mixed with streptavidin to prepare the poly-streptavidin. The activator includes a carbodiimide. In an alternative specific example, the activator is at least one of dicyclohexylcarbodiimide, 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC), and N, N' -diisopropylcarbodiimide. In some embodiments, the activator further comprises at least one of N-hydroxysuccinimide (NHS) and N-hydroxysulfosuccinimide (Sulfo-NHS). The reaction efficiency is increased by N-hydroxysuccinimide and/or N-hydroxysulfosuccinimide. It is understood that the preparation method of the poly-streptavidin is not limited to the above, but may be other methods.
In some embodiments, the conjugate pad 130 is further coated with a label-labeled quality control; the C line 142 is coated with a substance capable of specifically binding to the quality control substance. Optionally, the quality control material is a secondary antibody with a marker; the C line 142 is coated with a primary antibody. The labels are as described above. In an alternative specific example, the quality control material is sheep anti-chicken IgY-fluorescent microsphere, and the C-wire 142 is coated with chicken IgY. It is understood that the materials coated on the quality control material and the C-line 142 are not limited to the above.
Specifically, the water absorbing pad 150 is a power source of the chromatographic reaction, and controls the continuous flow direction of the sample to be detected in the chromatographic reaction. The absorbent pad 150 is generally made of high density cellulose.
In particular, the base plate 110 is used to provide rigid support for the stacked structure of the chromatographic test strip 10. Alternatively, the material of the base plate 110 is polystyrene or other plastic material.
In some embodiments, the chromatographic test strip is based on a sandwich method. It is understood in the art that the corresponding effect can be achieved by using the poly SA based on an indirect method, a capturing method, and the like.
Based on the above, an embodiment of the present application also provides a detection reagent, which includes a solid phase carrier and a poly-streptavidin coated on the solid phase carrier. The detection reagent can be used as a component of a detection kit, and a complex formed by an analyte, a labeling agent, the analyte and a capturing agent is captured by using a solid-phase carrier coated with the poly-streptavidin, so that detection is realized.
Specifically, the streptavidin is as described above.
In some embodiments, the solid support comprises at least one of a tube, plate, membrane, or microsphere, e.g., an Ep tube, multiwell plate, or the like.
In one embodiment, the multiwell plate is an elisa plate. In an alternative specific example, the holes of the porous plate are 16, 32, 48, 64, 96 or more.
As used herein, the term "microsphere" may be a sphere, a spheroid, a cube, a polyhedron, or an irregular shape. In some embodiments, the microspheres have a diameter of 10nm to 1mm. In an alternative specific example, the microsphere has a diameter of 100nm, 500nm, 1 μm, 10 μm, 100 μm or 500 μm. Further, the diameter of the microsphere is 400nm to 10 μm. In some embodiments, the microspheres are magnetic beads, the composition of which contains a magnetic substance. The magnetic substance can be metal (metal simple substance or alloy), nonmetal, or a compound formed by metal and nonmetal. Metals such as iron, alnico metals, and the like; non-metallic exampleSuch as ferrite nonmetallic (e.g. Fe 2 O 3 Or Fe (Fe) 3 O 4 Magnetic nanoparticles); composites of metals and non-metals such as neodymium iron boron rubber magnetic composites. In some embodiments, the surface of the microsphere is modified with one or more reactive functional groups. Alternatively, the reactive functional groups include-OH, -COOH, -NH 2 (s) -CHO and-SO 3 H, one or more of H. In some embodiments, the coated streptavidin is conjugated or bound to the microsphere by physical adsorption or direct chemical conjugation (e.g., bridging by a bridge). In some embodiments, the solid support is one or more of polystyrene, plastic, cellulose, polyacrylamide, polyethylene polypropylene, cross-linked dextran, glass, silicone rubber, and agarose gel.
Based on the above, an embodiment of the present application further provides a detection kit, which includes the detection reagent described above.
In some embodiments, the detection kit further comprises a capture agent to which biotin and/or a biotin derivative is attached; specifically, the capture reagent and the detection reagent are as described above. When the kit is used, qualitative or quantitative detection of the analyte is realized through the cooperation of the capture agent, the detection reagent and the labeling agent.
