Temperature-corrected analyte detection method

Abstract

The invention provides a temperature-corrected analyte detection method, and relates to the following steps: after a series of analyte calibrators with different concentrations are added into an immunoassay test strip, measuring a signal value generated after the analyte calibrator with each concentration is captured on a detection pad of the immunoassay test strip at each segmented temperature point by using an optical analyzer; drawing a corresponding equation by taking the T / C value at each segmented temperature point as an X axis and the concentration of the analyte calibrator as a Y axis; and then, according to the selected n segmented temperature points, carrying out temperature segmentation, and in different temperature segments, carrying out calculation by adopting different formulas. The analyte detection method subjected to temperature correction can be used for more accurately detecting myocardial markers, upper respiratory virus infection, metabolic hormones, digestive tract series markers, allergens and the like on the basis of a time-resolved fluorescence immunoassay test.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection and relates to a temperature-corrected analyte detection method. Background Art

[0002] Immunochromatography analysis technology is one of the commonly used technologies in clinical diagnosis today. Based on the principle of immunochromatography, the labeled signal substances are gathered on the T line and C line of the detection pad. Under the irradiation of the light emitted by the light source, the detection signal (T) and the control signal (C) will be 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] Immunochromatography 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] Most of the above clinical analytes require quantitative results to understand the degree of infection of the patient's disease or the degree of damage to the tissue (or organ). Immunochromatography is a testing technology based on antigen-antibody reaction. For most antigen-antibody reactions, temperature is an important influencing factor. Therefore, liquid antigen-antibody detection represented by chemiluminescence is carried out in a constant temperature environment. However, one of the most important advantages of immunochromatography analysis technology is that the analyzer is small in size and is used with dry analytical reagents, so 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, select some temperature points (15℃, 20℃, 25℃, 30℃) in the allowable temperature range of 15~30℃ as the experimental temperature, and then select a series of analytes for testing, for example, based on the clinical myoglobin test range of 20~1000mg / L, select 25, 50, 100, 300, 500, 800, 1000mg / L of myoglobin reference for testing. Select a certain temperature point as the reference temperature, such as 25℃, divide the T / C value measured at other temperature points by the T / C value measured at 25℃ to obtain the T / C ratio, calculate the average T / C ratio of different concentrations of myoglobin reference at a certain temperature point as its temperature correction coefficient relative to the reference temperature (note: the temperature correction coefficient of the reference temperature itself is defined as 1), and then use the selected temperature point as the X-axis and the average T / C ratio at each temperature point as the Y-axis to obtain the temperature correction equation. The standard curve equation is obtained by taking the T / C value measured at the reference temperature (25°C) as the X-axis and the analyte concentration value as the Y-axis. When calculating the results, first substitute the test temperature of the test card environment into the temperature correction equation to calculate the temperature correction coefficient, then divide the T / C value measured at the 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 analyte concentration value.

[0006] The premise of the temperature correction coefficient method is that the temperature correction coefficients calculated from analyte references of different concentrations must be similar and cannot vary too much. However, in the actual detection of some analytes, the temperature correction coefficients calculated using analyte references of different concentrations vary greatly, which can lead to inaccurate test results. Summary of the invention

[0007] In order to solve the shortcomings 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 segments the temperature according to the selected segmented temperature points. In 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 temperature correction coefficients calculated using analyte reference products with different concentrations can be significantly reduced.

[0008] The present invention provides a temperature-corrected analyte detection method, the method comprising the steps of: (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, namely t1, t2, …, t n , t1< t2<… <t n, where n is an integer and n≥3. After adding a series of analyte calibrators with different concentrations to the sample inlet of the test card at each segmented temperature point, a light analyzer is used to measure the measurement signals generated on the detection pad of the immunoassay strip after adding each concentration of the analyte calibrator; (3) Using the measurement signals at each segmented temperature point as the X-axis and the concentrations of the analyte calibrators as the Y-axis, a total of n equations are obtained. The 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 calibrators 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 calibrators with different concentrations at t n ; (4) When the test temperature after the test card is inserted into the light analyzer is set to t, according to the relationship between t and the n segmented temperature points, temperature segmentation is performed, 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, 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 )]; 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 )]; When t > t n ), y = y n .

[0009] In some cases of the present invention, the method further comprises the following steps: (5) providing a new immunoassay test strip belonging to the batch, adding a clinical sample to a sample inlet of the new immunoassay test strip, measuring a measurement signal obtained on a detection pad of the immunoassay test strip after the clinical sample is added using an optical analyzer, and providing a test temperature t after the test card is inserted into the optical analyzer; (6) Select an appropriate formula based on the temperature range in which t is located in (4), and calculate the analyte concentration in the clinical sample based on the measurement signal generated in (5).

