A temperature-corrected analyte detection method

Abstract

The present invention provides a temperature-corrected analyte detection method, which involves adding a series of analyte calibrators with different concentrations to an immunoassay strip, and then using an optical analyzer to measure the signal values generated after each concentration of analyte calibrator is captured on the detection pad of the immunoassay strip at each segmented temperature point. Then, with the T / C value at each segmented temperature point as the X-axis and the concentration of the analyte calibrator as the Y-axis, a corresponding equation is plotted; then, according to the selected n segmented temperature points, temperature segmentation is performed, and different formulas are used for calculation in different temperature segments. This temperature-corrected analyte detection method can be based on time-resolved fluorescence immunoassay and is used to more accurately detect myocardial markers, upper respiratory virus infections, metabolic hormones, digestive tract series markers, allergens, etc.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection and relates to an analyte detection method with temperature correction. Background Art

[0002] Immunochromatographic analysis technology is one of the commonly used technologies in current clinical diagnosis. Based on the principle of immunochromatography, labeled signal substances aggregate on the T line and C line of the test pad. Under the illumination of light emitted by a light source, detection signals (T) and control signals (C) are generated respectively. By calculating the ratio of these two signals (denoted as T / C), the concentration of the analyte in the sample can be quantitatively obtained.

[0003] Immunochromatographic technology can be used to detect a variety of clinical analytes, including myocardial markers, thyroid function markers, hormones, glycated hemoglobin, renal function markers, gastrointestinal disease markers, tumor markers, allergens, and respiratory disease markers.

[0004] For most of the above clinical analytes, quantitative results are required to understand the degree of infection of the patient's disease or the degree of damage to tissues (or organs). Immunochromatographic technology is a test technology based on antigen-antibody reactions. For most antigen-antibody reactions, temperature is an important influencing factor. Therefore, the detection of antigen-antibody reactions by liquid methods represented by chemiluminescence is carried out in a constant temperature environment. However, one of the most important advantages of immunochromatographic analysis technology is that the analyzer is small in size and is used in combination with dry analysis reagents. Therefore, temperature correction is very important.

[0005] The commonly used temperature correction method is the temperature correction coefficient method: Select several test temperatures within the temperature range allowed by the reagent for testing. For example, within the allowed temperature range of 15 - 30°C, select some temperature points (15°C, 20°C, 25°C, 30°C) as the experimental temperatures, and then select a series of concentrations of the analyte for testing. For example, based on the clinically tested range of myoglobin being 20 - 1000 mg / L, select myoglobin reference products with concentrations of 25, 50, 100, 300, 500, 800, 1000 mg / L for testing. Select a certain temperature point as the reference temperature, such as 25°C. Divide the T / C value measured at other temperature points by the T / C value measured at 25°C to obtain the T / C ratio. Calculate the average value of the T / C ratios of myoglobin reference products with different concentrations at a certain temperature point as the temperature correction coefficient relative to the reference temperature (Note: The temperature correction coefficient of the reference temperature itself is defined as 1). Then, use the selected temperature points as the X-axis and the average value of the T / C ratios at each temperature point as the Y-axis to obtain the temperature correction equation. Use the T / C value measured at the reference temperature (25°C) as the X-axis and the analyte concentration value as the Y-axis to obtain the standard curve equation. When calculating the results, first substitute the test temperature of the environment where the test card is located into the temperature correction equation to calculate the temperature correction coefficient, then divide the T / C value measured at this test temperature by the temperature correction coefficient to obtain the corrected T / C value, and finally substitute the corrected T / C value into the standard curve equation to obtain the concentration value of the analyte.

[0006] The prerequisite for the application of the temperature correction coefficient method is that the temperature correction coefficients calculated from reference products of the analyte with different concentrations must be similar and there should not be too large a variation. However, during the actual detection process of some analytes, the temperature correction coefficients calculated from reference products of the analyte with different concentrations vary greatly, which can lead to inaccurate detection results. Summary of the Invention

[0007] To solve the deficiencies of the temperature correction coefficient method in the prior art, the present invention selects multiple segmented temperature points within the actually allowed temperature range, and then performs temperature segmentation based on the selected segmented temperature points. Within different temperature segments, different calculation methods are selected. Therefore, the calculated analyte concentration is more accurate, and the measurement error caused by the large difference in the temperature correction coefficients calculated from reference products of the analyte with different concentrations can be significantly reduced.

[0008] The present invention provides a temperature-corrected method for detecting an analyte. The steps of the method include:

[0009] (1) Insert a test card containing a batch of immunoassay strips into the test card insertion slot of the optical analyzer;

[0010] (2) Select n segmented temperature points, namely t1, t2,..., t n, t1 < t2 < … < t n , where n is an integer and n ≥ 3. After adding a series of analyte calibration standards with different concentrations to the sample inlet of the test card at each segmented temperature point, the measurement signals generated on the detection pad of the immunoassay strip after adding each concentration of the analyte calibration standard are measured using an optical analyzer;

[0011] (3) Using the measurement signals at each segmented temperature point as the X-axis and the concentrations of the analyte calibration standards as the Y-axis, a total of n equations are obtained, and their general formula is y m = f(x m ), where m is an integer and 1 ≤ m ≤ n. The equation at the segmented temperature point t1 is y1 = f(x1), and x1 is the measurement signal obtained when measuring a series of analyte calibration standards with different concentrations at t1; the equation at the segmented temperature point t n is y n = f(x n ), and x n is the measurement signal obtained when measuring a series of analyte calibration standards with different concentrations at t n ;

[0012] (4) When the test temperature after inserting the test card into the optical analyzer is set to t, temperature segmentation is performed based on the relationship between t and the n segmented temperature points, and different formulas are used for calculation in different temperature segments, as follows:

[0013] When t ≤ t1, the analyte concentration y = y1; when t1 < t ≤ t2, y = y1 * [(t2 - t) / (t2 - t1)] + y2 * [(t - t1) / (t2 - t1)];

[0014] When 2 < m < n and t m-1 < t ≤ t m , y = y m-1 * [(t m - t) / (t m - t m-1 )] + y m * [(t - t m-1 ) / (t m - t m-1 )];

[0015] When t n-1 < t ≤ t n , y = y n-1 * [(t n - t) / (t n - t n-1 )] + y n [(t - t n-1 ) / (t n - t n-1 )];

[0016] When t > t n then, y = y n .

