Detection reagent for fully liquid phase reaction chromatography, method, and system

Abstract

A chromatographic detection method, in which the capture and binding reaction of an object to be tested are completed in a liquid phase medium, achieving high-precision control of the dose response, temperature, and time of a reaction process so as to improve detection performance. By means of a test strip adapted to the method, enrichment of a reaction complex is performed and subsequent detection is performed. Moreover, a membrane structure adapted to the test strip is improved and optimized so that the membrane structure is standardized, thereby achieving universality of the chromatographic carrier for different projects, which not only solves the problems of complex preparation processes of traditional test strips and the difficulty of controlling batch-to-batch differences, but also significantly improves the detection performance of the reagent. The detection method further provides an efficient sample pretreatment solution, comprising sample pretreatment, such as lysis, dilution, anti-interference, blood cell separation, and correction parameter detection, and further provides a preparation and test method for a dry reagent, so as to achieve stable storage and transportation of the reagent. The detection method can be adapted to different detection samples, detection items, detection requirements, or detection scenarios, and significantly improves detection performance.

Description

Liquid-phase reaction chromatographic detection reagent, method and system TECHNICAL FIELD

[0001] The present application relates to the field of medical testing, in particular to a liquid-phase reaction chromatographic detection reagent, method and system. BACKGROUND

[0002] The fluorescent immuno-chromatographic technology belongs to the field of POCT (point-of-care testing), which has the advantages of convenient and fast detection, low comprehensive cost, and normal temperature storage and transportation. The main technical principle is to use a nitrocellulose membrane with fixed antigen / antibody detection lines and a quality control line as a chromatographic carrier, and a fluorescently labeled antigen / antibody fixed combination pad, which flows with the sample to be tested, to form a fluorescently labeled immune complex detection strip. The fluorescence signal intensity is detected by an instrument to convert the concentration.

[0003] The existing fluorescent immuno-chromatographic technology is mostly a one-step chromatographic method, that is, the untreated sample to be tested is directly added to the chromatographic test strip for chromatographic reaction. However, the fluorescent immuno-chromatographic technology has the main shortcomings of poor repeatability, large batch-to-batch difference, insufficient anti-interference ability, and poor manufacturability. Based on the deficiencies of the chromatographic technology, Lin Yuan et al. proposed a method for improving the anti-interference ability of the immuno-chromatographic reagent by using an anti-interference reagent cup and an anti-interference coating in the reagent cup (CN 109187943 B). However, this method has obvious limitations in solving the key performance of repeatability and batch-to-batch difference in existing reagents.

[0004] In addition, the existing fluorescent immuno-chromatographic technology has two methods of dilution and liquid phase. The dilution method requires pretreatment of the sample, and then the sample is added to the chromatographic test strip. The dilution method significantly improves the anti-interference ability of the sample, but it still has other disadvantages of the one-step chromatographic method. The liquid phase method is a process of pre-reacting fluorescent microsphere markers with the sample to be tested, and then adding the pre-reacted fluorescent microsphere marker immune complex solution to the test strip for color development reaction. Du Zhongran et al. disclosed a hs-TnI detection method based on quantum dot microspheres (CN118409098A), which improves the sensitivity and precision through the liquid phase reaction process and the high affinity of streptavidin-biotin. This improves the possibility of improving the precision of the fluorescent immuno-chromatographic technology. However, the streptavidin-biotin system is easily disturbed by free organisms, and there is no effective solution to the batch-to-batch difference caused by the difference in coating of the NC membrane single detection line.

[0005] Therefore, there is an urgent need for a new POCT detection mode to solve the inherent problems of existing chromatographic technology. SUMMARY

[0006] The application provides a full liquid-phase reaction chromatographic detection reagent, method and system, which can detect a target substance by using a "sandwich method" and a "competition method" principle respectively, solves the technical problems of complex preparation process, large batch difference and poor detection accuracy of a traditional test strip by independently and prepositioning a "characterization reaction".

[0007] In a first aspect, the application provides a full liquid-phase reaction chromatographic sandwich method detection reagent, comprising a first reagent and a chromatographic carrier.

[0008] The first reagent comprises a first aptamer coupled with a tracer and a second aptamer coupled with a linker, and the first aptamer and the second aptamer specifically recognize and / or bind to the same target substance.

[0009] The chromatographic carrier comprises at least two detection lines coated with a capture agent that can specifically recognize and / or bind to the linker.

[0010] In a second aspect, the application provides a full liquid-phase reaction chromatographic sandwich method detection reagent, comprising a first reagent and a chromatographic carrier.

[0011] The first reagent comprises a first aptamer coupled with a tracer and a second aptamer coupled with a linker, and the first aptamer and the second aptamer specifically recognize and / or bind to the same target substance.

[0012] The detection line is coated with a capture agent that can specifically recognize and / or bind to the linker, and the mass ratio of the capture agent to the second aptamer is ≥1 / 5, and it should be understood that the mass of the second aptamer here is the mass of the second aptamer participating in the chromatographic reaction, that is, the mass of the second aptamer in the reaction solution after the first reagent and the target substance complete the reaction and are added to the chromatographic carrier, and the mass of the capture agent is the mass of the capture agent coated on the detection line of the chromatographic carrier.

[0013] In some specific embodiments, the first aptamer coupled with a tracer and the second aptamer coupled with a linker in the first reagent can be packaged separately.

[0014] The first aptamer and the second aptamer can be any molecule that can specifically recognize and bind to the target substance. Non-limiting examples of the first aptamer and the second aptamer include antibodies, antigen-binding fragments, antigens, proteins, polypeptides, multi-protein complexes, exosomes, microbial particles / pieces / fragments, oligonucleotides, aptamers, modified aptamers (such as low dissociation rate modified aptamers or aptibodies), and low molecular weight compounds.

[0015] In some embodiments, the first aptamer is an antibody or an antigen-binding fragment, and the analyte is an antigen, a protein, a polypeptide, a multi-protein complex, a hormone or an exosome. In other embodiments, the first aptamer is an antigen, a protein, a polypeptide, a multi-protein complex or an exosome, and the analyte is an antibody or an antigen-binding fragment.

[0016] Similarly, the second aptamer is an antibody or an antigen-binding fragment, and the analyte is an antigen, a protein, a polypeptide, a multi-protein complex, a hormone or an exosome. In other embodiments, the second aptamer is an antigen, a protein, a polypeptide, a multi-protein complex or an exosome, and the analyte is an antibody or an antigen-binding fragment.

[0017] The first aptamer and the second aptamer can be the same or different, as long as they both bind to the analyte simultaneously to form a triple complex, the aforementioned complex is captured by the detection zone, and the content or presence or absence of the analyte is calculated by the signal change generated by the tracer. In some embodiments, the first aptamer and the second aptamer are both antibodies, which can be the same antibody or different antibodies. They can be different antibodies that bind to the same epitope on the analyte, or they can be different antibodies that bind to different epitopes on the analyte. In the case where the first aptamer and the second aptamer bind to the same epitope on the analyte, the analyte should have two or more repeats of the same epitope, and the first aptamer and the second aptamer generally bind to different repeats of the same epitope on the analyte.

[0018] In some embodiments, the first aptamer and / or the second aptamer are antibodies, specifically at least one of a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a chimeric antibody, a humanized antibody or a camelid antibody.

[0019] In some embodiments, the first aptamer and / or the second aptamer are antigen-binding fragments, specifically at least one of a Fab', a Fab, a F(ab')2, a Fv and a scFV fragment.

[0020] It should be understood that the "sandwich method" described in the present application cannot be limitedly understood as "double antibody sandwich method", but can also be "indirect method" or "capture method", as long as the first aptamer and the second aptamer can specifically recognize and / or bind to the analyte simultaneously to form a "tracer-first aptamer-analyte-second aptamer-linker" sandwich-type multiple complex, and then the multiple complex is captured by the detection zone of the chromatographic carrier, and further, a signal is generated by the tracer in the multiple complex, the signal intensity of which is positively related to the concentration of the analyte to a certain extent. By measuring the signal generated by the tracer in the detection zone, it is determined whether the analyte exists in the sample or not, or the concentration of the analyte is calculated.

[0021] In a third aspect, the present application provides a full liquid phase reaction chromatographic competition assay reagent, comprising a first reagent and a chromatographic carrier;

[0022] The first reagent comprises a first aptamer coupled to a tracer and a second aptamer coupled to a linker, one of the first aptamer or the second aptamer competes with the analyte for binding and / or recognition of the other aptamer.

[0023] The chromatographic carrier comprises at least two detection lines coated with a capture capable of specifically recognizing and / or binding to the linker.

[0024] In a fourth aspect, the present application provides a full liquid phase reaction chromatographic competition assay reagent, comprising a first reagent and a chromatographic carrier;

[0025] The first reagent comprises a first aptamer coupled to a tracer and a second aptamer coupled to a linker, one of the first aptamer or the second aptamer competes with the analyte for recognition and / or binding of the other aptamer.

[0026] The detection line is coated with a capture capable of specifically recognizing and / or binding to the linker, and the mass ratio of the capture to the second aptamer is ≥ 1 / 5. It should be understood that the mass of the second aptamer here refers to the mass of the second aptamer participating in the chromatographic reaction, i.e., the mass of the second aptamer in the reaction solution after the first reagent and the analyte complete the reaction and are added to the chromatographic carrier, and the mass of the capture refers to the mass of the capture coated on the detection line of the chromatographic carrier.

[0027] One of the first aptamer or the second aptamer is any molecule that can compete with the analyte for recognition and / or binding of the other aptamer. It should be understood that the first aptamer can compete with the analyte for recognition and / or binding of the second aptamer, or the second aptamer can compete with the analyte for recognition and / or binding of the first aptamer. Non-limiting examples of the first aptamer or the second aptamer include antibodies, antigen-binding fragments, antigens, proteins, polypeptides, multi-protein complexes, exosomes, oligonucleotides, aptamers, modified aptamers (such as low off-rate modified aptamers or ligands), and low molecular weight compounds.

[0028] In some specific embodiments, the first aptamer and the analyte are antibodies or antigen-binding fragments, and the second aptamer can be an antigen, a protein, a polypeptide, a multi-protein complex, a hormone, or an exosome. In other embodiments, the first aptamer and the analyte are an antigen, a protein, a polypeptide, a multi-protein complex, a hormone, or an exosome, and the second aptamer is an antibody or an antigen-binding fragment.

[0029] More specifically, the first aptamer and the analyte can be the same or different substances, as long as they can specifically recognize and / or bind to the second aptamer, and the amount of the tracer-first aptamer complex is known in the system, the content or presence of the analyte can be calculated by detecting the signal change of the detection zone.

[0030] Similarly, the first aptamer is an antibody or an antigen-binding fragment, and the second aptamer and the analyte can be an antigen, a protein, a polypeptide, a multi-protein complex, a hormone or an exosome. Alternatively, the first aptamer is an antigen, a protein, a polypeptide, a multi-protein complex, a hormone or an exosome, and the second aptamer and the analyte are an antibody or an antigen-binding fragment.

[0031] More specifically, the second aptamer and the analyte can be the same or different substances, as long as they can specifically recognize and / or bind to the first aptamer, and the amount of the tracer-first aptamer complex is known in the system, the content or presence of the analyte can be calculated by detecting the signal change of the detection zone.

[0032] It should be understood that the "competition method" described in the present application cannot be limited to the competition between an antigen / antibody and an analyte for a limited / quantitative antibody / antigen binding site, as long as one of the first aptamer or the second aptamer can compete with the analyte for binding to the other analyte, and form a "tracer-first aptamer-second aptamer-linker" multiple complex, and then the multiple complex is captured by the detection zone of the chromatographic carrier, and further, a signal is generated by the tracer in the multiple complex, and the signal intensity is inversely proportional to the concentration of the analyte to a certain extent, and the presence or absence of the analyte in the sample can be determined or the concentration of the analyte can be calculated by measuring the signal generated by the tracer in the detection zone.

