Chromatographic reaction reagent with internal reference mark assisted quantification

Abstract

According to the chromatographic reaction reagent and the detection method provided by the invention, the effective coating state of the NC membrane capture substance is represented by coating the detection region with the first internal reference, and meanwhile, the chromatographic process is dynamically monitored through the second internal reference; the first internal reference and / or the second internal reference are / is introduced to carry out auxiliary correction on a detection result, so that high-precision detection of an analyte is realized.

Description

Technical Field

[0001] This invention relates to the field of medical testing, specifically to a chromatographic reaction reagent with internal reference labeling for quantification. Background Technology

[0002] With the continuous advancement of biomedical technology, the requirements for the detection accuracy of disease biomarkers are becoming increasingly stringent. Chromatographic techniques, as a rapid, simple, and cost-effective detection method, have been widely used in clinical diagnosis and biomedical research. Traditional chromatographic detection methods mainly rely on the binding of target molecules to specific antibodies or antigens, with results determined visually or using simple instruments. However, these methods typically only provide qualitative or semi-quantitative results and are easily affected by operating conditions and sample variations, limiting their application in precision medicine.

[0003] To improve the precision and accuracy of immunochromatographic detection, researchers have developed various chromatography-assisted quantitative detection techniques. Among them, Zheng Jingxian et al. proposed a method and immunoassay strip (WO 2024 / 088374) for detecting analytes in samples. This method uses the T' signal at the same position to correct the T signal, obtaining a corrected T signal, thus improving the precision of immunochromatographic detection from 15% to less than 10%, achieving a technological breakthrough. However, it failed to further improve the detection of the effective coating amount of the membrane and the dynamic state of the chromatographic reaction, thus failing to achieve truly accurate quantification.

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

[0005] To address the methodological shortcomings of immunochromatography, this application provides a chromatographic reaction reagent and detection method with internal reference labeling for assisted quantification.

[0006] In a first aspect, the present invention provides a chromatographic reaction reagent with internal reference labeling-assisted quantification, comprising a chromatographic carrier and a second internal reference;

[0007] The chromatography carrier includes a detection zone, on which the capture material and a first internal control are coated;

[0008] The capture material is used to directly and / or indirectly capture tracers. The tracer can generate a signal T1 to indicate the presence and / or content of the analyte and generate a signal calibration value T5.

[0009] The first internal parameter is used to indicate the amount of coating of the captured object in the detection zone and generate signal T2, and to know the effective amount of coating of the captured object in the detection zone and generate signal T3.

[0010] The second internal reference is used to indicate the state of the chromatography reaction and generate signal T4;

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

[0012] In a second aspect, the present invention provides a chromatographic reaction reagent with internal reference labeling-assisted quantification, comprising a chromatographic carrier, the chromatographic carrier including a detection region, the detection region being coated with a capture substance and a first internal reference;

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

[0014] The first internal parameter is used to indicate the coating state of the captured object in the detection area and generate signal T2, and to indicate the effective coating state of the captured object in the detection area and generate signal T3.

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

[0016] Thirdly, the present invention provides a chromatographic reaction reagent with internal reference labeling-assisted quantification, including a chromatographic carrier and a second internal reference, wherein the chromatographic carrier includes a detection region and the detection region is coated with a capture substance;

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

[0018] The second internal reference is used to indicate the state of the chromatography reaction and generate signal T4;

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

[0020] In some specific implementations, signal T2 is the signal generated by the first internal control in the detection zone before the chromatographic reaction reaches the detection zone. Specifically, signal T2 can be detected by the manufacturer before the reagent leaves the factory and pre-embedded in the reagent's data information, such as in the reagent's QR code / barcode information, so that the relevant reagent information can be obtained by scanning the code during detection. Alternatively, it can be the signal generated by the second internal control in the detection zone at any time during the terminal detection process before the chromatographic reaction reaches the detection zone. It can be understood that signal T2 can directly or indirectly indicate the coating content and uniformity of the captured analyte in the detection zone.

[0021] In some specific implementations, signal T3 is the signal generated by the first internal reference in the detection zone after detection is completed, for example, after acquiring signal T4, or simultaneously with acquiring signal T4. Specifically, it can be the signal generated by the first internal reference in the detection zone acquired after sample loading is completed and a specific time has elapsed; more specifically, it can be the signal generated by the first internal reference in the detection zone acquired within 15 minutes after sample loading is completed. It should be understood that signal T3 can directly or indirectly indicate the effective coating status of the captures on the detection zone (including the effective coating content and the uniformity of the coating), where the effective coating content is the actual effective coating of the captures on the detection zone after excluding the captures lost on the detection zone during chromatography.

[0022] In some specific implementations, the capture and the first internal control can be independently coated on the chromatography carrier, that is, the capture and the first internal control are coated on the detection area of ​​the chromatography carrier in a mixed manner.

[0023] In some specific embodiments, the capture and the first internal control are substances with similar properties, such that the capture and the first internal control coated on the detection region have similar coating stability, chromatography rate, or bleed rate, for example, they may have similar molecular weights and / or isoelectric points, and the capture and the first internal control may be the same substance, and the first internal control may generate a detectable signal to indicate its real-time status during chromatography (including coating content and coating uniformity). A non-limiting example is that the capture is SA, and the first internal control is SA coupled with a fluorescent protein tag or SA co-expressed fluorescent tag.

[0024] In some specific implementations, the first internal reference can be directly and / or indirectly bound to the trap, for example, by covalent bonds or intermolecular forces.

[0025] The second internal reference is used to indicate the state of the chromatographic reaction and generate signal T4. Signal T4 can be used to assist in correcting the calibration value T5. Specifically, the state of the chromatographic reaction can include the dynamic form of the chromatographic reaction in real time / specific region / specific time, and the chromatographic rate of the entire reaction process / specific region / specific time. Specifically, the chromatographic rate (i.e., dynamic mobility) can be calculated 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 state of the chromatographic reaction can include all dynamic processes occurring on the chromatographic support, including the chromatographic process, physical / chemical / biological reactions occurring on the membrane, etc. Specifically, by setting a threshold, when the chromatographic rate is lower or higher than the threshold, a chromatographic abnormality is detected, indicating that the detection result is not feasible. Secondly, the dynamic form of the chromatographic reaction process can also directly indicate whether there is an abnormality in the reaction process. For example, when obvious non-uniformity / peak phenomenon appears at the front end of the chromatographic liquid, it can directly indicate that the chromatographic reaction is abnormal, and the signal T4 at this time can be used to indicate that the detection result is unreliable.