[0017] In some cases of the present invention, the method further comprises the following steps:

[0018] (5) Provide a new immunoassay strip belonging to the batch, add a clinical sample to the sample inlet of the new immunoassay strip, measure the measurement signal obtained on the detection pad of the immunoassay strip after adding the clinical sample by using an optical analyzer, and provide a test temperature t after the test card is inserted into the optical analyzer;

[0019] (6) Select a suitable formula according to the temperature range in which t is located in (4), and calculate the analyte concentration in the clinical sample according to the measurement signal generated in (5).

[0020] In some cases of the present invention, the measurement signal obtained in step (2) is the value obtained by arithmetic calculation of the signal T generated on the T line of the detection pad and the signal C generated on the C line of the detection pad after adding the analyte calibrator of each concentration, or the value obtained by arithmetic calculation of the signal T generated on the T line of the detection pad and the signal B generated in the background area of the detection pad after adding the analyte calibrator of each concentration, or the value obtained by arithmetic calculation of the signal T generated on the T line of the detection pad, the signal C generated on the C line of the detection pad and the signal B generated in the background area of the detection pad after adding the analyte calibrator of each concentration. In some cases of the present invention, the measurement signal obtained in step (2) is the T / C or T / (T + C) ratio. In the present invention, the background area of the detection pad is the area on the detection pad other than the T line and the C line.

[0021] In some cases of the present invention, among these n equations with the general formula y m = f(x m ), one piecewise temperature point corresponds to one equation, the functional relationships of each equation are the same, but all the equation coefficients in the two equations corresponding to any two different piecewise temperature points are not all the same, which means that there may be some that are the same, but not all, or they may all be different. For example, when n = 4, there are 4 equations at this time, that is, the equation at t1 is y1 = f(x1), the equation at t2 is y2 = f(x2), the equation at t3 is y3 = f(x3), and the equation at t4 is y4 = f(x4). Each equation is closely related to the T / C value and the analyte concentration value at each piecewise temperature point, and can be selected from a double logarithmic trinomial equation, a cubic polynomial equation and other equations. When it is a double logarithmic trinomial equation, let the general formula of the equation y m = f(x m ) = , where a, b, c, and d are the coefficients of the equation, and the coefficients in the equations at different segmented temperature points are not all the same; when it is a cubic polynomial equation, let the general equation be y m =f(x m )=a + b x + c x 2 + d x 3 , where a, b, c, and d are the coefficients of the equation, and the coefficients in the equations at different segmented temperature points are not all the same. In some other cases of the present invention, among these n general equations of y m =f(x m ), one segmented temperature point corresponds to one equation, and the functional relationships of each equation are not all the same. For example, the equation at one segmented temperature point is a double logarithmic trinomial equation, and the equation at another segmented temperature point is a cubic polynomial equation.

[0022] In some cases of the present invention, when detecting multiple analytes simultaneously, such as detecting multiple respiratory viruses, according to needs, temperature segmentation is performed only when detecting one of the analytes, or temperature segmentation can also be performed when detecting each analyte.

[0023] In some cases of the present invention, n is selected from 3, 4, 5, 6, 7, or 8. In some cases of the present invention, n = 4 or 5. In some cases of the present invention, n = 4.

[0024] In some cases of the present invention, 2°C ≤ the difference between any two adjacent segmented temperature points ≤ 6°C. In some cases of the present invention, 3°C ≤ the difference between any two adjacent segmented temperature points ≤ 5°C.

[0025] In some cases of the present invention, 4°C ≤ each selected segmented temperature point ≤ 42°C. In some cases of the present invention, 15°C ≤ each selected segmented temperature point ≤ 30°C. In some cases of the present invention, the analyte is selected from myoglobin.

[0026] In some cases of the present invention, n = 4, y m =f(x m ) = , and all the coefficients in the two equations corresponding to any two different segmented temperature points are not all the same.

[0027] In some cases of the present invention, n = 4, t1 = 15.1 °C, t2 = 19.9 °C, t3 = 24.4 °C, t4 = 29.5 °C, and the concentrations of a series of myoglobin calibration standards are 25.78, 47.33, 101.6, 306.2, 502.1, 734.0, and 1010.0 ng / mL respectively;

[0028] Equation at t1:

[0029] y1 = ;

[0030] Equation at t2:

[0031] y2 = ;

[0032] Equation at t3:

[0033] y3 = ;

[0034] Equation at t4:

[0035] y4 = ;

[0036] The selection of the formula is as follows:

[0037] When t ≤ t1, y = y1;

[0038] When t1 < t ≤ t2, y = y1 * [(t2 - t) / (t2 - t1)] + y2 * [(t - t1) / (t2 - t1)];

[0039] When t2 < t ≤ t3, y = y2 * [(t3 - t) / (t3 - t2)] + y3 * [(t - t2) / (t3 - t2)];

[0040] When t3 < t ≤ t4, y = y3 * [(t4 - t) / (t4 - t3)] + y4 * [(t - t3) / (t4 - t3)];

[0041] When t > t4, y = y4.

[0042] In some cases of the present invention, the test temperature after the test card is inserted into the optical analyzer can be the ambient temperature near the test card insertion port of the optical analyzer, or the temperature of the internal environment of the test card where the immunoassay strip is located. The former can be measured by setting a thermistor near the test card insertion port of the optical analyzer, and the latter can be measured by setting a thermistor inside the test card. In other cases of the present invention, the test temperature after the test card is inserted into the optical analyzer is the ambient temperature of the place where the optical analyzer is located. For example, when the optical analyzer is located in a laboratory, the test temperature is the ambient temperature of the laboratory. This can be measured by using an external thermometer to measure the ambient temperature of the place where it is located, and then inputting the measured ambient temperature into the optical analyzer through the user interface, or it can be measured by a thermistor set on the surface of the optical analyzer.

[0043] In the present invention, this analyte detection method with temperature correction can be used to more accurately detect myocardial markers, internationally public health transmitted diseases, metabolic hormones, digestive tract series markers, respiratory tract series markers, and allergens. Brief Description of the Drawings

[0044] Figure 1 Is an exploded schematic view of the test card used in the present invention.

[0045] Figure 2 Is a schematic view of the immunoassay strip in the test card used in the present invention.

[0046] Figure 3 Is a temperature correction curve obtained with temperature as the X-axis and the average value of signal ratios at different concentrations at each temperature as the Y-axis.

[0047] Figure 4 Is a signal curve obtained with the signal value measured at the reference temperature of 24.4 °C as the X-axis and the myoglobin concentration values of different myoglobin calibrators as the Y-axis.

