Temperature-corrected analyte detection method

Abstract

The present 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, using an optical analyzer to measure, at each segmented temperature point, a signal value generated after the analyte calibrator of each concentration is captured on a detection pad of the immunoassay test strip; drawing a corresponding equation by taking a 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, performing temperature segmentation according to n selected segmented temperature points, and in different temperature segments, using different formulas to perform calculation. The temperature-corrected analyte detection method can be used for more accurately detecting myocardial markers, upper respiratory tract viral infections, metabolic hormones, gastrointestinal panel markers, allergens, and the like on the basis of time-resolved fluorescence immunoassay.

Description

A temperature-corrected method for analyte detection Technical Field

[0001] This invention belongs to the field of biological detection technology and relates to a temperature-corrected method for detecting analytes. Background Technology

[0002] Immunochromatographic analysis is one of the commonly used techniques in clinical diagnosis today. Based on the principle of immunochromatography, labeled signal substances accumulate on the T and C lines of the detection pad. Under the illumination of light emitted by a light source, a detection signal (T) and a control signal (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] 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 extent of infection or tissue (or organ) damage in patients. Immunochromatography is a testing technique based on antigen-antibody reactions, and temperature is a significant influencing factor for most antigen-antibody reactions. Therefore, liquid-based antigen-antibody detection methods, such as chemiluminescence, are performed in a constant-temperature environment. However, one of the most important advantages of immunochromatographic analysis is the small size of the analyzer and its use with dry analytical reagents, making temperature calibration extremely important.

[0005] A commonly used temperature correction method is the temperature correction coefficient method: Several test temperatures are selected within the allowable temperature range of the reagent. For example, within the allowable temperature range of 15–30℃, several temperature points (15℃, 20℃, 25℃, 30℃) are selected as experimental temperatures. Then, a series of analyte concentrations are selected for testing. For instance, based on the clinical testing range of myoglobin (20–1000 mg / L), myoglobin reference standards of 25, 50, 100, 300, 500, 800, and 1000 mg / L are selected for testing. A specific temperature point is selected as the reference temperature, such as 25℃. The T / C ratio is obtained by dividing the T / C value measured at other temperature points by the T / C value measured at 25℃. The average T / C ratio of different concentrations of myoglobin reference standards at a specific temperature point is calculated as its temperature correction coefficient relative to the reference temperature (note: the temperature correction coefficient for the reference temperature itself is defined as 1). The selected temperature point is then used as the X-axis, and the average T / C ratio at each temperature point is used as the Y-axis to obtain the temperature correction equation. The standard curve equation is obtained by using the T / C value measured at the reference temperature (25℃) as the X-axis and the analyte concentration value as the Y-axis. In calculating the results, the temperature of the environment where the test card is located is first substituted into the temperature correction equation to calculate the temperature correction coefficient. Then, the T / C value measured at that test temperature is divided by the temperature correction coefficient to obtain the corrected T / C value. Finally, the corrected T / C value is substituted into the standard curve equation to obtain the analyte concentration value.

[0006] The prerequisite for using the temperature correction factor method is that the temperature correction factors calculated from analyte references of different concentrations must be similar and not vary significantly. However, in actual testing of some analytes, the temperature correction factors calculated using analyte references of different concentrations differ considerably, which can lead to inaccurate test results. Summary of the Invention

[0007] To address the shortcomings of the existing temperature correction coefficient method, this invention selects multiple segmented temperature points within the actual allowable temperature range, and then segments the temperature based on the selected segmented temperature points. Different calculation methods are selected in different temperature segments, thus the calculated analyte concentration is more accurate, and the measurement error caused by the large difference in temperature correction coefficients calculated using analyte references of different concentrations can be significantly reduced.

[0008] This invention provides a temperature-corrected method for detecting analytes, the method comprising the following steps:

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

[0014] When t1 < t ≤ t2, y = y1 * [(t2 - t) / (t2 - t1)] + y2 * [(t - t1) / (t2 - t1)];

[0015] 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 )];

[0016] 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 ) / (tn -t n-1 )];

[0017] When t>t n When, y = y n .

[0018] In some aspects of the present invention, the method further includes the following steps:

[0019] (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 test pad of the immunoassay strip after the clinical sample is added using a light analyzer, and provide the test temperature t after the test card is inserted into the light analyzer;

[0020] (6) Select an appropriate formula based on the temperature range of t in (4), and calculate the concentration of analyte in the clinical sample based on the measurement signal generated in (5).

[0021] In some cases of the present invention, the measurement signal obtained in step (2) is the arithmetic 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 analyte calibrator, or the arithmetic 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 analyte calibrator, or the arithmetic 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 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 other than the T line and the C line.