[0010] In some cases of the present invention, the measurement signal obtained in step (2) is the numerical value 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 the addition of each concentration of the analyte calibrator, or the numerical value 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 the addition of each concentration of the analyte calibrator, or the numerical value 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 the addition of each concentration of the analyte calibrator. In some cases of the present invention, the measurement signal obtained in step (2) is the ratio of T / C or T / (T+C). In the present invention, the background area of ​​the detection pad is the area on the detection pad excluding the T line and the C line.

[0011] In some cases of the present invention, in the n general formula y m =f(x m ), one segmented temperature point corresponds to one equation, and the functional relationship of each equation is the same, but all the equation coefficients in the two equations corresponding to any two different segmented temperature points are not all the same, which means that there may be some similarities, but not all the same, or all of them may be different. For example, when n=4, there are 4 equations, namely, 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 segmented temperature point, and can be selected from double logarithmic trinomial equations, cubic polynomial equations and other equations. When it is a double logarithmic trinomial equation, assume that the general formula of the equation y m =f(x m )= , where a, b, c and d are equation coefficients. The equation coefficients in the equations at different segmented temperature points are not all the same; when it is a cubic polynomial equation, let the general formula of the equation y m =f(x m )=a+b x+c x 2 +d x 3 , where a, b, c and d are equation coefficients, and the equation coefficients in the equations at different segmented temperature points are not all the same. In other cases of the present invention, in the n general formula y m =f(x m ), one segmented temperature point corresponds to one equation, and the functional relationship of each equation is 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.

[0012] In some cases of the present invention, when multiple analytes are detected simultaneously, such as detecting multiple respiratory viruses, temperature segmentation can be performed when only one of the analytes is detected, or temperature segmentation can be performed when each analyte is detected.

[0013] In some instances of the invention, n is selected from 3, 4, 5, 6, 7 or 8. In some instances of the invention, n=4 or 5. In some instances of the invention, n=4.

[0014] 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.

[0015] 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.

[0016] In some cases of the present invention, n=4, y m =f(x m )= , all the equation coefficients in the two equations corresponding to any two different segmented temperature points are not all the same.

[0017] In some cases of the 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 calibrators are 25.78, 47.33, 101.6, 306.2, 502.1, 734.0, and 1010.0 ng / mL, respectively; The equation at t1: y1= ; The equation at t2: y2= ; Equation at t3: y3 = ; Equation at t4: y4 = ; The selection of the formula is as follows: When t ≤ t1, y = y1; When t1 < t ≤ t2, y = y1*[(t2 - t) / (t2 - t1)] + y2*[(t - t1) / (t2 - t1)]; When t2 < t ≤ t3, y = y2*[(t3 - t) / (t3 - t2)] + y3*[(t - t2) / (t3 - t2)]; When t3 < t ≤ t4, y = y3*[(t4 - t) / (t4 - t3)] + y4*[(t - t3) / (t4 - t3)]; When t > t4, y = y4.

[0018] 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 measure the ambient temperature of the place where it is located using an external thermometer, and then input the measured ambient temperature into the optical analyzer through the user interface, or it can be measured by a thermistor provided on the surface of the optical analyzer.

[0019] In the present invention, this temperature-corrected analyte detection method can be used to more accurately detect myocardial markers, international public health communication diseases, metabolic hormones, digestive tract series markers, respiratory tract series markers, and allergens. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Exploded schematic view of the test card used in the present invention.

[0021] Figure 2 Schematic view of the immunoassay strip in the test card used in the present invention.

[0022] Figure 3 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.

[0023] Figure 4 The signal curve is obtained with the signal 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.

[0024] Figure 5 The logarithmic signal curve is obtained by taking the logarithmic value of the signal 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. DETAILED DESCRIPTION

[0025] like Figure 1 and Figure 2 As 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, including a sample pad 11, a marking pad 12, a detection pad 13 and a sample absorption pad 14 which are overlapped in sequence. The detection pad 13 is made of materials such as nitrocellulose, glass fiber, polyethersulfone or nylon, 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 pad 11 is made of a water-absorbing material, and glass fiber or non-woven fabric can be selected. The marking pad 12 is also made of a water-absorbing material, and polyester film, glass fiber or non-woven fabric can be selected.

[0026] The number of detection lines set on the detection pad 13 can be adjusted according to actual needs. For example, when detecting one analyte, only one detection line is needed. When detecting two or more analytes, a corresponding number of detection lines should be set, such as setting 4 T lines to jointly detect multiple upper respiratory tract viruses, such as 4 upper respiratory tract viruses, namely, new coronavirus, influenza A virus, influenza B virus, adenovirus and respiratory syncytial virus. The area in the detection pad except the T line and the C line is the background area of ​​the detection pad.

[0027] The test strip 15 also includes a bottom support layer 17, which is made of common hydrophobic materials such as polyvinyl chloride to ensure that the sample cannot leak out of the bottom support layer 17. The detection pad 13 is arranged on the bottom support layer 17. The sample loading pad 11 is arranged on the bottom support layer 17, and one end of the sample loading pad 11 partially overlaps with the marking pad 12; the marking pad 12 is arranged on the bottom support layer 17, and one end of the marking pad 12 partially overlaps with the sample loading pad 11, and the other end of the marking pad 12 partially overlaps with the detection pad 13; the sample absorption pad 14 is arranged 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 mm long.