[0026] It should be noted that the chromatographic reaction reagent using internal reference labeling for assisted quantification described in this invention is also applicable to traditional dry chromatography reagents, semi-liquid chromatography reagents, and total liquid chromatography reagents.

[0027] When the aforementioned internal reference labeling-assisted quantification scheme is applied to total liquid chromatography reagents, it further includes a first reagent, wherein the first reagent includes a first aptamer of a coupled tracer and a second aptamer of a coupled linker, and the capture analyte 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 and / or second aptamers include at least one of antibodies, antigen-binding fragments, aptamers, modified aptamers, aptamers, affinity molecules, antigens, proteins, peptides, multi-protein complexes, exosomes, microbial particles / fragments / parts, oligonucleotides, or low molecular weight compounds.

[0029] More specifically, when the first aptamer and the second aptamer specifically recognize and / or bind to the same analyte, the first aptamer and the 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 sandwich-like multiplex complex of "tracer-first aptamer-analyte-second aptamer-connector". This complex is captured by the detection zone, and the content or presence of the analyte is calculated from the signal change generated by the tracer. For example, both the first and second aptamers are antibodies; they can be the same antibody or different antibodies. They can be different antibodies binding to the same epitope on the analyte, or they can be different antibodies binding to different epitopes on the analyte. When the first and second aptamers bind to the same epitope of the analyte, the analyte should have two or more repetitions of the same epitope. The first and second aptamers generally bind to different repetitions of the same epitope on the analyte. Furthermore, the signal generated by the tracer in the multiplex complex is proportional to the concentration of the analyte to a certain extent. By measuring the signal generated by the tracer in the detection zone, the presence of the analyte in the sample can be determined, or the concentration of the analyte can be calculated.

[0031] When one of the first aptamers or the second aptamer competes with the analyte to recognize and / or bind to the other aptamer, one of the first aptamers 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 multiple complex of "tracer-first aptamer-second aptamer-connector" during the competitive reaction with the analyte. The first aptamer and the analyte can be the same or different substances, as long as they can both specifically recognize and / or bind to the second aptamer. By competing with the first aptamer to bind to the second aptamer, and given the known amount of the coupled tracer-first aptamer in the system, the content or presence of the analyte can be calculated from the signal in the detection zone and changes in a preset signal value. Alternatively, the second aptamer and the analyte can be the same or different substances, as long as they can both specifically recognize and / or bind to the first aptamer. By competing with the second aptamer to bind to the first aptamer, and given the known amount of the coupled tracer-first aptamer in the system, the content or presence of the analyte can be calculated from the signal in the detection zone and changes in a preset signal value. Furthermore, by generating signals through tracers in multiple complexes, the signal intensity is inversely proportional to the concentration of the analyte to a certain extent. By measuring the signal generated by the tracers in the detection area, the presence of the analyte in the sample can be determined, or the concentration of the analyte can be calculated.

[0033] The capture and the linker are bound together with certain specificity and affinity. For example, you can choose "biotin-avidin, primary antibody-secondary antibody, antibody-protein, DNP (2,4-dinitrophenyl)-anti-DNP antibody, FITC (fluorescein isothiocyanate)-anti-FITC antibody", etc. Specifically, when the linker is biotin, the capture substance corresponds to avidin and / or streptavidin and / or anti-biotin antibodies; when the linker is avidin and / or streptavidin, the capture substance corresponds to biotin. When the linker is a primary antibody, the capture substance corresponds to a secondary antibody; when the linker is a secondary antibody, the capture substance corresponds to a primary antibody. When the linker is an antibody, the capture substance corresponds to a protein specifically recognized and / or bound by that antibody; when the linker is a protein, the capture substance corresponds to an antibody that can specifically recognize and / or bind to it. When the linker is DNP, the capture substance corresponds to anti-DNP antibodies; when the linker is anti-DNP, the capture substance corresponds to DNP. When the linker is FITC, the capture substance corresponds to anti-FITC antibodies; when the linker is anti-FITC antibodies, the capture substance corresponds to FITC.

[0034] In some specific implementations, the linker and trap connection system is a biotin-streptavidin linker system. It should be understood that one avidin molecule can bind four biotin molecules, and the binding is very stable. Furthermore, avidin and biotin can be conjugated to proteins, enzymes, luciferins, and other molecules without affecting their biological activity. One antibody molecule can conjugate multiple biotin molecules, and avidin can conjugate multiple biotin-antibody conjugates. This multi-stage amplification effect can further improve the sensitivity of the detection system.

[0035] In some specific implementation schemes, the chromatography carrier is also coated with control lines, 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 implementations, the chromatography carrier also includes a sample loading area. More specifically, the sample loading area is located at one end of the chromatography carrier and has a buffer zone with the detection zone. The purpose of setting the buffer zone is to allow the sample to pass through a certain distance of lateral chromatography buffer on the chromatography carrier, so that the subsequent chromatography can be carried out at a more uniform speed, and the chromatography reaction can be carried out at the desired rate when it reaches the detection zone, so as to obtain better binding efficiency.

[0037] In some specific implementations, at least one of a sample pad, an absorbent pad, a substrate, or a cassette is also included. More specifically, the sample pad is attached above the chromatography carrier to support the reaction complex and provide a buffering effect during the sample loading process. More specifically, the absorbent pad is attached above the chromatography carrier to utilize its absorbent properties to guide the solution through the chromatography process on the chromatography carrier. More specifically, the substrate is attached below the chromatography carrier to provide solid-phase support for the chromatography carrier. More specifically, the cassette is used to load the chromatography carrier and has a sample loading port and a detection window.

[0038] In some specific implementation schemes, the second internal control may be present in the first reagent or encapsulated in the sample loading area / sample pad of the chromatography carrier.