[0033] The "specific recognition" or "specific binding" described herein can refer to the interaction between an antibody, a protein or a peptide and a second chemical substance, wherein the interaction depends on the presence of a specific structure (e.g. an antigenic structure) or a determinant / epitope on the chemical substance, such as the recognition and binding of a specific antigenic structure by an antibody, the mutual recognition and binding of a receptor and a ligand, or the mutual recognition and binding of biotin and avidin, etc.

[0034] "Coupled" as described herein broadly refers to the stable association between two substances resulting from any chemical, physical, or physicochemical interaction (e.g., covalent bond, hydrogen bond, electrostatic interaction, polar attraction, van der Waals attraction, hydrophobic interaction, or adsorption). Unless otherwise indicated, the term is intended to encompass both: direct binding / conjugation between the two substances, e.g., direct binding / conjugation of an antibody to an antigen on a protein, and indirect binding / conjugation between the two substances through one or more intermediaries, e.g., association between an antibody and a polynucleotide by way of one or more oligonucleotides and / or labels. Thus, "coupled" when used in reference to two substances means bound / conjugated by any such direct or indirect means.

[0035] The analyte should not be particularly limited and can be any biomolecule or biological cell that needs to be detected and / or quantified in a sample. Non-limiting examples of analytes include antigens, antibodies, antigen-binding fragments, proteins, polypeptides, multi-protein complexes, hormones, exosomes, oligonucleotides, or low molecular weight compounds. The analyte can be detected in any sample of interest and is not particularly limited to biological samples, such as, without limitation, body fluids (e.g., urine, saliva, blood, serum, plasma, sweat), extracts (e.g., cell extracts), and solutions containing proteins and / or DNA (e.g., reaction mixtures).

[0036] The capture and the linker are bound together with certain specificity and affinity, for example, "biotin-avidin, primary antibody-secondary antibody, antibody-protein, DNP (2,4-dinitrophenyl)-anti-DNP antibody, FITC (fluorescein isothiocyanate)-anti-FITC antibody" and the like can be selected. Specifically, when the linker is biotin, the capture corresponds to avidin and / or streptavidin and / or anti-biotin antibody, when the linker is avidin and / or streptavidin, the capture corresponds to biotin; when the linker is a primary antibody, the capture corresponds to a secondary antibody, when the linker is a secondary antibody, the capture corresponds to a primary antibody; when the linker is an antibody, the capture corresponds to a protein specifically recognized and / or bound by the antibody, when the linker is a protein, the capture corresponds to an antibody that can specifically recognize and / or bind; when the linker is DNP, the capture corresponds to an anti-DNP antibody, when the linker is an anti-DNP, the capture corresponds to DNP; when the linker is FITC, the capture corresponds to an anti-FITC antibody, when the linker is an anti-FITC antibody, the capture corresponds to FITC.

[0037] In some specific embodiments, the linker and the capture system is a "biotin-streptavidin" linkage system, it should be understood that one avidin molecule can bind four biotin molecules, the binding is very stable, and the avidin and biotin can be coupled with protein, enzyme, fluorescein and other molecules, and do not affect the biological activity of the latter. One antibody molecule can be coupled with multiple biotin molecules, and avidin can be coupled with multiple biotin-antibody conjugates, and this multi-level amplification can further improve the sensitivity of the detection system.

[0038] The tracer can be any detectable marker substance that can generate a signal that can be detected by visual or instrumental means, examples of which include at least one of a fluorescent signal substance, an enzyme catalytic substance, a chemiluminescent signal substance, an electromagnetic signal substance, or a colored substance. Depending on the reaction principle, as the complex of the tracer-containing analyte to be detected flows on the chromatographic carrier, the tracer will be captured and aggregated on the detection line or not, and according to the signal of the tracer, such as color signal, fluorescence signal or electromagnetic signal, etc., it is determined whether the analyte exists or its concentration.

[0039] In some specific embodiments, the fluorescent signal substance can be at least one of a fluorescent microsphere, a quantum dot, a fluorescent protein, or a fluorescein molecule; the enzyme catalytic substance can be at least one of horseradish peroxidase, alkaline phosphatase, alkaline peroxidase, glucose 6-phosphate dehydrogenase, etc.; the chemiluminescent signal substance can be at least one of acridinium ester, thioester or sulfonamide, luminol, isoluminol or phenanthridinium ester, etc.; the electromagnetic signal substance can be at least one of a magnetic particle or a radioactive substance, the radioactive substance can be at least one of 3 H、 125 I、 35 S、 14 C、 32 P、or 33 P, etc.; the colored substance can be at least one of a metal colloidal particle or a colored particle, more specifically, the metal colloidal particle can be at least one of colloidal gold or colloidal platinum.

[0040] The chromatographic carrier is used to carry the detection area, which is a porous material with liquid capillary action, and the specific material can be at least one of nitrocellulose membrane, cellulose acetate membrane or nitrocellulose, nylon, polyvinylidene fluoride, polyethylene or glass fiber.

[0041] In some specific real-time embodiments, the capture detection line is at least two, when there are multiple detection lines, the same concentration of capture can be coated on different detection lines, or different concentrations of capture can be coated.

[0042] In some embodiments, the carrier is coated with a quality control line for determining whether the detection is valid or for positioning when the instrument reads the result.

[0043] In some embodiments, the chromatography carrier further comprises a sample loading area, more specifically, the sample loading area is located at one end of the chromatography carrier and is separated from the detection area by a buffer area. The purpose of the buffer area is to allow the sample to be laterally chromatographed for a certain distance to allow the subsequent chromatography to proceed at a more uniform speed, and when the sample reaches the detection area, the chromatography reaction proceeds at a desired speed to achieve better binding efficiency.

[0044] In some embodiments, at least one of a sample pad, a wicking pad, a backing or a cassette is further included. More specifically, the sample pad is attached above the chromatography carrier to carry the reaction complex and provide a buffer for the sample loading process. More specifically, the wicking pad is attached above the chromatography carrier to pull the solution through the chromatography carrier by its water absorption property. More specifically, the backing is attached below the chromatography carrier to provide solid support for the chromatography carrier. More specifically, the cassette is used to load the chromatography carrier, and the cassette is provided with a sample loading hole and a detection window.

[0045] In some embodiments, the first reagent can be in a liquid phase or a solid phase. More specifically, when the first reagent is in a solid phase, it can be in a lyophilized form.

[0046] In some embodiments, a second reagent is further included. The second reagent can be used for separation, dissociation, filtration or dilution of the sample, and is generally used for pretreatment of the sample. The second reagent can be any reagent that separates, dissociates, filters or dilutes the sample to be analyzed. It should be understood that, as described herein, not all samples need to be pretreated, which is determined by the sample type, the analyte to be detected and the subsequent reaction environment, etc. The second reagent can be homogeneous (i.e. no separation step is needed) or heterogeneous (a separation step is needed). If the second reagent is heterogeneous, pretreatment of the sample with the second reagent can remove any interfering components from the sample before the next step of the assay, or extract / isolate the analyte component. Non-limiting examples of the second reagent include: ① one or more solvents and salts; ② one or more solvents, salts and surfactants; ③ a surfactant; ④ a surfactant and a salt; ⑤ one or more solvents and surfactants; ⑥ solid particles (e.g. magnetic microparticles coated with anti-red blood cell antibodies for separation of blood plasma); ⑦ any reagent suitable for lysing, diluting, separating the sample and / or cells. It should be understood that the corresponding sample reaction step is: after pretreatment of the sample with the second reagent, the treated solution is mixed with the first reagent to react and generate a reaction complex, and then the foregoing reaction solution is loaded onto the chromatography carrier.

[0047] In some specific embodiments, the first reagent and the second reagent can be coexisting, i.e. the first reagent further comprises reagent components suitable for separating, dissociating, diluting and / or filtering the sample. It should be understood that the corresponding sample reaction procedure is: reacting the sample with the first reagent (the first reagent can pre-treat the sample and generate a reaction complex with the analyte), and then loading the reaction solution into the chromatographic carrier.

[0048] In some specific embodiments, when the first reagent is in a lyophilized form, a third reagent is further included for reconstituting the first reagent, which can be any solution suitable for reconstitution of the lyophilized solution, non-limiting examples including: one or more solvents, aqueous solutions or purified water, etc. It should be understood that the corresponding sample reaction procedure can be: after reconstituting the lyophilized first reagent with the third reagent, reacting the sample with the reconstituted first reagent to generate a reaction complex, and then loading the aforementioned reaction solution into the chromatographic carrier; or, reconstituting the lyophilized first reagent with the third reagent, and pre-treating the sample with the second reagent, mixing and reacting the aforementioned reconstituted first reagent and the pre-treated sample solution to produce a reaction complex, and then loading the aforementioned reaction solution into the chromatographic carrier.

[0049] In some specific embodiments, the second and third reagents can be coexisting in liquid phase, i.e. the second reagent is not only suitable for separating, dissociating, diluting and / or filtering the sample, but also suitable for reconstituting the lyophilized first reagent in liquid phase. The reaction procedure is: pre-treating the sample with the second reagent, then reconstituting the lyophilized first reagent with the pre-treated sample solution while reacting to produce a reaction complex, and then loading the aforementioned reaction solution into the chromatographic carrier.

[0050] In some specific embodiments, a fourth reagent is further included, which is used to pre-eliminate interfering factors in the sample;

[0051] In some specific embodiments, when the capture and the linker are selected from the "avidin or streptavidin and biotin" specific binding system, the interfering factor is free biotin in the sample. It should be understood that when the avidin-biotin specific binding system is used for immunoassay, if there is a high concentration of free biotin in the sample to be tested, it will compete with the biotinylated antibody for the binding site of avidin, thereby affecting the detection result, so it is necessary to pre-treat the sample for anti-biotin interference.

[0052] In some specific embodiments, the solution to the problem of biotin interference is to add a fourth reagent, and the fourth reagent comprises magnetic particles coated with avidin and / or streptavidin, the sample is pre-treated with the fourth reagent, and the free biotin is enriched on the magnetic particles by the specific binding reaction between biotin and avidin / streptavidin, and then the biotin interference in the sample is removed by magnetic separation. It should be understood that the removal of biotin interference is not limited to the above-mentioned solution, but can also include the addition of anti-biotin antibodies in the first and / or second reagents.

[0053] In some specific embodiments, the fourth reagent can be co-present with the second reagent.

[0054] In some specific embodiments, when the sample is whole blood, a fifth reagent is further included, which is used to dilute the sample, and the absorbance, turbidity, transmittance or colorimetric value of the diluted sample is detected to correct the detection result. More specifically, since the whole blood sample has many interference factors compared with the plasma sample, including but not limited to bilirubin, blood lipids and other impurities, and especially in some special patient samples, there are often more interference substances that can affect the detection result, such as anemia samples, bilirubin or albumin abnormal samples, etc. Therefore, in order to obtain more accurate detection results in clinical practice, plasma is often selected as the determination sample, but this requires an additional sample pretreatment step (such as centrifugation) to increase the complexity of the detection process. In the present application, the inventors found that the detection values of whole blood samples and homologous plasma samples have a correlation with the absorbance, turbidity, transmittance or colorimetric value of the whole blood sample. By constructing an algorithm based on the aforementioned measurement values and the detection values of whole blood samples and homologous plasma samples, a correction coefficient can be fitted to correct the detection value of the whole blood sample and improve the accuracy of the detection result.

