Filter paper dried blood spot thyroxine determination kit and detection method thereof

Abstract

The invention belongs to the technical field of detection, and particularly discloses a filter paper dried blood spot thyroxine determination kit and a detection method thereof, and the filter paper dried blood spot thyroxine determination kit and the detection method provided by the invention are used for quantitative determination of neonatal thyroxine. Through the reconstitution fluid of the T4 antigen dry powder, the uniformity and the stability of manufacturing of the filter paper dried blood spot calibrator and the quality control product are realized; by optimizing antigen and antibody pairing, the detection sensitivity and precision of the reagent are effectively improved, and the linear range is widened; by optimizing a reaction carrier and a tracer agent process, the detection sensitivity of the reagent is improved, and the working range of a curve is widened; the elution release buffer solution is invented, so that elution, release and detection of TT4 in the filter paper dried blood spot are realized; and a full-automatic intelligent detection method is adopted, so that the detection speed, efficiency and precision are improved.

Description

Technical Field

[0001] The present invention relates to the field of detection technology, and in particular to a filter paper dried blood spot thyroxine determination kit and a detection method thereof. Background Art

[0002] Thyroxine (T4) is one of the thyroid hormones (TH), composed of amino acids and iodine. T4 promotes cellular metabolism, increases oxygen consumption, stimulates tissue growth, maturation, and differentiation, and aids in intestinal glucose absorption. Thyroid-stimulating hormone (TSH) from the anterior pituitary gland promotes its secretion. In medical testing, T4 measurements include total thyroxine (TT4) and free thyroxine (FT4). These tests help doctors assess normal thyroid function and the presence of conditions such as hyperthyroidism or hypothyroidism. T4 measured in human serum is typically L-T4 (L-thyroxine). L-T4 is the primary form naturally secreted by the thyroid gland and plays an important role in human metabolism, growth, and development. D-T4 (D-thyroxine), an enantiomer of L-T4, is uncommon in the human body and is inactive. Therefore, routine thyroid function tests primarily target L-T4.

[0003] Newborn screening, one of the most effective public health initiatives, has been in operation for nearly 50 years. Beginning in 1962 with a single screening test for phenylketonuria, advancements in screening technology have expanded to include screening for over 50 diseases, including endocrine disorders (congenital hypothyroidism, congenital adrenal hyperplasia), hemoglobinopathies, infectious diseases (toxoplasmosis, human immunodeficiency virus infection), cystic fibrosis, and inborn errors of metabolism. As a tertiary preventive measure for reducing birth defects, newborn screening is a crucial component of improving population quality and reducing intellectual disability in children, becoming a pioneering public health initiative of the 20th century. Congenital hypothyroidism (CH) is the most common endocrine disorder in pediatrics. It is caused by developmental or functional abnormalities in the hypothalamic-pituitary-thyroid axis, resulting in insufficient TH production and an inability to maintain normal tissue and function development. Newborn screening (NBS) allows for early diagnosis and treatment of this condition.

[0004] T4 is an iodinated hormone produced and secreted by the thyroid gland. The fetal thyroid gland and pituitary-thyroid axis are not secretory during early pregnancy. Prior to this, fetal TH is derived from the maternal circulation. Immediately after birth, a newborn's blood TSH concentration rises rapidly—this is known as the TSH spike. If the newborn's thyroid function is normal, the concentration stabilizes at a low level within the first few days of life. T4 levels do not rise as rapidly as TSH levels in the first few days of life. The 2023 American Academy of Pediatrics publication, "Congenital Hypothyroidism: Guidelines for Screening and Management," proposes three different screening protocols: screening for TSH first, then T4; screening for T4 first, then TSH; and screening for both T4 and TSH simultaneously. While the three protocols have similar detection rates for moderate to severe primary hypothyroidism, they have different detection rates for mild primary hypothyroidism, late-onset primary hypothyroidism ("delayed TSH elevation"), and central CH.