[0039] When the aforementioned internal reference labeling-assisted quantification scheme is applied to dry / semi-liquid chromatography reagents, it also includes a linker for the conjugated tracer, wherein the linker and the capture specifically recognize and / or bind to the same analyte; or, the linker competes with the analyte to recognize and / or bind to the capture, or the capture competes with the analyte to recognize and / or bind to the linker.

[0040] Non-limiting examples of linkers and / or traps include at least one of antibodies, antigen-binding fragments, aptamers, modified aptamers, aptamers, affinity molecules, antigens, proteins, peptides, multi-protein complexes, exosomes, microbial particles / fragments / discs, oligonucleotides, or low molecular weight compounds.

[0041] In some specific embodiments, at least one of a sample pad, a conjugate pad, an absorbent pad, a substrate, or a retainer is also included. Specifically, the sample pad, conjugate pad, and / or absorbent pad are attached above the chromatography carrier, the substrate is attached below the chromatography carrier, and the retainer is used to mount the chromatography carrier.

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

[0043] In some specific implementations, the conjugate of the analyte is coated on a conjugate pad located between the sample pad / sample loading area and the detection area. The chromatographic liquid first passes through the sample pad / sample loading area, then through the conjugate pad to complete the mixing between the analyte and the conjugate of the analyte, and finally is chromatographically deposited into the detection area.

[0044] In some specific implementations, the second internal reference is packaged on the sample pad or binding pad.

[0045] In some specific implementations, a second reagent is also included, in which the conjugate of the coupled tracer and / or the second internal control are present. That is, the sample first reacts with the second reagent to complete the mixing of the analyte with the conjugate of the coupled tracer / second internal control, and then it is added to the sample pad / sample application area.

[0046] The tracer, first internal control, and / or second internal control can be any detectable labeled substance that can generate a signal detectable by visual or instrumental means. Exemplary examples include at least one of the following: fluorescent signaling substances, enzyme-catalyzed substances, chemiluminescent signaling substances, electromagnetic signaling substances, or colored substances. Depending on the reaction principle, as the analyte complex containing the tracer flows on the chromatography support, the tracer may be captured and aggregated or not captured on the detection line. The presence or concentration of the analyte is determined based on the tracer's signal, such as a color signal, fluorescence signal, or electromagnetic signal.

[0047] In some specific implementation schemes, the fluorescent signaling substance may be at least one of fluorescent microspheres, quantum dots, fluorescent proteins, or luciferin molecules; the enzyme catalytic substance may be at least one of horseradish peroxidase, alkaline phosphatase, glucose-6-phosphate dehydrogenase; the chemiluminescent signaling substance may be at least one of acridinium ester, thioester or sulfonamide, luminol, isoluminol or phenanthrene ester; and the electromagnetic signaling substance may be at least one of magnetic particles or radioactive materials. More specifically, the radioactive material may be... 3 H, 125 I, 35 S, 14 C 32 P, or 33 At least one of P, etc.; the colored substance may be at least one of metal colloidal particles or colored particles, more specifically, the metal colloidal particles may be selected from at least one of colloidal gold or colloidal platinum.

[0048] In some specific implementation schemes, the first internal control, the second internal control, and / or the tracer can be the same substance or different substances, as long as the signals they produce can be distinguished from each other. It should be understood that they can be distinguished by using different signaling substances to produce different signals, or by different detection methods, such as detecting and obtaining signals at different reaction stages or time points.

[0049] In some specific implementations, the second internal reference may be coupled to the connector together with the tracer, or the second internal reference may exist as an integral part of the tracer, with the tracer being the second internal reference.

[0050] In some specific implementations, the second internal control may be present in the first reagent, the second reagent, or coated on the chromatographic carrier. Specifically, when the second internal control is present in the first reagent, it may be mixed with the first aptamer of the coupled tracer and the second aptamer of the coupled linker, or it may be coupled to the first aptamer and / or the second aptamer, or it may be integrally present with the tracer, where the tracer is the second internal control. Specifically, when the second internal control is present in the second reagent, it may be mixed with the linker of the coupled tracer, or it may be coupled together with the tracer to the linker, or it may be integrally present with the tracer, where the tracer and the second internal control are both present. Specifically, when the second internal control is coated on the chromatographic carrier, it should be coated before the detection zone, such as in the sample loading zone.

[0051] The term "specific recognition" or "specific binding" as used in this article can refer to the interaction between an antibody, protein, or peptide and a second chemical substance, wherein the interaction depends on the presence of a specific structure (e.g., an antigenic structure) or a determinant / epitope on the chemical substance, such as an antibody recognizing and binding to a specific antigenic structure, the mutual recognition and binding of a receptor and ligand, or the mutual recognition and binding of biotin and avidin.

[0052] The term "coupled" as used herein broadly refers to a stable bond between two substances resulting from any chemical, physical, or physicochemical interaction (e.g., covalent bond, hydrogen bond, electrostatic interaction, polar attraction, van der Waals attraction, hydrophobic interaction, or adsorption). Unless otherwise stated, this term is intended to encompass both: direct binding / conjugation between two substances, such as the direct binding / conjugation of an antibody to an antigen on a protein, and indirect binding / conjugation between two substances through one or more intermediate means, such as the association between an antibody and a polynucleotide by means of one or more oligonucleotides and / or labels. Therefore, when used with respect to two substances, "coupled" refers to two substances bound / conjugated by 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 analytes include antigens, antibodies, antigen-binding fragments, proteins, peptides, multi-protein complexes, hormones, exosomes, oligonucleotides, or low-molecular-weight compounds. The analyte can be detected in any sample of interest and is not particularly limited to biological samples, such as, but not limited to, bodily 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 applicable to dry chromatography, half-liquid chromatography, and total liquid chromatography. The following is a non-limiting example of one form of detection step:

[0055] Simultaneously, a first internal control and a second internal control are used to assist in the correction of the detection results. When applied to total liquid chromatography reactions, the following steps are included:

[0056] (1) A chromatographic reaction reagent card is provided, comprising a first reagent, a chromatographic carrier and a second internal control, wherein the first reagent comprises a first aptamer of a coupled tracer and a second aptamer of a coupled linker; the chromatographic carrier comprises a sample loading area and a detection line, wherein the detection line is coated with a capture and the first internal control; the first aptamer and the second aptamer specifically recognize and / or bind to the analyte;

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

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

[0059] (4) The aforementioned reaction solution is added to the sample application area. The complex I is captured to the detection area by the specific binding of the capture substance to the linker, generating signal T1 and generating signal calibration value T5.