[0022] In some cases of this invention, in these n general formulas y m =f(x) m In the equations, each segmented temperature point corresponds to one equation. The functional relationship of each equation is the same, but the coefficients of the equations corresponding to any two different segmented temperature points are not all the same. This means that some coefficients may be identical, but not all, or they may be completely different. For example, when n=4, there are four equations: y1=f(x1) at t1, y2=f(x2) at t2, y3=f(x3) at t3, and y4=f(x4) at t4. Each equation is closely related to the T / C value and analyte concentration 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, let the general formula be... Where a, b, c, and d are equation coefficients, and the equation coefficients are not all the same at different segmented temperature points; when it is a cubic polynomial equation, let the general formula be y. m =f(x) m ) = a + bx + cx 2 +dx 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 these n general formulas y m =f(x) m In the equations, each piecewise temperature point corresponds to one equation, and the functional relationships of each equation are not all the same. For example, the equation at one piecewise temperature point is a double logarithmic trinomial equation, while the equation at another piecewise temperature point is a cubic polynomial equation.

[0023] In some aspects of this invention, when multiple analytes are detected simultaneously, such as multiple respiratory viruses, temperature segmentation can be performed when only one analyte is detected, or temperature segmentation can be performed when each analyte is detected, depending on the need.

[0024] In some cases of this invention, n is selected from 3, 4, 5, 6, 7, or 8. In some cases of this invention, n = 4 or 5. In some cases of this invention, n = 4.

[0025] In some cases of the present invention, 2℃ ≤ the difference between any two adjacent segmented temperature points ≤ 6℃. In some cases of the present invention, 3℃ ≤ the difference between any two adjacent segmented temperature points ≤ 5℃.

[0026] 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.

[0027] In some cases of this invention, n = 4. Not all coefficients in the two equations corresponding to any two different segmented temperature points are the same.

[0028] In some cases of this invention, n = 4, t1 = 15.1℃, t2 = 19.9℃, t3 = 24.4℃, t4 = 29.5℃, 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;

[0029] Equation at time t1:

[0030] Equation at t2:

[0031] Equation at t3:

[0032] Equation at t4:

[0033] The formula is selected as follows:

[0034] When t ≤ t1, y = y1;

[0035] When t1 < t ≤ t2, y = y_{1}*[(t2 - t) / (t2 - t1)] + y_{2}*[(t - t1) / (t2 - t1)];

[0036] When t2 < t ≤ t3, y = y_{2}*[(t3 - t) / (t3 - t2)] + y_{3}*[(t - t2) / (t3 - t2)];

[0037] [[ID=???]]When t3 < t ≤ t4, y = y_{3}*[(t4 - t) / (t4 - t3)] + y_{4}*[(t - t3) / (t4 - t3)];

[0038] When t > t4, y = y4.

[0039] 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 by 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 set on the surface of the optical analyzer.

[0040] 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

[0041] FIG. 1 is an exploded schematic view of the test card used in the present invention.

[0042] FIG. 2 is a schematic view of the immunoassay strip in the test card used in the present invention. Note: There seems to be a formatting issue in the original text where the equation in line 25 is missing some subscript formatting in the English translation template. It should be "y = y₃*[(t4 - t) / (t4 - t3)] + y₄*[(t - t3) / (t4 - t3)]" for proper mathematical representation. The above translation attempts to maintain the original structure as closely as possible while making the English text grammatically correct.

[0043] Figure 3 shows the temperature correction curve obtained with temperature as the X-axis and the average value of the signal ratio using different concentrations at each temperature as the Y-axis.

[0044] Figure 4 shows the signal curves obtained with the signal value measured at the reference temperature of 24.4℃ on the X-axis and the myoglobin concentration value of different myoglobin calibrators on the Y-axis.

[0045] Figure 5 shows the logarithmic signal curve obtained with the logarithmic value of the signal measured at the reference temperature of 24.4℃ as the X-axis and the myoglobin concentration values ​​of different myoglobin calibrators as the Y-axis. Detailed Implementation

[0046] As shown in Figures 1 and 2, the test card used in this invention includes a test strip 1, a card cover 2, and a card holder 3. The test strip 1 is an immunochromatographic test strip, comprising a sample application pad 11, a labeling pad 12, a detection pad 13, and a sample absorption pad 14, which are sequentially overlapped. The detection pad 13 is made of materials such as nitrocellulose, glass fiber, polyethersulfone, or nylon, preferably a nitrocellulose membrane. The detection pad 13 has a detection line (also called a T line) 15 and a control line (also called a C line) 16. The sample application pad 11 is made of an absorbent material, such as glass fiber or non-woven fabric. The labeling pad 12 is also made of an absorbent material, such as polyester film, glass fiber, or non-woven fabric.

[0047] The number of test lines on the test pad 13 can be adjusted according to actual needs. For example, when testing one analyte, only one test line is needed. When testing two or more analytes, a corresponding number of test lines should be set, such as four T lines to jointly detect multiple upper respiratory tract viruses, such as SARS-CoV-2, influenza A virus, influenza B virus, adenovirus, and respiratory syncytial virus. The area of ​​the test pad other than the T and C lines is the background area of ​​the test pad.

[0048] The test strip 15 also includes a bottom support layer 17, which is made of a common hydrophobic material such as polyvinyl chloride (PVC) to ensure that the sample does not leak out. A detection pad 13 is disposed on the bottom support layer 17. A sample application pad 11 is disposed on the bottom support layer 17, with one end of the sample application pad 11 partially overlapping with the marking pad 12; the marking pad 12 is disposed on the bottom support layer 17, with one end of the marking pad 12 partially overlapping with the sample application pad 11 and the other end of the marking pad 12 partially overlapping with the detection pad 13; a sample absorption pad 14 is disposed 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. Furthermore, in some cases, the overlap area between any two adjacent pads is 0.5–5 mm long.