[0048] Figure 5 Is a logarithmic signal curve obtained with the logarithm of the signal value measured at the reference temperature of 24.4 °C as the X-axis and the myoglobin concentration values of different myoglobin calibrators as the Y-axis. Detailed Description of the Invention

[0049] Such as Figure 1 And Figure 2As shown, the test card used in the present invention includes a test strip 1, a card cover 2 and a card holder 3. The test strip 1 used is an immunochromatographic test strip, which includes a sample application pad 11, a labeling pad 12, a detection pad 13 and a sample absorption pad 14 that are successively overlapped. The detection pad 13 is made of materials such as nitrocellulose, glass fiber, polyethersulfone or nylon, and is preferably a nitrocellulose membrane. A detection line (also called T line) 15 and a control line (also called C line) 16 are provided on the detection pad 13. The sample application pad 11 is made of a water-absorbent material, and glass fiber or non-woven fabric can be selected. The labeling pad 12 is also made of a water-absorbent material, and polyester film, glass fiber or non-woven fabric can be selected.

[0050] The number of detection lines provided on the detection pad 13 can be adjusted according to actual needs. For example, when detecting one analyte, only one detection line needs to be set. When detecting two or more analytes, the corresponding number of detection lines should be set. For example, 4 T lines are set to jointly detect multiple upper respiratory viruses, such as 4 upper respiratory viruses, namely novel coronavirus, influenza A virus, influenza B virus, adenovirus and respiratory syncytial virus. The area of the detection pad other than the T line and the C line is the background area of the detection pad.

[0051] The test strip 15 further includes a bottom support layer 17, which is made of a common hydrophobic material such as polyvinyl chloride to ensure that the sample cannot leak out from the bottom support layer 17. The detection pad 13 is provided on the bottom support layer 17. The sample application pad 11 is provided on the bottom support layer 17, and one end of the sample application pad 11 partially overlaps with the labeling pad 12; the labeling pad 12 is provided on the bottom support layer 17, one end of the labeling pad 12 partially overlaps with the sample application pad 11, and the other end of the labeling pad 12 partially overlaps with the detection pad 13; the sample absorption pad 14 is provided on the bottom support layer 17 and is made of a hydrophilic material, preferably filter paper; one end of the sample absorption pad 14 partially overlaps with the detection pad 13. In addition, in some cases, the overlapping area between any two adjacent pads is 0.5 to 5 millimeters.

[0052] The test strip 1 is located in a housing, and the housing is composed of a card cover 2 and a card holder 3 combined by ultrasonic welding, snap or glue bonding methods. In some cases, the card cover 2 and the card holder 3 are made of plastic. A test strip slot 32 is provided in the middle of the card holder 3 for placing the test strip 1. Preferably, the card cover 2 has a plurality of downwardly extending snaps (not shown in the figure), and the card holder 3 has a plurality of upwardly extending slots 31. The snaps provided on the card cover 2 and the slots 31 provided on the card holder 3 correspond one by one, so that when the card cover 2, the test strip 1 and the card holder 3 are assembled together, the card cover 2 and the card holder 3 can be firmly fixed together, and the test strip 1 is fixed in the test strip slot 32.

[0053] The card cover 2 is also provided with an observation window 21 and a sample inlet 22. After the test card is inserted into the test card insertion port of the optical analyzer, when a clinical sample is added through the sample inlet 22, the sample enters the sample application pad 11 located below the sample inlet 22, and under the capillary action, the sample migrates along the length direction of the test strip 1 towards the sample absorption pad 14. The observation window 21 is arranged above the test line 15 and the control line 16 of the detection pad 13. The light emitted by the light source in the optical analyzer can irradiate the test line 15 and the control line 16 of the test strip 1 through the transparent or semi-transparent observation window 21. After being irradiated by the light from the light source, the optical signals generated by the test line 15 and the control line 16 can be detected by the detector in the optical analyzer. The optical analyzer used in the present invention can be selected from commercially available optical analyzers, such as the dry fluorescence immunoassay analyzer iFIA-100 (Alcon Biotechnology (Hangzhou) Co., Ltd.).

[0054] According to the differences in the analyte to be detected (such as antigen, antibody or hapten) and the immunoassay principles (double antigen sandwich method, double antibody sandwich method, competitive method, indirect method, capture method), the substances coated on the labeling pad 12 and the test line 15 will vary. Here, taking the analyte as myoglobin and the detection principle as the double antibody sandwich method as an example for illustration. The labeling pad 12 is coated with a first reagent (first anti-myoglobin antibody) labeled with a signal label and a first quality control reagent (such as rabbit IgG) labeled with a signal label. The test line 15 is coated with a second reagent (second anti-myoglobin antibody). The first reagent and the second reagent specifically bind to myoglobin in the clinical sample. Thus, when a clinical sample is added to the sample application pad 11 through the sample inlet 22, the clinical sample flows along the length direction of the test strip 1. When the clinical sample reaches the labeling pad 12, the first reagent labeled with a signal label specifically binds to myoglobin (if present) in the clinical sample, and the formed signal label-first reagent-myoglobin complex continues to flow and specifically binds to the second reagent on the test line 15, thereby capturing the signal label-first reagent-myoglobin complex on the test line 15. After being irradiated by the light from the light source of the optical analyzer, a detection signal related to the concentration of myoglobin can be generated. At the same time, the first quality control reagent labeled with a signal label continues to flow. When it flows to the control line 16, the second quality control reagent (such as goat anti-rabbit IgG antibody) coated on the control line 16 can capture the first quality control reagent labeled with a signal label on the control line 16. After being irradiated by the light from the light source, the signal label captured on the control line 16 generates a control signal. The generated detection signal T and the control signal can be optical signals such as reflected light, transmitted light, fluorescence, phosphorescence, etc. generated by the T line and the C line after being irradiated by the light from the light source.