[0055] In a fifth aspect, the present application provides a whole liquid-phase reaction chromatographic detection method, comprising the following steps:

[0056] (1) treating the sample with a first reagent to obtain a reaction complex, the first reagent comprising a first aptamer coupled to a tracer and a second aptamer coupled to a linker, the first aptamer and the second aptamer specifically recognizing and / or binding to the analyte and forming a reaction complex of "tracer-first aptamer-analyte-second aptamer-linker";

[0057] (2) adding the aforementioned reaction complex to a chromatographic carrier, and coating a capture on the detection line, the capture specifically recognizing and / or binding to the linker, and the capture trapping the aforementioned reaction complex through the linker;

[0058] (3) result interpretation: interpreting the signal of the tracer in the detection zone to obtain the detection result;

[0059] Further, the chromatography carrier is coated with at least two detection lines;

[0060] Further, the mass ratio of the capture agent / second adaptor is ≥1 / 5.

[0061] In a sixth aspect, the present application provides another full liquid phase reaction chromatography detection method, comprising the following steps:

[0062] (1) treating the sample with a first reagent to obtain a reaction complex, the first reagent comprising a first adaptor coupled with a tracer and a second adaptor coupled with a linker, one of the first adaptor or the second adaptor competes with the analyte to recognize and / or bind to another adaptor, and forms a reaction complex I of "tracer-first adaptor-analyte" or "linker-second adaptor-analyte", and a reaction complex II of "tracer-first adaptor-second adaptor-linker";

[0063] (2) adding the aforementioned reaction complex to a chromatography carrier, the detection line of which is coated with a capture agent that specifically recognizes and / or binds to the linker, and the capture agent captures the aforementioned reaction complex II through the linker;

[0064] (3) result reading: interpreting the signal of the tracer in the detection area to obtain the detection result;

[0065] Further, the chromatography carrier is coated with at least two detection lines;

[0066] Further, the mass ratio of the capture agent / second adaptor is ≥1 / 5.

[0067] In some specific embodiments, before step (1), the sample is further pretreated with a second reagent, which can separate, dissociate, filter or dilute the sample.

[0068] In some specific embodiments, before step (1), the first reagent is further reconstituted with a third reagent.

[0069] In some specific embodiments, the second reagent and the third reagent can be in a liquid phase coexistence form.

[0070] In some specific embodiments, before step (1), the sample is further pretreated with a fourth reagent, which is used to pre-exclude interference factors in the sample.

[0071] In some specific embodiments, the second reagent and the fourth reagent can be in a coexistence form.

[0072] In some specific embodiments, the measurement value of absorbance, turbidity, transmittance or chroma of the sample is further obtained, and the detection result of the whole blood sample is corrected by the aforementioned measurement value.

[0073] In some specific embodiments, the measured value is absorbance, more specifically, the absorbance value of the whole blood sample is obtained by a spectrophotometer.

[0074] In some specific embodiments, the measured value is obtained after the sample is diluted by the fifth reagent.

[0075] In some specific embodiments, the step of diluting the sample by the fifth reagent to obtain the measured value can be parallel to the aforementioned reaction steps, i.e., part of the sample is taken for dilution and detection to obtain the measured value, and the rest of the sample completes the aforementioned pretreatment steps of the second reagent, the third reagent, and / or the fourth reagent, and the subsequent reaction step after the addition of the first reagent to obtain the detection result, which is corrected by the measured value; the step of obtaining the measured value can also be serial to the aforementioned reaction steps, i.e., the sample sequentially completes the treatment of the second reagent, the third reagent, and / or the fourth reagent, and the fifth reagent in a certain order to obtain the measured value, and the detection result is obtained after the subsequent reaction step after the addition of the first reagent, which is corrected by the measured value.

[0076] In some specific embodiments, the fifth reagent can coexist with the second reagent, the third reagent, and / or the fourth reagent.

[0077] In some specific embodiments, the first reagent, the second reagent, the third reagent, the fourth reagent, the fifth reagent, and the chromatographic carrier can be integrated and loaded on a one-piece card structure, can also be completely in the form of independent packaging, or can be integrated and loaded in any two, any three, or any four, and when integrated and loaded, the arrangement order of different structures can be arranged according to the sample flow order.

[0078] It should be understood that the presence or absence of the second reagent, the third reagent, the fourth reagent, and the fifth reagent in the reaction system is affected by the detection item, the sample type, and the detection scene, and at the same time, the addition order of the aforementioned four reagents and whether they coexist with each other are also affected by the detection item, the sample type, and the detection scene, and the following are non-limiting examples (the integrated reagent card is shown in FIG. 1):

[0079] (1) Take part of the whole blood sample and add it to the fifth reagent storage cavity, dilute the sample with the fifth reagent, and measure the absorbance value of the sample;

[0080] (2) Take part of the whole blood sample and add it to the fourth reagent storage cavity, and perform anti-biotin interference treatment in the fourth reagent, for example, the fourth reagent can be avidin-coated magnetic beads, which can enrich free biotin in the sample, and then separate by an external magnetic field;

[0081] (3) absorbing the third reagent in the third reagent storage cavity into the first reagent storage cavity to complete the reconstitution of the first reagent in the lyophilized form;

[0082] (4) adding the solution obtained in step (2) to the reconstituted first reagent and mixing, then adding the reaction solution to the sample loading site of the chromatographic carrier, and after the completion of the chromatographic reaction, calculating the detection result by obtaining the signal of the detection zone, and correcting the detection result using the absorbance value measured in step (1), to obtain the final detection result.

[0083] In some specific embodiments, step (1) can be at least one of temperature control, reaction time control, and mixing operation. It should be understood that, in addition to the chromatographic reaction on the chromatographic carrier, the rest of the reactions in the present application can be carried out in liquid phase, including the series of sample pretreatment, the acquisition of correction parameters, and the reaction process of the first reagent and the analyte. By carrying out the foregoing reactions in liquid phase, at least one of time control, temperature control, or mixing operation can be performed according to the requirements of the reaction conditions, so as to achieve more precise control of the reaction efficiency.

[0084] In some specific embodiments, the result interpretation can be at least one of naked eye recognition or instrument recognition. It should be understood that when the tracer is a substance that can be recognized by the naked eye, such as a colored substance, more specifically colloidal gold, colored microspheres, etc., the detection result can be recognized by the human eye.

[0085] In a seventh aspect, the present application provides a full-liquid-phase reaction chromatographic detection system, comprising:

[0086] a detection device, which can load a chromatographic carrier, the chromatographic carrier having a detection zone, the detection zone capturing a complex reaction product of "tracer-first aptamer-analyte-second aptamer-linker-captor", and the detection result is obtained by detecting the content change of the tracer.

[0087] In some specific embodiments, it further comprises a reaction pool for receiving the sample and the first reagent, so that the sample and the first reagent are mixed in the reaction pool to prepare a reaction complex, wherein the first reagent comprises a first aptamer coupled with a tracer and a second aptamer coupled with a linker, and the first aptamer and the second aptamer specifically recognize and / or bind to the analyte to form a complex of "tracer-first aptamer-analyte-second aptamer-linker".

[0088] In some specific embodiments, it further comprises a pipetting device for sample collection and transfer and reaction complex transfer.

[0089] In some specific embodiments, the pipetting device is divided into a sampling module and a pipetting module.

[0090] More specifically, the sampling module has a sampling tube and a driving device for driving the sampling tube to quantitatively suck the sample and move to the reaction pool.

[0091] More specifically, the pipetting module has a pipetting tube and a driving device for driving the pipetting tube to quantitatively suck the reagent and move to the detection area.

[0092] In some specific embodiments, a reagent supply part is further included, which provides the first reagent to the reaction pool.

[0093] In some specific embodiments, the detection device is an optical detection device, which includes a light source and a detector, the light source emits light to irradiate the detection area to make the tracer generate optical information, and the detector is used to collect the optical information.

[0094] In some specific embodiments, a data processing device is further included, which is electrically connected with the detection device and includes a processor and a computer readable storage medium storing a computer program.

[0095] In view of this, the present application provides a full liquid phase reaction chromatographic detection reagent, method and system, for the first time, the reaction sites of the two components of the immune reaction, i.e., the first aptamer and the second aptamer, are transferred from the traditional heterogeneous solid phase carrier to the liquid phase reaction process, and secondly, the solid phase coated capture carrier is quantitatively characterized, the incubated fluorescent immune complex is separated and enriched by the solid phase carrier, the performance of the fluorescent immune chromatographic reagent is improved, such as precision, linear range and anti-interference ability, and the limitation of the difference in the solid phase carrier coating amount is reduced.

[0096] The present application has the beneficial effects of providing a new chromatographic reaction detection method, completing the capture and binding reaction of the to-be-measured substance in a liquid phase medium, realizing high-precision control of the reaction process in terms of amount, temperature and time to improve the detection performance, enriching the reaction complex by the adapted test strip and performing subsequent detection. At the same time, the present application optimizes the film structure of the adapted test strip, standardizes it and realizes the universality of the chromatographic carrier in different projects, solves the problems of complex preparation process and difficult control of batch difference of the traditional test strip, and significantly improves the detection performance of the reagent. The detection method protected by the present application also provides an efficient sample pretreatment scheme, including sample pretreatment such as lysis, dilution, anti-interference, blood cell separation and correction parameter detection, and the present application also provides a preparation and detection method of a dry reagent to realize stable storage and transportation of the reagent. The comprehensive detection method provided by the present application can adapt to different detection samples, detection projects, detection requirements or detection scenes, and significantly improve the detection performance. BRIEF DESCRIPTION OF DRAWINGS

[0097] Figure 1: Schematic diagram of integrated reaction reagent. Legend: 1, fifth reagent storage cavity; 2, fourth reagent storage cavity; 3, second reagent storage cavity; 4, first reagent storage cavity; 5, sample adding site; 6, chromatographic carrier; 7, reagent card shell;

[0098] Figure 2: Reaction kinetics curve of two groups of detection reagents in Example 7;

[0099] Figure 3: Precision CV mean distribution graph of two groups of detection reagents in Example 7;

[0100] Figure 4: Reaction kinetics curve of six groups of detection reagents in Example 8;

[0101] Figure 5: Precision CV mean distribution graph of six groups of detection reagents in Example 8;

[0102] Figure 6: Reaction linearity graph of twelve groups of detection reagents in Example 9.

[0103] Figure 7: Correlation comparison graph of detection results of whole blood samples before and after OD value correction in Example 11;

[0104] Figure 8: Deviation analysis graph of detection results of whole blood samples before and after OD value correction in Example 11. DETAILED DESCRIPTION

[0105] In order to make the objectives, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. The described embodiments are some of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative effort fall within the scope of protection of the present application.

[0106] As used herein, "and / or" includes any and all combinations of one or more of the associated listed items.

[0107] As used herein, "plurality" means two or more, that is, it includes two, three, four, five, etc.

[0108] It should be noted that, in this document, the terms "comprising", "containing" or any other variant thereof are intended to cover non-exclusive inclusion, so that a process, method, article or apparatus that includes a list of elements not only includes those elements, but also includes other elements not explicitly listed, or further includes elements inherent in such a process, method, article or apparatus. Without more limitations, the element defined by the statement "comprising a" does not exclude the presence of additional identical elements in the process, method, article or apparatus that includes the element.

[0109] In this specification, certain embodiments can be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and that one of skill in the art will understand that the range format is applicable to each and every specification, example, and embodiment of the present application. For example, a range format may be described as 10 C to 15 C, however, it is to be understood that individual numbers, such as 11 C, 12 C, 13 C, and 14 C, within the range are also specifically disclosed.

