Chromatography reaction reagent with internal reference marker assisted quantification

Abstract

A chromatography reaction reagent with internal reference marker assisted quantification, comprising a chromatography carrier and a second internal reference. The chromatography carrier comprises a detection area, and the detection area is coated with a capture agent and a first internal reference. The first internal reference is coated on the detection area to characterize the effective coating state of the NC membrane capture agent, and the second internal reference is used to dynamically monitor the chromatography reaction state; the introduction of the first internal reference and / or the second internal reference enables auxiliary correction of detection results, thereby realizing high-precision analyte detection.

Description

A chromatographic reaction reagent with internal reference marker assisted quantification TECHNICAL FIELD

[0001] The present application relates to the field of medical testing, in particular to a chromatographic reaction reagent with internal reference marker assisted quantification. BACKGROUND

[0002] With the continuous progress of biomedical technology, the detection accuracy of disease markers is increasingly demanding. Chromatography technology, as a rapid, simple and efficient detection method, has been widely used in clinical diagnosis and biomedical research. Traditional chromatographic detection methods mainly rely on the binding of target molecules and specific antibodies or antigens, and the results are judged by the naked eye or simple instruments. However, these methods can usually only provide qualitative or semi-quantitative results, and are easily affected by operating conditions and sample differences, limiting their application in precision medicine.

[0003] In order to improve the precision and accuracy of immunochromatographic detection, researchers have developed various chromatographic auxiliary quantitative detection technologies. Among them, Zheng Jingxian et al. proposed a method for detecting analytes in a sample and an immunological analysis test strip (WO 2024 / 088374) uses the same position T' signal to correct the T signal to obtain a corrected T signal, which improves the detection precision of immunochromatography from 15% to within 10%, achieving a technological breakthrough; but it fails to further break through the detection of membrane effective coating amount and chromatographic dynamic reaction process state, and fails to achieve true quantitative effect.

[0004] Therefore, a technological breakthrough is urgently needed to further reduce the precision and achieve accurate quantification to solve the inherent problems of chromatography technology. SUMMARY

[0005] In order to solve the methodological defects of immunochromatography, the present application provides a chromatographic reaction reagent with internal reference marker assisted quantification and a detection method.

[0006] In a first aspect, the present application provides a chromatographic reaction reagent with internal reference marker assisted quantification, comprising a chromatographic carrier and a second internal reference.

[0007] The chromatographic carrier comprises a detection zone, and the detection zone is coated with a capture and a first internal reference;

[0008] The capture is used to directly and / or indirectly capture a tracer, and the tracer can generate a signal T1 for indicating the presence or absence and / or content of the analyte and generating a signal calibration value T5;

[0009] The first internal reference is used to indicate the coating amount of the capture in the detection zone and generate a signal T2, and to indicate the effective coating amount of the capture in the detection zone and generate a signal T3;

[0010] The second internal reference is used to indicate the chromatographic reaction state and generate signal T4.

[0011] Signals T2, T3 and T4 are used to assist in correcting signal calibration value T5.

[0012] In a second aspect, the present application provides a chromatographic reaction reagent with internal reference label assisted quantification, comprising a chromatographic carrier and a second internal reference, wherein the chromatographic carrier comprises a detection zone, and the detection zone is coated with a capture agent.

[0013] The capture agent is used to directly and / or indirectly capture a tracer, and the tracer can generate signal T1 to indicate the presence and / or content of the analyte and generate signal calibration value T5.

[0014] The first internal reference is used to indicate the coating state of the capture agent in the detection zone and generate signal T2, and to indicate the effective coating state of the capture agent in the detection zone and generate signal T3.

[0015] Signals T2 and T3 are used to assist in correcting signal calibration value T5.

[0016] In a second aspect, the present application provides a chromatographic reaction reagent with internal reference label assisted quantification, comprising a chromatographic carrier and a second internal reference, wherein the chromatographic carrier comprises a detection zone, and the detection zone is coated with a capture agent.

[0017] The capture agent is used to directly and / or indirectly capture a tracer, and the tracer can generate signal T1 to indicate the presence and / or content of the analyte and generate signal calibration value T5.

[0018] The second internal reference is used to indicate the chromatographic reaction state and generate signal T4.

[0019] Signal T4 is used to assist in correcting signal calibration value T5.

[0020] In some specific embodiments, signal T2 is the signal generated by the first internal reference in the detection zone before the chromatographic reaction reaches the detection zone. Specifically, the detection of signal T2 can be uniformly detected by the manufacturer before the reagent leaves the factory, and the signal T2 can be pre-built in the data information of the reagent, for example, it can be built in the two-dimensional code / bar code information of the reagent. When the detection is performed, the relevant information of the reagent can be obtained by scanning the code. Alternatively, the signal generated by the second internal reference in the detection zone can be detected at any time before the chromatographic reaction reaches the detection zone during the detection process at the terminal. It can be understood that signal T2 can directly or indirectly indicate the coating amount and uniformity of the capture agent in the detection zone.

[0021] In some embodiments, signal T3 is the signal generated by the first internal control in the detection zone after the completion of the detection, for example, after the signal T4 is obtained, or the signal T3 is obtained at the same time when the signal T4 is obtained. Specifically, the signal T3 can be the signal generated by the first internal control in the detection zone after the completion of the sample loading and at a specific time, more specifically, the signal generated by the first internal control in the detection zone within 15 minutes after the completion of the sample loading. It should be understood that the signal T3 can directly or indirectly indicate the effective coating state of the capture on the detection zone (including the effective coating amount and the uniformity of the coating), wherein the effective coating amount is the actual effective coating of the capture on the detection zone after excluding the loss of the capture on the detection zone during the chromatography.

[0022] In some embodiments, the capture and the first internal control can be independently coated on the chromatography carrier, i.e., the capture and the first internal control are coated on the detection zone of the chromatography carrier in a mixed manner.

[0023] In some embodiments, the capture and the first internal control are similar substances, so that the capture and the first internal control coated on the detection zone have similar coating stability, chromatography speed or loss rate, etc., for example, can have similar molecular weight and / or isoelectric point, etc., and the capture and the first internal control can be the same substance, and the first internal control can generate a detectable signal to indicate its real-time state (including the coating amount and the uniformity of the coating) during the chromatography. Non-limiting examples are: the capture is SA, and the first internal control is SA conjugated with a fluorescent protein tag or SA co-expressed with a fluorescent tag.

[0024] In some embodiments, the first internal control can be directly and / or indirectly bound to the capture, for example, the binding mode can be covalent bond or intermolecular force, etc.

[0025] The second internal reference is used to indicate the chromatography reaction state and generate a signal T4, which can be used to assist in correcting the signal calibration value T5. Specifically, the chromatography reaction state can include the dynamic form of the chromatography reaction in real time / specific region / specific time, the chromatography rate of the entire reaction process / specific region / specific time. Specifically, the chromatography rate (i.e., dynamic mobility) can be converted from the signals detected at two specific time points, or from the signals detected at one specific time point, or from the signals detected at multiple time points. It should be understood that the chromatography reaction state can include all dynamic processes occurring on the chromatography carrier, including the chromatography process, physical / chemical / biological reaction processes occurring on the membrane, etc. Specifically, a threshold value can be set, and when the chromatography rate is below or exceeds the threshold value, the reaction chromatography is abnormal and indicates that the detection result is not reliable. Secondly, the dynamic form presented by the chromatography reaction process can also directly indicate whether the reaction process is abnormal. For example, when the front end of the chromatography liquid appears obvious uneven / peak head phenomenon, the chromatography reaction can be directly abnormal, and the signal T4 can be used to indicate that the detection result is not reliable.

[0026] It should be noted that the chromatography reaction reagent for assisting in quantitative labeling by the internal reference described in the present application is suitable for traditional dry chromatography reagents, semi-liquid chromatography reagents, and full-liquid chromatography reagents.

[0027] When the aforementioned internal reference labeling assisted quantitative scheme is applied to a full-liquid chromatography reagent, a first reagent is further included, wherein the first reagent includes a first aptamer coupled with a tracer and a second aptamer coupled with a linker, and the capture can specifically recognize and / or bind to the linker; the first aptamer and the second aptamer specifically recognize and / or bind to the same analyte; or, one of the first aptamer or the second aptamer competes with the analyte to recognize and / or bind to the other aptamer.

[0028] Non-limiting examples of the first aptamer and / or the second aptamer include at least one of an antibody, an antigen binding fragment, an aptamer, a modified aptamer, an aptamer, an affimer, an antigen, a protein, a polypeptide, a multi-protein complex, an exosome, a microbial particle / piece / fragment, an oligonucleotide, or a low molecular weight compound.

[0029] More specifically, when the first aptamer and the second aptamer specifically recognize and / or bind to the same analyte, the first / second aptamer can be any molecule capable of specifically recognizing and binding to the analyte.

[0030] It should be understood that the first and second aptamers can be the same or different, as long as they both bind to the analyte, forming a "tracer-first aptamer-analyte-second aptamer-linker" sandwich-type multiplex complex, the aforementioned complex is captured by the detection zone, and the content or presence of the analyte is calculated by the signal change generated by the tracer. For example, the first and second aptamers 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 and second aptamers bind to the same epitope of the analyte, the analyte should have two or more repeats of the same epitope, and the first and second aptamers generally bind to different repeats of the same epitope on the analyte. Further, the signal generated by the tracer in the multiplex complex is proportional to the concentration of the analyte to a certain extent, and the presence or absence of the analyte in the sample is determined by measuring the signal generated by the tracer in the detection zone, or the concentration of the analyte is calculated.

[0031] When one of the first aptamer or the second aptamer competes with the analyte to recognize and / or bind to the other aptamer, one of the first aptamer or the second aptamer is any molecule that can compete with the analyte to recognize and / or bind to the other aptamer.

[0032] It should be understood that the first aptamer can compete with the analyte to recognize and / or bind to the second aptamer, or the second aptamer can compete with the analyte to recognize and / or bind to the first aptamer, forming a "tracer-first aptamer-second aptamer-linker" multiplex complex in the competition reaction with the analyte. The first aptamer and the analyte can be the same or different, as long as they can both specifically recognize and / or bind to the second aptamer, and the amount of tracer-first aptamer complex in the system is known, the content or presence of the analyte can be calculated by the signal change of the detection zone. Alternatively, the second aptamer and the analyte can be the same or different, as long as they can both specifically recognize and / or bind to the first aptamer, and the amount of tracer-first aptamer complex in the system is known, the content or presence of the analyte can be calculated by the signal change of the detection zone. Further, the signal generated by the tracer in the multiplex complex is inversely proportional to the concentration of the analyte to a certain extent, and the presence or absence of the analyte in the sample is determined by measuring the signal generated by the tracer in the detection zone, or the concentration of the analyte is calculated.

[0033] The capture and the linker are combined 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 combined by the antibody, when the linker is a protein, the capture corresponds to an antibody that can specifically recognize and / or combine; when the linker is DNP, the capture corresponds to anti-DNP antibody, when the linker is anti-DNP, the capture corresponds to DNP; when the linker is FITC, the capture corresponds to anti-FITC antibody, and when the linker is anti-FITC antibody, the capture corresponds to FITC.

[0034] In some specific embodiments, the linker and capture connection system is a "biotin-streptavidin" connection system, it should be understood that one avidin molecule can bind 4 biotin molecules, the binding is very stable, and avidin and biotin can be coupled with protein, enzyme, fluorescein and other molecules, and do not affect the biological activity of the latter. An 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.

[0035] In some specific embodiments, the chromatography carrier is also coated with a quality control line, which can be used to determine whether the detection is effective or for positioning when the instrument reads the detection results.