[0055] The signal markers in the present invention can be selected from time-resolved luminescent markers, colored luminescent microspheres, colored colloidal particles (such as latex, colloidal gold, colloidal carbon, colloidal selenium), magnetic nanoparticles, and luminescent compounds. Time-resolved luminescent markers have the property of luminescence delay, that is, when the excitation light emitted by the light source is turned off, they can still continuously emit light for a certain period of time. Time-resolved luminescent markers can exist in molecular form, called time-resolved luminescent molecules, and can be selected from lanthanide elements such as samarium (Sm(III)), dysprosium (Dy(III)), europium (Eu(III)), terbium (Tb(III)) and their chelates; platinum / palladium porphyrin compounds that can emit phosphorescence after excitation; up-conversion luminescent materials. A suitable lanthanide chelate is N-(p-isothiocyanatobenzene)-diethylenetriamine tetraacetic acid-Eu +3 . Time-resolved luminescent markers can also exist in another form: time-resolved luminescent microspheres, that is, time-resolved luminescent molecules are wrapped inside or on the surface of natural or synthetic microspheres or microbeads. Given that each time-resolved luminescent microsphere can encapsulate thousands of time-resolved luminescent molecules, effectively improving the detection sensitivity, therefore, the time-resolved luminescent marker is preferably a time-resolved luminescent microsphere. Time-resolved luminescent microspheres that produce fluorescence after excitation are called time-resolved fluorescent microspheres.

[0056] The time-resolved luminescent markers in the present invention have the property of luminescence delay, that is, when the excitation light emitted by the light source is turned off, they can still continuously generate emission light for a certain period of time. The wavelength of the emission light can be greater than or less than the wavelength of the excitation light. This property of luminescence delay makes the emission light generated by the time-resolved luminescent markers after excitation have a longer lifespan. Thus, in actual detection, after the excitation light from the light source excites the time-resolved luminescent markers, the light source can be turned off and waited for a period of time, and then the emission light signal emitted by the time-resolved luminescent markers can be detected, thereby eliminating the interference of background light signals with shorter lifespans and scattered excitation light.

[0057] Time-resolved luminescent markers can exist in molecular form, known as time-resolved luminescent molecules, and can be selected from lanthanide elements such as samarium (Sm(III)), dysprosium (Dy(III)), europium (Eu(III)), terbium (Tb(III)) and their chelates, as well as upconversion luminescent materials; platinum / palladium porphyrin compounds that can emit phosphorescence after excitation. The delay time of europium chelate is about 2 ms, and the emitted fluorescence signal gradually decays with time within 2 ms. Given that some proteins in clinical samples can also produce fluorescence under the light generated by the light source, but without the delay characteristic, most proteins produce fluorescence that completely disappears 200 μs after the light source is turned off. Therefore, time-resolved fluorescence detection based on europium chelate usually selects to collect fluorescence signals at a time of 200 - 400 μs after the light source is turned off. A suitable europium chelate is N-(p-isothiocyanatophenyl)-diethylenetriamine tetraacetic acid-Eu +3 .

[0058] Time-resolved luminescent markers can also exist in another form: time-resolved luminescent microspheres, that is, time-resolved luminescent molecules are encapsulated inside or on the surface of natural or synthetic microspheres or microbeads. Given that each time-resolved luminescent microsphere can encapsulate thousands of time-resolved luminescent molecules, effectively improving the detection sensitivity, therefore, time-resolved luminescent markers are preferably time-resolved luminescent microspheres. Time-resolved luminescent microspheres that produce fluorescence after being irradiated by light are called time-resolved fluorescence microspheres.

[0059] Upconversion luminescent materials refer to materials that emit high-energy light when irradiated by low-energy light. The optical signals generated by upconversion luminescent materials after being irradiated by light have strong stability, long lifespan and high sensitivity, and are also suitable for time-resolved immunoassay. Common upconversion luminescent materials can be selected from Y2O3, Y2O2S, LaF3, NaYF4, NaGdF4, NaYF4:Yb 3+ / Nd 3+ / Ho 3+ , NaGdF4:Yb 3+ / Nd 3+ / Ho 3+ , Y2O3:Er,Yb, etc.

[0060] The colored luminescent microspheres in the present invention refer to microspheres or microbeads in which luminescent compounds such as quantum dots and fluorescent dyes are encapsulated on the surface or inside, and can generate optical signals without luminescence delay characteristics after being irradiated by light of a suitable wavelength, and can be selected from green fluorescent microspheres, blue fluorescent microspheres, red fluorescent microspheres, yellow fluorescent microspheres and colored fluorescent microspheres (emitting multiple specific colors of fluorescence).

[0061] The luminescent compounds in the present invention refer to those that can produce light without luminescence delay characteristics when irradiated with light of a suitable wavelength, and can be selected from quantum dots; fluorescein and its derivatives, such as fluorescein isothiocyanate (FITC); fluorescent proteins that can emit fluorescence after being irradiated with light and their improved variants, such as green fluorescent protein, red fluorescent protein, blue fluorescent protein, yellow fluorescent protein, orange fluorescent protein, etc.; chemiluminescent markers, which can be selected from luminol, isoluminol and its derivatives, 1,2-dioxetane derivatives (commonly including AMPPD, CSPD, CDP and CDP-Star, as well as PPD, Lumi-Phos and Lumi-Plus of Lumigen company), and acridinium ester or acridinium sulfonamide.

[0062] The colored colloidal particles in the present invention refer to colloidal particles that can produce colored aggregates after aggregating on the T line and / or C line during the immunochromatographic reaction, and can be selected from latex, colloidal gold, colloidal carbon and colloidal selenium. After being irradiated with light from a light source, the reflected light generated by the colored colloidal particles on the T line or C line can be detected as a detection signal or a control signal.

[0063] In time-resolved luminescent microspheres and colored luminescent microspheres, the polymers forming the microspheres or microbeads can be selected from polystyrene, butadiene styrene, styrene acrylic-ethylene terpolymer, polymethyl methacrylate, polyethyl methacrylate, styrene-maleic anhydride copolymer, polyvinyl acetate, polyvinyl pyridine, polydivinyl benzene, polybutylene terephthalate, acrylonitrile, vinyl chloride-acrylate, etc., or their aldehyde group, carboxyl group, amino group, hydroxyl group, hydrazide derivatives, or mixtures thereof. In addition, the surface of the microspheres or microbeads usually carries groups such as hydroxyl group, carboxyl group, amino group, aldehyde group, sulfonic group, etc., and can be coupled and bound together with antibodies or antigens or hapten-carrier protein conjugates through conventional chemical coupling reagents. In some cases, the particle size of the time-resolved luminescent microspheres is 20 nm to 100 μm.

[0064] When detecting one or more analytes in a test sample, according to the different immunodetection principles, the intensity of the detection signal generated by the time-resolved luminescent marker on the T line is positively or negatively correlated with the concentration of the analyte. Among them, except for the competitive method, the intensity of the detection signal generated by the time-resolved luminescent marker on the T line is positively correlated with the concentration of the analyte. However, regardless of which immunodetection principle is adopted, the control line 16 (if present) should generate a control signal, otherwise it indicates that there is a problem with the test strip, or it is invalid.