[0060] (5) Obtain the signals T2 and T3 generated by the first internal reference in the detection zone before and after the chromatography reaction. Use signals T2, T3 and T4 to correct the signal calibration value T5 and output the detection result.

[0061] Or:

[0062] (1) A chromatographic reaction reagent card is provided, comprising a first reagent, a chromatographic carrier and a second internal control, wherein the first reagent comprises a first aptamer of a coupled tracer and a second aptamer of a coupled linker; the chromatographic carrier comprises a sample loading area and a detection line, wherein the detection line is coated with a trap and the first internal control; one of the first aptamer or the second aptamer competes with the analyte 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-analyte” or “linker-second aptamer-analyte” and a reaction complex III of “tracer-first aptamer-second aptamer-linker”;

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

[0065] (4) The aforementioned reaction solution is added to the sample application area. The complex III is captured to the detection area by the specific binding of the capture substance to the linker, generating signal T1 and generating signal calibration value T5.

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

[0067] It should be understood that the labels before the aforementioned reaction steps should not be interpreted as restrictions on the specific timing of operations. As long as the steps of the chromatographic reaction are followed, signals are acquired sequentially in each signal generation region or at a specific time, and the signal calibration value T5 is calibrated to obtain the final detection result, the results can be obtained.

[0068] The beneficial effects of this application are as follows: This invention provides a chromatographic reaction reagent and detection method with internal reference labeling for assisted quantification. By coating the detection zone with a first internal reference, the effective coating state of the NC membrane trap is characterized. Simultaneously, a second internal reference enables dynamic monitoring of the chromatographic process. By introducing the first and / or second internal references to assist in correcting the detection results, high-precision detection of the analyte is achieved, the difference between raw material rolls of the NC membrane is reduced, the complexity of production is lowered, and the requirements for the detection environment (such as temperature and humidity) are reduced. Furthermore, the immunochromatographic analysis method of this application is applicable to traditional dry immunochromatographic analysis, semi-liquid immunochromatographic analysis, and full-liquid immunochromatographic analysis, possessing a wide range of applicability scenarios and effectively improving the detection accuracy of various immunochromatographic modes. Specific Implementation

[0069] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

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

[0071] In this article, "multiple" means two or more, that is, it includes two, three, four, five, etc.

[0072] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0073] In this specification, certain embodiments may be disclosed in a range-bound format. It should be understood that this "range-bound" description is merely for convenience and brevity and should not be construed as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered as having specifically disclosed all possible subranges and independent numerical values ​​within those ranges. For example, range The description should be considered as having specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, such as 1, 2, 3, 4, 5, and 6. The above rules apply regardless of the breadth of the range.

[0074] Example 1

[0075] 1. Preparation of test strips

[0076] (1) Preparation of the first reagent

[0077] ① Coupling between the first aptamer and the tracer

[0078] Add cTnI antibody 1 (Hytest catalog number 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 minutes, add 5% BSA, block and stir for 60 minutes, centrifuge at 14000 r / min for 20 minutes, discard the supernatant, and restore the volume of the precipitate with fluorescent antibody preservation solution (100 mM Tris, 1% Tween 20, 5 mg / ml bovine serum albumin, 2% sucrose), and store at 2-8℃.

[0079] ② Coupling between the second aptamer and the connector

[0080] Weigh 0.001g of biotin and dissolve it in 175μL LDMSO 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.

[0081] ③ Preparation of the first reagent

[0082] The first reagent was prepared by mixing equal volumes of the two solutions (20 mM Tris, 1% Tween 20, 5 mg / mL sodium caseinate, 5% trehalose) with the first aptamer of the conjugate diluted at 0.34 mg / mL and the second aptamer of the conjugate diluted at 0.032 mg / mL, and stored at 2-8°C for later use.

[0083] (2) Preparation of chromatography support

[0084] ①Preparation of the detection line

[0085] The first internal reference is marked on the captured item:

[0086] Streptavidin was desalted using a desalting column, and the fluorescent substance (ThermoFisher Alexa Fluor) was used. TM Add 175 μL LDMSO to 647 (item number A20006) to dissolve into a 10 mg / mL working solution. 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.

[0087] 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.

[0088] ② Assembly of test strips

[0089] 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.

[0090] 2. Detection Method

[0091] The prepared liquid chromatography reagent containing the first internal control was used to detect troponin I in blood samples. The specific process is 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. Then, 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.

[0092] 3. Result Calculation

[0093] The formula for calculating the effective coating rate of the first internal reference detected by the above detection method is as follows:

[0094] The effective coverage rate of the first internal reference η = T3 / T2

[0095] T2 - Before sample addition (before reaction), the intensity of the first internal reference fluorescence signal on the detection line;

[0096] At T3, 3 minutes after sample addition (after reaction), the intensity of the first internal reference fluorescence signal on the detection line is measured.

[0097] 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:

[0098] Table 1 Effective coating rate before and after the first internal control reaction.

[0099]

[0100] The formula for the first internal reference auxiliary quantitative calibration of the above cTnI detection method is as follows:

[0101] Signal calibration value T5 = T1 / η

[0102] T1 - Fluorescence intensity of the detection result;

[0103] η - Effective coating rate.

[0104] Table 2 Comparison of auxiliary quantitative detection before and after the first internal control reaction.

[0105]

[0106]

[0107] As shown in Table 1, the effective coating rates of samples with different concentrations are comparable and are not affected by the fluorescence signal values ​​of the analyte. As shown in Table 2, the detection precision of samples with different concentrations can be improved by 0.2%-1.2% and reached within 4% by calibrating the results through the effective coating rate.

[0108] Example 2

[0109] 1. Preparation of test strips

[0110] (1) Preparation of the first reagent

[0111] ① Coupling between the first aptamer and the tracer

[0112] Add cTnI antibody 1 (Hytest catalog number 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 minutes, add 5% BSA, block and stir for 60 minutes, centrifuge at 14000 r / min for 20 minutes, discard the supernatant, and restore the volume of the precipitate with fluorescent antibody preservation solution (100 mM Tris, 1% Tween 20, 5 mg / ml bovine serum albumin, 2% sucrose), and store at 2-8℃.