[0028] The test strip 1 is located in a shell, and the shell is composed of a card cover 2 and a card base 3 by ultrasonic welding, snapping or gluing. In some cases, the card cover 2 and the card base 3 are made of plastic. A test strip groove 32 is provided in the middle of the card base 3 for placing the test strip 1. Preferably, the card cover 2 is provided with a plurality of snaps extending downward (not shown in the figure), and the card base 3 is provided with a plurality of card grooves 31 extending upward. The snaps provided on the card cover 2 and the card grooves 31 provided on the card base 3 correspond to each other one by one, so that when the card cover 2, the test strip 1 and the card base 3 are assembled together, the card cover 2 and the card base 3 can be firmly fixed together, and the test strip 1 is fixed in the test strip groove 32.

[0029] The card cover 2 is also provided with an observation window 21 and a sample addition port 22. After the test card is inserted into the test card insertion port of the optical analyzer, when the clinical sample is added through the sample addition port 22, the sample enters the sample addition pad 11 located below the sample addition port 22, and under the capillary action, the sample migrates to the sample absorption pad 14 along the length direction of the test strip 1. The observation window 21 is arranged above the detection line 15 and the control line 16 of the detection pad 13. The light emitted by the light source in the optical analyzer can be irradiated onto the detection line 15 and the control line 16 of the test strip 1 through the transparent or translucent observation window 21. After being irradiated by the light from the light source, the light signal generated by the detection 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 select a commercially available optical analyzer, such as a dry fluorescent immunoassay analyzer iFIA-100 (Aikang Biotechnology (Hangzhou) Co., Ltd.).

[0030] Depending on the different analytes to be tested (such as antigens, antibodies or haptens) and the different immunoassay principles (double antigen sandwich method, double antibody sandwich method, competitive method, indirect method, capture method), the substances coated on the label pad 12 and the detection line 15 will vary. Here, the analyte is myoglobin and the detection principle is the double antibody sandwich method as an example for explanation. The label pad 12 is coated with a first reagent (a first anti-myoglobin antibody) labeled with a signal marker and a first quality control reagent (such as rabbit IgG) labeled with a signal marker, and the detection line 15 is coated with a second reagent (a second anti-myoglobin antibody). The first reagent and the second reagent specifically bind to myoglobin in the clinical sample, so that when the clinical sample is added to the sample pad 11 through the sample addition port 22, the clinical sample flows along the length direction of the test strip 1. When the clinical sample reaches the label pad 12, the first reagent labeled with the signal marker specifically binds to the myoglobin (if present) in the clinical sample, and the signal marker-first reagent-myoglobin complex formed continues to flow and specifically binds to the second reagent on the detection line 15, thereby capturing the signal marker-first reagent-myoglobin complex on the detection line 15, and generating a detection signal related to the concentration of myoglobin after being irradiated by the light from the light source of the optical analyzer. At the same time, the first quality control reagent labeled with the signal marker continues to flow, and 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 the signal marker on the control line 16, and after being irradiated by the light from the light source, the signal marker captured on the control line 16 generates a control signal. The generated detection signal T and control signal can be light 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.

[0031] The signal marker 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. The time-resolved luminescent marker has the characteristic of delayed luminescence, that is, when the excitation light emitted by the light source is turned off, it can still continue to emit light for a certain period of time. The time-resolved luminescent marker can exist in molecular form, called time-resolved luminescent molecules, which can be selected from lanthanides such as samarium (Sm(III)), dysprosium (Dy(III)), europium (Eu(III)) and 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-isothiocyanatophenyl)-diethylenetriaminetetraacetic acid-Eu +3Time-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. In view of the fact that each time-resolved luminescent microsphere can encapsulate thousands of time-resolved luminescent molecules, the detection sensitivity is effectively improved. 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.

[0032] The time-resolved luminescent markers in the present invention have the characteristic of delayed luminescence, that is, when the excitation light emitted by the light source is turned off, they can still continue to generate emission light for a certain period of time. The wavelength of the emission light can be greater than the wavelength of the excitation light, or it can be less than the wavelength of the excitation light. This characteristic of delayed luminescence makes the emission light generated by the time-resolved luminescent marker after excitation have a longer lifespan. In this way, in actual detection, after the excitation light from the light source excites the time-resolved luminescent marker, the light source can be turned off and wait for a period of time, and then the emission light signal emitted by the time-resolved luminescent marker can be detected, thereby eliminating the interference of the background light signal with a shorter lifespan and the scattered excitation light.