[0036] In some specific 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 setting the buffer area is to make the sample undergo lateral chromatography buffer for a certain distance on the chromatography carrier, so that the subsequent chromatography is carried out in a more uniform manner, and when reaching the detection area, the chromatography reaction is carried out at the expected speed, so as to obtain better binding efficiency.

[0037] In some embodiments, at least one of a sample pad, an absorbent pad, a backing, or a cassette is further included. More specifically, a sample pad is attached on top of the chromatography carrier to carry the reaction complex and to provide a buffer for the sample loading process. More specifically, an absorbent pad is attached on top of the chromatography carrier to draw the solution through the chromatography carrier by its absorbent property. More specifically, a backing is attached on the bottom of the chromatography carrier to provide a solid support for the chromatography carrier. More specifically, a cassette is used to hold the chromatography carrier, and the cassette has a sample loading hole and a detection window.

[0038] In some embodiments, the second internal reference can be present in the first reagent or coated on the sample pad of the chromatography carrier.

[0039] When the aforementioned internal reference labeling assisted quantification scheme is applied to a dry / semi-liquid chromatography reagent, a linker coupled to a tracer is further included, wherein the linker and the capture specifically recognize and / or bind to the same analyte; or, the linker competes with the analyte for recognition and / or binding to the capture, or the capture competes with the analyte for recognition and / or binding to the linker.

[0040] Non-limiting examples of the linker and / or the capture include at least one of an antibody, an antigen binding fragment, an aptamer, a modified aptamer, an aptide, an affibody, an antigen, a protein, a polypeptide, a multi-protein complex, an exosome, a microbial particle / piece / fragment, an oligonucleotide, or a low molecular weight compound.

[0041] In some embodiments, at least one of a sample pad, a binding pad, an absorbent pad, a backing, or a cassette is further included. Specifically, the sample pad, the binding pad, and / or the absorbent pad are attached on top of the chromatography carrier, the backing is attached on the bottom of the chromatography carrier, and the cassette is used to hold the chromatography carrier.

[0042] In some embodiments, the linker coupled to a tracer and / or the second internal reference can be independent of the chromatography carrier or coated on the chromatography carrier. Specifically, the linker coupled to a tracer and the second internal reference can be both coated on the chromatography carrier, one of them can be coated on the chromatography carrier, or both of them can be independent of the chromatography carrier.

[0043] In some embodiments, the linker coupled to a tracer is coated on a binding pad, and the binding pad is located between the sample pad / sample loading area and the detection area. The chromatography liquid first passes through the sample pad / sample loading area, then passes through the binding pad to complete the mixing between the analyte and the linker coupled to a tracer, and finally passes through the detection area.

[0044] In some embodiments, the second internal reference is coated on the sample pad or the binding pad.

[0045] In some specific embodiments, the second reagent, the linker of the tracer and / or the second internal reference are present in the second reagent, i.e. the sample is first reacted with the second reagent, and then the analyte and the linker of the tracer / second internal reference are mixed, and then added to the sample pad / sample area.

[0046] The tracer, the first internal reference and / or the second internal reference can be any detectable labeling substance, which can generate a signal detectable by visual or instrumental means, and examples 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 analyte complex containing the tracer flows on the chromatographic carrier, the tracer can be captured and aggregated on the detection line or not, and the presence or concentration of the analyte is determined according to the signal of the tracer, such as color signal, fluorescent signal or electromagnetic signal, etc.

[0047] 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, and more specifically, 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, and more specifically, the metal colloidal particle can be at least one of colloidal gold or colloidal platinum.

[0048] In some specific embodiments, the first internal reference, the second internal reference and / or the tracer can be the same substance or different substances, as long as the signals generated by them can be distinguished from each other, and it should be understood that the different signals generated by different signal substances can be distinguished, or the signals can be distinguished by different detection forms, such as detection at different reaction stages or time points.

[0049] In some specific embodiments, the second internal reference can be coupled to the linker together with the tracer, or the second internal reference and the tracer are integrally present, and the tracer is the second internal reference.

[0050] In some specific embodiments, the second internal control can be present in the first reagent, the second reagent, or coated on the chromatographic support. Specifically, when the second internal control is present in the first reagent, it can be present mixed with the first aptamer conjugated to the tracer and the second aptamer conjugated to the linker, it can be conjugated to the first aptamer and / or the second aptamer, or it can be present as one piece with the tracer, i.e., the second internal control is the tracer. Specifically, when the second internal control is present in the second reagent, it can be present mixed with the linker conjugated to the tracer, it can be conjugated to the linker together with the tracer, or it can be present as one piece with the tracer and the second internal control. Specifically, when the second internal control is coated on the chromatographic support, it should be coated before the detection zone, e.g., the loading zone.

[0051] "Specifically recognizes" or "specifically binds" as described herein can refer to the interaction of an antibody, protein, or peptide with a second chemical entity, wherein the interaction is dependent upon the presence of a particular structure (e.g., an antigenic structure) or a determinant / epitope on the chemical entity, e.g., the recognition and binding of a specific antigenic structure by an antibody, the specific recognition and binding of a ligand by a receptor, or the specific recognition and binding of biotin by avidin, etc.

[0052] "Coupled" as described herein broadly refers to the stable association between two entities resulting from any chemical, physical, or physico-chemical 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 a direct binding / conjugation between the two entities, e.g., the direct binding / conjugation of an antibody to an antigen on a protein, and an indirect binding / conjugation between the two entities through one or more intermediaries, e.g., the association between an antibody and a polynucleotide via one or more oligonucleotides and / or labels. Thus, "coupled" when used in relation to two entities refers to the two entities being bound / conjugated, either directly or indirectly, through any such direct or indirect means.

[0053] 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 the analyte include an antigen, an antibody, an antigen-binding fragment, a protein, a polypeptide, a multi-protein complex, a hormone, an exosome, an oligonucleotide, or a low-molecular-weight compound. The analyte can be detected in any sample of interest and is not particularly limited to biological samples, such as, but not limited to, 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).

[0054] The aforementioned reagents are suitable for dry chromatographic reactions, semi-liquid chromatographic reactions, and liquid chromatographic reactions. Herein, a non-limiting example of the detection step in one form is as follows:

[0055] Meanwhile, the first internal reference and the second internal reference are used for auxiliary correction of the detection result, and when applied to a whole liquid chromatography reaction, the method comprises the following steps:

[0056] (1) A chromatography reagent card is provided, comprising a first reagent, a chromatography carrier and a second internal reference, the first reagent comprising a first aptamer coupled with a tracer and a second aptamer coupled with a linker; the chromatography carrier comprising a sample adding area and a detection line, the detection line being coated with a capture and a first internal reference; the first aptamer and the second aptamer specifically recognizing and / or binding to a target substance;

[0057] (2) The sample is reacted with the first reagent, and the target substance specifically binds to the first aptamer and the second aptamer to form a complex I of “tracer-first aptamer-target substance-second aptamer-linker”;

[0058] (3) During the chromatography process, a signal T4 generated by the second internal reference is obtained;

[0059] (4) The reaction solution is added to the sample adding area, and the complex I is captured to the detection area by the specific binding of the capture to the linker to generate a signal T1 and a signal calibration value T5;

[0060] (5) The signals T2 and T3 generated by the first internal reference on the detection area before and after the chromatography reaction are obtained, the signal calibration value T5 is corrected by using the signals T2, T3 and T4, and a detection result is output.

[0061] Or:

[0062] (1) A chromatography reagent card is provided, comprising a first reagent, a chromatography carrier and a second internal reference, the first reagent comprising a first aptamer coupled with a tracer and a second aptamer coupled with a linker; the chromatography carrier comprising a sample adding area and a detection line, the detection line being coated with a capture and a first internal reference; one of the first aptamer or the second aptamer competes with the target substance to recognize and / or bind to the other aptamer;

[0063] (2) The sample is reacted with the first reagent to form a reaction complex II of “tracer-first aptamer-target substance” or “linker-second aptamer-target substance” and a reaction complex III of “tracer-first aptamer-second aptamer-linker”;

[0064] (3) During the chromatography process, a signal T4 generated by the second internal reference is obtained;

[0065] (4) The reaction solution is added to the sample adding area, and the complex III is captured to the detection area by the specific binding of the capture to the linker to generate a signal T1 and a signal calibration value T5;

[0066] (5) Obtain the signal T2 generated by the first internal reference in the detection area before and after the chromatographic reaction and the signal T3, correct the signal calibration value T5 using the signals T2, T3 and T4, and output the detection result.

[0067] It should be understood that the labels before the aforementioned reaction steps should not be understood as limiting the specific operation timing, as long as the signals are obtained in the signal generation areas or at specific times in sequence according to the chromatographic reaction steps, and the final detection result is obtained by auxiliary correction of the signal calibration value T5.

[0068] The application has the beneficial effects that the application provides a chromatographic reaction reagent and a detection method with internal reference labeling auxiliary quantification, the effective coating state of the NC membrane capture is characterized by coating the first internal reference on the detection area, the dynamic monitoring of the chromatographic process is realized by the second internal reference, the high-precision detection of the analyte is realized by introducing the first internal reference and / or the second internal reference for auxiliary correction of the detection result, the raw material roll difference of the NC membrane is reduced, the production complexity and the requirements for the detection environment (such as temperature and humidity) are reduced. In addition, the immuno-chromatographic analysis method of the application can be applied to traditional dry immuno-chromatographic analysis, semi-liquid immuno-chromatographic analysis and full-liquid immuno-chromatographic analysis, has a wide range of application scenarios, and can effectively improve the detection accuracy of various immuno-chromatographic modes. Specific embodiments

[0069] To make the objectives, technical solutions and advantages of the embodiments of the application clearer, the technical solutions in the embodiments of the application will be described below in detail with reference to the drawings in the embodiments of the application. Obviously, the described embodiments are some of the embodiments of the application, rather than all the embodiments of the application. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the application.

[0070] In this document, "and / or" includes any and all combinations of one or more of the associated items.

[0071] In this document, "a plurality of" means two or more, that is, it includes two, three, four, five, etc.

[0072] It has to be understood that the terms "including", "containing" or any other similar term are used inclusively so that a process, method, article, or apparatus that includes a list of elements is not limited to those elements but can include other elements not expressly listed or inherent to such process, method, article, or apparatus. An element proceeded by "comprises a", "has a", "includes a", or "contains a", does not, without more constraints, exclude the presence of additional identical elements in the process, method, article, or apparatus that includes the element.

[0073] In this specification, certain embodiments can be disclosed in a format that is a range. It is to be understood that such a range format is used only for convenience and brevity and should be interpreted in the context of the description. Therefore, the description of a range should be considered to have specifically disclosed all possible sub-ranges as well as individual numerical values within that range. For example, a description of a range such as from 1 to 5 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 5, from 3 to 4, from 3 to 5, and also individual numbers within that range, such as 1, 2, 3, 4, and 5. This same principle applies to ranges reciting the same principles applied to all ranges, whether wide or narrow, that are inherently disclosed in a description.

[0074] In a first type of embodiment, the immuno-chromatographic analysis method corresponds to a dry immuno-chromatographic analysis, comprising: providing an immuno-chromatographic analysis reagent card, the immuno-chromatographic analysis reagent card comprising a test strip, the test strip comprising a sample pad, a conjugate pad, an NC membrane and a water absorption pad arranged in sequence, the NC membrane being provided with a detection zone; the detection zone being coated with a capture and a first internal reference;

[0075] providing a first reagent, the first reagent being disposed on the conjugate pad, the first reagent being a first aptamer coupled with a tracer, the tracer being used to indicate the presence or absence and / or content of the analyte and generate a signal T1; obtaining a detection result based on the signal T1; correcting the detection result with a correction parameter to obtain a corrected detection result.