[0005] Previously, T4 was often measured using serum assays, which require large sample volumes and are difficult to store and transport, especially in newborns. Neonatal genetic metabolic disease screening (NES) is performed using laboratory methods to detect parameters on filter paper blood spots. Filter paper blood spots are convenient, inexpensive, and easy to store and transport. However, the NES laboratory uses 3 mm blood spots, and the serum volume is equivalent to (1.5 ± 0.17) μL. This requires ultra-high sensitivity for the detection of parameters in these blood spots. 99.95% of T4 in human blood is reversibly bound to transport proteins. This specific binding makes quantitative T4 measurement difficult. Therefore, a release agent must be used to release all bound T4 molecules to accurately determine T4 levels. Releasers can both release T4 from its bound state and release it into free T4 molecules, and they can also block the specific immune response between antigens and antibodies. Therefore, selecting the appropriate releaser, ratio, and dosage is crucial. Previous releasers were developed based on serum testing methods and are not suitable for filter paper blood spot methods. It is necessary to develop an elution releaser suitable for the filter paper blood spot reaction system. This releaser must fully elute the analyte from the filter paper blood spot into the buffer solution while simultaneously releasing T4 from its bound state in the buffer solution to release it into free T4 molecules for immune response. Given the specificity of filter paper blood spot sample testing and the principle of competitive assays, sensitivity, linearity, and precision are also challenges that need to be addressed. This requires consideration of process optimization for the preparation of filter paper blood spot calibrators, quality controls, reaction carriers, and tracers, as well as optimization of the reaction experimental procedures. Summary of the Invention

[0006] The invention provides a filter paper dried blood spot thyroxine determination kit and a detection method thereof. The kit has high sensitivity, a wide curve working range and good precision.

[0007] The present invention solves its technical problems by adopting the following technical solutions: A filter paper dried blood spot thyroxine assay kit, comprising: a filter paper dried blood spot calibrator, a filter paper dried blood spot quality control product, a reaction carrier, a tracer, an elution release buffer, a cleaning agent, and an inducing agent; The elution and release buffer is a 20-100 mmol / L tris-hydrochloric acid buffer containing an eluent, a release agent, a preservative and a blocking agent.

[0008] As a preferred embodiment of the present invention, the concentration of the eluent in the elution release buffer is 0.01-1 wt %, and the eluent is at least one of Tween 20, Tween 40, Tween 60, Tween 80, disodium edetate, and sodium chloride; The concentration of the release agent in the elution release buffer is 0.01-1 wt %, and the release agent is at least one of 8-anilino-1-naphthalenesulfonic acid, sodium salicylate, sodium thimerosal, trichloroacetic acid, phenytoin sodium, 5-bromo-2-methylpyridine, 3-bromo-5-hydroxypyridine and pepsin; The concentration of the preservative in the elution release buffer is 0.01-1 wt %, and the preservative is at least one of Proclin® 300, sodium azide, and gentamicin; The concentration of the blocking agent in the elution release buffer is 0.5-2 wt %, and the blocking agent is at least one of bovine serum albumin, calf serum, newborn calf serum, fetal bovine serum, fish gelatin, casein and polyvinyl pyrrolidone.

[0009] As a preferred embodiment of the present invention, the eluent is a mixture of Tween 40, disodium EDTA and sodium chloride, and the contents of the three in the elution release buffer are 0.01 wt%, 0.074 wt% and 0.85 wt% respectively.

[0010] As a preferred embodiment of the present invention, the release agent is a mixture of 8-anilino-1-naphthalenesulfonic acid and sodium salicylate, and the contents of the two in the elution release buffer are 0.075 wt % and 0.05 wt %, respectively.

[0011] As a preferred embodiment of the present invention, the preservative is a mixture of Proclin® 300 and sodium azide, and the contents in the elution release buffer are 0.01 wt % and 0.05 wt %, respectively.

[0012] As a preferred embodiment of the present invention, the blocking agent is bovine serum albumin, and its content in the elution release buffer is 1 wt %.