[0049] The test strip 1 is located within a housing, which is assembled from a cover 2 and a base 3 by ultrasonic welding, snap fastening, or adhesive bonding. In some cases, the cover 2 and base 3 are made of plastic. The base 3 has a test strip groove 32 in the middle for inserting the test strip 1. Preferably, the cover 2 has multiple downward-extending snaps (not shown in the figure), and the base 3 has multiple upward-extending grooves 31. The snaps on the cover 2 and the grooves 31 on the base 3 correspond one-to-one, so that when the cover 2, test strip 1, and base 3 are assembled together, the cover 2 and base 3 can be firmly fixed together, and the test strip 1 can be fixed in the test strip groove 32.

[0050] The card cover 2 is also equipped 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 pad 11 located below the sample inlet 22. Under the action of capillary action, the sample migrates along the length of the test strip 1 towards the sample absorption pad 14. The observation window 21 is located above the detection line 15 and 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 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 light signals generated by the detection line 15 and control line 16 can be detected by the detector in the optical analyzer. The optical analyzer used in this invention can be a commercially available optical analyzer, such as the iFIA-100 dry fluorescence immunoassay analyzer (Aicon Biotechnology (Hangzhou) Co., Ltd.).

[0051] Depending on the analyte (e.g., antigen, antibody, or hapten) and the immunoassay principle (double antigen sandwich method, double antibody sandwich method, competitive method, indirect method, capture method), the coatings on the label pad 12 and the test line 15 will vary. Here, we will use myoglobin as the analyte and the double antibody sandwich method as an example. The label pad 12 is coated with a first reagent (first anti-myoglobin antibody) labeled with a signal marker and a first quality control reagent (e.g., rabbit IgG) labeled with a signal marker. The test line 15 is coated with a second reagent (second anti-myoglobin antibody). The first and second reagents specifically bind to myoglobin in the clinical sample. Thus, when the clinical sample is added to the sample pad 11 through the sample inlet 22, the clinical sample flows along the length of the test strip 1. When the clinical sample reaches the labeling pad 12, the first reagent labeled with the signal marker specifically binds to myoglobin in the clinical sample (if present). The resulting signal marker-first reagent-myoglobin complex 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. After irradiation by light from the light source of the optical analyzer, a detection signal related to the concentration of myoglobin is generated. Simultaneously, the first quality control reagent labeled with the signal marker continues to flow. When it reaches the control line 16, the second quality control reagent coated on the control line 16 (e.g., goat anti-rabbit IgG antibody) can capture the first quality control reagent labeled with the signal marker on the control line 16. After irradiation by 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, and phosphorescence generated by the T and C lines after irradiation by light from the light source.

[0052] The signal markers in this invention can be selected from time-resolved luminescent markers, colored luminescent microspheres, colored colloidal particles (such as latex, colloidal gold, colloidal carbon, and colloidal selenium), magnetic nanoparticles, and luminescent compounds. Time-resolved luminescent markers have the characteristic of delayed emission, meaning that even after the excitation light emitted by the light source is turned off, they can still 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 lanthanides such as samarium (Sm(III)), dysprosium (Dy(III)), europium (Eu(III)), and terbium (Tb(III)) and their chelates; platinum / palladium porphyrin compounds that emit phosphorescence after excitation; and upconversion luminescent materials. A suitable lanthanide chelate is N-(p-isothiocyanophenyl)-diethylenetriaminetetraacetic acid-Eu... +3Time-resolved luminescent markers can also exist in another form: time-resolved luminescent microspheres, which are made by encapsulating time-resolved luminescent molecules inside or on the surface of natural or synthetic microspheres or beads. Given that each time-resolved luminescent microsphere can encapsulate thousands of time-resolved luminescent molecules, effectively improving detection sensitivity, time-resolved luminescent markers are preferably time-resolved luminescent microspheres. Time-resolved luminescent microspheres that produce fluorescence after excitation are called time-resolved fluorescent microspheres.

[0053] The time-resolved luminescent markers of this invention possess a luminescence delay characteristic, meaning that even after the excitation light emitted by the light source is turned off, they can still continuously emit light for a certain period of time. The wavelength of the emitted light can be greater than or less than the wavelength of the excitation light. This luminescence delay characteristic gives the emitted light generated by the time-resolved luminescent markers after excitation a long lifetime. Thus, in actual detection, after the time-resolved luminescent markers are excited by the excitation light from the light source, the light source can be turned off and a period of time can be waited before detecting the emitted light signal from the time-resolved luminescent markers, thereby eliminating interference from short-lived background light signals and scattered excitation light.