In some embodiments, the detection kit further comprises a labeling agent. Specifically, the marking agent is specifically as described above. It will be appreciated that in other embodiments, the marking agent may be omitted. When the kit containing no labeling agent is used, it may be used in combination with another labeling agent (for example, another commercially available one).
In some embodiments, the analyte, label are as described above.
Based on the above, an embodiment of the present application further provides a chromatography kit, which includes a capture agent and a chromatography test strip. Specifically, the capture agent is as described above for immunochromatographic reagents and will not be described here. The chromatographic test strip in the chromatographic kit is approximately the same as the chromatographic test strip above, namely the chromatographic test strip in the chromatographic kit also comprises a bottom plate, a sample pad, a binding pad, a chromatographic membrane and a water absorption pad which are positioned on the bottom plate, wherein the binding pad is provided with a marker agent marked by a marker, the marker agent marked by the marker is used for forming a compound with an analyte and a capturing agent connected with biotin and/or biotin derivatives, the chromatographic membrane is provided with a T line, and the T line is provided with streptavidin which is used for binding the compound. The difference is that the sample pad of the chromatographic test strip in the chromatographic kit is not provided with a capturing agent. In use, the capture agent is mixed with the sample to be tested and then applied to the sample pad. By setting the capture agent independent of the chromatographic test strip, the capture agent and the sample to be tested can be mixed more uniformly, so that the capture agent and the analyte in the sample to be tested are combined more fully, and the influence on the detection sensitivity caused by less combination of the capture agent and the analyte due to high chromatographic speed is reduced.
In some embodiments, the chromatography kit further comprises a blocking agent. In some embodiments, the blocking agent is independent of the chromatographic test strip. In use, the blocking agent is mixed with the sample to be tested and then applied to the sample pad of the chromatographic test strip. In other embodiments, the blocking agent is coated on a sample pad of a chromatographic test strip. Of course, the composition of the blocking agent is as described above at the chromatographic test strip.
The chromatographic test strip of the chromatographic kit is approximately the same as the immunochromatographic reagent strip, and the same advantages as the immunochromatographic reagent strip are achieved by arranging the polymeric streptavidin on a T line for detection.
In addition, an embodiment of the present application provides an immunoassay method using a complex of a polymeric streptavidin-conjugated analyte coated on a solid support, a labeling agent and a capturing agent, wherein the capturing agent is a capturing agent to which biotin and/or a biotin derivative is attached. Alternatively, the detection method utilizes a double-antibody sandwich method, a double-antigen sandwich method, an indirect method or a capture method to detect the analyte in the sample to be detected. It will be appreciated that the solid support herein may be the solid support described above for the detection reagent or may be a chromatographic membrane in a chromatographic strip as described above. The polymeric streptavidin, capture agent, labeling agent, analyte, label may be as described herein above.
In some embodiments, the detection method comprises the steps of: after mixing the sample to be measured, the capturing agent, the labeling agent and the solid phase carrier coated with the poly-streptavidin, detecting the signal of the complex formed on the solid phase carrier to determine the amount of the analyte in the sample to be measured.
In some embodiments, the detection method is performed by using the chromatographic test strip of any of the embodiments described above. Optionally, the detecting method includes the steps of: after dropping the sample to be measured onto the sample pad of the chromatographic test strip of any of the above embodiments, the signal on the T-line is observed or detected, thereby determining the amount of the analyte in the sample to be measured.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The following is a detailed description of specific embodiments. The following examples are not specifically described but do not include other components than the unavoidable impurities. Reagents and apparatus used in the examples, unless otherwise specified, are all routine choices in the art. The experimental methods without specific conditions noted in the examples were carried out according to conventional conditions, such as those described in the literature, books, or recommended by the manufacturer.
Example 1
Preparation of Polystreptavidin (PolySA)
Mixing 10mg/mL Streptavidin (SA) and 20mg/mL EDC according to the volume of 1:1, reacting for 1h, blocking with glycine, dialyzing and quantifying to obtain the poly SA. Molecular weight determination was performed by Size Exclusion Chromatography (SEC), the SEC chromatogram being shown in FIG. 2, and the resulting multimeric SA had a molecular weight of approximately > 150kDa and ranging from 150kDa to 1340kDa.