[0110] Example 1

[0111] (1) Preparation of the first reagent

[0112] ① Coupling of the first aptamer and the tracer

[0113] Add NT-proBNP antibody 1 (Xiamen Tongrenxin, item number XJ29) to the fluorescent microspheres in an amount of 60 μg NT-proBNP monoclonal antibody / 300 μg fluorescent microspheres, add 6 μg EDC, stir at room temperature for 120 minutes, add 5% BSA, block and stir for 60 minutes, centrifuge at 14000 r / min for 20 minutes, discard the supernatant, and restore the volume of the precipitate with fluorescent antibody storage solution (100 mM Tris, 1% Tween 20, 5 mg / ml bovine serum albumin, 2% sucrose), and store at 2-8°C.

[0114] ② Coupling of the second aptamer and the linker

[0115] Dissolve 0.001 g of biotin in 175 μL DMSO to obtain a biotin working solution, take 0.5 mg of NT-proBNP antibody 2 (Xiamen Tongrenxin, item number XJ28) and add 7 μl of the biotin working solution, mix immediately, rotate for labeling at room temperature in the dark, and then dialyze or pass through a column to remove free biotin.

[0116] ③ Preparation of the first reagent

[0117] Dilute the first aptamer coupled with the tracer to 0.34 mg / mL and the second aptamer coupled with the linker to 0.032 mg / mL with the first reagent storage solution (20 mM Tris, 1% Tween 20, 5 mg / ml sodium casein, 5% trehalose), mix equal volumes of the two solutions to prepare the first reagent, and store at 2-8°C for standby use.

[0118] (2) Preparation of the chromatographic carrier

[0119] ① Preparation of the test line

[0120] Dilute streptavidin to 0.2 mg / mL with PBS buffer, and draw two capture detection lines on the left end of the nitrocellulose membrane at a distance of 5-15 mm.

[0121] ② Assembly of test strip

[0122] The backing, sample pad and absorbent pad are common materials in the art. The sample pad, nitrocellulose membrane and absorbent pad are sequentially and tightly overlapped on the backing to assemble a common chromatographic carrier. The assembled common chromatographic carrier is cut into a predetermined width by a cutting machine and loaded into a card shell.

[0123] (3) Reaction and detection method

[0124] ① Immune reaction

[0125] Mix 70 uL of the serum sample to be tested with 10 uL of the first reagent at room temperature and incubate for 3 min. The first aptamer and the second aptamer specifically recognize and bind to the analyte to form a reaction complex of "fluorescent microspheres-NT-proBNP antibody 1-analyte NT-proBNP-NT-proBNP antibody 2-biotin".

[0126] ② Chromatographic reaction and result processing

[0127] Transfer 40 uL of the above reaction solution to the sample loading area of the chromatographic carrier. The reaction solution moves to the detection area by capillary action. The streptavidin on the detection line captures the above reaction complex by specific binding reaction with biotin. The detection device collects the tracer signal on the detection line and processes the data to output the content of the analyte.

[0128] Example 2

[0129] (1) Preparation of the second reagent

[0130] Preparation of magnetic beads-anti-red blood cell antibody

[0131] Mix the biotinylated anti-red blood cell antibody (Guangzhou Greiner, product number G0093R) and streptavidin magnetic beads at a mass ratio of 1:24 to form a magnetic bead-anti-red blood cell antibody complex. React at room temperature for 30 min. Remove the supernatant by magnetic separation and wash twice with PBS buffer. Store the magnetic beads in a magnetic bead storage solution (20 mM Tris, 1% Tween 20, 10 mg / ml bovine serum albumin, 5% mannitol) as the second reagent and store at 2-8°C for future use.

[0132] (2) Preparation of the first reagent

[0133] ① Coupling of the first aptamer and the tracer

[0134] NT-proBNP antibody 1 (Xiamen Tongrenxin, item number XJ29) was added to the fluorescent microspheres at an amount of 60 μg NT-proBNP monoclonal antibody / 300 μg fluorescent microspheres, 6 μg EDC was added, and the reaction was stirred at room temperature for 120 minutes. 5% BSA was added, blocked and stirred for 60 minutes, centrifuged at 14000 r / min for 20 minutes, the supernatant was discarded, and the precipitate was restored to volume with fluorescent antibody storage solution (100 mM Tris, 1% Tween 20, 5 mg / ml bovine serum albumin, 2% sucrose) and stored at 2-8°C.

[0135] ②Coupling of the second aptamer and the linker

[0136] 0.001 g of biotin was weighed and dissolved in 175 μL DMSO to form a biotin working solution. 0.5 mg of NT-proBNP antibody 2 (Xiamen Tongrenxin, item number XJ28) was added to 7 μl of the biotin working solution, immediately mixed, and then rotated for labeling at room temperature in the dark. Free biotin was removed by dialysis or column chromatography.

[0137] ③Preparation of the first reagent

[0138] The first reagent was prepared by mixing equal volumes of the first reagent storage solution (20 mM Tris, 1% Tween 20, 5 mg / ml sodium casein, 5% trehalose) and the first aptamer coupled with the tracer diluted to 0.34 mg / mL, and the second aptamer coupled with the linker diluted to 0.032 mg / mL, and stored at 2-8°C for standby use.

[0139] (3) Preparation of the chromatographic carrier

[0140] ①Preparation of the test line

[0141] The streptavidin was diluted with PBS buffer to 0.2 mg / mL, and 3 capture test lines were drawn on the left end of the nitrocellulose membrane at a distance of 5-15 mm.

[0142] ②Assembly of the test strip

[0143] The backing, sample pad and water absorption pad are common materials in the art. The sample pad, nitrocellulose membrane and water absorption pad were sequentially and tightly lapped on the backing to assemble a general chromatographic carrier. The assembled general chromatographic carrier was cut into a predetermined width with a cutting machine and loaded into a card shell.

[0144] (4) Reaction and detection method

[0145] ①Pre-treatment

[0146] Take 100 uL volume of whole blood sample to be tested and mix with 20 uL volume of second reagent at room temperature. Red blood cells in whole blood bind with "magnetic bead-anti red blood cell antibody" complex in the second reagent. Through the application of magnetic field, "magnetic bead-anti red blood cell antibody-red blood cell" is enriched and separated, and the rapidly separated plasma is obtained.

[0147] ②Immune reaction

[0148] Mix 70 uL volume of separated plasma sample with 10 uL volume of first reagent at room temperature and incubate for 3 min. The first aptamer and the second aptamer specifically recognize and bind the analyte to form a reaction complex of "fluorescent microspheres-NT-proBNP antibody 1-analyte NT-proBNP-NT-proBNP antibody 2-biotin".

[0149] ③Chromatographic reaction and result processing

[0150] Transfer 40 uL of the above reaction solution to the sample loading area of the chromatographic carrier. The reaction solution moves to the detection area by capillary action. The streptavidin on the detection line captures the above reaction complex on the detection line through specific binding reaction with biotin. The detection device collects the tracer signal on the detection line and processes the data to output the content result of the analyte.

[0151] Example 3

[0152] (1) Preparation of third reagent

[0153] Prepare TBST buffer solution (30 mM Tris, 5% Tween 20, 0.5% Proclin 300).

[0154] (2) Preparation of second reagent

[0155] Preparation of magnetic bead-anti red blood cell antibody

[0156] Mix biotinylated anti red blood cell antibody (Guangzhou Gelin, product number G0093R) and streptavidin magnetic beads in a mass ratio of 1:24 to form a magnetic bead-anti red blood cell antibody complex. React at room temperature for 30 min. Remove the supernatant by magnetic separation. Wash twice with PBS buffer solution. Store the magnetic beads in magnetic bead storage solution (20 mM Tris, 1% Tween 20, 10 mg / ml bovine serum albumin, 5% mannitol) as the second reagent. Freeze dry in a freeze dryer.

[0157] (3) Preparation of first reagent

[0158] ①Coupling of first aptamer and tracer

[0159] Add NT-proBNP antibody 1 (Xiamen Tongrenxin, item number XJ29) into fluorescent microspheres at an amount of 60 μg NT-proBNP monoclonal antibody / 300 μg fluorescent microspheres, add 6 μg EDC, stir at room temperature for 120 minutes, add 5% BSA, block and stir for 60 minutes, centrifuge at 14000 r / min for 20 minutes, discard the supernatant, and restore the volume of the precipitate with fluorescent antibody storage solution (100 mM Tris, 1% Tween 20, 5 mg / ml bovine serum albumin, 2% sucrose) for storage at 2-8°C.

[0160] ②Coupling of the second aptamer and the linker

[0161] Dissolve 0.001 g of biotin in 175 μL DMSO to obtain a biotin working solution, take 0.5 mg of NT-proBNP antibody 2 (Xiamen Tongrenxin, item number XJ28) and add 7 μl of the biotin working solution, mix immediately, rotate for labeling at room temperature in the dark, and then dialyze or pass through a column to remove free biotin.

[0162] ③Preparation of the first reagent

[0163] Dilute the first aptamer coupled with the tracer to 0.34 mg / mL and dilute the second aptamer coupled with the linker to 0.032 mg / mL with the first reagent storage solution (20 mM Tris, 1% Tween 20, 5 mg / ml sodium casein, 5% trehalose), mix the two solutions in equal volumes to prepare the first reagent, and freeze-dry the first reagent in a freeze-dryer.

[0164] (4) Preparation of the chromatographic carrier

[0165] ①Preparation of the test line:

[0166] Dilute streptavidin to 0.2 mg / mL with PBS buffer, and draw two capture test lines on the left end of the nitrocellulose membrane at a distance of 5-15 mm.

[0167] ②Assembly of the test strip

[0168] The backing, sample pad, and water absorption pad are common materials in the art. The sample pad, nitrocellulose membrane, and water absorption pad are tightly overlapped on the backing in sequence to assemble a general chromatographic carrier. The assembled general chromatographic carrier is cut into a predetermined width with a cutting machine and loaded into a card shell.

[0169] (5) Reaction and detection method

[0170] ①Reconstitution of the first reagent

[0171] Take 10 uL of the third reagent and mix it with the freeze-dried first reagent to perform the first reagent reconstitution step.

[0172] ② Second reagent reconstitution

[0173] A volume of 20 uL of the third reagent is taken up and mixed with the freeze-dried second reagent to perform the second reagent reconstitution step.

[0174] ③ Pretreatment

[0175] A volume of 100 uL of the whole blood sample to be tested is taken up and mixed with a volume of 20 uL of the reconstituted second reagent. The red blood cells in the whole blood bind to the "magnetic bead-anti-red blood cell antibody" complex in the second reagent. By applying a magnetic field, the "magnetic bead-anti-red blood cell antibody-red blood cell" is enriched and separated, and the rapidly separated plasma is obtained.

[0176] ④ Immune reaction

[0177] A volume of 70 uL of the separated plasma sample is taken up and mixed with a volume of 10 uL of the reconstituted first reagent at room temperature and incubated for 3 min. The first aptamer and the second aptamer specifically recognize and bind to the analyte to form a reaction complex of "tracer-NT-proBNP antibody 1-analyte NT-proBNP-NT-proBNP antibody 2-biotin".

[0178] ⑤ Chromatographic reaction and result processing

[0179] A volume of 40 uL of the foregoing reaction solution is transferred to the sample addition area of the chromatographic carrier. The reaction solution moves to the detection area by capillary action. The streptavidin on the detection line captures the foregoing reaction complex on the detection line through specific binding reaction with biotin. The detection device collects the tracer signal on the detection line and processes the data to output the content result of the analyte.