[0065] Clinical samples in the present invention can be selected from serum, plasma, whole blood, cerebrospinal fluid, urine, bronchoalveolar lavage fluid, nasopharyngeal swabs, sputum, feces, skin lesion samples (including swabs of rash / pustule exudate; pustule fluid; scabs, etc.). Depending on the type of clinical sample and the analyte, some clinical samples need to be pretreated (such as lysed with a lysis solution to release the analyte to be detected) before being added to the test card for detection.

[0066] The analytes detectable by the present invention include antigens or antibodies, and even haptens, such as inflammatory markers, such as CRP, IL-6, procalcitonin (PCT), and SAA, etc.; heart failure markers, such as BNP, NT-proBNP, cTnI, CK-MB, myoglobin, and D-dimer, etc.; tumor markers, such as alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), ferritin, prostate specific antigen (PSA), neuron specific enolase (NSE), CYFRA21-1, CA19-9, CA50, CA125, CA153, CA724, etc.; bone metabolism series markers, such as 25-hydroxyvitamin D, β-CrossLaps, bone alkaline phosphatase, calcitonin, parathyroid hormone, human N-terminal mid-region osteocalcin, and total type I procollagen amino-terminal propeptide, etc.; sex markers, such as follicle stimulating hormone (FSH), luteinizing hormone (LH), prolactin, progesterone, testosterone, estradiol, estriol, β-human chorionic gonadotropin (β-HCG); thyroid function markers, such as total triiodothyronine (TT3), total thyroxine (TT4), free triiodothyronine (FT3), free thyroxine (FT4), thyroid stimulating hormone (TSH), thyroglobulin, thyroglobulin antibody, and thyroid peroxidase antibody, etc.; infectious disease markers, such as upper respiratory virus (such as novel coronavirus, influenza A virus, influenza B virus, respiratory syncytial virus, adenovirus, rhinovirus, parainfluenza virus, etc.) antigen / antibody, monkeypox virus antigen / antibody, gonococcus antigen / antibody, chlamydia antigen / antibody, mycoplasma antigen / antibody, hepatitis B five items (HBsAg, anti-HBs, anti-HBc, HBeAg, anti-HBe), HCV antigen / antibody, syphilis treponema antigen / antibody, TORCH (human cytomegalovirus, rubella virus, toxoplasma gondii, HSV-1 and HSV-2) IgG / IgM, HIV antigen / antibody, etc.; diabetes markers, such as insulin, C-peptide, insulin autoantibody, islet cell antibody, glutamic acid decarboxylase antibody; liver fibrosis markers, such as laminin, hyaluronic acid, type IV collagen, type III procollagen N-terminal peptide, and chitinase 3-like protein 1, etc.; allergy markers, such as allergens, allergen-specific IgE, and total IgE, etc.; intestinal health series markers, such as intestinal adenovirus antigen, rotavirus antigen, enterovirus 71 IgG / IgM, coxsackievirus A group 16 IgG / IgM, coxsackievirus B group IgG / IgM, calprotectin, Clostridium difficile toxin A, Clostridium difficile toxin B, Clostridium difficile glutamate dehydrogenase, etc.; cocaine, morphine, etc. for evaluating drug abuse.

[0067] Example 1: Preparation of the immunotest strip and test card of the present invention

[0068] A method for preparing an immunochromatographic strip and a test card for quantitatively detecting an analyte (such as IL-6, NT-proBNP, myoglobin, and upper respiratory virus antigens or antibodies, etc., taking myoglobin as an example for illustration) in a sample includes the following steps:

[0069] A. Antibody preparation: Monoclonal or polyclonal antibodies for detecting myoglobin can be screened out by immunizing animals such as mice, rats, rabbits, etc. with antigens or by hybridoma cell counting, or commercially available paired myoglobin antibodies (one is a myoglobin capture antibody, and the other is a myoglobin detection antibody) can be selected. In this embodiment, commercially available paired myoglobin antibodies (purchased from Jiangsu Dongkang Biopharmaceutical Technology Co., Ltd., product numbers C3601: lot 20221107 and C3604: lot 20210326) are taken as an example for illustration. Rabbit IgG antibody and goat anti-rabbit IgG antibody are prepared by oneself or purchased from the market.

[0070] B. Spotting the detection pad

[0071] Add the myoglobin capture antibody to 0.02M phosphate buffer (pH 7.2) to obtain the T-line solution, and the concentration of the myoglobin capture antibody in the T-line solution is 1mg / ml. Add the goat anti-rabbit IgG antibody to 0.02M phosphate buffer (pH 7.2) to obtain the C-line solution, and the concentration of the goat anti-rabbit IgG antibody in the C-line solution is 0.3mg / ml. Then use a quantitative spotting device (AUTOKUN continuous membrane scribing machine spotting instrument HGS101 of Hangzhou Fenghang Technology Co., Ltd.) to coat the T-line solution and the C-line solution on a 2.5-cm-wide nitrocellulose membrane (purchased from Satorious company, model 1UN95ER100025NT) used as the detection pad at a rate of 1μl / cm with an interval of 0.8 cm, so as to form the T-line and the C-line respectively. Dry at 45°C for 18 hours, and add a desiccant for storage and standby.

[0072] C. Preparation of fluorescent microspheres:

[0073] Selection of fluorescent microspheres: Time-resolved fluorescent microspheres (Merk company, product number F1-XC010) with europium chelate encapsulated inside, whose excitation wavelength is 365nm and emission wavelength is 615nm.

[0074] Preparation of MES buffer: Add morpholineethanesulfonic acid sodium salt to pure water and mix evenly to make the concentration of morpholineethanesulfonic acid sodium salt 1.0% (w / v).

[0075] Preparation of storage buffer: 50mM Tris-HCl buffer (pH 8.0).

[0076] Preparation of myoglobin detection antibody labeled with time-resolved fluorescent microspheres: The fluorescent microspheres were washed with MES buffer, and carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were added to make the final concentrations of 0.4 mg / ml and 0.1 mg / ml, respectively. The time-resolved fluorescent microspheres were activated by reaction at room temperature for 20 minutes. After the activated microspheres were fully washed with MES buffer, myoglobin detection antibody was added at a ratio of 1 mg:0.1 mg (mass ratio) and reacted at room temperature for 1.5 hours. After being fully washed with MES buffer, 0.05 M Tris-HCl buffer (pH 7.0) containing 10% BSA (w / v) was added. 8.0), blocked at room temperature for 1 hour, washed with MES buffer, and re-dissolved with storage buffer to detect time-resolved fluorescent microspheres to a final concentration of 5 mg / ml, and stored at 4°C for use; in the same manner as the preparation of time-resolved fluorescent microsphere-labeled myoglobin detection antibody, prepared time-resolved fluorescent microsphere-labeled rabbit IgG antibody, and re-dissolved with storage buffer to a final concentration of 5 mg / ml, and stored at 4°C for use.