[0113] ② Coupling between the second aptamer and the connector

[0114] Weigh 0.001g of biotin and dissolve it in 175μL LDMSO 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.

[0115] ③ Preparation of the first reagent

[0116] The first reagent was prepared by mixing equal volumes of the two solutions (20 mM Tris, 1% Tween 20, 5 mg / mL sodium caseinate, 5% trehalose) with the first aptamer of the conjugate diluted at 0.34 mg / mL and the second aptamer of the conjugate diluted at 0.032 mg / mL, and stored at 2-8°C for later use.

[0117] (2) Preparation of chromatography support

[0118] ①Preparation of the detection line

[0119] The first internal reference is independent of the catch:

[0120] Streptavidin-R-phycoerythrin (Agilent product number PJ32S-1) was diluted with PBS buffer to a final concentration of 0.05 mg / mL, and the final concentration of the capture streptavidin was adjusted to 0.2 mg / mL. The first internal agent was mixed with streptavidin and coated with a capture detection line drawn at a distance of 5-15 mm from the left end of the nitrocellulose membrane.

[0121] ② Assembly of test strips

[0122] 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.

[0123] 2. Detection Method

[0124] The prepared liquid chromatography reagent containing the first internal control was used to detect troponin I in blood samples. The specific process is 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. Then, 40 μL of the aforementioned reaction solution was added to the sample well of the reagent card. After incubation for 5 min, the fluorescence signal T3 of the first internal control and the fluorescence signal T1 of the detection result were read on the detection line.

[0125] 3. Result Calculation

[0126] The formula for calculating the effective coating amount of the first internal reference detected by the above detection method is as follows:

[0127] The effective coverage rate of the first internal reference η = T3 / T2

[0128] T2 - Before sample addition, detect the intensity of the first internal reference fluorescence signal on the online test line;

[0129] The intensity of the first internal reference fluorescence signal on the detection line was measured 5 minutes after sample addition at T3.

[0130] 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:

[0131] Table 3 Effective coating rate of the first internal control independent of the pre- and post-capsule reaction.

[0132]

[0133] The formula for the first internal reference auxiliary quantitative calibration of the above cTnI detection method is as follows:

[0134] Signal calibration value T5 = T1 / η

[0135] T1 - Fluorescence signal intensity of the detection result;

[0136] η - Effective coating rate.

[0137] Table 4 Comparison of auxiliary quantitative detection before and after the first internal control response independent of the capture reaction.

[0138]

[0139]

[0140] A comparison of the experimental data in Tables 3 and 1 shows that there is a difference in the coating binding rate of the first internal control independent of the trap and that of the non-independent first internal control, with the effective coating rate of the independent first internal control being slightly higher than that of the non-independent first internal control. Meanwhile, the effective coating rate is not affected by the concentration of the sample. As shown in Table 4, calibrating the detection results for different concentration samples using the effective coating rate independent of the trap can improve the detection precision by 0.7%-1.8%, reaching within 3%.

[0141] Example 3

[0142] 1. Preparation of test strips

[0143] (1) Preparation of the first reagent

[0144] ① Coupling between the first aptamer and the tracer

[0145] Add cTnI antibody 1 (Hytest catalog number 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 minutes, add 5% BSA, block and stir for 60 minutes, centrifuge at 14000 r / min for 20 minutes, discard the supernatant, and restore the volume of the precipitate with fluorescent antibody preservation solution (100 mM Tris, 1% Tween 20, 5 mg / ml bovine serum albumin, 2% sucrose), and store at 2-8℃.

[0146] ② Coupling between the second aptamer and the connector

[0147] Weigh 0.001g of biotin and dissolve it in 175μL LDMSO 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.

[0148] ③ Marking of the second internal reference

[0149] Fluorescent materials (ThermoFisher Alexa Fluor) TM Add 175 μL LDMSO to BSA (item number 488 A20000) to dissolve it into a 10 mg / mL working solution. Add fluorescent material to BSA at a molar ratio of 1:1, spin-label at room temperature in the dark, and then desalt using a desalting column.

[0150] ④ Preparation of the first reagent

[0151] 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.

[0152] (2) Preparation of chromatography support

[0153] ①Preparation of the detection line

[0154] The first internal reference is marked on the captured item:

[0155] Streptavidin was desalted using a desalting column, and the fluorescent substance (ThermoFisher Alexa Fluor) was used. TM Add 175 μL LDMSO to 647 (item number A20006) to dissolve into a 10 mg / mL working solution. 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.

[0156] 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.

[0157] ② Assembly of test strips

[0158] 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.

[0159] 2. Detection Method

[0160] The prepared liquid chromatography reagent containing the first internal control was used to detect troponin I in blood samples. The specific process is 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. Simultaneously, the reagent card was placed in an immunofluorescence quantitative analyzer (Q20, Zhongyuan Huiji Biotechnology Co., Ltd.). The fluorescence signal of the first internal control on the detection line before sample addition was read as T2. Then, 40 μL of sample was added to the sample well of the reagent card. The fluorescence signal T3 generated by the first internal control on the detection line was obtained after 3 min of incubation. The fluorescence signals t3 generated by the second internal control at the detection line were obtained after 1 min and 3 min of incubation, respectively. 1min and t 3minThe fluorescence signal T1 of the detection result was obtained after incubation for 4 minutes.

[0161] 3. Result Calculation

[0162] The formula for calculating the effective coating rate of the first internal reference detected by the above detection method is as follows:

[0163] The effective coverage rate of the first internal reference η = T3 / T2

[0164] T2 - Before sample addition, detect the intensity of the first internal reference fluorescence signal on the online test line;

[0165] T3 - 3 minutes after sample addition, the intensity of the first internal reference fluorescence signal on the detection line is measured.

[0166] The second intrinsic parameter, dynamic mobility ν = t 1min / t 3min

[0167] t 1min - Baseline fluorescence signal value of the second internal reference 1 minute after sample addition;

[0168] t 3min - Baseline fluorescence signal value of the second internal reference 3 minutes after sample addition;

[0169] The dynamic migration rate of the second internal reference refers to its ability to migrate between the stationary and mobile phases during chromatography. In chromatography, dynamic migration rate is an important parameter describing the rate at which the second internal reference moves within the chromatography column.