[0013] As a preferred embodiment of the present invention, the tracer preparation method is as follows: T4 antibody is first subjected to antibody storage liquid replacement treatment using 50 mmol / L, pH 9.6 carbonate buffer, T4 antibody and lanthanum salt are uniformly mixed at a mass ratio of 1:1 to 3:1, incubated with shaking at 2 to 8°C for 24 to 72 hours, and purified by chromatography using a gel column filled with polyacrylamide gel S-200. The collected tracer mother liquor is diluted with 20 mmol / L, pH 7.8 3-(N-morpholino)propanesulfonic acid-triethanolamine buffer containing 2 g / L bovine serum albumin at a dilution ratio of 1:(1000 to 6000) to form a semi-finished product; The lanthanum salt includes at least one of a europium salt and a samarium salt; preferably, sodium europium N1-(P-isothiocyanatobenzyl)-diethylenetriaminetetraacetate.

[0014] As a preferred embodiment of the present invention, the T4 antibody (thyroxine antibody) is a mouse monoclonal antibody.

[0015] As a preferred embodiment of the present invention, the reaction carrier is a 96-well polystyrene enzyme-labeled microplate, and the reaction carrier contains a coating, which is T4 antigen, T4 antigen derivative or a ligand that binds to a T4 antigen derivative; The coating material in the reaction carrier is diluted with a coating buffer to a concentration of 1-10 μg / mL, coated in the reaction carrier with a coating volume of 50-200 μL, incubated at 2-8°C for 20±2 hours, then washed once with a washing solution with a volume of 100-300 μL / well, and then 100-250 μL of blocking solution is added to each well and incubated at 37°C for 1-3 hours. The blocking solution is discarded, the wells are spun and dried, vacuum-sealed, and stored at 2-8°C for later use; The coating buffer is 50 mmol / L carbonate buffer with a pH of 9.5-9.7, 50 mmol / L Tris-HCl buffer with a pH of 7.7-7.9, 50 mmol / L phosphate buffer with a pH of 7.1-7.3, and 20 mmol / L phosphate buffer with a pH of 4.2-4.4.

[0016] As a preferred embodiment of the present invention, the thyroxine antigen derivative is characterized in that a linker with a molecular weight of not less than 244.31 is connected to the T4 antigen, and the linker includes but is not limited to biotin, peptides and proteins. The T4 antigen derivative includes but is not limited to: thyroxine-bovine serum albumin complex (T4-BSA), thyroxine-mouse serum albumin complex (T4-MSA), thyroxine-keyhole limpet hemocyanin complex (T4-KLH), thyroxine-bovine serum gamma globulin complex (T4-BSG), thyroxine-chicken ovalbumin complex (T4-OVA), thyroxine-bovine serum gamma globul ... Thyroxine-human serum albumin complex (T4-HSA), thyroxine-keyhole limpet hemocyanin-bovine serum albumin complex (T4-KLH-BSA), thyroxine-keyhole limpet hemocyanin-mouse serum albumin complex (T4-KLH-MSA), thyroxine-keyhole limpet hemocyanin-bovine serum gamma globulin complex (T4-KLH-BSG), thyroxine-keyhole limpet hemocyanin-chicken ovalbumin complex (T4-KLH-OVA), thyroxine-keyhole limpet hemocyanin-human serum albumin complex (T4-KLH-HSA), thyroxine-biotin complex ( T4-Bio), thyroxine-bovine serum albumin-biotin complex (T4-BSA-Bio), thyroxine-mouse serum albumin-biotin complex (T4-MSA-Bio), thyroxine-keyhole limpet hemocyanin-biotin complex (T4-KLH-Bio), thyroxine-bovine serum gamma globulin-biotin complex (T4-BSG-Bio), thyroxine-hen ovalbumin-biotin complex (T4-OVA-Bio), thyroxine-human serum albumin-biotin complex (T4-HSA-Bio), thyroxine-keyhole limpet hemocyanin One of the following biotin complexes: thyroxine-keyhole limpet hemocyanin-mouse serum albumin-biotin complex (T4-KLH-MSA-Bio), thyroxine-keyhole limpet hemocyanin-bovine serum gamma globulin-biotin complex (T4-KLH-BSG-Bio), thyroxine-keyhole limpet hemocyanin-hen ovalbumin-biotin complex (T4-KLH-OVA-Bio), and thyroxine-keyhole limpet hemocyanin-human serum albumin-biotin complex (T4-KLH-HSA-Bio). The ligand for biotin is streptavidin or avidin.