[0180] Example 4

[0181] (1) Preparation of the first reagent

[0182] ① Coupling of the first aptamer and the tracer

[0183] NT-proBNP antibody 1 (Xiamen Tongren, item number XJ29) is added to the fluorescent microspheres at an amount of 60 ug NT-proBNP monoclonal antibody / 300 ug fluorescent microspheres. 6 ug EDC is added, and the reaction is stirred at room temperature for 120 min. 5% BSA is added, and the stirring is blocked for 60 min. Centrifugation is performed at 14000 r / min for 20 min, the supernatant is discarded, and the precipitate is restored to volume with fluorescent antibody storage solution (100 mM Tris, 1% Tween 20, 5 mg / ml bovine serum albumin, 2% sucrose). Storage is performed at 2-8°C.

[0184] ② Coupling of the second aptamer and the linker

[0185] Take 0.001 g of biotin and add it to 175 μL of DMSO to dissolve it into a biotin working solution. Take 0.5 mg of NT-proBNP antibody 2 (Xiamen Tongren, item number XJ28) and add 7 μl of the biotin working solution. Mix immediately, rotate for labeling at room temperature in the dark, and then dialyze or pass through a column to remove free biotin.

[0186] ③Preparation of magnetic beads-anti-red blood cell antibody

[0187] Mix the biotinylated anti-red blood cell antibody (Guangzhou Gelin, item number G0093R) and the streptavidin magnetic beads according to a mass ratio of 1:24 to form a magnetic bead-anti-red blood cell antibody complex. React at room temperature for 30 minutes, remove the supernatant by magnetic separation, wash twice with PBS buffer, and add magnetic bead storage solution (20 mM Tris, 1% Tween 20, 10 mg / ml bovine serum albumin, 5% mannitol) to store as the second reagent. Store at 2-8°C for standby use.

[0188] ④Preparation of the first reagent

[0189] Dilute the first aptamer coupled with the tracer to 0.34 mg / mL, dilute the second aptamer coupled with the linker to 0.032 mg / mL, and dilute the magnetic bead-anti-red blood cell antibody to 1.0 mg / mL with the first reagent storage solution (20 mM Tris, 1% Tween 20, 5 mg / ml sodium casein, 5% trehalose). Mix the above three liquids in a volume ratio of 5:5:20 to prepare the first reagent. Store at 2-8°C for standby use.

[0190] (2) Preparation of the chromatographic carrier

[0191] ①Preparation of the detection line

[0192] Dilute the streptavidin to 0.2 mg / mL with PBS buffer and draw 3 capture detection lines on the left end of the nitrocellulose membrane at a distance of 5-15 mm.

[0193] ②Assembly of the test strip

[0194] The backing, sample pad, and water absorption pad are general materials in the art. Assemble the backing, sample pad, nitrocellulose membrane, and water absorption pad in that order on the backing to form a general chromatographic carrier. Cut the assembled general chromatographic carrier into a predetermined width with a cutting machine and load it into a card shell.

[0195] (3) Reaction and detection method

[0196] ①Pre-treatment + immune reaction

[0197] Take 100 uL of the whole blood sample to be tested and 30 uL of the first reagent and mix them at room temperature for 5 min. The magnetic bead-anti-erythrocyte antibody binds to the red blood cells in the whole blood. A magnetic field is applied to enrich and separate the "magnetic bead-anti-erythrocyte antibody-erythrocyte", which separates the red blood cells. At the same time, the first aptamer and the second aptamer specifically recognize and bind to the analyte to form a reaction complex of "tracer-NT-proBNP antibody 1-analyte NT-proBNP-NT-proBNP antibody 2-linker biotin".

[0198] ② Chromatographic reaction and result processing

[0199] Take 40 uL of the above reaction solution and transfer it to the sample addition area of the chromatographic carrier. The reaction complex moves to the detection area by capillary action, and the streptavidin capture binds specifically to the linker biotin. The detector in the detection device collects the tracer signal, and through data processing, the content of the analyte is output.

[0200] Example 5

[0201] (1) Preparation of the second reagent

[0202] Preparation of magnetic bead-anti-erythrocyte antibody

[0203] Mix the biotinylated anti-erythrocyte antibody (Guangzhou Gelin, product number G0093R) and the streptavidin magnetic beads in a mass ratio of 1:24 to form a magnetic bead-anti-erythrocyte antibody complex. React at room temperature for 30 minutes, remove the supernatant by magnetic separation, wash twice with PBS buffer, and store in magnetic bead storage solution (20 mM Tris, 1% Tween 20, 10 mg / mL bovine serum albumin, 5% mannitol). Further dilute the magnetic beads to 0.5 mg / mL with TBST buffer (20 mM Tris, 2% Tween 20, 0.02% Proclin 300) and store at 2-8°C for future use.

[0204] (2) Preparation of the first reagent

[0205] ① Coupling of the first aptamer and the tracer

[0206] Add NT-proBNP antibody 1 (Xiamen Tongren, product number XJ29) to the fluorescent microspheres at a dose of 60 ug NT-proBNP monoclonal antibody / 300 ug fluorescent microspheres, add 6 ug EDC, stir at room temperature for 120 minutes, add 5% BSA, block and stir for 60 minutes, centrifuge at 14000 r / min for 20 minutes, discard the supernatant, and restore the volume of the precipitate with fluorescent antibody storage solution (100 mM Tris, 1% Tween 20, 5 mg / mL bovine serum albumin, 2% sucrose), and store at 2-8°C.

[0207] ② Coupling of the second aptamer and the linker

[0208] Take 0.001 g of biotin and dissolve it in 175 μL of DMSO to obtain a biotin working solution. Take 0.5 mg of NT-proBNP antibody 2 (Xiamen Tongren, item number XJ28) and add 7 μL of the biotin working solution. Mix immediately, rotate for labeling at room temperature in the dark, and then dialyze or pass through a column to remove free biotin.

[0209] ③ Preparation of the first reagent

[0210] Dilute the first aptamer coupled with the tracer to 0.34 mg / mL and the second aptamer coupled with the linker to 0.032 mg / mL with the first reagent storage solution (20 mM Tris, 1% Tween 20, 5 mg / mL sodium casein, 5% trehalose). Mix the two solutions in equal volumes to prepare the first reagent, and freeze-dry it in a freeze-dryer to obtain a freeze-dried form.

[0211] (3) Preparation of the chromatographic carrier

[0212] ① Preparation of the detection line

[0213] Dilute the streptavidin to 0.2 mg / mL with PBS buffer, and draw two capture detection lines on the left end of the nitrocellulose membrane at a distance of 5-15 mm.

[0214] ② Assembly of the test strip

[0215] The backing, sample pad, and water-absorbing pad are common materials in the art. The sample pad, nitrocellulose membrane, and water-absorbing pad are tightly overlapped in sequence on the backing to obtain a general chromatographic carrier. The assembled general chromatographic carrier is cut into a predetermined width with a cutting machine and loaded into a card shell.

[0216] (4) Reaction and detection method

[0217] ① Pretreatment

[0218] Mix 50 uL of the whole blood sample to be tested with 50 uL of the second reagent. The red blood cells in the whole blood bind to the magnetic bead-anti-red blood cell antibody complex in the second reagent. By applying a magnetic field, the "magnetic bead-anti-red blood cell antibody-red blood cell" complex is enriched and separated to obtain rapidly separated plasma.

[0219] ② Immune reaction

[0220] Mix 70 uL of the separated plasma sample with the freeze-dried form of the first reagent at room temperature and incubate for 3 min. The first aptamer and the second aptamer specifically recognize and bind to the analyte to form a reaction complex of "tracer-NT-proBNP antibody 1-analyte NT-proBNP-NT-proBNP antibody 2-linker biotin".

[0221] ③ Chromatographic reaction and result processing

[0222] Take 40 uL of the foregoing reaction solution to the sample loading area of the chromatographic carrier, and the reaction complex moves to the detection area by capillary action. The capture streptavidin specifically binds to the linker biotin. The detector in the detection device collects the tracer signal, and through data processing, the content result of the measured substance is output.

[0223] Example 6

[0224] (1) Preparation of the first reagent

[0225] ① Coupling of the first aptamer and the tracer

[0226] Add NT-proBNP antibody 1 (Xiamen Tongrenxin, item number XJ29) to the fluorescent microspheres at an amount of 60 ug NT-proBNP monoclonal antibody / 300 ug fluorescent microspheres, add 6 ug EDC, stir at room temperature for 120 minutes, add 5% BSA, block and stir for 60 minutes, centrifuge at 14000 r / min for 20 minutes, discard the supernatant, and restore the volume of the fluorescent antibody storage solution (100 mM Tris, 1% Tween 20, 5 mg / ml bovine serum albumin, 2% sucrose) to the precipitate. Store at 2-8°C.

[0227] ② Coupling of the second aptamer and the linker

[0228] Weigh 0.001 g of biotin and dissolve it in 175 uL of DMSO to obtain a biotin working solution. Take 0.5 mg of NT-proBNP antibody 2 (Xiamen Tongrenxin, item number XJ28) and add 7 uL of the biotin working solution. Mix immediately, rotate for labeling at room temperature in the dark, and then dialysis or column to remove free biotin.

[0229] ③ Preparation of the first reagent

[0230] Dilute the first aptamer coupled with the tracer to 0.34 mg / mL and the second aptamer coupled with the linker to 0.032 mg / mL with the first reagent storage solution (20 mM Tris, 1% Tween 20, 5 mg / ml sodium casein, 5% trehalose). Mix equal volumes of the two solutions to prepare the first reagent, and store it at 2-8°C for standby.

[0231] (2) Preparation of the chromatographic carrier

[0232] ① Preparation of the detection line

[0233] Dilute streptavidin to 0.2 mg / mL with PBS buffer, and draw a capture detection line on the left end of the nitrocellulose membrane at a distance of 5-15 mm.

[0234] ② Assembly of the test strip

[0235] The substrate, sample pad, and absorbent pad are common materials in this field. The sample pad, nitrocellulose membrane, and absorbent pad are sequentially and tightly overlapped on the substrate to assemble a universal chromatography carrier. The assembled universal chromatography carrier is cut into a preset width using a chopper and loaded into a cartridge.

[0236] (3) Reaction and detection methods

[0237] ① Immune response

[0238] Take 70 μL of the serum sample to be tested and mix it with 10 μL of the first reagent at room temperature and incubate for 3 min. The first aptamer and the second aptamer specifically recognize and bind to the analyte and form a reaction complex of "fluorescent microsphere-NT-proBNP antibody 1-analyte NT-proBNP-NT-proBNP antibody 2-biotin".

[0239] ②Chromatographic reaction and result processing

[0240] Take 40 μL of the aforementioned reaction solution and transfer it to the sample loading area of ​​the chromatography carrier. The reaction solution moves to the detection area through capillary action. Streptavidin on the detection line captures the aforementioned reaction complex onto the detection line through a specific binding reaction with biotin. The detection device collects and acquires the tracer signal on the detection line, processes the data, and outputs the content result of the analyte.

[0241] Example 7

[0242] To demonstrate the performance difference between reagents with multiple detection lines and those with a single detection line on the chromatography carrier, the following two sets of experiments were conducted. Detection reagents prepared according to Examples 1 and 6 were selected respectively to verify different detection performances. The experimental results are as follows:

[0243] (1) Linear

[0244] NT-proBNP high and low value samples were selected and prepared according to the dilution ratio (L:H). Each test was repeated twice and the average value was recorded. The experimental results are shown in Table 1. The reaction kinetic curve was plotted based on the theoretical concentration-T / C signal value (as shown in Figure 2).