[0170] t 1min and t 3min The baseline fluorescence signal is the average fluorescence signal of 50-100 points out of 500 signal acquisition points in the chromatography detection.

[0171] 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:

[0172] Table 5. Dynamic migration rate of the second internal reference chromatography process.

[0173]

[0174] The formula for the combined auxiliary quantitative calibration of the first and second internal references for the above cTnI detection method is as follows:

[0175] Signal calibration value T5 = T1 / (η*ν)

[0176] T1 - Fluorescence signal intensity of the detection result;

[0177] η - Effective coating rate of the first internal reference;

[0178] ν - Second internal reference dynamic mobility.

[0179] Table 6 Comparison of Quantitative Analysis Before and After Using the Composite Correction Factor of the First and Second Internal References

[0180]

[0181] As can be seen from the comparison of the experimental data in Tables 2, 4 and 6, the quantitative effect of the combined calibration factor of the first internal reference and the second internal reference is significantly better than that of the single factor calibration of the first internal reference. By adopting the multi-factor composite calibration described in this embodiment, the detection precision can be improved by 1%-2.3%, reaching within 2%.

[0182] Example 4

[0183] The preparation of the test strip in this embodiment is the same as in Example 1, wherein the conjugate of the first aptamer and the tracer serves as both the detection signal and the second internal reference signal, and the detection method and result calculation are the same as in Example 3.

[0184] 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:

[0185] Table 7 Comparison of Quantitative Analysis Before and After Using the Composite Correction Factor of the First and Second Internal References

[0186]

[0187] 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%.

[0188] Example 5

[0189] (1) Preparation of the first reagent

[0190] ① Coupling between the first aptamer and the tracer

[0191] Add cTnI antibody 1 (Hytest catalog number 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 minutes, add 5% BSA, block and stir for 60 minutes, centrifuge at 14000 r / min for 20 minutes, discard the supernatant, and restore the volume of the precipitate with fluorescent antibody preservation solution (100 mM Tris, 1% Tween 20, 5 mg / ml bovine serum albumin, 2% sucrose), and store at 2-8℃.

[0192] ② Coupling between the second aptamer and the connector

[0193] Weigh 0.001g of biotin and dissolve it in 175μL LDMSO 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.

[0194] ③ Marking of the second internal reference

[0195] 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.

[0196] ④ Preparation of the first reagent

[0197] 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.

[0198] (2) Preparation of chromatography support

[0199] ①Preparation of the detection line

[0200] The first internal reference is marked on the captured item:

[0201] Streptavidin was desalted using a desalting column, and the fluorescent substance (ThermoFisher Alexa Fluor) was used. TM Add 175 μL LDMSO to 647 (item number A20006) to dissolve into a 10 mg / mL working solution. 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.

[0202] 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.

[0203] ② Assembly of test strips

[0204] 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.

[0205] The detection method and result calculation in this embodiment are the same as in Embodiment 3.

[0206] 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:

[0207] 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.

[0208]

[0209] A comparison of the experimental data in Tables 2, 4, and 8 shows that the combined calibration factor of the first internal reference and the second internal reference labeled with the connector has no significant effect on the auxiliary quantification of the detection results. A comparison of Table 8 with Tables 7 and 6 shows that the calibration effect of the combined factor of the second internal reference labeled with the connector is worse than the former two. The calibration result of the combined factor of the second internal reference labeled with the connector shows no significant improvement, but the precision can still be controlled within 5%.

[0210] Example 6

[0211] 1. Preparation of test strips

[0212] (1) Preparation of the first reagent

[0213] ① Coupling between the first aptamer and the tracer

[0214] Add cTnI antibody 1 (Hytest catalog number 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 minutes, add 5% BSA, block and stir for 60 minutes, centrifuge at 14000 r / min for 20 minutes, discard the supernatant, and restore the volume of the precipitate with fluorescent antibody preservation solution (100 mM Tris, 1% Tween 20, 5 mg / ml bovine serum albumin, 2% sucrose), and store at 2-8℃.

[0215] ② Coupling between the second aptamer and the connector

[0216] Weigh 0.001g of biotin and dissolve it in 175μL LDMSO 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.

[0217] ③ Preparation of the first reagent

[0218] The first reagent was prepared by mixing equal volumes of the two solutions (20 mM Tris, 1% Tween 20, 5 mg / mL sodium caseinate, 5% trehalose) with the first aptamer of the conjugate diluted at 0.34 mg / mL and the second aptamer of the conjugate diluted at 0.032 mg / mL, and stored at 2-8°C for later use.

[0219] (2) Preparation of chromatography support

[0220] ①Preparation of the detection line

[0221] Dilute streptavidin to 0.2 mg / mL with PBS buffer and draw a capture line 5-15 mm from the left end of the nitrocellulose membrane.

[0222] ② Assembly of test strips

[0223] 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.

[0224] 2. Detection Method

[0225] The above-prepared liquid-phase detection reagent was used to detect troponin I in blood samples. The specific process is 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. 40 μL of sample was added to the sample well of the reagent card and incubated for 3 min. The fluorescence signal intensity T1 of the detection result was then compared.

[0226] (3) Calculation of results

[0227] Using the detection reagent prepared in this embodiment, low-value, medium-value, and high-value samples were selected for detection. The T1 signal values ​​were obtained and the results were calculated according to the detection method. The experimental results are shown in the table below:

[0228] Table 9. Results of whole-liquid chromatography without internal control.

[0229]

[0230]

[0231] As can be seen from the experimental data in Table 9, the detection precision of the whole liquid chromatography reagent without internal control is <5%. Comparison of the experimental data in Table 9 with those in Tables 1-8 shows that the precision of the whole liquid chromatography reagent without internal control is significantly worse than that with the first internal control and / or the second internal control (independent or tracer-labeled), and is comparable to that of the whole liquid chromatography reagent with the second internal control labeled on the connector.

[0232] Example 7

[0233] The preparation method of the reagent in this embodiment is the same as that in Example 1.