[0033] Time-resolved luminescent markers can exist in molecular form, called time-resolved luminescent molecules, which can be selected from lanthanide elements such as samarium (Sm(III)), dysprosium (Dy(III)), europium (Eu(III)) and terbium (Tb(III)) and their chelates, as well as up-conversion luminescent materials; platinum / palladium porphyrin compounds that can emit phosphorescence after excitation. The delay time of europium chelates is about 2ms, and the emitted fluorescence signal gradually decays over time within 2ms. Considering that some proteins in clinical samples can also produce fluorescence under the irradiation of light generated by the light source, but have no delay characteristics, most proteins produce fluorescence and the fluorescence disappears completely 200μs after the light source is turned off. Therefore, time-resolved fluorescence detection based on europium chelates usually chooses to collect fluorescence signals 200 to 400μs after the light source is turned off. A suitable europium chelate is N-(p-isothiocyanatophenyl)-diethylenetriaminetetraacetic acid-Eu +3 .

[0034] 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. In view of the fact that each time-resolved luminescent microsphere can encapsulate thousands of time-resolved luminescent molecules, the detection sensitivity is effectively improved. Therefore, the time-resolved luminescent marker is preferably a time-resolved luminescent microsphere. Time-resolved luminescent microspheres that produce fluorescence after being irradiated with light are called time-resolved fluorescent microspheres.

[0035] Upconversion luminescent materials refer to materials that emit high-energy light when exposed to low-energy light. The light signal generated by the upconversion luminescent material after exposure to light has strong stability, long life and high sensitivity, and can also be 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.

[0036] The colored luminescent microspheres in the present invention refer to microspheres or microbeads with luminescent compounds such as quantum dots and fluorescent dyes wrapped on the surface or inside, which can generate light signals without luminescence delay characteristics when irradiated with 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 fluorescence of multiple specific colors).

[0037] The luminescent compound in the present invention refers to light that can generate no 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 and improved variants thereof that can emit fluorescence after being irradiated with light, 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-dioxyethane derivatives (common ones include AMPPD, CSPD, CDP and CDP-Star, as well as PPD, Lumi-Phos and Lumi-Plus of Lumigen, etc.) and acridinium esters or acridinium sulfonamides.

[0038] The colored colloidal particles in the present invention refer to colloidal particles that aggregate on the T line and / or C line during the immunochromatographic reaction to produce colored aggregates, and can be selected from latex, colloidal gold, colloidal carbon and colloidal selenium. After being irradiated by light from a light source, the reflected light generated by the colored colloidal particles on the T line or C line can be used as a detection signal or a control signal after detection.

[0039] In the time-resolved luminescent microspheres and colored luminescent microspheres, the polymer forming the microspheres or microbeads can be selected from polystyrene, butadiene styrene, styrene acrylic acid-ethylene terpolymer, polymethyl methacrylate, polyethyl methyl acrylate, styrene-maleic anhydride copolymer, polyvinyl acetate, polyvinyl pyridine, polydivinyl benzene, polybutylene terephthalate, acrylonitrile, vinyl chloride-acrylate, etc., or their aldehyde, carboxyl, amino, hydroxyl, hydrazide derivatives, or mixtures thereof. In addition, the surface of the microspheres or microbeads usually carries hydroxyl, carboxyl, amino, aldehyde, sulfonic and other groups, which can be coupled to 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 20nm to 100μm.

[0040] When detecting one or more analytes in a sample, 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 according to different immunoassay principles, wherein, 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 the immunoassay principle used, 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.

[0041] The clinical samples in the present invention may be selected from serum, plasma, whole blood, cerebrospinal fluid, urine, bronchoalveolar lavage fluid, nasopharyngeal swab, sputum, feces, skin lesion samples (including swabs of rash / acne exudate; acne fluid; acne scabs, etc.), etc. Depending on the type of clinical samples and analytes, some clinical samples can only be added to the test card for detection after being pre-treated (such as lysed with a lysing solution to release the analyte to be detected) before detection.

[0042] 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 markers, such as 25-hydroxyvitamin D, β-CrossLaps, bone alkaline phosphatase, calcitonin, parathyroid hormone, human N-terminal mid-section osteocalcin and total type I procollagen amino-terminal extended peptide, etc.; sex markers, such as follicle-stimulating hormone (FSH), luteinizing hormone (CEA), prostate-specific antigen (PSA), neuron-specific enolase (NSE), CYFRA21-1, CA19-9, CA50, CA125, CA153, CA724, etc. lutein (LH), prolactin, progesterone, testosterone, estradiol, estriol, beta-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 antibodies and thyroid peroxidase antibodies, etc.; infectious disease markers, such as upper respiratory tract virus (such as new coronavirus, influenza A virus, influenza B virus, respiratory syncytial virus, adenovirus, rhinovirus, parainfluenza virus, etc.) antigen / antibody, monkeypox virus antigen / antibody, gonococcal 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, 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 markers, such as intestinal adenovirus antigen, rotavirus antigen, enterovirus 71 IgG / IgM, coxsackievirus group A type 16 IgG / IgM, coxsackievirus group B IgG / IgM, calprotectin, Clostridium difficile toxin A, Clostridium difficile toxin B, Clostridium difficile glutamate dehydrogenase, etc.; cocaine and morphine for the assessment of drug abuse, etc.