[0054] Time-resolved luminescent markers can exist in molecular form, called time-resolved luminescent molecules, and can be selected from lanthanides such as samarium (Sm(III)), dysprosium (Dy(III)), europium (Eu(III)) and terbium (Tb(III)) and their chelates, as well as upconversion luminescent materials; and platinum / palladium porphyrin compounds that emit phosphorescence upon excitation. The delay time of europium chelates is approximately 2 ms, and the emitted fluorescence signal gradually decays over time within 2 ms. Given that some proteins in clinical samples can also fluoresce under light irradiation but lack delay characteristics, and that most proteins' fluorescence disappears completely 200 μs after the light source is turned off, time-resolved fluorescence detection based on europium chelates typically selects a time interval of 200–400 μs after the light source is turned off. A suitable europium chelate is N-(p-isothiocyanophenyl)-diethylenetriaminetetraacetic acid-Eu... +3 .

[0055] Time-resolved luminescent markers can also exist in another form: time-resolved luminescent microspheres, which are made by encapsulating time-resolved luminescent molecules inside or on the surface of natural or synthetic microspheres or beads. Given that each time-resolved luminescent microsphere can encapsulate tens of thousands of time-resolved luminescent molecules, effectively improving detection sensitivity, time-resolved luminescent markers are preferably time-resolved luminescent microspheres. Time-resolved luminescent microspheres that fluoresce upon illumination are called time-resolved fluorescent microspheres.

[0056] Upconversion luminescent materials are materials that emit high-energy light when exposed to low-energy light. The light signals generated by upconversion luminescent materials after illumination exhibit strong stability, long lifetime, and high sensitivity, making them suitable for time-resolved immunoassay. Common upconversion luminescent materials can be selected from Y₂O₃, Y₂O₂S, LaF₃, NaYF₄, NaGdF₄, and NaYF₄:Yb. 3+ / Nd 3+ / Ho 3+ NaGdF4:Yb 3+ / Nd 3+ / Ho 3+ Y2O3:Er,Yb, etc.

[0057] The colored luminescent microspheres in this invention refer to microspheres or microbeads that have quantum dots, fluorescent dyes, or other luminescent compounds encapsulated on their surface or inside. When irradiated with light of a suitable wavelength, they can produce light signals without luminescence delay. They can be selected from green fluorescent microspheres, blue fluorescent microspheres, red fluorescent microspheres, yellow fluorescent microspheres, and colored fluorescent microspheres (emitting fluorescence of various specific colors).

[0058] The luminescent compounds in this invention refer to those that can produce light without emission 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 their improved variants 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-dioxane derivatives (common ones include AMPPD, CSPD, CDP and CDP-Star, as well as PPD, Lumi-Phos and Lumi-Plus from Lumigen), and acridine esters or acridine sulfonamides.

[0059] The colored colloidal particles in this invention refer to colloidal particles that aggregate on the T-line and / or C-line during immunochromatographic reactions to produce colored aggregates. These particles can be selected from latex, colloidal gold, colloidal carbon, and colloidal selenium. After irradiation by a light source, the reflected light from the colored colloidal particles on the T-line or C-line can be detected and used as a detection signal or a control signal.

[0060] In time-resolved luminescent microspheres and colored luminescent microspheres, the polymers forming the microspheres or microbeads can be selected from polystyrene, butadiene-styrene, styrene-vinyl acrylate trimer, polymethyl methacrylate, polyethyl methyl acrylate, styrene-maleic anhydride copolymer, polyvinyl acetate, polyvinylpyridine, polydivinylbenzene, polybutylene terephthalate, acrylonitrile, vinyl chloride-acrylate, etc., or their aldehyde, carboxyl, amino, hydroxyl, hydrazide derivatives, or mixtures thereof. Furthermore, the surface of the microspheres or microbeads typically carries hydroxyl, carboxyl, amino, aldehyde, sulfonyl, or other groups, which can be coupled to antibodies, antigens, or haptens-carrier protein conjugates using conventional chemical coupling reagents. In some cases, the particle size of time-resolved luminescent microspheres ranges from 20 nm to 100 μm.

[0061] When detecting one or more analytes in a sample, depending on the immunoassay principle, the detection signal intensity of the time-resolved luminescent marker on the T line is positively or negatively correlated with the analyte concentration. Except for competitive methods, the detection signal intensity of the time-resolved luminescent marker on the T line is positively correlated with the analyte concentration. However, regardless of the immunoassay principle used, control line 16 (if present) should produce a control signal; otherwise, it indicates a problem with the test strip, or that it is ineffective.

[0062] The clinical samples used in this invention can be selected from serum, plasma, whole blood, cerebrospinal fluid, urine, bronchoalveolar lavage fluid, nasopharyngeal swabs, sputum, stool, and skin lesion samples (including swabs of rash / vesicle exudate; vesicle fluid; scabs, etc.). Depending on the type of clinical sample and the analyte, some clinical samples may be pre-treated before testing (e.g., lysed with a lysis buffer to release the analyte to be detected) before being added to the test card for detection.