[0077] Finally, the myoglobin detection antibody labeled with time-resolved fluorescent microspheres, the rabbit IgG antibody labeled with time-resolved fluorescent microspheres, and the fluorescent labeling diluent were thoroughly mixed at a volume ratio of 6:1:13 to obtain a fluorescent microsphere mixture. The fluorescent labeling diluent was 50 mM Tris-HCl buffer (pH 8.0) containing 15% sucrose (w / v), 5% trehalose (w / v), 2% Tween-20 (v / v), 0.5% PVP (w / v), and 0.5% BSA (w / v).

[0078] D. Spraying and drying of fluorescent microspheres

[0079] The prepared fluorescent microsphere mixture was evenly sprayed onto a 1.0 cm wide marking pad (glass fiber) using the dedicated streaking head of the AUTOKUN continuous streaking machine HGS101 at a rate of 2 μl / cm. The mixture was dried at 45°C for 18 h, and then sealed with a desiccant for later use.

[0080] E. Sample Pad Processing

[0081] Soak a 2.5 cm wide sample pad in sample pad treatment solution for 1 hour, then dry at 37°C overnight (12-24 hours). The sample pad treatment solution consists of 50 mM (pH 8.0) Tris-HCl buffer, 1% BSA (w / v), and 0.5% Tween-20 (v / v).

[0082] F. Assembly and cutting of test strips

[0083] Assembly of the test strip plate: Manually or by machine, paste a 2.5-cm-wide sample pad, a 1.0-cm-wide marker pad, a 2.5-cm-wide nitrocellulose membrane, and a 2.5-cm-wide absorbent paper (as a sample absorption pad) onto an 8-cm-long plastic bottom plate (as a bottom support layer) with adhesive, such that the 2.5-cm-wide sample pad, the 1.0-cm-wide marker pad, the 2.5-cm-wide nitrocellulose membrane, and the 2.5-cm-wide absorbent paper overlap each other alternately by 2.0 mm in sequence to assemble the test strip plate.

[0084] Cutting of the test strip plate: Use an AUTOKUN HGS201 strip cutter to cut the assembled test strip plate into single-person test strips with a width of 4 mm.

[0085] G. Assembly of the test card

[0086] Place the cut single-person test strip into the card slot on the plastic card holder, cover the card cover, and use a card press or manually press the card holder and the card cover tightly to ensure that the entire immunoassay test strip is in a taut state. Add a desiccant and seal it at room temperature for standby.

[0087] After the test card is assembled, it can be placed into a kit containing a desiccant and an instruction manual. In addition, according to needs, the kit can also contain a sampling tube, a test tube containing lysis buffer, a test tube containing sample diluent, a sampling swab, etc.

[0088] Example 2: Detection method

[0089] Use the test card prepared in Example 1 to detect analytes in a sample (for example, IL-6, NT-proBNP, myoglobin, upper respiratory virus antigen or antibody, etc., here taking myoglobin as an example for illustration). The specific process is as follows: Add 100 μl of the sample (here taking a whole blood sample as an example for illustration) to the sample inlet of the test card. After incubating for 15 min, insert the test card into an iFIA-100 dry fluorescence immunoassay analyzer (Alcon Biotechnology (Hangzhou) Co., Ltd.) for detection.

[0090] During detection, first let the light source emit light with a wavelength of 360 nm to irradiate the time-resolved fluorescence microspheres captured on the T line and the C line, then turn off the light source, and the time-resolved fluorescence microspheres captured on the T line and the C line emit time-resolved fluorescence signals with a wavelength of 615 nm. After turning off the light source for 300 μs, use the detector of the light analyzer to capture and count, and respectively obtain the time-resolved fluorescence signal (T signal) emitted on the T line and the time-resolved fluorescence signal (C signal) emitted on the C line.

[0091] To correct the adverse effects caused by manufacturing differences during the manufacturing process of test cards of the same batch, the T signal can be corrected, that is, the T signal is corrected using the C signal to obtain the ratio of the T signal to the C signal, denoted as T / C, or T / (T + C). According to the pre-determined calibration curve, the T / C or T / (T + C) obtained after adding clinical samples can be used to determine the concentration of myoglobin.

[0092] When preparing a series of myoglobin calibrators with different concentrations, serum samples with high-concentration myoglobin and serum samples with low-concentration myoglobin are collected and formulated into 7 different concentrations of calibrators. They are tested using a Roche Cobas e411 fully automated electrochemiluminescence analyzer (SN#8866 - 29) and a Roche supporting myoglobin detection kit (Lot#70743802). The measured myoglobin concentration values (25.78, 47.33, 101.6, 306.2, 502.1, 734.0, and 1010.0 ng / mL) are used as the concentration reference values for these 7 reference products. Then, these calibrators with different concentrations are separately added to test cards of the same batch. After 15 minutes of incubation, a dry fluorescence immunoassay analyzer iFIA - 100 (Acon Biotech (Hangzhou) Co., Ltd.) is used to detect the T signal and C signal of each test card, and the T / C or T / (T + C) ratio is calculated. A calibration curve is plotted based on each T / C or T / (T + C) ratio and its corresponding myoglobin concentration value, which is used to calculate the myoglobin concentration in clinical samples.

[0093] Of course, a series of myoglobin calibrators with different concentrations can also be prepared using a calibrator diluent (such as PBS buffer) and myoglobin.

[0094] In addition, when using an immunoassay analyzer to measure myoglobin calibrators with different concentrations, it is carried out at a certain temperature. To ensure that the immunoassay analyzer, test cards, and supporting sample diluent (20 mM PBS, 0.02% Proclin - 300, and 0.1% Tween - 20) and a series of myoglobin calibrators with different concentrations are at a specific temperature, they can be placed at this temperature for more than 1 hour to reach temperature equilibrium.