Example 2
Fluorescent microsphere platform-cardiac troponin I (cTnI) detection chromatography test strip
1. Preparation of detection antibody-fluorescent microsphere
Taking 200 microliters of yellow-green fluorescent microsphere mother liquor, centrifuging at 15000rpm at 4 ℃ for 5min, removing supernatant after centrifugation, adding an activation buffer solution for re-dissolution, and uniformly dispersing by ultrasonic waves; EDC with the final concentration of 0.2mg/mL and NHS solution with the final concentration of 5mg/mL are respectively added, uniformly mixed for 18min at room temperature and in the dark, and centrifuged at 15000rpm for 5min at 4 ℃, and the supernatant is removed; adding coupling buffer solution for redissolution, performing ultrasonic dispersion uniformly, centrifuging at 15000rpm at 4 ℃ for 5min, removing supernatant, redissolving in the coupling buffer solution, performing ultrasonic dispersion uniformly, adding 60 mug detection antibody Ab1 (namely cTnI Ab 1), coating aluminum foil paper in a dark place, and performing shake reaction for 2.5h in a mixer; centrifuging to remove supernatant after the reaction is completed, adding 200 mu L of sealing buffer solution, uniformly dispersing by ultrasonic, and then placing on a mixer for shake reaction for 45min; centrifuging at 15000rpm at 4deg.C for 5min after sealing, and removing supernatant; adding 200 microliter of washing solution for redissolution, uniformly dispersing by ultrasonic, centrifuging at 15000rpm at 4 ℃ for 5min, removing supernatant, finally adding 200 microliter of fluorescent microsphere preservation solution for redissolution, uniformly dispersing by ultrasonic, and preserving at 4 ℃.
2. Preparation of detection antibody Ab 1-fluorescent microsphere binding pad
And (3) preparing microsphere working solution according to the concentration of 15% by using the prepared detection antibody Ab 1-fluorescent microsphere, adding 1% concentration of goat anti-chicken IgY-fluorescent microsphere and 1% Evan blue dye solution into the microsphere working solution, uniformly spraying on glass fibers by using a gold spraying spot film instrument according to the condition of 4 mu L/cm, and drying at 50 ℃ for 2 hours to prepare the Ab 1-fluorescent microsphere bonding pad.
3. NC film preparation
A nitrocellulose membrane and absorbent paper are stuck on a PVC bottom plate, SA (namely streptavidin) or poly SA prepared in the embodiment 1 is respectively diluted to 1mg/mL by using a coating diluent, meanwhile, chicken IgY of a C line coating antibody is diluted to 1mg/mL, and the mixture is uniformly marked on an NC membrane by using a metal spraying spot film instrument according to the parameter of 1 mu L/cm, and is dried for 2 hours at 37 ℃.
4. Preparation of biotinylated Capture antibody
The capture antibody Ab2 (namely cTnI Ab 2) is mixed with biotin for reaction for 1h and then dialyzed in a dialysis bag for 4 hours, so as to prepare the biotinylated capture antibody Ab2.
5. Preparation of sample pad
The biotinylated capture antibody Ab2 is diluted to 2 mug/mL, and a blocking agent M009 with a final concentration of 0.4mg/mL is added to prepare a working solution, which is uniformly smeared on glass fibers and dried at 37 ℃ for 2 hours for later use.
6. Assembling and detecting of cTnI detection test strip
The marking pad, sample pad, NC film were assembled as shown in position 1 and cut uniformly 3 mm/bar with a slitter. After 50 mu L of sample is added, the sample is reacted on a test strip for 15min and then is placed in a fluorescence detection instrument to detect T, C signal value.
Control group: the preparation method of the test strip of the control group is approximately the same as that of the test strip, and is different in that the sample pad of the test strip of the control group is not coated with the biotinylated capture antibody Ab2, only is coated with the blocking agent, and the T line of the test strip of the control group is coated with the biotinylated capture antibody Ab2 instead of SA or poly SA.
The results of the detection are shown in Table 1 and FIG. 3. In Table 1, "T/C" indicates the ratio of the fluorescence intensities of the T line and the C line, and the faster the ratio increases, the better the gradient.
TABLE 1
During chromatography, the unpolymerized streptavidin readily diffuses after immobilization on the T-line. As can be seen from Table 1 and FIG. 3, the test strip coated with poly SA on the T line has a remarkable improvement in sensitivity and activity, while the test strip coated with SA on the T line has a lower overall activity and a poor sensitivity.