[0043] Example 1: Preparation of the Immunoassay Strips and Test Cards of the Present Invention A method for preparing an immunoassay strip and a test card for quantitatively detecting an analyte in a sample (e.g., IL-6, NT-proBNP, myoglobin, and upper respiratory tract virus antigens or antibodies, etc., myoglobin is used as an example for illustration) comprises the following steps: A. Antibody preparation: Paired monoclonal antibodies or polyclonal antibodies for detecting myoglobin can be screened out by antigen immunization of mice, rats, rabbits and other animals or hybridoma cell counting. Commercial myoglobin paired antibodies (one is myoglobin capture antibody and the other is myoglobin detection antibody) can also be selected. In this embodiment, commercial myoglobin paired antibodies (purchased from Jiangsu Dongkang Biopharmaceutical Technology Co., Ltd., item number C3601: lot 20221107 and C3604: lot 20210326) are used as an example for description. Rabbit IgG antibody and goat anti-rabbit IgG antibody are prepared by themselves or purchased from the market.

[0044] B. Detection pad liquid Myoglobin capture antibody was added to 0.02M phosphate buffer (pH 7.2) to obtain a T line solution, the concentration of myoglobin capture antibody in the T line solution was 1 mg / ml, goat anti-rabbit IgG antibody was added to 0.02M phosphate buffer (pH7.2) to obtain a C line solution, the concentration of goat anti-rabbit IgG antibody in the C line solution was 0.3 mg / ml, and then a quantitative liquid spotting device (AUTOKUN continuous film spotting device HGS101, Hangzhou Fenghang Technology Co., Ltd.) was used to coat the T line solution and the C line solution at intervals of 0.8 cm on a 2.5 cm wide nitrocellulose membrane used as a detection pad (purchased from Satorious, model 1UN95ER100025NT) at 1 μl / cm, thereby forming a T line and a C line, respectively, and dried at 45°C for 18 hours, and sealed with a desiccant for later use.

[0045] C. Preparation of fluorescent microspheres: Selection of fluorescent microspheres: Time-resolved fluorescent microspheres with europium chelate wrapped inside (Merk Company, product number F1-XC010), with an excitation wavelength of 365nm and an emission wavelength of 615nm.

[0046] Preparation of MES buffer: Add sodium morpholineethanesulfonic acid salt into pure water and mix well to make the concentration of sodium morpholineethanesulfonic acid salt 1.0% (w / v).

[0047] Preparation of storage buffer: 50 mM Tris-HCl buffer (pH 8.0).

[0048] 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 their final concentrations 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 the reaction was carried out at room temperature for 1.5 hours. After being fully washed with MES buffer, 0.05 M Tris-HCl buffer (pH 5.0) containing 10% BSA (w / v) was added. 8.0), blocked at room temperature for 1 hour, washed the time-resolved fluorescent microspheres with MES buffer, and re-dissolved the time-resolved fluorescent microspheres with storage buffer to make the final concentration of the detection time-resolved fluorescent microspheres 5 mg / ml, and stored at 4°C for later use; in the same manner as the preparation of the time-resolved fluorescent microsphere-labeled myoglobin detection antibody, prepared the time-resolved fluorescent microsphere-labeled rabbit IgG antibody, and used the storage buffer to make the final concentration of the time-resolved fluorescent microsphere-labeled rabbit IgG antibody 5 mg / ml, and stored at 4°C for later use.

[0049] 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, wherein the fluorescent labeling diluent was 50mM 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).

[0050] D. Spraying and drying of fluorescent microspheres Use the special streaking head of AUTOKUN continuous streaking machine HGS101 to spray the prepared fluorescent microsphere mixture evenly on a 1.0 cm wide marking pad (glass fiber) at a rate of 2 μl / cm, dry at 45°C for 18 hours, add desiccant and seal for later use.

[0051] E. Sample Pad Treatment Place a 2.5 cm wide sample pad in the sample pad treatment solution and soak for 1 hour, then take it out and dry it at 37°C overnight (12 to 24 hours). The sample pad treatment solution is a buffer containing 50 mM (pH 8.0) Tris-HCl buffer, 1% BSA (w / v) and 0.5% Tween-20 (v / v).

[0052] F. Assembly and cutting of test strips Assembly of the test strip: manually or by machine, adhesively adhere a 2.5 cm wide sample pad, a 1.0 cm wide marking pad, a 2.5 cm wide nitrocellulose membrane, and a 2.5 cm wide absorbent paper (as a sample absorption pad) to an 8 cm long plastic base plate (as a bottom support layer), so that the 2.5 cm wide sample pad, the 1.0 cm wide marking pad, the 2.5 cm wide nitrocellulose membrane, and the 2.5 cm wide absorbent paper are staggered and overlapped by 2.0 mm in sequence to form a test strip.