[0245] Table 1

[0246] (2) Precision

[0247] Serum samples within the linear range of NT-proBNP (10-35000 pg / mL) were selected. Each sample was tested 10 times using the reagents described in Examples 1 and 6 above, and the precision CV was calculated. The experimental results are shown in the table below and Figure 3:

[0248] Table 2

[0249] (1) Anti-free biotin interference

[0250] Select clinical samples and add 0, 100, 300 ng / mL free biotin interferent, respectively, using the detection reagent prepared according to the preparation scheme of Example 1 and Example 6, respectively, to detect 3 groups of samples, repeat 3 times for each sample, and calculate the relative deviation, and the test results are shown in Table 3:

[0251] Table 3

[0252] (4) NC membrane batch difference

[0253] Select 10 different batches of NC membranes A-J, respectively, and prepare the detection reagent according to the methods of Example 1 and 6, select serum samples within the linear range of NT-proBNP, repeat 3 times to record the average value and calculate the relative deviation of the test results of the NC membranes of batches B-J and A, and the test results are as follows:

[0254] Table 4

[0255] According to the test results, compared with Example 6 which coats a single detection line, Example 1 which coats multiple detection lines is superior to Example 6 in linearity and precision, especially in anti-free biotin interference and reducing the batch difference of NC membrane.

[0256] The plurality of detection lines enhances the binding capacity and repeatability of the reaction complex, and improves the linearity and precision of the reagent. Taking the biotin-streptavidin system as an example, the detection line is coated with streptavidin, and when free biotin in the sample interferes, it will compete with the second adapter linker biotin for the binding site of streptavidin on the detection line. When multiple detection lines are coated, the front-end T2 detection line can bind the free biotin interferent in the sample, while the rear-end T1 detection line can still function to bind the second adapter linker biotin, thereby unexpectedly resisting the biotin interference. However, the binding capacity of the front-end T2 detection line to the interferent is limited, and it still needs to be combined with Example 10 to completely eliminate the interference. Secondly, the relative deviation of the total of 10 batches of NC membranes A-J in Example 1 is within ±5%, which indicates that the 10 batches of NC membranes can be combined into one batch, while in Example 6, only 3 batches of NC membranes D, I and J meet the batch combination requirement, which indicates that the use of multiple detection lines for the chromatography carrier can significantly reduce the batch-to-batch difference of the NC membrane and realize the mass production of universal chromatography materials. It is theoretically speculated that the use of multiple detection lines for the capture and recognition of the linker compensates for the batch-to-batch difference of the NC membrane.

[0257] Example 8

[0258] To prove the difference in detection performance of reagents using different pretreatment schemes for different samples, the following 6 groups of tests were set up, and the detection reagents prepared according to Examples 1-5 and the commercially available NT-proBNP fluorescent immunochromatographic detection reagent (Zhongyuanhuiji Biotechnology Co., Ltd.) were selected, respectively. According to their respective detection methods (the commercially available reagent was detected according to its product instructions), the different detection performances were verified, and the test results are as follows:

[0259] (1) Linearity

[0260] Select NT-proBNP high and low value samples according to the dilution ratio (L:H) for preparation, and record the average value of each repetition 3 times, and the test results are as shown in Table 4, calculate the linear correlation coefficient r, and draw the reaction kinetics curve according to the theoretical concentration-T / C signal value (as shown in FIG. 4):

[0261] Table 5

[0262] (2) Precision

[0263] Select NT-proBNP serum samples within the linear range of 10-35000 pg / mL, and test each sample 10 times using the aforementioned 6 groups of reagents, and calculate the precision CV (CV mean distribution graph as shown in FIG. 5):

[0264] Table 6

[0265] According to the test results, the linear correlation coefficients r of the above 6 groups of reagents meet the acceptance standard (r>0.99), the reaction kinetics curves of examples 1-5 are better than those of the commercial test paper, the precision CVs meet the acceptance standard, examples 1-5 are better than example 6 and better than the conventional commercial test paper, and the differences of examples 1-5 are not obvious, and the detection performance is optimal.

[0266] The commercial reagent is limited by its reaction methodology, which is to perform an immune reaction on a nitrocellulose membrane. After sample addition, the liquid dissolves the fluorescent microsphere-labeled antibody on the test strip, and the immune reaction is performed at the same time. Therefore, the fullness and uniformity of the reaction cannot be guaranteed. The full-liquid-phase detection method proposed in the present application can fully guarantee the efficiency and uniformity of the reaction by performing independent and front-end immune reactions, and the nitrocellulose membrane can intercept the signal by using the high binding force of biotin-streptavidin.

[0267] Examples 1-5 use different pretreatment modes corresponding to different sample types and application scenarios, and the test results are not affected.

[0268] Example 9

[0269] To prove the optimal proportion of the capture on the detection line and the second aptamer in the reaction system, 11 groups of reagents were set up for verification. The preparation scheme of the 11 groups of reagents is different from that of example 1 only in that the mass ratio of the sum of the capture (SA) on the detection line T1 and the detection line T2 to the second aptamer (Bio-NT) participating in the chromatographic reaction, and the specific information is shown in the following table:

[0270] Table 7

[0271] *T1-SA is the mass of SA coated on the detection line T1, T2-SA is the mass of SA coated on the detection line T2, SA total is the total mass of SA coated on the detection lines T1 and T2, Bio-NT is the mass of the second aptamer NTpro-BNP antibody added to the reaction solution on the chromatographic carrier, and the mass ratio (SA: Bio) is the proportional relationship between the total mass of SA and the mass of NTpro-BNP antibody.

[0272] The above 12 groups of reagents were used to test serum samples in the linear range of 10-35000 pg / mL, and the test results are shown in the following table and in FIG. 6.

[0273] Table 8

[0274] According to the test results, the adapt relationship between the capture and the second aptamer is the key factor affecting the performance of the reagent. Only when the mass ratio of the capture (i.e. the sum of SA coated on the detection line T1 and the detection line T2) to the second aptamer in the first reagent is ≥1 / 5, can the ideal detection performance be obtained. At the same time, the test results show that the concentration of the coated capture on different detection lines has no special limitation, as long as the mass adaptation ratio of the total sum of the capture coated to the second aptamer in the first reagent meets the requirements.

[0275] Example 10

[0276] (1) Preparation of the fourth reagent

[0277] The streptavidin magnetic beads were washed twice with PBS buffer, and were stored in a magnetic bead storage solution (20 mM Tris, 1% Tween 20, 10 mg / ml BSA, 5% mannitol). The magnetic beads were diluted to 1.0 mg / mL using a TBST buffer (20 mM Tris, 2% Tween 20, 0.02% Proclin 300), and were stored at 2-8°C.

[0278] (2) Preparation of the first reagent

[0279] ① Coupling of the first aptamer and the tracer

[0280] NT-proBNP antibody 1 (Xiamen Tongrenxin, item number XJ29) was added to the fluorescent microspheres at an amount of 60 μg NT-proBNP monoclonal antibody / 300 μg fluorescent microspheres, 6 μg EDC was added, and the reaction was stirred at room temperature for 120 minutes. 5% BSA was added, and the stirring was blocked for 60 minutes. Centrifugation was performed at 14000 r / min for 20 minutes, the supernatant was discarded, and the precipitate was restored to the volume with a fluorescent antibody storage solution (100 mM Tris, 1% Tween 20, 5 mg / ml bovine serum albumin, 2% sucrose), and was stored at 2-8°C.

[0281] ② Coupling of the second aptamer and the linker

[0282] 0.001 g of biotin was dissolved in 175 μL DMSO to obtain a biotin working solution. 0.5 mg of NT-proBNP antibody 2 (Xiamen Tongrenxin, item number XJ28) was added to 7 μl of the biotin working solution, and was immediately mixed, and was labeled by rotation at room temperature in the dark. Free biotin was removed by dialysis or column.

[0283] ③ Preparation of the first reagent

[0284] ​The first reagent was prepared by mixing the first aptamer diluted with the first reagent storage solution (20 mM Tris, 1% Tween 20, 5 mg / ml sodium casein, 5% trehalose) at 0.34 mg / mL and the second aptamer diluted with the second reagent storage solution (20 mM Tris, 1% Tween 20, 5 mg / ml sodium casein, 5% trehalose) at 0.032 mg / mL in equal volumes.

[0285] (3) Preparation of the chromatography carrier

[0286] ① Preparation of the detection line

[0287] The streptavidin was diluted with the PBS buffer to 0.2 mg / mL, and two capture detection lines were drawn on the left end of the nitrocellulose membrane at a distance of 5-15 mm.

[0288] ② Assembly of the test strip

[0289] The backing, sample pad and water absorption pad were common materials in the art. The sample pad, nitrocellulose membrane and water absorption pad were sequentially and closely overlapped on the backing to assemble a common chromatography carrier. The assembled common chromatography carrier was cut into a predetermined width by a cutting machine and loaded into a card shell.

[0290] (4) Reaction and detection method

[0291] ① Anti-interference treatment

[0292] 80 uL of the serum sample to be tested was mixed with the fourth reagent. The interference biotin in the sample was combined with the streptavidin magnetic beads, and the "magnetic bead-streptavidin-interference biotin" was enriched and separated by a magnetic field to quickly remove the biotin interference.

[0293] ② Immune reaction

[0294] 70 uL of the serum sample after the interference was removed was mixed with 10 uL of the first reagent at room temperature for 3 min. The first aptamer and the second aptamer specifically recognized and combined the analyte to form a reaction complex of "tracer-NT-proBNP antibody 1-analyte NT-proBNP-NT-proBNP antibody 2-linker biotin".

[0295] ③ Chromatography reaction and result processing

[0296] 40 uL of the foregoing reaction solution was transferred to the sample addition area of the chromatography carrier. The reaction complex moved to the detection area by capillary action, and the capture streptavidin and the linker biotin were specifically combined together. The detector in the detection device collected the tracer signal, and the content of the analyte was output by data processing.

[0297] (5) Verification of anti-interference ability

[0298] Select clinical samples and add 0, 100, 1200 ng / mL biotin interferent, respectively, use the detection reagent prepared according to the preparation scheme of Example 1 and the detection reagent prepared according to the preparation scheme of the present example, respectively, according to the respective detection methods, detect the aforementioned three groups of samples, repeat the test for each sample three times, and calculate the relative deviation, and the test results are shown in the following table:

[0299] Table 9

[0300] According to the results, after removing the biotin interference by Example 10, the relative deviation meets the requirements, and the biotin interference can be effectively resisted to ensure the detection accuracy.

[0301] Example 11

[0302] (1) Preparation of the fifth reagent

[0303] 5.0 g / L Tris buffer, 3.0 g / L Tris hydrochloride, 0.02 mM 3-cyclohexylaminopropanesulfonic acid, 0.05 g / L triton X-100, 0.08 g / L quaternary ammonium salt surfactant, 1.7 g / L EDTA tetrasodium salt, 9 g / L NaCl and 0.5 g / L isothiazolinone preservative were successively added into pure water and stirred to dissolve, and then filtered with a 0.22 um PES filter membrane to obtain the fifth reagent, which was stored at 2-8℃ for standby use.

[0304] (2) Preparation of the third reagent

[0305] TBST buffer solution (30 mM Tris, 5% Tween 20, 0.5% Proclin 300) was prepared.

[0306] (3) Preparation of the first reagent

[0307] ① Coupling of the first aptamer and the tracer

[0308] NT-proBNP antibody 1 (Xiamen Tongren, product number XJ29) was added to the fluorescent microspheres at an amount of 60 μg NT-proBNP monoclonal antibody / 300 μg fluorescent microspheres, 6 μg EDC was added, and the reaction was stirred at room temperature for 120 minutes, 5% BSA was added, and the stirring was blocked for 60 minutes, centrifuged at 14000 r / min for 20 minutes, the supernatant was discarded, and the precipitate was restored to volume with fluorescent antibody storage solution (100 mM Tris, 1% Tween 20, 5 mg / ml bovine serum albumin, 2% sucrose), and stored at 2-8℃ for standby use.