[0076] In an embodiment, the correction parameter is related to the coating amount of the capture on the detection zone. In other embodiments, the correction parameter is related to the coating uniformity of the capture on the detection zone. In still other embodiments, the correction parameter is related to the speed of the chromatographic reaction.

[0077] In an embodiment, the correction parameter is a combination of a first capture coating amount correction parameter a related to the coating amount of the capture on the detection zone before the chromatographic reaction reaches the detection zone and a second capture coating amount correction parameter b related to the coating amount of the capture on the detection zone after the chromatographic reaction is completed, i.e. (a, b) combination.

[0078] ​As an example, the correction parameter is taken as (a, b) combination.

[0079] The signal T1 of the tracer after the completion of the chromatographic reaction (in this example, the time point after the completion of the chromatographic reaction is taken as 5 min after the sample addition) is obtained, and the detection result X of the analyte is obtained based on the signal T1.

[0080] The first signal T2 of the first internal reference before the sample addition is obtained, and the second signal T3 of the first internal reference after the completion of the chromatographic reaction (in this example, the time point after the completion of the chromatographic reaction is taken as 5 min after the sample addition) is obtained. The fluorescence intensity of the first signal T2 is taken as a, and the fluorescence intensity of the second signal T3 is taken as b. Then, the effective coating rate n of the first internal reference before and after the chromatographic reaction is b / a.

[0081] As an example, the method for correcting the detection result by using the correction parameter is: the corrected detection result is Y=f(a, b, X)=X*a / b.

[0082] As an example, the correction parameter is taken as a or b.

[0083] The signal T1 of the tracer after the completion of the chromatographic reaction (in this example, the time point after the completion of the chromatographic reaction is taken as 5 min after the sample addition) is obtained, and the detection result X of the analyte is obtained based on the signal T1.

[0084] The first signal T1 of the first internal reference before the sample addition is obtained, and the second signal T2 of the first internal reference after the completion of the chromatographic reaction (in this example, the time point after the completion of the chromatographic reaction is taken as 5 min after the sample addition) is obtained. The fluorescence intensity of the first signal T1 is taken as a, and the fluorescence intensity of the second signal T2 is taken as b.

[0085] The factory fluorescence intensity of the capture on the detection area of the reagent card of this batch is obtained, and is recorded as a0.

[0086] As an example, the method for correcting the detection result by using the correction parameter is: the corrected detection result is Y=f(a, X)=X*a0 / a, or Y=f(b, X)=X*a0 / b.

[0087] As an example, the correction parameter is taken as (a, b, d) combination.

[0088] The signal T1 of the tracer after the completion of the chromatographic reaction (in this example, the time point after the completion of the chromatographic reaction is taken as 5 min after the sample addition) is obtained, and the detection result X of the analyte is obtained based on the signal T1.

[0089] The first signal T2 of the first internal reference before sample loading is obtained, and the second signal T3 of the first internal reference after the chromatographic reaction is completed (in this example, the time point after the sample loading is 5 min) is obtained. The fluorescence intensity of the first signal T2 is taken as a, the fluorescence intensity of the second signal T3 is taken as b, and the uniformity of the signal T3 is taken as d (for example, the fluorescence signal region can be divided into 100 small rectangular regions of the same size, the fluorescence signal intensity values in each small rectangular region are obtained, and then the uniform distribution standard deviation of the fluorescence signal intensity values is calculated, and the uniform distribution standard deviation is taken as the uniformity d).

[0090] As an example, the method for correcting the detection result by using the correction parameter is: the corrected detection result is Y=f(a, b, d, X)=X*a(b-d) / (b*b).

[0091] The following is an example, taking the combination of correction parameters (a, b, c, d) as an example.

[0092] The signal T1 of the tracer after the chromatographic reaction is completed (in this example, the time point after the sample loading is 5 min) is obtained, and the detection result X of the analyte based on the signal T1 is obtained.

[0093] The signal T2 of the first internal reference before sample loading is obtained, and the signal T3 of the first internal reference after the chromatographic reaction is completed (in this example, the time point after the sample loading is 5 min) is obtained. The fluorescence intensity of the signal T2 is taken as a, the fluorescence intensity of the signal T3 is taken as b, the uniformity of the signal T2 is taken as c (for example, the fluorescence signal region can be divided into 100 small rectangular regions of the same size, the fluorescence signal intensity values in each small rectangular region are obtained, and then the uniform distribution standard deviation of the fluorescence signal intensity values is calculated, and the uniform distribution standard deviation is taken as the uniformity c), and the uniformity of the signal T3 is taken as d (for example, the fluorescence signal region can be divided into 100 small rectangular regions of the same size, the fluorescence signal intensity values in each small rectangular region are obtained, and then the uniform distribution standard deviation of the fluorescence signal intensity values is calculated, and the uniform distribution standard deviation is taken as the uniformity d).

[0094] As an example, the method for correcting the detection result by using the correction parameter is: the corrected detection result is Y=f(a, b, c, d, X)=X*a*a(b-d) / [b*b*(a-c)].

[0095] In a preferred embodiment, a second internal reference is further provided, the second internal reference is used to indicate the speed of the chromatographic reaction and generate a light signal; the signal T4 of the second internal reference during the chromatographic reaction is obtained, and the chromatographic speed correction parameter e is obtained based on the signal T4.

[0096] As an example, the combination of the correction parameters (a, b, c, d, e) is taken as an example.

[0097] The signal T1 of the tracer is obtained after the completion of the chromatographic reaction (in this example, the time point after the completion of the chromatographic reaction is taken as the 5th minute after the sample is added). The detection result X of the analyte is obtained based on the signal T1.

[0098] The signal T2 of the first internal reference before sample addition is obtained, and the signal T3 of the first internal reference after the completion of the chromatographic reaction (in this example, the time point after the completion of the chromatographic reaction is taken as the 5th minute after the sample is added). The fluorescence intensity of the signal T2 is taken as a, the fluorescence intensity of the signal T3 is taken as b, the uniformity of the signal T2 is taken as c (for example, the fluorescence signal region can be divided into 100 small rectangular regions of the same size, the fluorescence signal intensity values in each small rectangular region are obtained, and then the uniform distribution standard deviation of these fluorescence signal intensity values is calculated, and the uniform distribution standard deviation is taken as the uniformity c), and the uniformity of the signal T3 is taken as d (for example, the fluorescence signal region can be divided into 100 small rectangular regions of the same size, the fluorescence signal intensity values in each small rectangular region are obtained, and then the uniform distribution standard deviation of these fluorescence signal intensity values is calculated, and the uniform distribution standard deviation is taken as the uniformity d).

[0099] In this embodiment, the dynamic mobility of the second internal reference is taken as the chromatographic speed correction parameter e. The dynamic mobility of the second internal reference refers to the ability of the second internal reference to migrate between the stationary phase and the mobile phase. In chromatographic technology, the dynamic mobility is an important parameter for describing the moving rate of the second internal reference in the chromatographic column,

[0100] The baseline fluorescence signal T4 of the second internal reference at 1 minute of incubation during the chromatographic reaction is obtained 1min and the baseline fluorescence signal T4 at 3 minutes of incubation is obtained 3min . Among them, the baseline fluorescence signal is the average fluorescence signal of 50-100 points of the 500 signal acquisition points of the chromatographic detection. In this embodiment, as an example, e = T4 1min / T4 3min .

[0101] As an example, the method for correcting the detection result by using the correction parameters is: the corrected detection result is Y = f(a, b, c, d, e, X) = X*a*a(b-d) / [b*b*(a-c)*e].

[0102] In a preferred embodiment, the first internal reference and the tracer are different fluorescence signal substances.

[0103] In some alternative embodiments, the capture is at least one of an antibody, an antigen binding fragment, an aptamer, a modified aptamer, an aptide, an affimer, an antigen, a protein, a polypeptide, a polyprotein complex, an exosome, a microbial particle / piece / fragment, an oligonucleotide, or a low molecular weight compound.

[0104] In some preferred embodiments, the capture and the first internal reference are similar in nature; more preferably, the capture and the first internal reference have similar molecular weight and / or isoelectric point, and the first internal reference can generate a detectable signal; most preferably, the capture and the first internal reference are the same substance.

[0105] In some alternative embodiments, the completion of the chromatographic reaction generally refers to a specific time point after sample loading, for example, the completion of the chromatographic reaction can be any time point between the 30th second and the 15th minute after sample loading. In a preferred embodiment, the time interval between obtaining the signal T1 of the tracer and obtaining the signal T3 of the first internal reference is less than or equal to 5 minutes. For example, it can be 4 minutes, 3 minutes, 2 minutes, 1 minute, 30 seconds, 20 seconds, 10 seconds, 5 seconds, or 0 seconds.

[0106] In the second type of embodiments, the immunochromatographic assay method corresponds to a semi-liquid phase immunochromatographic assay, comprising: providing an immunochromatographic assay reagent card, the immunochromatographic assay reagent card comprising a test strip, the test strip comprising a sample pad, an NC membrane, and a water absorption pad arranged in sequence, the NC membrane being provided with a detection line; the detection line being coated with a capture and a first internal reference;

[0107] providing a first reagent, the first reagent being located in a premixed pool at the tail of the reagent card, being independent of the test strip of the reagent card, the first reagent being a first aptamer coupled with a tracer, the tracer being used to indicate the presence or absence and / or content of the analyte and generate a signal T1; obtaining a detection result based on the signal T1; correcting the detection result with a correction parameter to obtain a corrected detection result.

[0108] In one embodiment, the correction parameter is related to the coating amount of the capture on the detection line. In other embodiments, the correction parameter is related to the coating uniformity of the capture on the detection line. In still other embodiments, the correction parameter is related to the speed of the chromatographic reaction.

[0109] In one embodiment, the correction parameter can be any one of a, b, c, d, e, or a combination of (a, b), (a, c), (a, d), (a, e), (b, c), (b, d), (b, e), (c, d), (c, e), or (d, e), (a, b, c), (a, b, d), (a, b, e), (a, c, d), (a, c, e), (a, d, e), (b, c, d), (b, c, e), (b, d, e), or (c, d, e), (a, b, c, d), (a, b, c, e), (a, b, d, e), (a, c, d, e), (b, c, d, e), or (a, b, c, d, e).

[0110] As an example, the correction parameter is taken as a combination of (a, b).

[0111] The signal T1 of the tracer after the completion of the chromatographic reaction (in this example, the time point after the completion of the chromatographic reaction is taken as the 4th minute after the sample is added) is obtained, and the detection result X of the analyte is obtained based on the signal T1.

[0112] The signal T2 of the first internal reference before the sample is added is obtained, and the signal T3 of the first internal reference after the completion of the chromatographic reaction (in this example, the time point after the completion of the chromatographic reaction is taken as the 4th minute after the sample is added) is obtained. The fluorescence intensity of the signal T2 is taken as a, and the fluorescence intensity of the signal T3 is taken as b. Then, the effective coating rate n of the first internal reference before the chromatographic reaction and after the completion of the chromatographic reaction is b / a.

[0113] As an example, the method for correcting the detection result by using the correction parameter is: the corrected detection result is Y = f(a, b, X) = K * X * a / b. Wherein, K is a temperature compensation factor.

[0114] As an example, the correction parameter is taken as a or b.

[0115] The signal T1 of the tracer after the completion of the chromatographic reaction (in this example, the time point after the completion of the chromatographic reaction is taken as the 4th minute after the sample is added) is obtained, and the detection result X of the analyte is obtained based on the signal T1.