[0053] Cutting of test strips: Use AUTOKUN HGS201 type strip cutter to cut the assembled test strips into single-serving test strips with a width of 4 mm.

[0054] G. Assembly of test card Place the cut single-dose 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 card cover to ensure that the entire immunoassay test strip is in a taut state. Add desiccant and seal at room temperature for later use.

[0055] After the test card is assembled, it can be placed in a test kit containing a desiccant and instructions for use. In addition, the test kit can also be filled with a sampling tube, a test tube containing a lysate, a test tube containing a sample diluent, a sampling swab, etc. as needed.

[0056] Example 2: Detection method The test card prepared in Example 1 is used to detect analytes in samples (e.g., IL-6, NT-proBNP, myoglobin, and upper respiratory virus antigens or antibodies, etc., myoglobin is used as an example for illustration here), and the specific process is as follows: 100 μl of sample (here a whole blood sample is used as an example for illustration) is added to the sample addition port of the test card. After incubation for 15 minutes, the test card is inserted into the iFIA-100 dry fluorescent immunoassay analyzer (Aicon Biotechnology (Hangzhou) Co., Ltd.) for detection.

[0057] During the detection, the light source is first allowed to emit light with a wavelength of 360nm to illuminate the time-resolved fluorescent microspheres captured on the T line and the C line, and then the light source is turned off. The time-resolved fluorescent microspheres captured on the T line and the C line emit a time-resolved fluorescent signal with a wavelength of 615nm. After the light source is turned off for 300μs, the detector of the optical analyzer is used to capture and count, and the time-resolved fluorescent signal (T signal) emitted on the T line and the time-resolved fluorescent signal (C signal) emitted on the C line are obtained respectively.

[0058] In order to correct the adverse effects of manufacturing differences in the manufacturing process of the same batch of test cards, 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, recorded as T / C, or T / (T+C). According to the pre-determined calibration curve, the T / C or T / (T+C) obtained after adding the clinical sample can be used to determine the concentration of myoglobin.

[0059] When preparing a series of different concentrations of myoglobin calibrators, serum samples with high myoglobin concentration and serum samples with low myoglobin concentration were collected to prepare 7 different concentrations of calibrators, which were tested using the Roche Cobas e411 fully automatic electrochemiluminescence analyzer (SN#8866-29) and the Roche matching 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) were used as the concentration reference values ​​of the 7 reference products. Then, these different concentrations of calibrators were added to the same batch of test cards, and after incubation for 15 minutes, the T signal and C signal of each test card were detected using the dry fluorescent immunoassay analyzer iFIA-100 (Aicon Biotechnology (Hangzhou) Co., Ltd.), and the T / C or T / (T+C) ratio was calculated. A calibration curve is drawn according to each T / C or T / (T+C) ratio and its corresponding myoglobin concentration value, which is used to calculate the myoglobin concentration in the clinical sample.

[0060] Of course, a series of myoglobin calibrators of different concentrations can also be prepared using calibrator diluent (such as PBS buffer) and myoglobin.

[0061] In addition, the measurement of myoglobin calibrators of different concentrations by immunoassay analyzer is carried out at a certain temperature. In order to ensure that the immunoassay analyzer, test card and supporting sample diluent (20mM PBS, 0.02% Proclin-300 and 0.1% Tween-20) and a series of myoglobin calibrators of different concentrations are at a specific temperature, they can be placed at this temperature for more than 1 hour to allow their temperature to reach equilibrium.

[0062] Example 3: Comparative Method Using the test card in Example 1 and the measurement method in Example 2, different concentrations of myoglobin calibrators (concentrations were 25.78, 47.33, 101.6, 306.2, 502.1, 734.0 and 1010.0 ng / mL, respectively) were measured at four selected temperature points (15.1°C, 19.9°C, 24.4°C and 29.5°C), and the results are shown in Table 1.

[0063] Table 1

[0064] 24.4℃ was selected as the reference temperature, and the T / C ratio was obtained by dividing the T / C value measured at each temperature point (15.1℃, 19.9℃ and 29.5℃) by the T / C value measured at 24.4℃. The average T / C ratio of different myoglobin concentrations at each temperature point was calculated as the temperature correction coefficient of each temperature point relative to the reference temperature. The results are shown in Table 2.

[0065] Table 2

[0066] Next, based on the data in Table 2, the temperature point is used as the X-axis and the average value of the T / C ratio is used as the Y-axis to obtain the temperature correction curve (eg Figure 3 ). Figure 3 It can be seen that the temperature correction coefficient is not linear with temperature, and the temperature correction equation obtained is: tcf=-0.4299+0.1120t -0.002373t 2 +0.000007617t 3 , where t is temperature and tcf is the temperature correction coefficient corresponding to each temperature point.

[0067] The signal curve is drawn with 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, such as Figure 4 shown.