7. Clinical verification
The test strips of T-wire coated multimeric SA were validated using 77 clinical samples with a conventional test strip (i.e., the control group test strip mentioned in step 6) as a control, and the results are shown in table 2.
TABLE 2
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As can be seen from Table 2, the test strips coated with poly SA on the T line are more excellent in clinical relevance, and the low-value relevance is more obvious.
Example 3
Colloidal gold chromatographic platform-new crown N antigen detection chromatographic test strip
1. Antibody 1 to the N protein of SARS-CoV-2 (i.e. 19covna 1 or neocrown N antibody 1) tag: 10mL of 4/ten thousand (40 nm) colloidal gold was added to 60. Mu.L of 0.2. 0.2M K 2 CO 3 (pH is approximately equal to 6.8-7.2), stirring for 5min, adding 1 μg of the novel crown N antibody (volume of the added antibody=100 μg/antibody concentration), stirring for 10min, adding 100 μl of 10% BSA, blocking, and stirring for 10min; centrifuging at 10000rpm for 5min, removing supernatant, precipitating with gold complex solution, re-dissolving, and finally fixing volume to 1mL (i.e. 1/10 volume of colloidal gold solution) with gold complex solution to obtain colloidal gold labeled new crown N antibody 1 solution.
2. Preparing a working solution of the golden seeds: and (3) finally diluting the colloidal gold-labeled new crown N antibody 1 solution prepared in the step (1) to 10OD by using gold seed diluent to prepare gold seed working solution, and paving the gold seed working solution on glass fibers.
3. Preparing dried golden seeds: the paved golden seeds are put into a freeze dryer for freeze drying (2 h) or are put into a drying room for drying overnight at 37 ℃.
4. And (3) coating a film: the polymeric SA or SA (i.e., streptavidin) prepared in example 1 was diluted to 2.0mg/mL coating using a coating dilution; placing in a 37 ℃ incubator for 60min.
5. Sample pad treatment: the biotin-labeled antibody 2 to the N protein of SARS-CoV-2 (i.e., 19COVNAB2 or neocrown N antibody 2) was diluted to 5. Mu.g/mL with the sample pad treatment solution (or neocrown lysate) and the sample pad was treated. The sample pad was 1.6cm wide by 10cm long and 800. Mu.L.
6. Preparing a gold mark strip: and cutting the gold standard strips according to the required width by using a strip cutting machine, and adding samples for detection after assembly. The results are shown in Table 3. Note that, the readings of the colloid Jin Seka include 10 gradients of C1, C2, C3, C4, C5, C6, C7, C8, C9, and B, the 10 gradients sequentially lighten from left to right, C1 represents the color of the T line is the darkest, C9 represents the color of the T line is the lightest, B represents no color development, and b+ is almost no color development; "C3+" in Table 3 refers to a color that is slightly darker than C3 but does not reach C2, the other bands "+" and so on.
Control group: the preparation method of the test strip of the control group is approximately the same as that of the test strip, and is different in that the sample pad of the test strip of the control group is not coated with the biotinylated capture antibody Ab2, and the T line of the test strip of the control group is coated with the biotinylated capture antibody Ab2 instead of SA or poly SA.
TABLE 3 Table 3
During chromatography, the unpolymerized streptavidin readily diffuses after immobilization on the T-line. As is clear from Table 3, the activity of the SA multimer was 0.5 to 2C higher than that of SA and 0.5 to 1C higher than that of the SA-BIO amplification system without SA.
Example 4
Colloidal gold chromatographic platform-HCV antibody detection chromatographic test strip
Labeling colloidal gold with HCV antigen 1 (HCV-Ag 1), biotinylating HCV antigen 2 (HCV-Ag 2), and preparing the same by the method of example 3; the T-line sets up a poly SA or SA.
The results of detecting the HCV antibody sample show that the activity of the SA polymer detection sample is higher than that of SA by more than 0.5C on an HCV colloidal gold chromatography platform.