[0116] The signal T2 of the first internal reference before the sample is added is obtained, and the signal T3 of the first internal reference after the completion of the chromatographic reaction (in this example, the time point after the completion of the chromatographic reaction is taken as the 4th minute after the sample is added) is obtained. The fluorescence intensity of the signal T2 is taken as a, and the fluorescence intensity of the signal T3 is taken as b.

[0117] The fluorescence intensity of the capture on the detection line of the reagent card of the batch is obtained, and is recorded as a0.

[0118] As an example, the method for correcting the detection result with the correction parameter is: the corrected detection result is Y=f(a, X)=X*a0 / a, or Y=f(b, X)=X*a0 / b.

[0119] The following is an example, taking the combination of correction parameters (a, b, d) as an example, and also including HCT correction parameters.

[0120] Obtain the signal T1 of the tracer after the chromatographic reaction is completed (in this example, the time point after the chromatographic reaction is completed is 4 min after the sample is added), and obtain the detection result X of the analyte based on the signal T1.

[0121] Obtain the signal T2 of the first internal reference before the sample is added, and obtain the signal T3 of the first internal reference after the chromatographic reaction is completed (in this example, the time point after the chromatographic reaction is completed is 4 min after the sample is added). The fluorescence intensity of signal T2 is taken as a, the fluorescence intensity of signal T3 is taken as b, and the uniformity of signal T3 is taken as d (for example, the fluorescence signal region can be divided into 100 small rectangular regions of the same size, the fluorescence signal intensity values in each small rectangular region are obtained, and then the uniform distribution standard deviation of these fluorescence signal intensity values is calculated, and the uniform distribution standard deviation is taken as the uniformity d).

[0122] As an example, the method for correcting the detection result with the correction parameter is: the corrected detection result is Y=f(a, b, d, X)=i*X*a(b-d) / (b*b). i is the HCT correction parameter of the sample.

[0123] The following is an example, taking the combination of correction parameters (a, b, c, d) as an example.

[0124] Obtain the signal T1 of the tracer after the chromatographic reaction is completed (in this example, the time point after the chromatographic reaction is completed is 4 min after the sample is added), and obtain the detection result X of the analyte based on the signal T1.

[0125] The signal T2 of the first internal reference before sample addition is obtained, and the signal T3 of the first internal reference after the chromatographic reaction is completed (in this example, the time point after the chromatographic reaction is completed is taken as the 4th minute after sample addition) is obtained. The fluorescence intensity of the first light signal T2 is taken as a, the fluorescence intensity of the signal T3 is taken as b, the uniformity of the first light signal T2 is taken as c (for example, the fluorescence signal region can be divided into 100 small rectangular regions of the same size, the fluorescence signal intensity values in each small rectangular region are obtained, and then the uniform distribution standard deviation of these fluorescence signal intensity values is calculated, and the uniform distribution standard deviation is taken as the uniformity c), and the uniformity of the signal T3 is taken as d (for example, the fluorescence signal region can be divided into 100 small rectangular regions of the same size, the fluorescence signal intensity values in each small rectangular region are obtained, and then the uniform distribution standard deviation of these fluorescence signal intensity values is calculated, and the uniform distribution standard deviation is taken as the uniformity d).

[0126] As an example, the method for correcting the detection result by the correction parameter is: the corrected detection result is Y=f(a, b, c, d, X)=X*a*a(b-d) / [b*b*(a-c)].

[0127] In a preferred embodiment, a second internal reference is further provided, the second internal reference is used to indicate the speed of the chromatographic reaction and generate a light signal; the signal T4 of the second internal reference during the chromatographic reaction is obtained, and based on the signal T4, the chromatographic speed correction parameter e is obtained.

[0128] The following is an example, taking the combination of correction parameters (a, b, c, d, e) as an example.

[0129] The signal T1 of the tracer after the chromatographic reaction is completed (in this example, the time point after the chromatographic reaction is completed is taken as the 4th minute after sample addition) is obtained, and based on the signal T1, the detection result X of the target substance is obtained.

[0130] The signal T2 of the first internal reference before sample addition is obtained, and the signal T3 of the first internal reference after the chromatographic reaction is completed (in this example, the time point after the chromatographic reaction is completed is taken as the 4th minute after sample addition) is obtained. The fluorescence intensity of the signal T2 is taken as a, the fluorescence intensity of the signal T3 is taken as b, the uniformity of the signal T2 is taken as c (for example, the fluorescence signal region can be divided into 100 small rectangular regions of the same size, the fluorescence signal intensity values in each small rectangular region are obtained, and then the uniform distribution standard deviation of these fluorescence signal intensity values is calculated, and the uniform distribution standard deviation is taken as the uniformity c), and the uniformity of the signal T3 is taken as d (for example, the fluorescence signal region can be divided into 100 small rectangular regions of the same size, the fluorescence signal intensity values in each small rectangular region are obtained, and then the uniform distribution standard deviation of these fluorescence signal intensity values is calculated, and the uniform distribution standard deviation is taken as the uniformity d).

[0131] In this embodiment, the dynamic mobility of the second internal reference is used as the correction parameter e for the chromatographic speed. The dynamic mobility of the second internal reference refers to the ability of the second internal reference to migrate between the stationary phase and the mobile phase. In chromatographic technology, the dynamic mobility is an important parameter for describing the moving speed of the second internal reference in the chromatographic column,

[0132] The baseline fluorescence signal T4 of the second internal reference at 1 min of incubation is obtained during the chromatographic reaction process 1min and the baseline fluorescence signal T4 at 3 min of incubation 3min . In this embodiment, as an example, e = T4 1min / T4 3min .

[0133] As an example, the method for correcting the detection result using the correction parameter is: the corrected detection result is Y = f(a, b, c, d, e, X) = X * a * a(b-d) / [b * b * (a-c) * e].

[0134] In a preferred embodiment, the first internal reference and the tracer are different fluorescent signal substances.

[0135] In some alternative embodiments, the capture is at least one of an antibody, an antigen-binding fragment, an aptamer, a modified aptamer, an aptide, an affimer, an antigen, a protein, a polypeptide, a multi-protein complex, an exosome, a microbial particle / piece / fragment, an oligonucleotide, or a low-molecular-weight compound.

[0136] In some preferred embodiments, the capture and the first internal reference are similar substances in nature; more preferably, the capture and the first internal reference have similar molecular weights and / or isoelectric points, and the first internal reference can generate a detectable signal; most preferably, the capture and the first internal reference are the same substance.

[0137] In some alternative embodiments, the completion of the chromatographic reaction generally refers to a specific time point after sample addition, for example, the completion of the chromatographic reaction can be any time point between the 30th second and the 15th minute after sample addition. In a preferred embodiment, the time interval between obtaining the signal T1 of the tracer and obtaining the second light signal T3 of the first internal reference is less than or equal to 5 minutes. For example, it can be 4 minutes, 3 minutes, 2 minutes, 1 minute, 30 seconds, 20 seconds, 10 seconds, 5 seconds, or 0 seconds.

[0138] In the third type of embodiment, the immunochromatographic analysis method corresponds to a full liquid immunochromatographic analysis, comprising: providing an immunochromatographic analysis reagent card, the immunochromatographic analysis reagent card comprising a test strip, the test strip comprising a sample pad, an NC membrane and a water absorption pad arranged in sequence, the NC membrane being provided with a detection line; the detection line being coated with a capture agent and a first internal reference;

[0139] providing a first reagent, the first reagent being a first aptamer coupled with a tracer; the first reagent being independent of the reagent card; providing a second reagent, the second reagent being a second aptamer coupled with a linker; the second reagent being independent of the reagent card; the first aptamer and the second aptamer specifically binding to the same analyte.

[0140] the tracer being used to indicate the content of the analyte and generate a signal T1; obtaining a detection result based on the signal T1; correcting the detection result with a correction parameter to obtain a corrected detection result.

[0141] In one embodiment, the correction parameter is related to the coating amount of the capture agent on the detection line. In other embodiments, the correction parameter is related to the coating uniformity of the capture agent on the detection line. In still other embodiments, the correction parameter is related to the speed of the chromatographic reaction.

[0142] In one embodiment, the correction parameter can be any one of a, b, c, d, e, or a combination of (a, b), (a, c), (a, d), (a, e), (b, c), (b, d), (b, e), (c, d), (c, e), or (d, e), (a, b, c), (a, b, d), (a, b, e), (a, c, d), (a, c, e), (a, d, e), (b, c, d), (b, c, e), (b, d, e), or (c, d, e), (a, b, c, d), (a, b, c, e), (a, b, d, e), (a, c, d, e), (b, c, d, e), or (a, b, c, d, e).

[0143] The following is an example, taking the correction parameter as a combination of (a, b) as an example.

[0144] obtaining the signal T1 of the tracer after the chromatographic reaction is completed (in this example, taking the 6th minute after sample addition as the time point after the chromatographic reaction is completed), and obtaining the detection result X of the analyte based on the signal T1.

[0145] Obtain the signal T2 of the first internal reference before sample addition, and obtain the signal T3 of the first internal reference after the chromatographic reaction is completed (in this example, the time point after the chromatographic reaction is completed is the 6th minute after sample addition). Take the fluorescence intensity of the signal T2 as a, and take the fluorescence intensity of the signal T3 as b. Then, the effective coating rate n of the first internal reference before and after the chromatographic reaction is completed is b / a.

[0146] As an example, the method for correcting the detection result by using the correction parameter is: the corrected detection result is Y=f(a, b, X)=K*X*a / b. Wherein, K is a temperature compensation factor.

[0147] As an example, the correction parameter is a or b.

[0148] Obtain the signal T1 of the tracer after the chromatographic reaction is completed (in this example, the time point after the chromatographic reaction is completed is the 6th minute after sample addition), and obtain the detection result X of the measured object based on the signal T1.

[0149] Obtain the signal T2 of the first internal reference before sample addition, and obtain the signal T3 of the first internal reference after the chromatographic reaction is completed (in this example, the time point after the chromatographic reaction is completed is the 6th minute after sample addition). Take the fluorescence intensity of the signal T2 as a, and take the fluorescence intensity of the signal T3 as b.

[0150] Obtain the fluorescence intensity of the capture on the detection line of the reagent card of the batch, denoted as a0.

[0151] As an example, the method for correcting the detection result by using the correction parameter is: the corrected detection result is Y=f(a, X)=X*a0 / a, or Y=f(b, X)=X*a0 / b.

[0152] As an example, the correction parameter is a combination of (a, b, d), and also includes an HCT correction parameter.

[0153] Obtain the signal T1 of the tracer after the chromatographic reaction is completed (in this example, the time point after the chromatographic reaction is completed is the 4th minute after sample addition), and obtain the detection result X of the measured object based on the signal T1.

[0154] Obtain the signal T2 of the first internal reference before sample addition, and obtain the signal T3 of the first internal reference after the chromatographic reaction is completed (in this example, the time point after the chromatographic reaction is completed is the 6th minute after sample addition). Take the fluorescence intensity of the signal T2 as a, take the fluorescence intensity of the signal T3 as b, and take the uniformity of the signal T3 as d (for example, the fluorescence signal region can be divided into 100 small rectangular regions of the same size, the fluorescence signal intensity values in each small rectangular region are obtained, and then the uniform distribution standard deviation of these fluorescence signal intensity values is calculated as the uniformity d).

[0155] As an example, the method for correcting the detection result with the correction parameter is: the corrected detection result is Y = f(a, b, d, X) = i * X * a(b-d) / (b*b). i is the HCT correction parameter of the sample.

[0156] As an example, the combination of the correction parameter is (a, b, c, d).

[0157] Obtain the signal T1 of the tracer after the chromatographic reaction is completed (in this example, the time point after the chromatographic reaction is completed is 6 min after the sample is added), and obtain the detection result X of the analyte based on the signal T1.