[0068] Figure 4 Each test point in the curve is basically exponentially distributed. To this end, the logarithmic signal curve is drawn with the logarithmic value of the T / C value measured at the reference temperature of 24.4°C as the X-axis and the logarithmic value of the myoglobin concentration of different myoglobin calibrators as the Y-axis, as shown in Figure 5 shown.

[0069] Depend on Figure 5 The standard curve equation for the myoglobin concentration in the sample is as follows: y= .

[0070] When actually measuring clinical samples (such as plasma, serum or whole blood) with unknown analyte concentrations, after adding the clinical samples, the ambient temperature (which can be measured 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) provided in the optical analyzer. Here, the external air temperature of the optical analyzer is measured by a thermometer as an example for explanation. At the same time, according to the measurement method in Example 2, the T / C value after adding the clinical sample at the test temperature is obtained. When calculating the results, the measured test temperature is first substituted into the temperature correction equation to calculate the temperature correction coefficient, and then the obtained T / C value is divided by the temperature correction coefficient to obtain the T / C correction value, as shown in Table 3.

[0071] Table 3

[0072] 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) were added, and the myoglobin calibrator of different concentrations was measured at these three verification temperature points using the test card in Example 1 and the measurement method in Example 2, and the measured T / C values ​​are shown in Table 4. At the same time, each verification temperature point was substituted into the temperature correction equation to calculate the temperature correction coefficient, and then the measured T / C value was divided by the temperature correction coefficient to obtain the T / C correction value, and the corrected T / C value is shown in Table 4.

[0073] Table 4

[0074] Finally, the T / C correction values ​​in Tables 3 and 4 were substituted into the standard curve equation to obtain the concentration values ​​of myoglobin, as shown in Table 5.

[0075] Table 5

[0076] Using the data in Table 5, the deviation between the temperature-corrected myoglobin concentration value and the actual concentration can be calculated = (temperature-corrected myoglobin concentration value - actual concentration) / actual concentration. The calculated deviation results are shown in Table 6.

[0077] Table 6

[0078] It can be seen from Table 6 that the deviation range of the myoglobin concentration measurement results obtained by the temperature correction coefficient method is -13.0%~+12.0%, and the average value of the absolute value of the deviation is 5.7%. In addition, it can be seen from Table 6 that for 17.3℃ used for result verification, the deviation range of the measurement results is -11.9%~8.7%; for 22.1℃ used for result verification, the deviation range of the measurement results is -13.0%~7.3%; for 27.2℃ used for result verification, the deviation range of the measurement results is -5.6%~12.0%.

[0079] Example 4: Testing method of the present invention It can be seen from Table 2 in Example 3 that for the same concentration of myoglobin calibrator, when the temperature increases from 15.1°C to 29.5°C, the temperature correction coefficient at different temperatures generally shows an upward trend under the higher concentration of myoglobin calibrator. However, when the myoglobin calibrator is a lower concentration (such as 25.78ng / mL and 47.33ng / mL), the temperature correction coefficient at different temperatures shows a trend of first increasing and then decreasing. In addition, at the same temperature, the temperature correction coefficients measured for myoglobin calibrators of different concentrations show different trends. For example, at 15.1°C and 19.9°C, as the concentration of myoglobin increases, the temperature correction coefficient shows a trend of first decreasing and then increasing; at 29.5°C, as the concentration of myoglobin increases, the temperature correction coefficient generally shows an upward trend.

[0080] It can be seen that the temperature correction coefficient shows different trends at different temperatures and different concentrations of myoglobin calibrators, and the calculated temperature correction coefficients are not similar under different concentrations of myoglobin calibrators, and there are large fluctuations. As a result, the deviation range of the myoglobin concentration measurement results obtained by the temperature correction coefficient method is -12.5% ​​to +15.1% (see Table 6), which leads to inaccurate test results.

[0081] Based on this, the present invention selects n segmented temperature points (n is an integer and n≥3) within the temperature range for measuring myoglobin concentration (usually 4 to 42°C, in some cases, 15 to 30°C). For ease of comparison, the selection scheme of the segmented temperature points is the same as that in Implementation 3 (the 4 segmented temperature points are selected as follows: t1=15.1°C, t2=19.9°C, t3=24.4°C and t4=29.5°C), and the T / C values ​​of the initial measurement are shown in Table 1. At the same time, for the purpose of result verification, in addition to the selected 4 segmented temperature points, 3 verification temperature points (17.3°C, 22.1°C and 27.2°C) are added, and the test card in Example 1 and the measurement method in Example 2 are used to measure different concentrations of myoglobin calibrators at these three verification temperature points, and the T / C values ​​of the initial measurement are shown in Table 4.

[0082] Then, the T / C value measured at each selected segmented temperature point is taken as the X-axis, and the myoglobin concentration value is taken as the Y-axis to obtain the general formula of the standard curve equation at each segmented temperature point: m =f(x m )= , where m is an integer and 1≤m≤n, a, b, c and d are equation coefficients, which are different at different segmented temperature points, and x m is the segment temperature point t m T / C value measured under m is the myoglobin concentration.