Example 5
An HCV antigen detection kit comprising a multimeric SA coated ELISA plate as a detection reagent and HCV antibody 1 (HCV-Ab 1) labeled biotin as a capture reagent; HCV antibody 2 (HCV-Ab 2) labeled HRP as a labeling agent. Specifically, mixing poly SA with coating liquid, coating on an ELISA plate, taking out the plate after coating at 4 ℃, balancing, washing the plate for 2 times, closing overnight at 4 ℃ after beating, and drying for later use to obtain a detection reagent; mixing HCV-Ab1 with biotin solution, crosslinking at 2-8deg.C, and dialyzing to remove free biotin to obtain capture agent; the HCV-Ab2 was labeled with HRP using sodium periodate to obtain a labeling agent. The test sample is tested by the kit, and the result shows that compared with the coated SA serving as a detection reagent, the whole detection activity of the coated poly SA serving as the detection reagent is improved.
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples merely represent a few embodiments of the present application, which facilitate a specific and detailed understanding of the technical solutions of the present application, but are not to be construed as limiting the scope of the application. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the application, which are all within the scope of the application. It should be understood that, based on the technical solutions provided by the present application, those skilled in the art can obtain technical solutions through logical analysis, reasoning or limited experiments, which are all within the scope of protection of the appended claims. The scope of the patent is therefore intended to be covered by the appended claims, and the description and drawings may be interpreted as illustrative of the contents of the claims.
Claims (10)
1. The chromatographic test strip is characterized by comprising a bottom plate, a sample pad, a binding pad, a chromatographic membrane and a water absorption pad, wherein the sample pad, the binding pad, the chromatographic membrane and the water absorption pad are arranged on the bottom plate, a marker marked by a marker is arranged on the binding pad, the marker marked by the marker can form a compound with an analyte and a capturing agent connected with biotin and/or biotin derivatives, a T line is arranged on the chromatographic membrane, and the T line is provided with poly-streptavidin.
2. The chromatographic test strip according to claim 1, wherein the capture agent to which biotin and/or a biological derivative is attached is provided on the sample pad;
and/or the label comprises at least one of a chromophore, a digoxin-labeled probe, an electron dense substance, a metal particle, and an enzyme that produces a detectable signal.
3. The chromatographic test strip according to claim 1, wherein the molecular weight of the poly-streptavidin is above 150 kDa;
optionally, the molecular weight of the poly-streptavidin is 150 kDa-1340 kDa.
4. A chromatographic test strip according to any of claims 1 to 3 wherein the analyte is an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimitope or aptamer.
5. A chromatographic test strip according to any of claims 1-3 characterized in that the analyte is one of SARS-CoV-2, SARS-CoV-2N protein, SARS-CoV-2 antibody, C-reactive protein, serum amyloid a, procalcitonin, interleukin 6, urinary microalbumin, beta 2-microglobulin, retinol binding protein, cystatin C, human neutrophil gelatinase-associated lipocalin, D-dimer, troponin I, troponin T, N terminal brain natriuretic peptide, N-terminal brain natriuretic peptide precursor, brain natriuretic peptide, creatine kinase isozymes or myoglobin;
and/or, the chromatographic test strip is based on a sandwich method.
6. A detection reagent comprising a solid support and a polymeric streptavidin coated on the solid support.
7. The detection reagent according to claim 6, wherein the molecular weight of the streptavidin is 150kDa or higher;
optionally, the molecular weight of the poly-streptavidin is 150 kDa-1340 kDa;
optionally, the solid support comprises at least one of a tube, a plate, a membrane, and a microsphere.
8. A test kit comprising the test reagent of claim 6 or 7;
optionally, the detection kit further comprises a capture agent to which biotin and/or a biotin derivative is attached;
optionally, the detection kit further comprises a labeling agent labeled with a label;
optionally, the analyte of the detection kit is an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimitope, or aptamer;
optionally, the label comprises at least one of a chromophore, a digoxin-labeled probe, an electron dense substance, a metal particle, and an enzyme that produces a detectable signal.
9. A chromatography kit comprising:
a capture agent to which biotin and/or a biotin derivative is attached; and
The chromatographic test strip according to any one of claims 1 to 5.
10. An immunoassay method, characterized in that the assay is performed using a complex of a streptavidin-conjugated analyte coated on a solid support, a labeling agent and a capturing agent; the capture agent is a capture agent with biotin and/or a biotin derivative attached thereto.
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