[0158] Obtain the signal T2 of the first internal reference before the sample is added, and obtain the signal T3 of the first internal reference after the chromatographic reaction is completed (in this example, the time point after the chromatographic reaction is completed is 6 min after the sample is added). The fluorescence intensity of signal T2 is taken as a, the fluorescence intensity of signal T3 is taken as b, the uniformity of signal T2 is taken as c (for example, the fluorescence signal region can be divided into 100 small rectangular regions of the same size, the fluorescence signal intensity values in each small rectangular region are obtained, and then the uniform distribution standard deviation of these fluorescence signal intensity values is calculated, and the uniform distribution standard deviation is taken as the uniformity c), and the uniformity of signal T3 is taken as d (for example, the fluorescence signal region can be divided into 100 small rectangular regions of the same size, the fluorescence signal intensity values in each small rectangular region are obtained, and then the uniform distribution standard deviation of these fluorescence signal intensity values is calculated, and the uniform distribution standard deviation is taken as the uniformity d).

[0159] As an example, the method for correcting the detection result with the correction parameter is: the corrected detection result is Y = f(a, b, c, d, X) = K * X * a * a(b-d) / [b*b*(a-c)]. Wherein, K is a temperature compensation factor.

[0160] In a preferred embodiment, it further comprises providing a second internal reference, the second internal reference is used to indicate the speed of the chromatographic reaction and generate a signal; obtain the signal T4 of the second internal reference during the chromatographic reaction, and obtain the chromatographic speed correction parameter e based on the signal T4.

[0161] As an example, the combination of the correction parameter is (a, b, c, d, e).

[0162] Obtain the signal T1 of the tracer after the chromatographic reaction is completed (in this example, the time point after the chromatographic reaction is completed is 6 min after the sample is added), and obtain the detection result X of the analyte based on the signal T1.

[0163] The signal T2 of the first internal reference before sample loading is obtained, and the signal T3 of the first internal reference after the completion of the chromatographic reaction (in this example, the time point after the completion of the chromatographic reaction is 6 min after sample loading) is obtained. The fluorescence intensity of the signal T2 is taken as a, the fluorescence intensity of the signal T3 is taken as b, the uniformity of the signal T2 is taken as c (for example, the fluorescence signal region can be divided into 100 small rectangular regions of the same size, the fluorescence signal intensity values in each small rectangular region are obtained, and then the uniform distribution standard deviation of the fluorescence signal intensity values is calculated, and the uniform distribution standard deviation is taken as the uniformity c), and the uniformity of the signal T3 is taken as d (for example, the fluorescence signal region can be divided into 100 small rectangular regions of the same size, the fluorescence signal intensity values in each small rectangular region are obtained, and then the uniform distribution standard deviation of the fluorescence signal intensity values is calculated, and the uniform distribution standard deviation is taken as the uniformity d).

[0164] In this embodiment, the dynamic mobility of the second internal reference is taken as the chromatographic speed correction parameter e. The dynamic mobility of the second internal reference refers to the ability of the second internal reference to migrate between the stationary phase and the mobile phase. In chromatographic technology, the dynamic mobility is an important parameter for describing the moving speed of the second internal reference in the chromatographic column,

[0165] The baseline fluorescence signal T4 of the second internal reference at 1 min during the chromatographic reaction is obtained 1min and the baseline fluorescence signal T4 at 3 min during incubation 3min . Among them, the baseline fluorescence signal is the average fluorescence signal of 50-100 points of the 500 signal collection points of the chromatographic detection. In this embodiment, as an example, e = T4 1min / T4 3min .

[0166] As an example, the method for correcting the detection result by using the correction parameter is: the corrected detection result is Y = f(a, b, c, d, e, X) = X*a*a(b-d) / [b*b*(a-c)*e].

[0167] In a preferred embodiment, the first internal reference and the tracer are different fluorescent signal substances.

[0168] In some optional embodiments, the capture is selected from at least one of avidin, streptavidin, anti-biotin antibody, anti-FITC antibody, or anti-DNP antibody.

[0169] In some preferred embodiments, the capture and the first internal reference are similar substances; more preferably, the capture and the first internal reference have similar molecular weight and / or isoelectric point, and the first internal reference can generate a detectable signal; most preferably, the capture and the first internal reference are the same substance.

[0170] In some alternative embodiments, the completion of the chromatographic reaction generally refers to a specific time point after sample loading, for example, the completion of the chromatographic reaction can be any time point between 30 seconds and 15 minutes after sample loading. In a preferred embodiment, the time interval between obtaining the signal T1 of the tracer and obtaining the signal T3 of the first internal reference is less than or equal to 5 minutes. For example, it can be 4 minutes, 3 minutes, 2 minutes, 1 minute, 30 seconds, 20 seconds, 10 seconds, 5 seconds, 0 seconds.

[0171] The technical solutions of the present application are further illustrated below through some specific embodiments.

[0172] Embodiment 1

[0173] 1. Preparation of test strip

[0174] (1) Preparation of first reagent

[0175] ① Coupling of first aptamer and tracer

[0176] According to the amount of 60 μg cTnI monoclonal antibody / 300 μg fluorescent microspheres, cTnI antibody 1 (Hytest, 4T21-19C7cc) is added to the fluorescent microspheres, 6 μg EDC is added, and the reaction is stirred at room temperature for 120 minutes. 5% BSA is added, blocked and stirred for 60 min, centrifuged 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), and stored at 2-8°C.

[0177] ② Coupling of second aptamer and linker

[0178] 0.001 g of biotin is weighed and dissolved in 175 μL DMSO to form a biotin working solution. 0.5 mg of cTnI antibody 2 (Hytest, 4T21-16A11cc) is added to 7 μl of the biotin working solution, mixed immediately, and labeled by rotation at room temperature in the dark. Free biotin is removed by dialysis or column.

[0179] ③ Preparation of first reagent

[0180] The first aptamer coupled with the tracer is diluted to 0.34 mg / mL with the first reagent storage solution (20 mM Tris, 1% Tween 20, 5 mg / ml sodium casein, 5% trehalose), and the second aptamer coupled with the linker is diluted to 0.032 mg / mL. The two solutions are mixed in equal amounts to prepare the first reagent, which is stored at 2-8°C for standby.

[0181] (2) Preparation of chromatographic carrier

[0182] ① Preparation of detection line

[0183] The first internal reference is marked on the captured item:

[0184] Streptavidin was desalted using a desalting column, and the fluorescent substance (ThermoFisher Alexa Fluor) was used. TM Add 175 μL of DMSO to dissolve (647, catalog number A20006) to make a working solution of 10 mg / mL. Add fluorescent material to streptavidin at a molar ratio of 1:1, spin-label at room temperature in the dark, and then desalt using a desalting column.

[0185] Dilute the first internal control labeled streptavidin to 0.2 mg / mL with PBS buffer, and draw a capture detection line 5-15 mm away from the left end of the nitrocellulose membrane.

[0186] ② Assembly of test strips

[0187] 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.

[0188] 2. Detection Method

[0189] The prepared liquid chromatography reagent containing the first internal control was used to detect troponin I in blood samples. The specific procedure was as follows: 50 μL of sample was added to 150 μL of sample diluent (30 mM Tris, 5% Tween 20, 0.5% Proclin 300) and mixed thoroughly. The diluted sample was then mixed with the first reagent and reacted for 5 min. The reagent card was placed in an immunofluorescence quantitative analyzer (Q20, Zhongyuan Huiji Biotechnology Co., Ltd.) to read the fluorescence signal T2 generated by the first internal control before sample addition. 40 μL of the aforementioned reaction solution was added to the sample well of the reagent card. After incubation for 3 min, the fluorescence signal T3 of the first internal control and the fluorescence signal T4 of the detection result were read on the detection line.

[0190] 3. Result Calculation

[0191] The formula for calculating the effective coating rate of the first internal reference detected by the above detection method is as follows:

[0192] The effective coverage rate of the first internal reference η = T3 / T2

[0193] T2 - Before sample addition (before reaction), the intensity of the first internal reference fluorescence signal on the detection line;

[0194] At T3, 3 minutes after sample addition (after reaction), the intensity of the first internal reference fluorescence signal on the detection line is measured.

[0195] The test reagent prepared by the embodiment is used to detect low, medium and high value samples, and each signal value is obtained according to the detection method and the result is calculated, and the test results are shown in the following table:

[0196] Table 1 Effective coating rate before and after first internal reference reaction

[0197] The first internal reference assisted quantitative calibration formula of the above cTnI detection method is as follows:

[0198] Signal calibration value T5 = T1 / η

[0199] T1- detection result fluorescence intensity;

[0200] η- effective coating rate.

[0201] Table 2 Comparison before and after first internal reference reaction assisted quantitative detection

[0202] From the data in Table 1, the effective coating rate of different concentrations of samples is comparable and is not disturbed by the fluorescence signal value of the analyte. From the data in Table 2, the detection results of different concentrations of samples are calibrated by the effective coating rate, and the detection precision can be improved by 0.2%-1.2%, reaching within 4%.

[0203] Example 2

[0204] 1. Preparation of test strip

[0205] (1) Preparation of first reagent

[0206] ① Coupling of first aptamer and tracer

[0207] According to the amount of 60 μg cTnI monoclonal antibody (Hytest, 4T21-19C7cc) / 300 μg fluorescent microspheres, the cTnI antibody 1 (Hytest, 4T21-19C7cc) is added to the fluorescent microspheres, 6 μg 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 at 14000 r / min for 20 min, discard the supernatant, 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℃.

[0208] ② Coupling of second aptamer and linker

[0209] Take 0.001 g of biotin and add it to 175 μL of DMSO to prepare a biotin working solution. Take 0.5 mg of cTnI antibody 2 (Hytest, product number 4T21-16A11cc) and add 7 μl of the biotin working solution. Mix immediately, rotate at room temperature in the dark, and then dialyze or pass through a column to remove free biotin.

[0210] ③ Preparation of the first reagent

[0211] 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 in 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 later use.

[0212] (2) Preparation of the chromatographic carrier

[0213] ① Preparation of the detection line

[0214] The first internal reference is independent of the capture:

[0215] Dilute streptavidin-R-phycoerythrin (Agilent, product number PJ32S-1) to a final concentration of 0.05 mg / mL in PBS buffer, and dilute the capture streptavidin to a final concentration of 0.2 mg / mL. Mix the first internal reference and the streptavidin, and coat them on the left end of the nitrocellulose membrane at a distance of 5-15 mm to draw a capture detection line.

[0216] ② Assembly of the test strip

[0217] The backing, sample pad, and absorbent pad are common materials in the art. Assemble the backing, sample pad, nitrocellulose membrane, and absorbent pad in that order on the backing to form a general chromatographic carrier. Cut the assembled general chromatographic carrier into a predetermined width using a cutting machine, and load it into a card shell.

[0218] 2. Detection method

[0219] Use the above-prepared full liquid-phase detection reagent containing the first internal reference to detect troponin I in a blood sample. Specifically, add 50 μL of the sample to 150 μL of sample diluent (30 mM Tris, 5% Tween 20, 0.5% Proclin 300) and mix well. Then, mix the diluted sample with the first reagent and react for 5 min,

[0220] Place the reagent card into an immunofluorescence quantitative analyzer (Q20, Zhongyuanhuiji Biotechnology Co., Ltd.) to read the fluorescence signal T2 of the first internal reference before sample addition. Take 40 μL of the above-mentioned reaction solution and add it to the sample addition hole of the reagent card. Incubate for 5 min, and then read the fluorescence signal T3 of the first internal reference on the detection line and the fluorescence signal T1 of the detection result, respectively.