[0234] 2. Detection Method

[0235] The prepared liquid chromatography reagent containing the first internal control was used to detect troponin I in blood samples. The specific process is 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. Then, 40 μL of sample was added to the sample well of the reagent card and incubated for 3 min. The fluorescence signal T3 after the reaction of the first internal control and the fluorescence signal T1 of the detection result were read respectively.

[0236] 3. Result Calculation

[0237] The formula for the first internal reference auxiliary quantitative calibration of the cTnI detection method is as follows:

[0238] Signal calibration value T5 = T1 / T3

[0239] T1 - Fluorescence intensity of the detection result; T3;

[0240] Fluorescence signal after the first internal reference reaction.

[0241] 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:

[0242] Table 10. Auxiliary quantitative results of fluorescence signal after the first internal control reaction.

[0243]

[0244] As can be seen from the experimental data in Table 10, the fluorescence signal after the first internal control reaction has no significant effect on the quantitative assistance of the whole liquid phase reagent. The comparison between the experimental data in Table 10 and Table 2 shows that the effective coating rate of the whole liquid phase reagent is significantly better than the quantitative assistance of the fluorescence signal after the reaction.

[0245] Example 8

[0246] This embodiment tests the performance of the reagents corresponding to Examples 1-7, and the test results are shown below:

[0247] (1) Precision

[0248] Serum samples within the linear range of 40-40000 pg / mL were tested, with each test repeated 10 times. The precision CV was calculated, and the experimental results are shown in the table below:

[0249] Table 11 Comparison of precision under different measurement modes

[0250]

[0251]

[0252] (2) Linear

[0253] High and low value samples were selected and prepared according to the dilution ratio (L:H). The reagents prepared according to the method described in this embodiment were used for testing. Each test was performed three times. The experimental results are shown in the table below:

[0254] Table 12 Linearity Comparison under Different Measurement Modes

[0255]

[0256] As shown in Tables 11 and 12, the precision results under different detection modes of the whole liquid phase in Examples 1-7 are as follows: Example 7 ≈ Example 6 ≈ Example 5 < Example 1 ≈ Example 2 < Example 3 ≈ Example 4; the composite correction factor is better for precision correction. The linearity results under different detection modes of the whole liquid phase in Examples 1-7 are comparable, and all can achieve the standard detection mode linearity of r>0.990.

[0257] Example 9

[0258] This embodiment verifies the effect of different acquisition times of the signal T3 generated by the first internal reference on the detection results. A total of 6 groups of reagents were set up, and the preparation method of each group of reagents was the same as that in Example 3. The only difference between the detection method and the calculation method in Example 3 was the acquisition time of the signal T3 generated by the first internal reference. The details are shown in the table below:

[0259] Table 13 Experimental Grouping Settings

[0260] A B C D E F T3 acquisition time 1min 2min 5min 10min 15min 20min

[0261] *In the table, 1 min represents the completion of sample addition. The fluorescence signal intensity T3 generated by the first internal reference on the detection line is obtained after 1 min of incubation of the reagent card.

[0262] Using the above six sets of reagents, low-value, medium-value, and high-value samples were selected for testing. Each signal value was obtained according to its respective detection method, and the results were calculated. The CV value calculation results are shown in the table below:

[0263] Table 14

[0264] A B C D E F L-low value 2.7％ 2.1％ 1.9％ 1.8％ 4.5％ 6.3％ M-median 3.2％ 3.0％ 2.2％ 1.7％ 3.4％ 5.2％ H-high value 2.6％ 1.8％ 1.6％ 2.0％ 2.9％ 8.4％

[0265] According to the experimental data in Table 14, when more than 15 minutes have passed since the sample was added, the fluorescence signal T3 generated by the first internal reference on the detection line has a significantly reduced auxiliary correction effect on the detection results.

[0266] Example 10

[0267] 1. Preparation of test strips

[0268] (1) Preparation of the first reagent

[0269] ① Coupling between the first aptamer and the tracer

[0270] Add CK-MB antibody 1 (Philips catalog number CK-MB-03) to fluorescent microspheres at a ratio of 60 μg CK-MB monoclonal antibody / 300 μg fluorescent microspheres, add 6 μg EDC, stir at room temperature for 120 minutes, add 5% BSA, block and stir for 60 minutes, centrifuge at 14000 r / min for 20 minutes, discard the supernatant, and restore the volume of the precipitate with fluorescent antibody preservation solution (100 mM Tris, 1% Tween 20, 5 mg / ml bovine serum albumin, 2% sucrose), and store at 2-8℃.

[0271] ② Coupling between the second aptamer and the connector

[0272] Weigh 0.001g of biotin and dissolve it in 175μL LDMSO to prepare biotin working solution. Take 0.5mg of CK-MB antibody 2 (Philips product number CK-MB-04) and add it to 7μl of biotin working solution. Mix well immediately, rotate label at room temperature in the dark, and then remove free biotin by dialysis or column chromatography.

[0273] ③ Preparation of the first reagent

[0274] The first reagent was prepared by mixing equal volumes of the two solutions (20 mM Tris, 1% Tween 20, 5 mg / mL sodium caseinate, 5% trehalose) with the first aptamer of the conjugate diluted at 0.34 mg / mL and the second aptamer of the conjugate diluted at 0.032 mg / mL, and stored at 2-8°C for later use.

[0275] (2) Preparation of chromatography support

[0276] ①Preparation of the detection line

[0277] The first internal reference is marked on the captured item:

[0278] Streptavidin was desalted using a desalting column, and the fluorescent substance (ThermoFisher Alexa Fluor) was used. TM Add 175 μL LDMSO to 647 (item number A20006) to dissolve into a 10 mg / mL working solution. 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.

[0279] 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.

[0280] ② Assembly of test strips

[0281] 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.

[0282] 2. Detection Method

[0283] The prepared total liquid chromatography reagent containing a first internal control and a second internal control was used to detect creatine kinase isoenzymes in blood samples. The specific process is as follows: 20 μL of sample was added to 180 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 of the first internal control on the detection line before sample addition as T2. Then, 40 μL of sample was added to the sample well of the reagent card, and the baseline signal value t of the second internal control on the detection line was obtained after 1 min and 3 min of incubation. 1min , t 3min After incubation for 3 minutes, the fluorescence signal T3 after the first internal control reaction and the fluorescence intensity T1 of the detection result were read simultaneously.