[0095] Example 3: Comparative Method

[0096] Using the test cards in Example 1 and the measurement method in Example 2, myoglobin calibrators with different concentrations (concentrations are 25.78, 47.33, 101.6, 306.2, 502.1, 734.0, and 1010.0 ng / mL) are measured at 4 selected temperature points (15.1 °C, 19.9 °C, 24.4 °C, and 29.5 °C). The results are shown in Table 1.

[0097] Table 1

[0098]

[0099] Select 24.4 °C as the reference temperature. Divide the T / C values measured at each temperature point (15.1 °C, 19.9 °C, and 29.5 °C) by the T / C value measured at 24.4 °C to obtain the T / C ratio. Calculate the average value of the T / C ratios at different myoglobin concentrations at each temperature point as the temperature correction coefficient for each temperature point relative to the reference temperature. The results are shown in Table 2.

[0100] Table 2

[0101]

[0102] Next, based on the data in Table 2, with the temperature point as the X-axis and the average value of the T / C ratio as the Y-axis, obtain the temperature correction curve (as shown in Figure 3 ). It can be seen from Figure 3 that the temperature correction coefficient is not linear with temperature. Thus, the obtained temperature correction equation is:

[0103] tcf = -0.4299 + 0.1120t - 0.002373t 2 + 0.000007617t 3 , where t is the temperature and tcf is the corresponding temperature correction coefficient at each temperature point.

[0104] Taking the T / C value measured at the reference temperature of 24.4 °C as the X-axis and the myoglobin concentration value of different myoglobin calibrators as the Y-axis, plot the signal curve, as shown in Figure 4 .

[0105] Figure 4 Each test point in Figure 5 basically shows an exponential distribution. For this reason, taking the logarithm of the T / C value measured at the reference temperature of 24.4 °C as the X-axis and the logarithm of the myoglobin concentration value of different myoglobin calibrators as the Y-axis, plot the logarithmic signal curve, as shown in

[0106] From Figure 5 , the standard curve equation for the myoglobin concentration in the sample is as follows:

[0107] y = .

[0108] When actually measuring a clinical sample (such as plasma, serum or whole blood) with an unknown analyte concentration, after adding the clinical sample, the ambient temperature (measurable by a thermometer) in the external environment (such as the external air temperature) or the local environment (such as the internal environment of the optical analyzer, the internal environment of the test card) where the optical analyzer or the test card is located is manually input, or the test temperature in the external environment or the local environment where the optical analyzer or the test card is located is measured by a temperature measurement module (such as a thermistor) set in the optical analyzer. Here, taking the measurement of the external air temperature of the optical analyzer using a thermometer as an example for illustration. At the same time, according to the measurement method in Example 2, the T / C value after adding the clinical sample at this test temperature is obtained. When calculating the result, first substitute the measured test temperature into the temperature correction equation to calculate the temperature correction coefficient, and then divide the obtained T / C value by the temperature correction coefficient to obtain the T / C corrected value, as shown in Table 3.

[0109] Table 3

[0110]

[0111] For the purpose of result verification, in addition to the selected 4 temperature points (15.1 °C, 19.9 °C, 24.4 °C and 29.5 °C), 3 different verification temperature points (17.3 °C, 22.1 °C and 27.2 °C) are added. Using the test card in Example 1 and the measurement method in Example 2, calibrators of different concentrations of myoglobin are measured at these three verification temperature points, and the measured T / C values are shown in Table 4. At the same time, substitute each verification temperature point into the temperature correction equation to calculate the temperature correction coefficient, and then divide the measured T / C value by the temperature correction coefficient to obtain the T / C corrected value. The corrected T / C values are shown in Table 4.

[0112] Table 4

[0113]

[0114] Finally, substitute the T / C corrected values in Table 3 and Table 4 into the standard curve equation to obtain the concentration value of myoglobin, as shown in Table 5.

[0115] Table 5

[0116]

[0117] Using the data in Table 5, the deviation between the temperature-corrected myoglobin concentration value and the actual concentration can be calculated as = (temperature-corrected myoglobin concentration value - actual concentration) / actual concentration. The calculated deviation results are shown in Table 6.

[0118] Table 6

[0119]

[0120] As can be seen from Table 6, the deviation range of the measured results of myoglobin concentration obtained by the temperature correction coefficient method is -13.0% to +12.0%, and the average value of the absolute deviation is 5.7%. In addition, as can also be seen from Table 6, for 17.3 °C used for result verification, the deviation range of the measured results is -11.9% to 8.7%; for 22.1 °C used for result verification, the deviation range of the measured results is -13.0% to 7.3%; for 27.2 °C used for result verification, the deviation range of the measured results is -5.6% to 12.0%.

[0121] Example 4: The test method of the present invention

[0122] As can be seen from Table 2 in Example 3, for myoglobin calibrators with the same concentration, when the temperature increases from 15.1 °C to 29.5 °C, at higher concentrations of myoglobin calibrators, the temperature correction coefficients at different temperatures generally show an upward trend. However, when using lower concentrations of myoglobin calibrators (such as 25.78 ng / mL and 47.33 ng / mL), the temperature correction coefficients at different temperatures show a trend of first increasing and then decreasing. In addition, at the same temperature, the temperature correction coefficients measured for myoglobin calibrators with different concentrations show different trends. For example, at 15.1 °C and 19.9 °C, as the myoglobin concentration increases, the temperature correction coefficient shows a trend of first decreasing and then increasing; at 29.5 °C, as the myoglobin concentration increases, the temperature correction coefficient generally shows an upward trend overall.

[0123] It can be seen from this that the change trends of the temperature correction coefficient under different temperatures and different concentrations of myoglobin calibrators show different trends, and the calculated temperature correction coefficients are not close under different concentrations of myoglobin calibrators, showing large fluctuations. The result of this is that the deviation range of the measured results of myoglobin concentration obtained by the temperature correction coefficient method is -12.5% to +15.1% (see Table 6), resulting in inaccurate detection results.

[0124] Based on this, within the temperature range for measuring myoglobin concentration in the present invention (usually 4 - 42°C, and in some cases, 15 - 30°C), n segmented temperature points are selected (n is an integer and n≥3). For the sake of comparison, the selection scheme of the segmented temperature points is the same as that in Example 3 (specifically, when selecting 4 segmented temperature points: t1 = 15.1°C, t2 = 19.9°C, t3 = 24.4°C, and t4 = 29.5°C), and the initially measured T / C values are shown in Table 1. Meanwhile, for the purpose of result verification, in addition to the 4 selected segmented temperature points, 3 more verification temperature points (17.3°C, 22.1°C, and 27.2°C) are added. Using the test card in Example 1 and the measurement method in Example 2, myoglobin calibration products with different concentrations are measured at these three verification temperature points, and the initially measured T / C values are shown in Table 4.