[0221] 3. Result calculation

[0222] The formula for calculating the effective coating amount of the first internal reference detected by the above detection method is:

[0223] The first internal reference effective coating rate η = T3 / T2

[0224] T2 - The first internal reference fluorescence signal intensity on the detection line before sample addition;

[0225] T3 - The first internal reference fluorescence signal intensity on the detection line at 5 min after sample addition.

[0226] The test reagent prepared in this embodiment was used to detect low, medium and high value samples, and the signal values were obtained according to the detection method and the results were calculated. The test results are shown in the following table:

[0227] Table 3 Effective coating rate of the first internal reference independent of the capture reaction before and after

[0228] The first internal reference auxiliary quantitative calibration formula of the above cTnI detection method is as follows:

[0229] Signal calibration value T5 = T1 / η

[0230] T1 - The fluorescence signal intensity of the detection result;

[0231] η - Effective coating rate.

[0232] Table 4 Comparison of auxiliary quantitative detection before and after the first internal reference independent of the capture reaction

[0233] As can be seen from the comparison of the test data in Table 3 and Table 1, the independent and non-independent first internal reference coating binding rates are different, and the effective coating rate of the independent first internal reference is slightly higher than that of the non-independent first internal reference. At the same time, the effective coating rate is not affected by the concentration of the sample. As can be seen from the data in Table 4, the detection results of different concentrations of samples can be calibrated by the effective coating rate independent of the capture, and the detection precision can be improved by 0.7%-1.8%, reaching within 3%.

[0234] Example 3

[0235] 1. Preparation of test strip

[0236] (1) Preparation of the first reagent

[0237] ① Coupling of the first aptamer and the tracer

[0238] Add cTnI antibody 1 (Hytest, 4T21-19C7cc) to fluorescent microspheres at a ratio of 60 μg cTnI monoclonal antibody / 300 μg fluorescent microspheres, add 6 μg EDC, stir at room temperature for 120 min, add 5% BSA, block and stir for 60 min, centrifuge at 14000 r / min for 20 min, discard the supernatant, restore the volume of the precipitate with fluorescent antibody preservation solution (100 mM Tris, 1% Tween 20, 5 mg / ml bovine serum albumin, 2% sucrose), and store at 2-8℃.

[0239] ② Coupling between the second aptamer and the connector

[0240] Weigh 0.001g of biotin and dissolve it in 175μL of DMSO to prepare biotin working solution. Add 0.5mg of cTnI antibody 2 (Hytest catalog number 4T21-16A11cc) to 7μL of biotin working solution, mix immediately, rotate label at room temperature in the dark, and then remove free biotin by dialysis or column chromatography.

[0241] ③ Marking of the second internal reference

[0242] Fluorescent materials (ThermoFisher Alexa Fluor) TM Add 175 μL of DMSO to dissolve (488, catalog number A20000) to prepare a 10 mg / mL working solution. Add fluorescent material to BSA at a molar ratio of 1:1, rotate label at room temperature in the dark, and then desalt using a desalting column.

[0243] ④ Preparation of the first reagent

[0244] The first reagent was prepared by mixing equal volumes of the three solutions (20 mM Tris, 1% Tween 20, 5 mg / mL casein sodium, 5% trehalose) with the first aptamer of the tracer diluted at 0.34 mg / mL, the second aptamer of the conjugate diluted at 0.032 mg / mL, and the second internal control of BSA diluted at 0.01 mg / mL. The reagent was then stored at 2-8°C for later use.

[0245] (2) Preparation of chromatography support

[0246] ① Preparation of the detection line

[0247] The first internal reference is marked on the captured item:

[0248] Streptavidin was desalted using a desalting column, and the fluorescent substance (ThermoFisher Alexa Fluor) was used. TM647 Stock No. A20006) was added 175 μL DMSO to make 10 mg / mL working solution, and the fluorescent substance was added to the streptavidin at a molar ratio of 1:1, and then the mixture was labeled at room temperature in the dark, and then desalted by a desalting column.

[0249] The first internal reference labeled streptavidin was diluted with PBS buffer to 0.2 mg / mL, and a capture detection line was drawn at a distance of 5-15 mm from the left end of the nitrocellulose membrane.

[0250] ② Assembly of test strips

[0251] The backing, sample pad and absorbent pad were general materials in the art, and the sample pad, nitrocellulose membrane and absorbent pad were sequentially and closely lapped on the backing to assemble a general chromatography carrier. The assembled general chromatography carrier was cut into a predetermined width by a cutting machine and loaded into a card shell.

[0252] 2. Detection method

[0253] The above-prepared full liquid-phase detection reagent containing the first internal reference was used to detect troponin I in a blood sample, and the specific process was as follows: 50 μL of sample was added to 150 μL of sample diluent (30 mM Tris, 5% Tween 20, 0.5% Proclin 300) and mixed thoroughly, and then the diluted sample was mixed with the first reagent for 5 min, while the reagent card was placed into an immunofluorescence quantitative analyzer (Q20, Zhongyuanhuiji Biotechnology Co., Ltd.), the fluorescence signal of the first internal reference on the detection line before sample addition was read as T2, and then 40 μL of sample was added to the sample addition hole of the reagent card, and the fluorescence signal T3 of the first internal reference on the detection line was obtained when incubated for 3 min, and the fluorescence signals t 1min and t 3min of the second internal reference on the detection line were obtained when incubated for 1 min and 3 min, respectively, and the detection result fluorescence signal T1 was obtained when incubated for 4 min.

[0254] 3. Result calculation

[0255] The calculation formula of the effective coating rate of the first internal reference detected by the above detection method was as follows:

[0256] The effective coating rate of the first internal reference η = T3 / T2

[0257] T2- fluorescence signal intensity of the first internal reference on the detection line before sample addition;

[0258] T3- fluorescence signal intensity of the first internal reference on the detection line after 3 min of sample addition.

[0259] The dynamic migration rate of the second internal reference v = t 1min / t 3min

[0260] t1min - the second internal reference baseline fluorescence signal value at 1 min after sample addition;

[0261] t 3min - the second internal reference baseline fluorescence signal value at 3 min after sample addition;

[0262] wherein the second internal reference dynamic mobility refers to the ability of the second internal reference to migrate between the stationary phase and the mobile phase during the chromatography process. In chromatography technology, dynamic mobility is an important parameter describing the moving rate of the second internal reference in the chromatography column.

[0263] t 1min and t 3min The baseline fluorescence signal is the average fluorescence signal of 50-100 points among the 500 signal collection points of the chromatography detection.

[0264] The test reagent prepared in this embodiment is used to detect low, medium and high value samples, and each signal value is obtained according to the detection method and the result is calculated. The test results are shown in the following table:

[0265] Table 5: Dynamic mobility of the second internal reference in the chromatography process

[0266] The first internal reference and the second internal reference compound auxiliary quantitative calibration formula of the above cTnI detection method is as follows:

[0267] Signal calibration value T5 = T1 / (η*ν)

[0268] T1- fluorescence signal intensity of the detection result;

[0269] η- effective coating rate of the first internal reference;

[0270] ν- dynamic mobility of the second internal reference.

[0271] Table 6: Comparison before and after the first internal reference and the second internal reference compound correction factor auxiliary quantification

[0272] As can be seen from the comparison of the test data in Table 2, Table 4 and Table 6, the first internal reference and the second internal reference compound calibration factor auxiliary quantification effect is significantly better than the first internal reference single factor calibration. By using the multi-factor compound correction described in this embodiment, the detection precision can be improved by 1%-2.3%, to within 2%.

[0273] Example 4

[0274] The test strip of this embodiment is prepared in accordance with Example 1, wherein the conjugate of the first aptamer and the tracer serves as both the detection signal and the second internal reference signal. The detection method and result calculation are consistent with Example 3.

[0275] Using the detection reagent prepared in this embodiment, low-value, medium-value, and high-value samples were selected for detection. The signal values ​​were obtained and calculated according to the detection method. The experimental results are shown in the table below:

[0276] Table 7 Comparison of Quantitative Analysis Before and After Using the Composite Correction Factor of the First and Second Internal References

[0277] A comparison of the experimental data in Tables 2, 4, and 7 shows that the quantitative effect of using the composite calibration factor of the first and second internal references is better than that of the single-factor calibration of the first internal reference. A comparison of the experimental data in Tables 7 and 6 shows that the independent second internal reference and the tracer-integrated internal reference are equally effective. The precision of the detection results can be improved by 0.4%-1.6% using the tracer-integrated second internal reference correction method, reaching within 2%.

[0278] Example 5

[0279] (1) Preparation of the first reagent

[0280] ① Coupling between the first aptamer and the tracer

[0281] Add cTnI antibody 1 (Hytest, 4T21-19C7cc) to fluorescent microspheres at a ratio of 60 μg cTnI monoclonal antibody / 300 μg fluorescent microspheres, add 6 μg EDC, stir at room temperature for 120 min, add 5% BSA, block and stir for 60 min, centrifuge at 14000 r / min for 20 min, discard the supernatant, restore the volume of the precipitate with fluorescent antibody preservation solution (100 mM Tris, 1% Tween 20, 5 mg / ml bovine serum albumin, 2% sucrose), and store at 2-8℃.

[0282] ② Coupling between the second aptamer and the connector

[0283] Weigh 0.001g of biotin and dissolve it in 175μL of DMSO to prepare biotin working solution. Add 0.5mg of cTnI antibody 2 (Hytest catalog number 4T21-16A11cc) to 7μL of biotin working solution, mix immediately, rotate label at room temperature in the dark, and then remove free biotin by dialysis or column chromatography.

[0284] ③ Marking of the second internal reference

[0285] Fluorescent materials (ThermoFisher Alexa Fluor) TM 488 (Catalog No. A20000) was dissolved in 175 μL of DMSO to prepare a 10 mg / mL working solution. Fluorescent material was added to the second aptamer coupled with the linker at a molar ratio of 1:1. The solution was then rotated at room temperature in the dark and desalted.

[0286] ④ Preparation of the first reagent

[0287] The first reagent was prepared by diluting the first aptamer of the tracer at 0.34 mg / mL with the first reagent preservation solution (20 mM Tris, 1% Tween 20, 5 mg / mL casein sodium, 5% trehalose) and the second aptamer at 0.032 mg / mL with the second internal reference labeled linker. The three solutions were mixed in equal volumes to prepare the first reagent, which was then stored at 2-8°C for later use.

[0288] (2) Preparation of chromatography support

[0289] ①Preparation of the detection line

[0290] The first internal reference is marked on the captured item:

[0291] Streptavidin was desalted using a desalting column, and the fluorescent substance (ThermoFisher Alexa Fluor) was used. TM Add 175 μL of DMSO to dissolve (647, catalog number A20006) to make a working solution of 10 mg / mL. Add fluorescent material to streptavidin at a molar ratio of 1:1, spin-label at room temperature in the dark, and then desalt using a desalting column.

[0292] Dilute the first internal control labeled streptavidin to 0.2 mg / mL with PBS buffer and draw a capture detection line 5-15 mm from the left end of the nitrocellulose membrane.

[0293] ② Assembly of test strips

[0294] 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.

[0295] The detection method and result calculation in this embodiment are the same as in Embodiment 3.

[0296] Using the detection reagent prepared in this embodiment, low-value, medium-value, and high-value samples were selected for detection. The signal values ​​were obtained and calculated according to the detection method. The experimental results are shown in the table below:

[0297] Table 8 Comparison of the results of the composite correction factor for the first internal reference and the second internal reference marked on the connector.

[0298] From the comparison of the test data in Table 2, Table 4 and Table 8, it can be seen that the first internal reference and the second internal reference labeled on the linker have no significant effect on the auxiliary quantitative detection results. From the comparison of Table 8 with Table 7 and Table 6, it can be seen that the correction effect of the second internal reference labeled on the linker is worse than the first two. The correction result of the second internal reference labeled on the linker has no significant improvement, but the precision can still be controlled within 5%.