[0284] The calculation method for the reagent results in this embodiment is the same as in Example 3. 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:

[0285] Table 15 Comparison of Quantitative Analysis Before and After Combination of First and Second Internal References

[0286]

[0287] As can be seen from the experimental data in Table 15, the combined auxiliary quantitative detection method of the first and second internal references is also applicable to other items.

[0288] Example 11

[0289] 1. Preparation of test strips

[0290] Preparation of cTnI antibody 1 labeled with fluorescent microspheres: cTnI antibody 1 was added to fluorescent microspheres at a ratio of 60 μg NT-proBNP monoclonal antibody / 300 μg fluorescent microspheres, 6 μg EDC was added, the mixture was stirred at room temperature for 120 minutes, 5% BSA was added, the mixture was blocked and stirred for 60 minutes, centrifuged at 14000 r / min for 20 minutes, the supernatant was discarded, the precipitate was restored to volume with fluorescent antibody preservation solution, and stored at 4℃.

[0291] (1) Preparation of chromatography support:

[0292] Preparation of the detection line:

[0293] The first internal reference is marked on the captured item:

[0294] cTnI antibody 2 was desalted using a desalting column, and the fluorescent substance (ThermoFisher Alexa Fluor) was used. TM Add 175 μL LDMSO to 647 (catalog number A20006) to dissolve it into a 10 mg / mL working solution. Add fluorescent material to cTnI antibody 2 at a molar ratio of 1:1, spin-label at room temperature in the dark, and then desalt using a desalting column.

[0295] The cTnI antibody 2 labeled with the first internal control was diluted to 1.0 mg / mL with PBS buffer and the capture line was drawn on the nitrocellulose membrane at the detection line position.

[0296] The fluorescent microsphere-labeled cTnI antibody 1 was sprayed onto the glass fiber of the binding pad and then dried.

[0297] The sample pad, conjugate pad, nitrocellulose membrane and absorbent pad are tightly overlapped in sequence on the base. The assembled intermediate is cut into a preset width using a chopper and loaded into a cartridge to make a reagent card.

[0298] 2. Detection Method

[0299] The dry detection reagent containing the first and second internal controls prepared above was used to detect troponin I in blood samples. The specific process is as follows: 60 μL of sample was added to 120 μL of sample diluent (30 mM Tris, 5% Tween 20, 0.5% Proclin 300) and mixed thoroughly. First, the reagent card containing the first internal control was placed in an immunofluorescence quantitative analyzer (Q20, Zhongyuan Huiji Biotechnology Co., Ltd.) to read the fluorescence signal after the first internal control reaction as T2. Then, 70 μL of sample was added to the sample well of the reagent card, and the baseline signal value t of the second internal control was obtained after 1 min and 3 min of incubation. 1min , t 3min After incubation for 3 minutes, the fluorescence signal T3 after the first internal control reaction and the fluorescence intensity T1 of the detection result were read simultaneously.

[0300] The calculation method for the reagent results in this embodiment is the same as that in Example 3.

[0301] 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:

[0302] Table 16 Comparison of Quantitative Analysis Before and After Combination of First and Second Internal References

[0303]

[0304]

[0305] As shown by the experimental data in Table 16, the combined auxiliary quantitative detection method of the first and second internal references is also applicable to traditional dry chromatography reagents.

[0306] Example 12

[0307] 1. Preparation of test strips

[0308] Preparation of cTnI antibody 1 labeled with fluorescent microspheres: cTnI antibody 1 was added to fluorescent microspheres at a ratio of 60 μg NT-proBNP monoclonal antibody / 300 μg fluorescent microspheres, 6 μg EDC was added, the mixture was stirred at room temperature for 120 minutes, 5% BSA was added, the mixture was blocked and stirred for 60 minutes, centrifuged at 14000 r / min for 20 minutes, the supernatant was discarded, the precipitate was restored to volume with fluorescent antibody preservation solution, and stored at 4℃.

[0309] (2) Preparation of chromatography support:

[0310] Preparation of the detection line:

[0311] The first internal reference is marked on the captured item:

[0312] cTnI antibody 2 was desalted using a desalting column, and fluorescent material (ThermoFisher Alexa Fluor) was used. TM Add 175 μL LDMSO to 647 (catalog number A20006) to dissolve it into a 10 mg / mL working solution. Add fluorescent material to cTnI antibody 2 at a molar ratio of 1:1, spin-label at room temperature in the dark, and then desalt using a desalting column.

[0313] The cTnI antibody 2 labeled with the first internal control was diluted to 1.0 mg / mL with PBS buffer and the capture line was drawn on the nitrocellulose membrane at the detection line position.

[0314] A blood filtration membrane with red blood cell filtration function is used as a sample pad. The sample pad, nitrocellulose membrane and absorbent pad are tightly overlapped on the backing in sequence. The assembled intermediate is cut into a preset width using a chopper and loaded into a cartridge to make a reagent card.

[0315] cTnI antibody 1 labeled with fluorescent microspheres was spotted in the reaction chamber and then dried.

[0316] 2. Detection Method

[0317] The dry detection reagent containing the first and second internal controls prepared above was used to detect troponin I in blood samples. The specific process is 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 thoroughly. First, the reagent card containing the first internal control was placed in an immunofluorescence quantitative analyzer (Q20, Zhongyuan Huiji Biotechnology Co., Ltd.) to read the fluorescence signal after the reaction of the first internal control as T2. Then, 120 μL of diluted sample was taken and mixed thoroughly with fluorescent microspheres in the reaction cell, and added to the sample well of the reagent card. The baseline signal value t of the second internal control was obtained after 1 min and 3 min of incubation. 1min , t 3min After incubation for 3 minutes, the fluorescence signal T3 after the first internal control reaction and the fluorescence intensity T1 of the detection result were read simultaneously.

[0318] The calculation method for the reagent results in this embodiment is the same as that in Example 3.

[0319] 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:

[0320] Table 17 Comparison of Quantitative Analysis Before and After Combination of First and Second Internal References

[0321]

[0322] As shown by the experimental data in Table 17, the combined auxiliary quantitative detection method of the first and second internal references is also applicable to semi-liquid chromatography detection reagents.

[0323] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention without departing from the principles and spirit of the present invention.

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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