[0063] The analytes detectable by this invention include antigens or antibodies, and even haptens, such as inflammatory markers like CRP, IL-6, procalcitonin (PCT), and SAA; heart failure markers like BNP, NT-proBNP, cTnI, CK-MB, myoglobin, and D-dimer; tumor markers like alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), ferritin, prostate-specific antigen (PSA), neuron-specific enolase (NSE), CYFRA21-1, CA19-9, CA50, CA125, CA153, and CA724; and bone metabolism markers, such as 25-hydroxylamine. Vitamin D, β-CrossLaps, bone alkaline phosphatase, calcitonin, parathyroid hormone, human N-terminal mid-segment osteocalcin, and total type I procollagen N-terminal elongated peptide, etc.; sex markers, such as follicle-stimulating hormone (FSH), luteinizing hormone (LH), prolactin, progesterone, testosterone, estradiol, estriol, and 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 agents. Disease biomarkers, such as upper respiratory tract virus (e.g., SARS-CoV-2, influenza A, influenza B, respiratory syncytial virus, adenovirus, rhinovirus, parainfluenza virus, etc.) antigens / antibodies, monkeypox virus antigens / antibodies, gonococcal antigens / antibodies, chlamydia antigens / antibodies, mycoplasma antigens / antibodies, hepatitis B five markers (HBsAg, anti-HBs, anti-HBc, HBeAg, anti-HBe), HCV antigens / antibodies, Treponema pallidum antigens / antibodies, TORCH (human cytomegalovirus, rubella virus, Toxoplasma gondii, HSV-1 and HSV-2) IgG / IgM, HIV antigens / antibodies, etc.; diabetes biomarkers, such as insulin, C-peptide. Insulin autoantibodies, islet cell antibodies, and glutamate decarboxylase antibodies; liver fibrosis markers, such as laminin, hyaluronic acid, type IV collagen, type III procollagen N-terminal peptide, and chitosanase 3-like protein 1; allergy markers, such as allergens, allergen-specific IgE, and total IgE; gut health markers, such as enterovirus antigens, rotavirus antigens, enterovirus 71 IgG / IgM, Coxsackievirus A16 IgG / IgM, Coxsackievirus B IgG / IgM, calprotectin, Clostridium difficile toxin A, Clostridium difficile toxin B, and Clostridium difficile glutamate dehydrogenase; and markers used to assess drug abuse, such as cocaine and morphine.

[0064] Example 1: Preparation of the immunoassay strips and test cards of the present invention

[0065] A method for preparing an immunoassay strip and test card for the quantitative detection of analytes (e.g., IL-6, NT-proBNP, myoglobin, and upper respiratory tract virus antigens or antibodies, with myoglobin as an example) in a sample includes the following steps:

[0066] A. Antibody Preparation: Paired monoclonal or polyclonal antibodies for detecting myoglobin can be screened by immunizing mice, rats, rabbits, or by counting hybridoma cells using antigens. Alternatively, commercially available myoglobin pairing antibodies (one type is a myoglobin capture antibody, and the other is a myoglobin detection antibody) can be used. This example uses commercially available myoglobin pairing antibodies (purchased from Jiangsu Dongkang Biomedical Technology Co., Ltd., catalog numbers C3601: lot 20221107 and C3604: lot 20210326) as an example. Rabbit IgG antibodies and goat anti-rabbit IgG antibodies were prepared in-house or purchased from the market.

[0067] B. Test pad fluid

[0068] T-line solution was obtained by adding myoglobin capture antibody to 0.02M phosphate buffer (pH 7.2), with a concentration of 1 mg / ml. C-line solution was obtained by adding goat anti-rabbit IgG antibody to 0.02M phosphate buffer (pH 7.2), with a concentration of 0.3 mg / ml. Then, using a quantitative dispensing device (AUTOKUN continuous dispensing machine HGS101 from Hangzhou Fenghang Technology Co., Ltd.), the T-line solution and C-line solution were coated onto a 2.5 cm wide nitrocellulose membrane (purchased from Satorious, model 1UN95ER100025NT) used as a detection pad at a volume of 1 μl / cm with a 0.8 cm interval, thus forming T-line and C-line respectively. The membrane was dried at 45°C for 18 hours and sealed with desiccant for later use.

[0069] C. Preparation of fluorescent microspheres:

[0070] Fluorescent microsphere selection: Time-resolved fluorescent microspheres (Merk, catalog number F1-XC 010) encapsulated with europium chelates, with an excitation wavelength of 365 nm and an emission wavelength of 615 nm.

[0071] Preparation of MES buffer: Add sodium morpholine ethanesulfonate to pure water and mix well to make the concentration of sodium morpholine ethanesulfonate 1.0% (w / v).

[0072] Preparation of the storage buffer: 50mM Tris-HCl buffer (pH 8.0).

[0073] Preparation of time-resolved fluorescent microsphere-labeled myoglobin detection antibody: Fluorescent microspheres were washed with MES buffer, and carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were added to final concentrations of 0.4 mg / ml and 0.1 mg / ml, respectively. The microspheres were activated by incubation at room temperature for 20 minutes. After thorough washing with MES buffer, myoglobin detection antibody was added at a ratio of 1 mg:0.1 mg (w / v). The mixture was incubated at room temperature for 1.5 hours, and after thorough washing with MES buffer, 0.05 M Tris-HCl buffer (pH 10.5) containing 10% BSA (w / v) was added. 8.0), block at room temperature for 1 hour, wash time-resolved fluorescent microspheres with MES buffer, reconstitute the detection time-resolved fluorescent microspheres with storage buffer to a final concentration of 5 mg / ml, and store at 4°C for later use; prepare rabbit IgG antibody labeled with time-resolved fluorescent microspheres in the same way as preparing the myoglobin detection antibody labeled with time-resolved fluorescent microspheres, and use storage buffer to make the final concentration of the rabbit IgG antibody labeled with time-resolved fluorescent microspheres 5 mg / ml, and store at 4°C for later use.