[0309] ② Coupling of the second aptamer and the linker

[0310] Take 0.001 g of biotin and dissolve it into 175 μL DMSO to form a biotin working solution. Take 0.5 mg of NT-proBNP antibody 2 (Xiamen Tongren, item number XJ28) and add 7 μL of the biotin working solution. Mix immediately, rotate for labeling at room temperature in the dark, and then dialyze or pass through a column to remove free biotin.

[0311] ③ Preparation of the first reagent

[0312] Dilute the first aptamer coupled with the tracer to 0.34 mg / mL and the second aptamer coupled with the linker to 0.032 mg / mL with the first reagent storage solution (20 mM Tris, 1% Tween 20, 5 mg / mL sodium casein, 5% trehalose). Mix the two solutions in equal volumes to prepare the first reagent, and freeze-dry it in a freeze dryer to form a lyophilized form.

[0313] (4) Preparation of the chromatographic carrier

[0314] ① Preparation of the detection line:

[0315] Dilute the streptavidin to 0.2 mg / mL with PBS buffer, and draw two capture detection lines on the left end of the nitrocellulose membrane at a distance of 5-15 mm.

[0316] ② Assembly of the test strip

[0317] The backing, sample pad, and absorbent pad are common materials in the art. The sample pad, nitrocellulose membrane, and absorbent pad are tightly lapped in sequence on the backing to form a general chromatographic carrier. The assembled general chromatographic carrier is cut into a predetermined width with a cutting machine and loaded into a card shell.

[0318] (6) Reaction and detection method

[0319] ① Resuspension of the first reagent

[0320] Take 10 uL of the third reagent and mix it with the lyophilized first reagent to perform the first reagent resuspension step.

[0321] ② Pretreatment

[0322] Take 6 uL of the sample whole blood to be tested and mix it with 2.0 mL of the fifth reagent for dilution. The dilution product is detected by a spectrophotometer at an absorbance value OD at 540 nm.

[0323] ③ Immune reaction

[0324] Mix and incubate 70 uL of whole blood sample with 10 uL of first reagent after reconstitution at room temperature for 3 min, and the first aptamer and the second aptamer specifically recognize and bind to the analyte to form a reaction complex of "tracer-NT-proBNP antibody 1-analyte NT-proBNP-NT-proBNP antibody 2-biotin".

[0325] (6) Chromatographic reaction and result processing

[0326] Transfer 40 uL of the above reaction solution to the sample addition area of the chromatographic carrier, and the reaction solution moves to the detection area by capillary action. The streptavidin on the detection line specifically binds to the biotin to capture the above reaction complex on the detection line. The detection device collects the tracer signal on the detection line, and through data processing, the content result of the analyte is output. The detection result is corrected using the absorbance OD value detected in step ③. The specific correction algorithm is as follows: Corrected result = whole blood result / (1-OD value / Δ)

[0327] Whole blood result: actual test result of whole blood sample;

[0328] Corrected result: result of whole blood sample after correction by OD value and algorithm;

[0329] OD value: absorbance value of whole blood sample after dilution by the fifth reagent;

[0330] Δ: algorithm correction coefficient, which varies with the detection item, detection process and reaction system.

[0331] In this embodiment, the establishment process of the algorithm correction coefficient is as follows:

[0332] Collect n cases of clinically homologous plasma and whole blood samples, obtain the detection results of plasma and whole blood samples according to the reagents and detection methods prepared according to the preparation scheme of the embodiment, obtain the OD value of the whole blood sample diluent by step ③, and calculate the correction coefficient Δ of all samples according to the formula i = OD i / (1- whole blood result i / plasma result i ). The average correction coefficient Δ = (Δ1+ Δ2+…Δ n ) / n of all samples is the algorithm correction coefficient, and 100 clinical samples are selected, and the results are as follows:

[0333] Table 10

[0334] (7) Performance evaluation results

[0335] Select 140 cases of clinical homologous plasma and whole blood samples, use the detection reagent and detection method prepared by the preparation scheme of the embodiment to detect homologous plasma and whole blood samples respectively, obtain the absorbance OD value of plasma results, whole blood results and whole blood sample dilution, and correct the results according to the foregoing method, analyze the consistency of the detection results of whole blood sample before and after correction with the results of homologous plasma sample, and the test results are shown in FIG. 7 and FIG. 8. According to the test results, after correction based on the OD value algorithm, the consistency of the whole blood sample results and the plasma sample results is significantly improved: the correlation coefficient R of the blood detection results and the plasma detection results 2 After correction by the algorithm (0.9988), it is significantly better than before correction (0.9787), and the relative deviation of the blood detection results and the plasma detection results after correction (-12% to +12%) is significantly better than before correction (-25% to -60%).

[0336] Example 12

[0337] To verify the universality of the detection method of the application, supplement the preparation method and performance detection of another item reagent.

[0338] (1) Preparation of the first reagent

[0339] ① Coupling of the first aptamer and the tracer

[0340] Add PCT antibody 1 (Feifeng Biological, item number PCT-Ab7#) to the fluorescent microspheres at an amount of 60 μg PCT monoclonal antibody / 300 μg fluorescent microspheres, add 6 μg EDC, stir at room temperature for 120 minutes, add 5% BSA, block and stir for 60 minutes, centrifuge at 14000 r / min for 20 minutes, discard the supernatant, and restore the volume of the precipitate with the fluorescent antibody storage solution (100 mM Tris, 1% Tween 20, 5 mg / ml bovine serum albumin, 2% sucrose), and store at 2-8°C.

[0341] ② Coupling of the second aptamer and the linker

[0342] Dissolve 0.001 g of biotin in 175 μL of DMSO to obtain a biotin working solution, take 0.5 mg of PCT antibody 2 (Feifeng Biological, item number PCT-Ab4#) and add 7 μl of the biotin working solution, mix immediately, rotate for labeling at room temperature in the dark, and then dialysis or column to remove free biotin.

[0343] ③ Preparation of the first reagent

[0344] The first reagent was prepared by mixing the first aptamer coupled with the tracer at 0.34 mg / mL and the second aptamer coupled with the linker at 0.032 mg / mL in equal volumes with the first reagent storage solution (20 mM Tris, 1% Tween 20, 5 mg / mL sodium caseinate, 5% trehalose), and stored at 2-8°C.

[0345] (2) Preparation of the chromatographic carrier

[0346] ① Preparation of the detection line

[0347] The streptavidin was diluted with PBS buffer to 0.2 mg / mL, and two capture detection lines were drawn on the left end of the nitrocellulose membrane at a distance of 5-15 mm.

[0348] ② Assembly of the test strip

[0349] The backing, sample pad, and absorbent pad were general materials in the art. The sample pad, nitrocellulose membrane, and absorbent pad were tightly overlapped on the backing in sequence to assemble a general chromatographic carrier. The assembled general chromatographic carrier was cut into a predetermined width with a cutting machine and loaded into a card shell.

[0350] (3) Reaction and detection method

[0351] ① Immune reaction

[0352] 70 uL of the serum sample to be tested was mixed with 10 uL of the first reagent at room temperature and incubated for 3 min. The first aptamer and the second aptamer specifically recognized and bound to the analyte to form a reaction complex of "tracer-PCT antibody 1-analyte PCT-PCT antibody 2-linker biotin".

[0353] ② Chromatographic reaction and result processing

[0354] 40 uL of the above reaction solution was transferred to the sample application area of the chromatographic carrier. The reaction complex moved to the detection area by capillary action, and the capture streptavidin specifically bound to the linker biotin. The detector in the detection device collected the tracer signal, and through data processing, the content of the analyte was output.

[0355] (4) Performance evaluation results

[0356] ① Linearity

[0357] High and low value samples were selected and prepared according to the dilution ratio (L:H). The reagent prepared according to the preparation scheme of the present embodiment was used for detection, and each test was repeated 3 times. The test results are shown in the following table:

[0358] Table 11

[0359] ②Precision

[0360] The precision CV was calculated by testing serum samples in the linear range of 0-100 pg / mL, 10 times for each repetition, and the test results are shown in the following table:

[0361] Table 12

[0362] According to the results, the PCT reagent prepared in Example 12 meets the requirements of linearity and precision, indicating that the scheme of the present application is universal in different projects.

[0363] Example 13

[0364] (1) Preparation of the first reagent

[0365] ① Coupling of the first aptamer and the tracer

[0366] Cortisol derivative (Biosynth, Code: 80-1060) was added to the fluorescent microspheres at a ratio of 60 μg cortisol derivative / 300 μg fluorescent microspheres, 6 μg EDC was added, and the reaction was stirred at room temperature for 120 minutes. 5% BSA was added, and the mixture was stirred for 60 minutes. Centrifugation was performed at 14000 r / min for 20 minutes, the supernatant was discarded, and the precipitate was resuspended to the original volume with fluorescent antibody storage solution (100 mM Tris, 1% Tween 20, 5 mg / ml bovine serum albumin, 2% sucrose). The mixture was stored at 2-8°C.

[0367] ② Coupling of the second aptamer and the linker

[0368] 0.001 g of biotin was dissolved in 175 μL DMSO to prepare a biotin working solution. 0.5 mg of cortisol-specific antibody (Biosynth, Code: 10-154) was added to 7 μl of the biotin working solution, and the mixture was immediately mixed, labeled at room temperature in the dark, and then dialyzed or passed through a column to remove free biotin.

[0369] ③ Preparation of the first reagent

[0370] The first reagent storage solution (20 mM Tris, 1% Tween 20, 5 mg / ml sodium casein, 5% trehalose) was diluted to 0.34 mg / mL to dilute the first aptamer coupled with the tracer, and the mixture was stored at 2-8°C for later use.

[0371] (2) Preparation of the chromatography carrier

[0372] ① Preparation of the detection line

[0373] Streptavidin was diluted with PBS buffer to 0.2 mg / mL, and two capture detection lines were drawn on the left end of the nitrocellulose membrane at a distance of 5-15 mm.

[0374] ② Test strip assembly

[0375] The backing, sample pad and absorbent pad are common materials in the art. The sample pad, nitrocellulose membrane and absorbent pad are sequentially and tightly overlapped on the backing to assemble a common chromatographic carrier. The assembled common chromatographic carrier is cut into a preset width by a cutting machine and loaded into a card shell.

[0376] (3) Reaction and detection method

[0377] ① Immune reaction

[0378] 70 uL of the serum sample to be tested is added to 10 uL of the first aptamer, and then 10 uL of the second aptamer is added. After mixing and incubating at room temperature for 3 min, the cortisol to be tested competes with the first aptamer (fluorescently labeled cortisol derivative) to bind to the second aptamer (biotinylated anti-cortisol antibody).

[0379] ② Chromatographic reaction and result processing

[0380] 40 uL of the reaction solution is transferred to the sample addition area of the chromatographic carrier. The reaction complex moves to the detection area by capillary action, and the capture streptavidin specifically binds to the linker biotin. The detector in the detection device collects the tracer signal, and through data processing, the content result of the to-be-tested substance is output.

[0381] (4) Performance evaluation

[0382] ① Linearity

[0383] High and low value samples are selected and prepared according to the dilution ratio (L:H). The reagents prepared according to the preparation scheme of the embodiment are used for detection, and each repetition is tested 3 times. The test results are shown in the following table:

[0384] Table 13

[0385] ② Precision

[0386] The serum samples in the linear range of 0-100 pg / mL are tested 10 times for each repetition, and the precision CV is calculated. The test results are shown in the following table:

[0387] Table 14

[0388] The test results prove that the cortisol reagent prepared in Example 13 meets the requirements in terms of linearity and precision, indicating that the method of the application is suitable for the competition method project.