[0299] Example 6

[0300] 1. Preparation of test strip

[0301] (1) Preparation of first reagent

[0302] ① Coupling of first aptamer and tracer

[0303] According to the amount of 60 μg cTnI monoclonal antibody / 300 μg fluorescent microspheres, cTnI antibody 1 (Hytest, 4T21-19C7cc) is added to the fluorescent microspheres, 6 μg EDC is added, and the reaction is stirred at room temperature for 120 minutes. 5% BSA is added, blocked and stirred for 60 min, centrifuged 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), and stored at 2-8°C.

[0304] ② Coupling of second aptamer and linker

[0305] 0.001 g of biotin is weighed and dissolved in 175 μL DMSO to form a biotin working solution. 0.5 mg of cTnI antibody 2 (Hytest, 4T21-16A11cc) is added to 7 μl of the biotin working solution, immediately mixed, and labeled at room temperature in the dark. Free biotin is removed by dialysis or column.

[0306] ③ Preparation of first reagent

[0307] The first reagent is prepared by diluting 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), mixing the two solutions in equal amounts, and storing at 2-8°C for standby.

[0308] (2) Preparation of chromatographic carrier

[0309] ① Preparation of detection line

[0310] The streptavidin is diluted with PBS buffer to 0.2 mg / mL, and a capture detection line is drawn on the left end of the nitrocellulose membrane at a distance of 5-15 mm.

[0311] ② Assembly of test strip

[0312] The bottom liner, 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 bottom liner to assemble a common chromatography carrier. The assembled common chromatography carrier is cut into a predetermined width by a cutting machine and loaded into a card case.

[0313] 2. Detection method

[0314] The prepared full liquid phase detection reagent containing the first internal reference is used for detecting troponin I in a blood sample. Specifically, 50 μL of sample is added to 150 μL of sample diluent (30 mM Tris, 5% Tween 20, 0.5% Proclin 300) and mixed thoroughly. Then, the diluted sample is mixed with the first reagent for 5 min. 40 μL of sample is added to the reagent card sample hole, incubated for 3 min, and the fluorescence signal intensity T1 of the detection result is read.

[0315] (3) Result calculation

[0316] The detection reagent prepared in this example is used to detect low, medium and high value samples. The T1 signal value is obtained according to the detection method, and the result is calculated. The test results are as follows:

[0317] Table 9 Full liquid phase detection results without internal reference

[0318] From the test data in Table 9, it can be seen that the precision of the full liquid phase reagent without internal reference is <5%. From the comparison of the test data in Table 9 and Tables 1-8, it can be seen that the precision of the full liquid phase reagent without internal reference is significantly poorer than that of the full liquid phase reagent containing the first internal reference and / or the second internal reference (independent or tracer labeled), and is comparable to that of the full liquid phase reagent with the second internal reference labeled on the linker.

[0319] Example 7

[0320] The preparation method of the reagent in this example is the same as that in Example 1.

[0321] 2. Detection method

[0322] The prepared full liquid phase detection reagent containing the first internal reference is used for detecting troponin I in a blood sample. Specifically, 50 μL of sample is added to 150 μL of sample diluent (30 mM Tris, 5% Tween 20, 0.5% Proclin 300) and mixed thoroughly. Then, the diluted sample is mixed with the first reagent for 5 min. 40 μL of sample is added to the reagent card sample hole, incubated for 3 min, and the fluorescence signal intensity T1 of the detection result is read.

[0323] 3. Result calculation

[0324] The first internal reference assisted quantitative calibration formula of cTnl detection method is as follows:

[0325] Signal calibration value T5 = T1 / T3

[0326] T1, detection result fluorescence intensity T3;

[0327] Fluorescence signal after the first internal reference reaction.

[0328] The detection reagent prepared in this embodiment was used to detect low, medium and high value samples, and each signal value was obtained according to the detection method and the result was calculated. The test results are shown in the following table:

[0329] Table 10: First internal reference reaction fluorescence signal assisted quantitative results

[0330] From the test data in Table 10, it can be seen that the fluorescence signal after the first internal reference reaction is not significant in the whole liquid phase reagent assisted quantitative effect. From the comparison of the test data in Table 10 and Table 2, it can be seen that the effective coating rate based on the whole liquid phase reagent is significantly better than the assisted quantitative effect of the fluorescence signal after the reaction.

[0331] Example 8

[0332] This embodiment detects the performance of the reagents corresponding to Examples 1-7, and the test results are as follows:

[0333] (1) Precision

[0334] Test the serum samples in the linear range of 40-40000 pg / mL, 10 times for each repetition, calculate the precision CV, and the test results are shown in the following table:

[0335] Table 11: Precision comparison under different measurement modes

[0336] (2) Linearity

[0337] Select high and low value samples according to the dilution ratio (L:H) for preparation, use the reagent prepared according to the preparation scheme of this embodiment for detection, 3 times for each repetition, and the test results are shown in the following table:

[0338] Table 12: Linearity comparison under different measurement modes

[0339] From the test data of Tables 11 and 12, the precision results of Examples 1-7 in different detection modes of full liquid phase are as follows: Example 7 ≈ Example 6 ≈ Example 5 < Example 1 ≈ Example 2 < Example 3 ≈ Example 4; the composite correction factor is more optimal for precision correction. The linear results of Examples 1-7 in different detection modes of full liquid phase are equivalent, and all can achieve the standard detection mode linear r > 0.990.

[0340] Example 9

[0341] This example verifies the influence of different acquisition times of the signal T3 generated by the first internal reference on the detection results. Six groups of reagents are set, and the preparation method of each group of reagents is consistent with that of Example 3. The difference between the detection method and the calculation of the record in Example 3 is only that the acquisition time of the signal T3 generated by the first internal reference is not the same. The specific information is shown in the following table:

[0342] Table 13 Test grouping setting

[0343] *1 min represents that the signal intensity T3 of the first internal reference generated on the detection line is acquired when the reagent card is incubated for 1 min after the sample is added.

[0344] The above-mentioned six groups of reagents are used to select low, medium and high value samples for detection. The signal values are acquired according to the respective detection methods, and the result calculation is shown in the following table:

[0345] Table 14

[0346] According to the test data in Table 14, when the sample addition is completed for more than 15 min, the fluorescence signal T3 of the first internal reference generated on the detection line greatly decreases the auxiliary correction effect on the detection results.

[0347] Example 10

[0348] 1. Preparation of test strip

[0349] (1) Preparation of the first reagent

[0350] ① Coupling of the first aptamer and the tracer

[0351] According to the amount of 60 μg CK-MB monoclonal antibody / 300 μg fluorescent microspheres, CK-MB antibody 1 (Feifeng, CK-MB-03) is added to the fluorescent microspheres, 6 μg EDC is added, and the reaction is stirred at room temperature for 120 min. 5% BSA is added, and the blocking is stirred for 60 min. Centrifugation is performed at 14000 r / min for 20 min, the supernatant is discarded, the sediment is restored in volume with the fluorescent antibody storage solution (100 mM Tris, 1% Tween 20, 5 mg / ml bovine serum albumin, 2% sucrose), and the storage is performed at 2-8°C.

[0352] 2. Coupling of the second adaptor with the linker

[0353] 0.001 g of biotin was weighed and dissolved in 175 μL of DMSO to obtain a biotin working solution. 0.5 mg of CK-MB antibody 2 (Feifeng, CK-MB-04) was added to 7 μL of the biotin working solution, and immediately mixed, and then labeled at room temperature in the dark, and then dialyzed or columned to remove free biotin.

[0354] 3. Preparation of the first reagent

[0355] 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 adaptor coupled with the tracer diluted to 0.34 mg / mL, and the second adaptor coupled with the linker diluted to 0.032 mg / mL, and then stored at 2-8°C for standby.

[0356] (2) Preparation of the chromatographic carrier

[0357] 1. Preparation of the detection line

[0358] The first internal reference was labeled on the capture:

[0359] The streptavidin was desalted using a desalting column, and the fluorescent substance (ThermoFisher Alexa Fluor 647, item number A20006) was dissolved in 175 μL of DMSO to obtain a 10 mg / mL working solution. The fluorescent substance was added to the streptavidin at a molar ratio of 1:1, and then labeled at room temperature in the dark, and then desalted using a desalting column. TM 647, item number A20006) was dissolved in 175 μL of DMSO to obtain a 10 mg / mL working solution. The fluorescent substance was added to the streptavidin at a molar ratio of 1:1, and then labeled at room temperature in the dark, and then desalted using a desalting column.

[0360] The first internal reference labeled streptavidin was diluted with PBS buffer to 0.2 mg / mL, and a capture detection line was drawn at a distance of 5-15 mm from the left end of the nitrocellulose membrane.

[0361] 2. Assembly of the test strip

[0362] The bottom liner, sample pad, and water absorption pad were general materials in the art. The sample pad, nitrocellulose membrane, and water absorption pad were sequentially and tightly lapped on the bottom liner to assemble a general chromatographic carrier. The assembled general chromatographic carrier was cut into a predetermined width using a cutting machine, and then loaded into a card shell.

[0363] 2. Detection method

[0364] The whole liquid phase detection reagent containing the first internal reference and the second internal reference prepared above is used for detecting creatine kinase isoenzyme in a blood sample, and the specific process is as follows: 20 μL of sample is added to 180 μL of sample diluent (30 mM Tris, 5% Tween 20, 0.5% Proclin 300) and mixed thoroughly, then the diluted sample is mixed with the first reagent for 5 min, the reagent card is placed into an immunofluorescence quantitative analyzer (Q20, Zhongyuanhuijibio), the fluorescence signal of the first internal reference on the detection line before sample addition is read as T2, then 40 μL of sample is added to the sample addition hole of the reagent card, and the baseline signal values t 1min , t 3min of the second internal reference on the detection line after incubation for 1 min and 3 min are obtained respectively, and the fluorescence signal T3 of the first internal reference after reaction and the fluorescence intensity T1 of the detection result are read simultaneously after incubation for 3 min.

[0365] The result calculation method of the reagent of this example is consistent with that of Example 3. The detection reagent prepared in this example is used to detect low, medium and high value samples, and the signal values are obtained according to the detection method and the results are calculated, and the test results are shown in the following table:

[0366] Table 15 Comparison of first internal reference and second internal reference before and after auxiliary quantitative

[0367] From the test data in Table 15, it can be seen that the first internal reference and the second internal reference auxiliary quantitative detection method is also applicable to other projects.

[0368] Example 11

[0369] 1. Preparation of test strip

[0370] Preparation of fluorescent microsphere labeled cTnI antibody 1: cTnI antibody 1 is added to fluorescent microspheres at an amount of 60 μg NT-proBNP monoclonal antibody / 300 μg fluorescent microspheres, 6 μg EDC is added, and the reaction is stirred at room temperature for 120 minutes, 5% BSA is added, and the stirring is blocked for 60 minutes, centrifuged at 14000 r / min for 20 minutes, the supernatant is discarded, the precipitate is restored to volume with fluorescent antibody storage solution, and stored at 4°C.

[0371] (1) Preparation of chromatography carrier:

[0372] Preparation of detection line:

[0373] The first internal reference is labeled on the capture:

[0374] cTnI antibody 2 is desalted by desalting column, and fluorescent substance (ThermoFisher Alexa Fluor TM647 Stock No. A20006) was added 175 μL DMSO to make 10 mg / mL working solution, and the fluorescent substance was added to the cTnl antibody 2 at a molar ratio of 1:1, and then the mixture was labeled at room temperature in the dark, and desalted by a desalting column.