[0074] Finally, the time-resolved fluorescent microsphere-labeled myoglobin detection antibody, the time-resolved fluorescent microsphere-labeled rabbit IgG antibody, and the fluorescent labeling diluent were thoroughly mixed at a volume ratio of 6:1:13 to obtain the fluorescent microsphere mixture. The fluorescent labeling diluent was a 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).

[0075] D. Spraying and drying of fluorescent microspheres

[0076] Using the dedicated spray head of the AUTOKUN HGS101 continuous coating machine, the prepared fluorescent microsphere mixture was uniformly sprayed onto a 1.0 cm wide marking pad (glass fiber) at a rate of 2 μL / cm. The mixture was then dried at 45°C for 18 hours and sealed with desiccant for later use.

[0077] E. Sample pad treatment

[0078] Immerse a 2.5 cm wide sample pad in the sample pad treatment solution for 1 hour, then remove and dry at 37°C overnight (12–24 h). The sample pad treatment solution is a buffer solution containing 50 mM (pH 8.0) Tris-HCl buffer, 1% BSA (w / v), and 0.5% Tween-20 (v / v).

[0079] F. Assembly and cutting of test strips

[0080] Assembly of the test strip: Manually or by machine, attach the 2.5cm wide sample pad, 1.0cm wide marking pad, 2.5cm wide nitrocellulose membrane, and 2.5cm wide absorbent paper (as the sample absorption pad) to the 8cm long plastic base plate (as the bottom support layer), so that the 2.5cm wide sample pad, 1.0cm wide marking pad, 2.5cm wide nitrocellulose membrane, and 2.5cm wide absorbent paper overlap each other by 2.0mm to assemble the test strip.

[0081] Cutting of test strips: Use an AUTOKUN HGS201 strip cutter to cut the assembled test strips into 4mm wide single-person test strips.

[0082] G. Assembly of the test card

[0083] Place the cut, single-use test strip into the slot on the plastic card holder, close the card cover, and use a card press or manually to tighten the card holder and card cover, ensuring the entire immunoassay strip is taut. Add desiccant and store at room temperature for later use.

[0084] After the test card is assembled, it can be placed into the kit containing desiccant and instructions for use. Additionally, the kit can be supplemented with sampling tubes, test tubes containing lysis buffer, test tubes containing sample diluent, and sampling swabs, as needed.

[0085] Example 2: Detection Method

[0086] The test card prepared in Example 1 was used to detect analytes in the sample (e.g., IL-6, NT-proBNP, myoglobin, and upper respiratory tract virus antigens or antibodies, with myoglobin as an example). The specific procedure is as follows: 100 μl of sample (whole blood sample as an example) was added to the sample inlet of the test card. After incubation for 15 min, the test card was inserted into an iFIA-100 dry fluorescence immunoassay analyzer (Acon Biotech (Hangzhou) Co., Ltd.) for detection.

[0087] During detection, light with a wavelength of 360 nm was first emitted from the light source to illuminate the time-resolved fluorescent microspheres captured on the T and C lines. Then, the light source was turned off, and the time-resolved fluorescent microspheres captured on the T and C lines emitted time-resolved fluorescence signals with a wavelength of 615 nm. After the light source was turned off for 300 μs, the time-resolved fluorescence signals emitted on the T line (T signal) and the time-resolved fluorescence signals emitted on the C line (C signal) were captured and counted using the detector of the optical analyzer.

[0088] To correct for the adverse effects of manufacturing variations within the same batch of test cards, the T signal can be corrected by using the C signal to obtain the ratio of the T signal to the C signal, denoted as T / C, or T / (T+C). Based on a 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.

[0089] When preparing a series of myoglobin calibrators with different concentrations, serum samples with high and low concentrations of myoglobin were collected to prepare seven calibrators with different concentrations. These calibrators were tested using a Roche Cobas e411 fully automated electrochemiluminescence analyzer (SN#8866-29) and a Roche matching myoglobin assay kit (Lot#70743802). The measured myoglobin concentrations (25.78, 47.33, 101.6, 306.2, 502.1, 734.0, and 1010.0 ng / mL) were used as reference values ​​for these seven calibrators. These calibrators with different concentrations were then added to test cards from the same batch. After incubation for 15 minutes, the T and C signals of each test card were detected using an iFIA-100 dry fluorescence immunoassay analyzer (Aicon Biotechnology (Hangzhou) Co., Ltd.), and the T / C or T / (T+C) ratio was calculated. A calibration curve is plotted based on each T / C or T / (T+C) ratio and its corresponding myoglobin concentration value to calculate the myoglobin concentration in clinical samples.

[0090] Of course, a series of myoglobin calibrators of different concentrations can also be prepared using calibrator diluent (such as PBS buffer) and myoglobin.