[0083] 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: 15.1℃:y1=f(x1)= ;

[0084] 19.9℃:y2=f(x2)= ;

[0085] 24.4℃:y3=f(x3)= ;

[0086] 29.5℃:y4=f(x4)= .

[0087] In view of the large differences in temperature correction coefficients at different temperatures in the prior art, the present invention divides the temperature into segments according to the relationship between the test temperature in the environment where the test card is located and the temperature points of different segments after the test card is inserted into the optical analyzer. Different formulas are used for calculation in different temperature segments. The specific segments are as follows: When the measured temperature t≤t1, y= y1; When t1<measured temperature t≤t2, y= y1*[(t2-t) / (t2-t1)]+y2*[(t-t1) / (t2-t1)]; When t2<measured temperature t≤t3, y= y2*[(t3-t) / (t3-t2)]+y3*[(t-t2) / (t3-t2)]; When t3<measured temperature t≤t4, y= y3*[(t4-t) / (t4-t3)]+y4*[(t-t3) / (t4-t3)]; When the measured temperature t >t4, y=y4.

[0088] Based on the initial T / C values ​​provided in Tables 1 and 4, the myoglobin concentration values ​​calculated at different test temperatures are shown in Table 7.

[0089] Table 7

[0090] Using the data in Table 7, the deviation between the myoglobin concentration value in Table 7 and the actual concentration can be calculated = (myoglobin concentration value in Table 7 - actual concentration) / actual concentration. The calculated deviation result is shown in Table 8.

[0091] Table 8

[0092] As shown in 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%~+9.6%, and the average value of the absolute value of the deviation is 3.2%, which is significantly better than the comparison 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%~3.4%; for 22.1°C used for result verification, the deviation range of the measurement result is -4.7%~7.0%; for 27.2°C used for result verification, the deviation range of the measurement result is -5.4%~5.3%. It can be seen that at these three verification temperature points for result verification, the measurement method of the present invention is significantly better than the comparison 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 a test card containing a batch of immunoassay strips into the test card insertion port of the optical analyzer; (2) Select n segmented temperature points, namely t1, t2, …, t n , t1< t2<… <t n , n is an integer and n≥3, after a series of analyte calibrators of different concentrations are added 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 each concentration of the analyte calibrator is measured by an optical analyzer; (3) With the measured 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 n =f(x n ), x n t n The measurement signal obtained when measuring a series of different concentrations of the analyte calibrator; (4) When the test temperature after the test card is inserted into the optical analyzer is set to t, according to the relationship between t and n segmented temperature points, perform temperature segmentation, and use different formulas for calculation in different temperature segments, specifically 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 , 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, characterized in that The method further includes the following steps: (5) Provide a new immunoassay strip belonging to the batch, add a clinical sample to the sample addition port of the new immunoassay strip, and use the optical analyzer to measure the measurement signal obtained on the detection pad of the immunoassay strip after adding the clinical sample and the test temperature t after the test card is inserted into the optical analyzer; (6) According to the temperature segment where t is located in (4), select an appropriate formula, and calculate the analyte concentration in the clinical sample according to the measurement signal generated in (5).

3. The method according to claim 1, characterized in that 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.

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

5. The method according to claim 1, characterized in that 2°C ≤ the difference between any two adjacent segmented temperature points ≤ 6°C.

6. The method according to claim 5, characterized in that 3°C ≤ the difference between any two adjacent segmented temperature points ≤ 5°C.

7. The method according to claim 1, characterized in that 4°C ≤ each selected segmented temperature point ≤ 42°C.

8. The method according to claim 7, characterized in that 15°C ≤ each selected segmented temperature point ≤ 30°C.

9. The method according to claim 1, characterized in that The analyte is selected from myoglobin.

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

11. The method according to claim 10, characterized in that n = 4, t1 = 15.1°C, t2 = 19.9°C, t3 = 24.4°C, t4 = 29.5°C, the analyte is selected from myoglobin, and the concentrations of a series of analyte calibrators are 25.78, 47.33, 101.6, 306.2, 502.1, 734.0, and 1010.0 ng / mL respectively; The equation at t1: y1= ； The equation at t2: y2= ； The equation at t3: y3= ； The equation at t4: y4= ； The selection of the formula is as follows: When t ≤ t1, y = y1; When t1 < t ≤ t2, y = y1 * [(t2 - t) / (t2 - t1)] + y2 * [(t - t1) / (t2 - t1)]; When t2 < t ≤ t3, y = y2 * [(t3 - t) / (t3 - t2)] + y3 * [(t - t2) / (t3 - t2)]; When t3 < t ≤ t4, y = y3 * [(t4 - t) / (t4 - t3)] + y4 * [(t - t3) / (t4 - t3)]; When t >t4, y=y4.

Images