[0375] The first internal reference labeled cTnl antibody 2 was diluted to 1.0 mg / mL with PBS buffer, and a capture detection line was drawn on the detection line position of the nitrocellulose membrane.

[0376] The fluorescent microsphere labeled cTnl antibody 1 was sprayed on the glass fiber of the conjugate pad, and then dried.

[0377] The sample pad, the conjugate pad, the nitrocellulose membrane and the absorbent pad were sequentially and closely overlapped on the backing, and the assembled intermediate was cut into a predetermined width by a cutting machine, and then loaded into a card shell to prepare a reagent card.

[0378] 2. Detection method

[0379] The dry detection reagent containing the first internal reference and the second internal reference prepared above was used to detect troponin I in a blood sample, and the specific process was as follows: 60 μL of the sample was added to 120 μL of sample diluent (30 mM Tris, 5% Tween 20, 0.5% Proclin 300) and mixed well, and then the reagent card of the first internal reference before sample addition was placed into an immunofluorescence quantitative analyzer (Q20, Zhongyuanhui Biological Technology Co., Ltd.) to read the fluorescence signal after the first internal reference reaction as T2 ， Then 70 μL of the sample was added to the sample addition hole of the reagent card, and the second internal reference baseline signal values t 1min , t 3min were obtained after 1 min and 3 min of incubation, respectively, and the fluorescence signal T3 after the first internal reference reaction and the detection result fluorescence intensity T1 were read at the same time after 3 min of incubation.

[0380] The result calculation method of the reagent of this example was consistent with that of Example 3.

[0381] The detection reagent prepared in this example was used to detect low, medium and high value samples, and the signal values were obtained according to the detection method and the results were calculated, and the test results were shown in the following table:

[0382] Table 16 Comparison of first internal reference and second internal reference before and after auxiliary quantitative

[0383] As shown by the test data in Table 16, the first internal reference and the second internal reference auxiliary quantitative detection method was also applicable to the traditional dry chromatography detection reagent.

[0384] Example 12

[0385] 1. Preparation of test strip

[0386] Preparation of fluorescent microsphere-labeled cTnI antibody 1: cTnI antibody 1 was added to fluorescent microspheres in 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, and then centrifuged at 14000 r / min for 20 minutes, the supernatant was discarded, the precipitate was restored to volume with fluorescent antibody storage solution, and stored at 4°C.

[0387] (2) Preparation of chromatography carrier:

[0388] Preparation of detection line:

[0389] First internal standard labeled on capture:

[0390] cTnI antibody 2 was desalted with a desalting column, and fluorescent substance (ThermoFisher Alexa Fluor 647, item number A20006) was dissolved in 175 μL DMSO to form a 10 mg / mL working solution, and the fluorescent substance was added to cTnI antibody 2 at a molar ratio of 1:1, and then the rotation labeling was carried out at room temperature in the dark, and then the desalting column was used for desalting. TM 647, item number A20006) was dissolved in 175 μL DMSO to form a 10 mg / mL working solution, and the fluorescent substance was added to cTnI antibody 2 at a molar ratio of 1:1, and then the rotation labeling was carried out at room temperature in the dark, and then the desalting column was used for desalting.

[0391] The first internal standard-labeled cTnI antibody 2 was diluted with PBS buffer to 1.0 mg / mL, and the capture detection line was drawn on the nitrocellulose membrane detection line position.

[0392] A blood filtering membrane with red blood cell filtering function was used as a sample pad, and the sample pad, nitrocellulose membrane and water absorption pad were sequentially and closely lapped on the bottom lining, the assembled intermediate was cut into a predetermined width with a cutting machine, and then loaded into a card shell to form a reagent card.

[0393] The fluorescent microsphere-labeled cTnI antibody 1 was spotted on the reaction pool and dried.

[0394] 2. Detection method

[0395] The above-prepared dry detection reagent containing the first internal standard and the second internal standard was used to detect troponin I in a blood sample, and the specific process was as follows: 40 μL of sample was added to 80 μL of sample diluent (30 mM Tris, 5% Tween 20, 0.5% Proclin 300) and mixed well, then the reagent card of the first internal standard before sample addition was first placed into an immunofluorescence quantitative analyzer (Q20, Zhongyuanhuiji Biotechnology Co., Ltd.) to read the fluorescence signal of the first internal standard after reaction as T2 ， Then 120 μL of the diluted sample was mixed well with fluorescent microspheres in the reaction pool and added to the sample addition hole of the reagent card, and the second internal standard baseline signal value t 1min , t 3minand the first internal reference reaction fluorescence signal T3 and the detection result fluorescence intensity T1 are read simultaneously after 3 minutes of incubation.

[0396] The result calculation method of the reagent of this example is consistent with that of Example 3.

[0397] The test reagent prepared in this example is used to detect low, medium and high value samples, and the signal values are obtained according to the detection method and the results are calculated. The test results are shown in the following table:

[0398] Table 17 Comparison of first internal reference and second internal reference compound auxiliary quantitative before and after

[0399] From the test data in Table 17, it can be seen that the first internal reference and the second internal reference compound auxiliary quantitative detection method is also suitable for semi-liquid phase chromatographic test reagent.

[0400] Although the embodiments of the present application have been shown and described above, it should be understood that the above-mentioned embodiments are exemplary and should not be construed as limiting the present application. Those skilled in the art can make changes, modifications, replacements and deformations to the above-mentioned embodiments without departing from the principles and purposes of the present application within the scope of the present application.

Claims

1. A chromatographic reaction reagent having an internal reference marker-assisted quantification, characterized in that, The chromatography carrier comprises a detection zone coated with a capture and a first internal reference; The capture is used to directly and / or indirectly capture a tracer which can generate a signal T1 for indicating the presence and / or content of the analyte and generating a signal calibration value T5; The first internal reference is used to indicate the coating state of the capture in the detection zone and generate a signal T2, and to indicate the effective coating state of the capture in the detection zone and generate a signal T3; The second internal reference is used to indicate the chromatography reaction state and generate a signal T4; The signals T2, T3 and T4 are used to assist in correcting the signal calibration value T5.

2. A chromatographic reaction reagent having internal reference marker assisted quantification, characterized in that, The chromatography carrier comprises a detection zone coated with a capture and a first internal reference; The capture is used to directly and / or indirectly capture a tracer which can generate a signal T1 for indicating the presence and / or content of the analyte and generating a signal calibration value T5; The first internal reference is used to indicate the coating state of the capture in the detection zone and generate a signal T2, and to indicate the effective coating state of the capture in the detection zone and generate a signal T3; The signals T2 and T3 are used to assist in correcting the signal calibration value T5.

3. A chromatographic reaction reagent having internal reference marker assisted quantification, characterized in that, The chromatography carrier comprises a detection zone coated with a capture and a first internal reference; The capture is used to directly and / or indirectly capture a tracer which can generate a signal T1 for indicating the presence and / or content of the analyte and generating a signal calibration value T5; The second internal reference is used to indicate the chromatography reaction state and generate a signal T4; The signal T4 is used to assist in correcting the signal calibration value T5.

4. The agent according to claim 1 or 2, characterized in that, The signal T2 is a signal generated by the first internal reference in the detection zone before the chromatography reaction proceeds to the detection zone; Preferably, the signal T2 is information loaded into the reagent data before the reagent is shipped.

5. The agent according to any one of claims 1, 2, 4, characterized in that, The signal T3 is a signal generated by the first internal reference in the detection zone after the detection is completed; Preferably, the signal T3 can be a signal generated by the first internal reference in the detection zone at a specific time after the sample is added; More preferably, the specific time is less than 15 minutes after the sample is added.

6. The agent according to any one of claims 1, 2, 4, 5, characterized in that, The capture and the first internal reference are independently coated on the chromatography carrier; Preferably, the capture and the first internal reference are similar substances in nature; More preferably, the capture and the first internal reference have similar molecular weights and / or isoelectric points, and the first internal reference can generate a detectable signal; Most preferably, the capture and the first internal reference are the same substance.

7. The agent according to any one of claims 1, 2, 4, 5, characterized in that, The first internal reference is directly and / or indirectly combined with the capture.

8. The agent of any one of claims 1, 3-7, wherein, The signal T4 can be a signal generated by the second internal reference in a specific area or at a specific time; Preferably, the signal T4 can indicate the chromatography reaction rate and / or chromatography reaction abnormalities.

9. The agent according to any one of claims 1 to 8, characterized in that, The first reagent comprises a first aptamer coupled to a tracer and a second aptamer coupled to a linker; the capture can specifically recognize and / or bind to the linker; the first aptamer and the second aptamer specifically recognize and / or bind to the same analyte; or, one of the first aptamer or the second aptamer competes with the analyte for recognition and / or binding to the other aptamer.

10. The agent of claim 9, wherein The linker is selected from at least one of biotin, FITC or DNP, and the capture is correspondingly 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 correspondingly selected from at least one of biotin, FITC or DNP.

11. The agent of claim 10, wherein The first and / or second aptamer is selected from at least one of an antibody, an antigen-binding fragment, an aptamer, a modified aptamer, an aptide, an affimer, an antigen, a protein, a polypeptide, a multi-protein complex, an exosome, a microbial particle / piece / fragment, an oligonucleotide or a low-molecular-weight compound.

12. The agent of any one of claims 1-11, wherein, The capture and / or the first internal reference is coated on the detection zone in the form of a detection line; Preferably, 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 12, wherein Further comprising at least one of a sample pad, an absorbent pad, a backing or a card shell; Preferably, the sample pad and / or the absorbent pad is attached above the chromatography carrier; Preferably, the backing is attached below the chromatography carrier; Preferably, the card shell is used to load the chromatography carrier.

15. The agent according to claim 13 or 14, characterized in that, The second internal reference is present in the first reagent, the sample loading zone or the sample pad.

16. The agent of any one of claims 1-8, wherein, Further comprising a linker coupled to a tracer, the linker and the capture specifically recognize and / or bind to the same analyte; or, the linker competes with the analyte for recognition and / or binding to the capture; or the capture competes with the analyte for recognition and / or binding to the linker.

17. The agent of claim 16, wherein The capture and / or the linker can be at least one of an antibody, an antigen-binding fragment, an aptamer, a modified aptamer, an aptide, an affimer, an antigen, a protein, a polypeptide, a multi-protein complex, an exosome, a microbial particle / piece / fragment, an oligonucleotide or a low-molecular-weight compound.

18. The agent of any one of claims 16, wherein Further comprising at least one of a sample pad, a binding pad, an absorbent pad, a backing or a card shell; Preferably, the sample pad, the binding pad and / or the absorbent pad is attached above the chromatography carrier; Preferably, the backing is attached below the chromatography carrier; Preferably, the card shell is used to load the chromatography carrier.

19. The agent of claim 18, wherein The linker coupled to a tracer and / or the second internal reference can be independent of the chromatography carrier, or coated on the chromatography carrier; Preferably, the linker coupled to a tracer is coated on the binding pad; Preferably, the second internal reference is coated on the sample pad or the binding pad.

20. The agent of claim 16, wherein Further comprising a second reagent, the linker coupled to a tracer and / or the second internal reference is present in the second reagent.

21. The agent of any one of claims 1-20, wherein, The tracer, the first internal reference and / or the second internal reference is selected from at least one of a fluorescent signaling substance, an enzyme catalytic substance, an electromagnetic signaling substance or a colored substance; Preferably, the first internal reference, the second internal reference and / or the tracer can be the same substance, or different substances; Preferably, the signals generated by the first internal reference, the second internal reference and / or the tracer can be distinguished from each other.

22. The agent of claim 21, wherein The second internal reference can be coupled to the linker, or can be integrally present with the tracer.

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