[0091] In addition, the measurement of myoglobin calibrators at different concentrations using an immunoassay analyzer is performed at a specific temperature. To ensure that the immunoassay analyzer, test cards, and accompanying sample diluents (20 mM PBS, 0.02% Proclin-300, and 0.1% Tween-20) and a range of myoglobin calibrators at different concentrations are at a specific temperature, they can be placed at that temperature for more than 1 hour to allow them to reach temperature equilibration.

[0092] Example 3: Comparison Method

[0093] Using the test card in Example 1 and the measurement method in Example 2, different concentrations of myoglobin calibrator (concentrations of 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℃, 19.9℃, 24.4℃ and 29.5℃). The results are shown in Table 1.

[0094] Table 1

[0095] 24.4℃ was selected as the reference temperature. The T / C value measured at each temperature point (15.1℃, 19.9℃ and 29.5℃) was divided by the T / C value measured at 24.4℃ to obtain the T / C ratio. 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.

[0096] Table 2

[0097] Next, based on the data in Table 2, a temperature correction curve was obtained with the temperature points as the X-axis and the average T / C ratio as the Y-axis (as shown in Figure 3). Figure 3 shows that the temperature correction coefficient is not linear with temperature, thus yielding the following temperature correction equation:

[0098] tcf=-0.4299+0.1120t-0.002373t 2 +0.000007617t 3 , where t is the temperature, and tcf is the temperature correction coefficient for each temperature point.

[0099] The signal curve was plotted with the T / C value measured at the reference temperature of 24.4℃ on the X-axis and the myoglobin concentration value of different myoglobin calibrators on the Y-axis, as shown in Figure 4.

[0100] The test points in Figure 4 generally exhibit an exponential distribution. Therefore, a logarithmic signal curve was plotted with the logarithmic value of the T / C value measured at the reference temperature of 24.4℃ 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.

[0101] As shown in Figure 5, the standard curve equation for the myoglobin concentration in the sample is as follows:

[0102] When measuring clinical samples (such as plasma, serum, or whole blood) with unknown analyte concentrations, after adding the clinical sample, the ambient temperature of the external environment (such as external air temperature) or local environment (such as the internal environment of the optical analyzer or test card) where the optical analyzer or test card is located is manually entered (which can be measured with a thermometer), or the test temperature in the external or local environment where the optical analyzer or test card is located is measured using a temperature measurement module (such as a thermistor) set in the optical analyzer. Here, we will use the measurement of the external air temperature of the optical analyzer with a thermometer as an example. Simultaneously, following 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, 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.

[0103] Table 3

[0104] For the purpose of result verification, in addition to the four selected temperature points (15.1℃, 19.9℃, 24.4℃, and 29.5℃), three more different verification temperature points (17.3℃, 22.1℃, and 27.2℃) were added. Using the test card from Example 1 and the measurement method from Example 2, different concentrations of myoglobin calibrator were measured at these three verification temperature points. The resulting T / C values ​​are shown in Table 4. Simultaneously, the temperature correction coefficient was calculated by substituting each verification temperature point into the temperature correction equation. The corrected T / C value was then obtained by dividing the measured T / C value by the temperature correction coefficient. The corrected T / C values ​​are shown in Table 4.

[0105] Table 4

[0106] Finally, the T / C correction values ​​from Tables 3 and 4 were substituted into the standard curve equation to obtain the concentration values ​​of myoglobin, as shown in Table 5.

[0107] Table 5

[0108] Using the data in Table 5, the deviation between the temperature-corrected myoglobin concentration and the actual concentration can be calculated as (temperature-corrected myoglobin concentration - actual concentration) / actual concentration. The calculated deviation results are shown in Table 6.

[0109] Table 6.

[0110] Table 6 shows that the deviation range of the myoglobin concentration measurement results obtained using the temperature correction coefficient method is -13.0% to +12.0%, with an average absolute deviation of 5.7%. Furthermore, Table 6 also shows that for the temperature used for result verification (17.3℃), the deviation range is -11.9% to 8.7%; for the temperature used for result verification (22.1℃), the deviation range is -13.0% to 7.3%; and for the temperature used for result verification (27.2℃), the deviation range is -5.6% to 12.0%.

[0111] Example 4: Test method of the present invention

[0112] As shown in Table 2 of Example 3, for the same concentration of myoglobin calibrator, when the temperature increases from 15.1°C to 29.5°C, the temperature correction coefficient generally shows an increasing trend at higher concentrations of myoglobin calibrator. However, when using lower concentrations of myoglobin calibrator (e.g., 25.78 ng / mL and 47.33 ng / mL), the temperature correction coefficient at different temperatures shows a trend of first increasing and then decreasing. Furthermore, at the same temperature, the temperature correction coefficients measured for different concentrations of myoglobin calibrator show different trends. For example, at 15.1°C and 19.9°C, the temperature correction coefficient shows a trend of first decreasing and then increasing with increasing myoglobin concentration; at 29.5°C, the temperature correction coefficient generally shows an increasing trend with increasing myoglobin concentration.