[0389] Although the embodiments of the present application have been shown and described above, it is understood that the above-described embodiments are exemplary and are not to be construed as limiting the present application, and that modifications, substitutions, replacements and changes can be made by those skilled in the art to the above-described embodiments without departing from the principles and spirit of the present application within the scope of the present application.

Claims

1. A fully liquid reaction chromatographic test reagent, characterized in that, The first reagent and the chromatography carrier are included; The first reagent includes a first aptamer coupled with a tracer and a second aptamer coupled with a linker, and the first aptamer and the second aptamer specifically recognize and / or bind to the same analyte; The chromatography carrier includes at least two detection lines coated with a capture reagent that can specifically recognize and / or bind to the linker.

2. A fully liquid reaction chromatographic test reagent, characterized in that The first reagent and the chromatography carrier are included; The first reagent includes a first aptamer coupled with a tracer and a second aptamer coupled with a linker, and one of the first aptamer or the second aptamer competes with the analyte to recognize and / or bind to the other aptamer; The chromatography carrier includes at least two detection lines coated with a capture reagent that can specifically recognize and / or bind to the linker.

3. A fully liquid reaction chromatographic test reagent, characterized in that, The first reagent and the chromatography carrier are included; The first reagent includes a first aptamer coupled with a tracer and a second aptamer coupled with a linker, and the first aptamer and the second aptamer specifically recognize and / or bind to the same analyte; The detection lines are coated with a capture reagent that can specifically recognize and / or bind to the linker, and the mass ratio of the capture reagent to the second aptamer is ≥1 / 5.

4. A fully liquid reaction chromatographic test reagent, characterized in that, The first reagent and the chromatography carrier are included; The first reagent includes a first aptamer coupled with a tracer and a second aptamer coupled with a linker, and one of the first aptamer or the second aptamer competes with the analyte to recognize and / or bind to the other aptamer; The detection lines are coated with a capture reagent that can specifically recognize and / or bind to the linker, and the mass ratio of the capture reagent to the second aptamer is ≥1 / 5.

5. The agent according to any one of claims 1 to 4, characterized in that, The first aptamer coupled with a tracer and the second aptamer coupled with a linker in the first reagent are in a split-packaging form.

6. The agent according to any one of claims 1 to 4, characterized in that, The first aptamer and / or the second aptamer are selected from at least one of the following categories: an antibody, an antigen-binding fragment, an aptamer, a modified aptamer, an aptide, an affibody, an antigen, a protein, a polypeptide, a polyprotein complex, an exosome, a microbial particle / piece / fragment, an oligonucleotide, or a low-molecular-weight compound; Preferably, the antibody is selected from at least one of a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a chimeric antibody, a humanized antibody, or a camelid antibody. Preferably, the antigen-binding fragment is selected from at least one of a Fab', a Fab, a F(ab')2, an Fv, and an scFV fragment.

7. The agent according to any one of claims 1 to 4, characterized in that, The first aptamer and the second aptamer are different categories of substances.

8. The agent according to any one of claims 1 to 4, characterized in that, The first aptamer and the second aptamer are the same category of substances. Preferably, the first aptamer and the second aptamer are both antibodies. More preferably, the first aptamer and the second aptamer are antibodies that can bind to different epitopes on the analyte. More preferably, the first aptamer and the second aptamer are antibodies that can bind to the same epitope on the analyte, the analyte has two or more repeats of the epitope, and the first aptamer and the second aptamer bind to different repeat epitopes on the analyte.

9. The reagent according to any one of claims 1-8, wherein The linker is selected from at least one of biotin, FITC or DNP, and the capture is selected from at least one of avidin / streptavidin / anti-biotin antibody, anti-FITC antibody or anti-DNP antibody; or, The linker is selected from at least one of avidin / streptavidin / anti-biotin antibody, anti-FITC antibody or anti-DNP antibody, and the capture is selected from at least one of biotin, FITC or DNP. Preferably, the linker and the capture are selected from biotin or streptavidin / avidin, respectively.

10. The agent according to any one of claims 1 to 9, characterized in that, The tracer is selected from at least one of fluorescent signal substance, enzyme catalytic substance, electromagnetic signal substance or colored substance. Preferably, the fluorescent signal substance is selected from at least one of fluorescent microspheres, quantum dots, fluorescent proteins or fluorescein molecules.

11. The agent according to any one of claims 1 to 10, characterized in that, The chromatography carrier is a porous material with liquid capillary action or chromatography effect.

12. The agent of claim 11, wherein The detection zone is further coated with a quality control line.

13. The agent of claim 12, wherein The chromatography carrier further comprises a sample loading zone. Preferably, the sample loading zone is located at one end of the chromatography carrier and is separated from the detection zone by a buffer zone.

14. The agent of claim 13, wherein Further comprising at least one of a sample pad, a water absorption pad, a bottom liner or a card shell. Preferably, the sample pad and / or the water absorption pad are attached above the chromatography carrier. Preferably, the bottom liner is attached below the chromatography carrier. Preferably, the card shell is used to contain the chromatography carrier.

15. The agent of any one of claims 1-14, wherein, The first reagent is in liquid or solid phase. Preferably, the first reagent is in solid phase.

16. The agent of claim 15, wherein The reagent further comprises a second reagent, which has the function of separating, dissociating, filtering and / or diluting the sample.

17. The agent of claim 15 or 16, wherein When the first reagent and / or the second reagent is in solid phase, a third reagent is further included, which is used to reconstitute the first reagent and / or the third reagent.

18. The agent of claim 16 or 17, wherein The first reagent and the second reagent are in a form that can coexist.

19. The agent of claim 17, wherein The second reagent and the third reagent are in a form that can coexist in liquid phase.

20. The agent of claim 15, wherein A fourth reagent is further included, which is used to pre-exclude interference factors in the sample. Preferably, when the capture and the linker are an "avidin or streptavidin and biotin" specific connection system, the interference factor is free biotin in the sample. More preferably, the fourth reagent comprises magnetic microparticles coated with avidin and / or streptavidin / anti-biotin antibody.

21. The agent of claim 15, wherein When the sample is whole blood, a fifth reagent is further included, which is used to dilute the sample and obtain the absorbance, turbidity, transmittance or colorimetric measurement value of the diluted sample, and the measurement value is used to correct the detection result. Preferably, the measurement value is the absorbance value.

22. The agent of claim 21, wherein The fifth reagent can coexist with the second reagent, the third reagent and / or the fourth reagent.

23. A fully liquid phase reaction chromatographic detection method, characterized in that, The method comprises the following steps: (1) treating the sample with the first reagent to obtain a reaction complex, wherein the first reagent comprises a first aptamer coupled with a tracer and a second aptamer coupled with a linker, and the first aptamer and the second aptamer specifically recognize and / or bind to the target substance to form a reaction complex I of "tracer-first aptamer-target substance-second aptamer-linker"; (2) adding the reaction complex to a chromatographic carrier, the detection line of which is coated with a capture specific to recognizing and / or binding the linker, the capture capturing the reaction complex I through the linker; (3) result interpretation: interpreting the signal of the tracer in the detection area to obtain the detection result; Preferably, the chromatographic carrier comprises at least two detection lines. Preferably, the mass ratio of the capture / the second aptamer is ≥1 / 5.

24. A fully liquid phase reaction chromatographic detection method, characterized in that, The method comprises the following steps: (1) treating the sample with a first reagent to obtain a reaction complex, the first reagent comprising a first aptamer coupled with a tracer and a second aptamer coupled with a linker, one of the first aptamer or the second aptamer competing with the analyte to recognize and / or bind the other aptamer and forming a reaction complex I of "tracer-first aptamer-analyte" or "linker-second aptamer-analyte" and a reaction complex II of "tracer-first aptamer-second aptamer-linker"; (2) adding the reaction complex to a chromatographic carrier, the detection line of which is coated with a capture specific to recognizing and / or binding the linker, the capture capturing the reaction complex II through the linker; (3) result interpretation: interpreting the signal of the tracer in the detection area to obtain the detection result; Preferably, the chromatographic carrier comprises at least two detection lines. Preferably, the mass ratio of the capture / the second aptamer is ≥1 / 5.

25. The method of claim 23 or 24, wherein, The step (1) further comprises pre-treating the sample with a second reagent, the second reagent having the function of separating, dissociating, filtering or diluting the sample.

26. The method of any one of claims 23-25, wherein, The step (1) further comprises reconstituting the first reagent with a third reagent.

27. The method of any one of claims 23-26, wherein, The step (1) further comprises treating the sample with a fourth reagent to remove interfering factors in the sample.

28. The method of any one of claims 21-27, wherein, The method further comprises obtaining the absorbance, turbidity, transmittance or colorimetric measurement value of the sample, and correcting the detection result through the measurement value; Preferably, the measurement value is the absorbance value.

29. The method of claim 28, wherein, The measurement value is obtained after the sample is diluted with a fifth reagent.

30. The method of any one of claims 23-29, wherein, The step (1) can be at least one of temperature control, reaction time control and mixing operation.

31. The method of any one of claims 23-30, wherein, The result interpretation is at least one of naked eye recognition or instrument recognition.

32. A fully liquid reaction detection system, characterized in that, The method comprises: a detection device, which can load a chromatographic carrier, the chromatographic carrier having a detection area, the detection area capturing a complex reaction I of "tracer-first aptamer-analyte-second aptamer-linker-capture", and obtaining the detection result by detecting the signal of the tracer.

33. A fully liquid reaction detection system characterized by, The method comprises: a detection device, which can load a chromatographic carrier, the chromatographic carrier having a detection area, the detection area capturing a reaction complex II of "tracer-first aptamer-second aptamer-linker", and obtaining the detection result by detecting the signal change of the tracer.

34. The detection system of claim 32 or 33, wherein, The reaction pool is used for receiving the sample and the first reagent, and mixing the sample and the first reagent in the reaction pool to prepare a reaction complex, wherein the first reagent comprises a first aptamer coupled with a tracer and a second aptamer coupled with a linker, and the first aptamer and the second aptamer specifically recognize and / or bind the analyte and form a reaction complex of "tracer-first aptamer-analyte-second aptamer-linker".

35. The detection system of claim 32 or 33, wherein, The reaction pool is used for receiving the sample and the first reagent, and mixing the sample and the first reagent in the reaction pool to prepare a reaction complex, wherein the first reagent comprises a first aptamer coupled with a tracer and a second aptamer coupled with a linker, and one of the first aptamer and the second aptamer competes with the analyte to recognize and / or bind the other aptamer and form a reaction complex I of "tracer-first aptamer-analyte" or "linker-second aptamer-analyte", and a reaction complex II of "tracer-first aptamer-second aptamer-linker".

36. The detection system of any one of claims 32-35, wherein, The pipetting device is used for collecting and transferring the sample and transferring the reaction complex.

37. The detection system of claim 36, wherein, The pipetting device is divided into a sampling module and a pipetting module. Preferably, the sampling module has a sampling tube and a driving device for driving the sampling tube to quantitatively suck the sample and move to the reaction pool. Preferably, the pipetting module has a pipetting tube and a driving device for driving the pipetting tube to quantitatively suck the reagent and move to the detection area.

38. The detection system of any one of claims 32-37, wherein, The reagent supply part provides the first reagent to the reaction pool.

39. The detection system of any one of claims 32-38, wherein, The detection device is an optical detection device, which comprises a light source and a detector, the light source emits light to irradiate the detection area to make the tracer generate optical information, and the detector is used to collect the optical information.

40. The detection system of any one of claims 32-39, wherein, The data processing device is electrically connected with the detection device and comprises a processor and a computer readable storage medium storing a computer program.

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