[0125] Then, taking the T / C value measured at each selected segmented temperature point as the X-axis and the myoglobin concentration value as the Y-axis, the general formula of the standard curve equation at each segmented temperature point is obtained: y m =f(x m )= , where m is an integer and 1≤m≤n, and a, b, c, and d are equation coefficients. These equation coefficients are different at different segmented temperature points. x m is the T / C value measured at the segmented temperature point t m , and y m is the myoglobin concentration value.

[0126] When n = 4, the standard curve equations for myoglobin concentration at t1, t2, t3, and t4 are y1 = f(x1), y2 = f(x2), y3 = f(x3), and y4 = f(x4) respectively:

[0127] 15.1°C: y1 = f(x1) =

[0128] ;

[0129] 19.9°C: y2 = f(x2) =

[0130] ;

[0131] 24.4°C: y3 = f(x3) =

[0132] ;

[0133] 29.5°C: y4 = f(x4) =

[0134] .

[0135] In view of the fact that there are significant differences in temperature correction coefficients at different temperatures in the prior art, the present invention conducts temperature segmentation based on the relationship between the test temperature in the environment where the test card is located and different segmented temperature points after the test card is inserted into the optical analyzer. Different formulas are used for calculation in different temperature segments, and the specific segmentation is as follows:

[0136] When the measured temperature t ≤ t1, y = y1;

[0137] When t1 < the measured temperature t ≤ t2, y = y1 * [(t2 - t) / (t2 - t1)] + y2 * [(t - t1) / (t2 - t1)];

[0138] When t2 < the measured temperature t ≤ t3, y = y2 * [(t3 - t) / (t3 - t2)] + y3 * [(t - t2) / (t3 - t2)];

[0139] When t3 < the measured temperature t ≤ t4, y = y3 * [(t4 - t) / (t4 - t3)] + y4 * [(t - t3) / (t4 - t3)];

[0140] When the measured temperature t > t4, y = y4.

[0141] Based on the initial T / C values provided in Table 1 and Table 4, the myoglobin concentration values calculated at different test temperatures are shown in Table 7.

[0142] Table 7

[0143]

[0144] Using the data in Table 7, the deviation between the myoglobin concentration value in Table 7 and the actual concentration can be calculated as = (myoglobin concentration value in Table 7 - actual concentration) / actual concentration, and the calculated deviation results are shown in Table 8.

[0145] Table 8

[0146]

[0147] As can be seen from Table 8, the deviation range of the myoglobin concentration value calculated by the measurement method of the present invention from the actual concentration is -8.0% to +9.6%, and the average value of the absolute deviation is 3.2%, which is significantly superior to the comparative method in Example 3 in terms of accuracy. In addition, for 17.3°C used for result verification, the deviation range of the measurement result is -2.4% to 3.4%; for 22.1°C used for result verification, the deviation range of the measurement result is -4.7% to 7.0%; for 27.2°C used for result verification, the deviation range of the measurement result is -5.4% to 5.3%. It can be seen that at these three verification temperature points used for result verification, the measurement method of the present invention is significantly superior to the comparative method in Example 3 in terms of measurement accuracy.

Claims

1. A temperature-corrected analyte detection method, characterized in that The steps of the method include: (1) Insert the test card containing a batch of immunoassay test strips into the test card insertion port of the optical analyzer; (2) Select n segmented temperature points, i.e. t1, t2, ..., t n , t1< t2<… <t n , n is an integer and n ≥ 3, after adding a series of analyte calibrators of different concentrations to the sample addition port of the test card at each segmented temperature point, the measurement signal generated on the detection pad of the immunoassay test strip after adding the analyte calibrator of each concentration is measured using an optical analyzer; (3) With the measurement signal at each segmented temperature point as the X-axis and the concentration of the analyte calibrator as the Y-axis, a total of n equations are obtained, the general formula of which is y m =f(x m ), m is an integer and 1≤m≤n, the equation at the segmented temperature point t1 is y1=f(x1), x1 is the measurement signal obtained when measuring a series of analyte calibrators with different concentrations at t1; the segmented temperature point t n The equation for y is n =f(x n ), x n t n The measurement signal obtained when measuring a series of analyte calibrators at different concentrations; (4) When the test temperature after the test card is inserted into the optical analyzer is set to t, temperature segmentation is performed according to the relationship between t and n segmented temperature points, and different formulas are used for calculation in different temperature segments, as follows: When t ≤ t1, the analyte concentration y = y1; When t1 < t ≤ t2, y = y1 * [(t2 - t) / (t2 - t1)] + y2 * [(t - t1) / (t2 - t1)]; When 2 < m < n and t m-1 < t ≤ t m then, y = y m-1 *[(t m - t) / (t m - t m-1 )] + y m * [(t - t m-1 ) / (t m - t m-1 )]; When t n-1 < t ≤ t n then, y = y n-1 *[(t n - t) / (t n - t n-1 )] + y n * [(t - t n-1 ) / (t n - t n-1 )]; When t>t n When y=y n .

2. The method according to claim 1, wherein The measurement signal obtained in step (2) is the value after arithmetic calculation of the signal T generated on the T line of the detection pad and the signal C generated on the C line of the detection pad after adding the analyte calibrator of each concentration, or the value after arithmetic calculation of the signal T generated on the T line of the detection pad and the signal B generated in the background area of the detection pad after adding the analyte calibrator of each concentration, or the value after arithmetic calculation of the signal T generated on the T line of the detection pad, the signal C generated on the C line of the detection pad, and the signal B generated in the background area of the detection pad after adding the analyte calibrator of each concentration.

3. The method according to claim 2, wherein The measurement signal obtained in step (2) is the T / C or T / (T + C) ratio.

4. The method according to claim 1, wherein 2°C ≤ the difference between any two adjacent segmented temperature points ≤ 6°C.

5. The method according to claim 4, wherein 3°C ≤ the difference between any two adjacent segmented temperature points ≤ 5°C.

6. The method according to claim 1, wherein 4°C ≤ each selected segmented temperature point ≤ 42°C.

7. The method according to claim 6, wherein 15°C ≤ each selected segmented temperature point ≤ 30°C.

8. The method according to claim 7, wherein n=4,y m =f(x m )= , the coefficients a, b, c and d in the two equations corresponding to any two different segmented temperature points are not all the same.

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