[0113] Therefore, it can be seen that the temperature correction coefficient exhibits different trends under different temperatures and concentrations of myoglobin calibrators. Moreover, the calculated temperature correction coefficients are not similar for different concentrations of myoglobin calibrators, showing significant fluctuations. As a result, the deviation range of the myoglobin concentration measurement results obtained using the temperature correction coefficient method is -12.5% ​​to +15.1% (see Table 6), leading to inaccurate detection results.

[0114] 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 4-42℃, and in some cases 15-30℃). For ease of comparison, the selection scheme for the segmented temperature points is the same as in Example 3 (the four segmented temperature points are: t1 = 15.1℃, t2 = 19.9℃, t3 = 24.4℃, and t4 = 29.5℃). The initial measured T / C values ​​are shown in Table 1. Meanwhile, for the purpose of result verification, in addition to the four selected segmented temperature points, three verification temperature points (17.3℃, 22.1℃, and 27.2℃) are added. Using the test card in Example 1 and the measurement method in Example 2, different concentrations of myoglobin calibrators are measured at these three verification temperature points. The initial measured T / C values ​​are shown in Table 4.

[0115] Then, using the T / C value measured at each selected temperature segment as the X-axis and the myoglobin concentration value as the Y-axis, the general formula for the standard curve equation at each temperature segment is obtained:

[0116] Where m is an integer and 1 ≤ m ≤ n, a, b, c, and d are equation coefficients, which are different at different piecewise temperature points, x m For segmented temperature points t m The measured T / C value, y m This represents the myoglobin concentration. When n = 4, the standard curve equations for myoglobin concentration at times t1, t2, t3, and t4 are y1 = f(x1), y2 = f(x2), y3 = f(x3), and y4 = f(x4), respectively:

[0117] 15.1℃:

[0118] 19.9℃:

[0119] 24.4℃:

[0120] 29.5℃:

[0121] Given the significant differences in temperature correction coefficients at different temperatures in existing technologies, this invention addresses the relationship between the test temperature in the environment where the test card is located and different temperature points after the test card is inserted into the optical analyzer. Temperature segmentation is implemented, and different formulas are used for calculation in different temperature segments. The specific segmentation is shown below.

[0122] When the measured temperature t ≤ t1, y = y1;

[0123] When t1 < the measured temperature t ≤ t2, y = y1*[(t2-t) / (t2-t1)] + y2*[(t-t1) / (t2-t1)];

[0124] When t2 < the measured temperature t ≤ t3, y = y2*[(t3-t) / (t3-t2)] + y3*[(t-t2) / (t3-t2)];

[0125] When t3 < the measured temperature t ≤ t4, y = y3*[(t4-t) / (t4-t3)] + y4*[(t-t3) / (t4-t3)];

[0126] When the measured temperature t > t4, y = y4.

[0127] 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.

[0128] Table 7

[0129] 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. The calculated deviation results are shown in Table 8.

[0130] Table 8

[0131] As shown in Table 8, the deviation range between the myoglobin concentration value calculated by the measurement method of the present invention and the actual concentration is -8.0% to +9.6%, with an average absolute deviation of 3.2%, which is significantly better than the comparative method in Example 3 in terms of accuracy. Furthermore, for the verification temperature of 17.3°C, the deviation range of the measurement results is -2.4% to 3.4%; for the verification temperature of 22.1°C, the deviation range is -4.7% to 7.0%; and for the verification temperature of 27.2°C, the deviation range is -5.4% to 5.3%. It can be seen that at these three verification temperature points, the measurement method of the present invention is significantly better than the comparative method in Example 3 in terms of measurement accuracy.

Claims

1. A temperature-corrected method for detecting analytes, 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. At each segmented temperature point, a series of analyte calibrators of different concentrations are added to the sample inlet of the test card. The measurement signal generated on the detection pad of the immunoassay strip after adding each concentration of analyte calibrator is measured by an optical analyzer. (3) Using 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 ), where m is an integer and 1≤m≤n, the equation at the segmented temperature point t1 is y1=f(x1), where x1 is the measurement signal obtained when measuring a series of analyte calibrators of different concentrations at t1; segmented temperature point t n The equation for time is y n =f(x) n ), x n For t n Measurement signals 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 , 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 as described in 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 the clinical sample is added and the test temperature t after the test card is inserted into the optical analyzer; (6) Select an appropriate formula according to the temperature segment where t is located in (4), and calculate the analyte concentration in the clinical sample according to the measurement signal generated in (5).

3. The method as described in 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 each concentration of analyte calibrator is added, 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 each concentration of analyte calibrator is added, 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 each concentration of analyte calibrator is added.

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

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

6. The method as described in claim 5, characterized in that, 7. The method as described in claim 1, characterized in that, 3°C ≤ the difference between any two adjacent segmented temperature points ≤ 5°C.

8. The method as described in claim 7, characterized in that, 9. The method as described in claim 1, characterized in that, 4°C ≤ each selected segmented temperature point ≤ 42°C.

10. The method as described in claim 8, characterized in that, n=4, The 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 as described in claim 10, characterized in that, Equation at time t1: Equation at time t2: Equation at t3: Equation at t4: 15°C ≤ each selected segmented temperature point ≤ 30°C. The analyte is selected from myoglobin. 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 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.

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