[0017] As a preferred embodiment of the present invention, the cleaning agent is a tris-hydrochloric acid buffer solution containing 1.125 g / L sodium chloride, 0.2 mL / L Tween-20, and 30 ppm Proclin® 300 preservative.

[0018] As a preferred embodiment of the present invention, the inducer is an enhancement solution containing 0.0494 g / L sodium acetate (anhydrous), 0.0043 g / L β-naphthylcarbonyltrifluoroacetone, 0.021 g / L tri-n-octylphosphine oxide, 1.143 ml / L glacial acetic acid, 0.5 ml / L anhydrous ethanol, 0.5 ml / L Triton X-100 and purified water.

[0019] As a preferred embodiment of the present invention, the preparation method of the filter paper dried blood spot calibrator is as follows: dilute thyroxine with a dissolving solution to a concentration of 2 to 30 mg / mL, then dilute it with a calibrator diluent to a concentration of 0 to 64 μg / dL, add an equal volume of blood cells, and mix to obtain a filter paper dried blood spot calibrator with a concentration of 0 to 32 μg / dL.

[0020] As a preferred embodiment of the present invention, the preparation method of the filter paper dried blood spot quality control product is as follows: dilute thyroxine with a dissolving solution to a concentration of 2 to 30 mg / mL, then dilute it with a calibrator diluent to a concentration of 6 to 24 μg / dL, add an equal volume of blood cells, and mix to obtain a filter paper dried blood spot calibrator with a concentration of 3 to 12 μg / dL.

[0021] As a preferred embodiment of the present invention, the calibrator diluent is preferably a 50 mmol / L Tris-HCl buffer containing 5 g / L bovine serum albumin; The preparation method of the dissolving solution is as follows: 80-120 μL of 20-25 mg / mL clear dimethyl sulfoxide stock solution is added to 300-500 μL of polyethylene glycol 300 and mixed evenly; 40-60 μL of Tween-80 is added to the above system and mixed evenly; then physiological saline is added to make the volume to 1 mL and mixed evenly to obtain the dissolving solution; or The dissolving solution is prepared by adding 80 to 120 μL of a 20 to 25 mg / mL clear dimethyl sulfoxide stock solution to 880 to 920 μL of a 15% to 25% sulfobutyl-β-cyclodextrin saline solution, and mixing the mixture to obtain a dissolving solution; or The dissolving solution is prepared by adding 80-120 μL of a 20-25 mg / mL clear dimethyl sulfoxide stock solution to 880-920 μL of corn oil, and mixing the mixture to obtain a dissolving solution.

[0022] As a preferred embodiment of the present invention, the filter paper dried blood spot thyroxine assay kit further includes a plate needle and a barcode.

[0023] As a preferred embodiment of the present invention, the plate needle is a detachable strip plate needle containing a plate needle holder body and fixed on the holder body, wherein the strip plate needle is a conical column with a needle tip at the front end and a slot 3 mm behind the needle tip. The conical column is an 8-pin or 12-pin type fixed on a strip colloid carrier. The plate needle spacing is adapted to the micropores of a 96-well microplate. The needle tip can pass through a 3 mm blood spot at the center and can fix the blood spot on the slot.

[0024] The barcode is a barcode containing item information.

[0025] The present invention also provides a detection method for a filter paper dried blood spot thyroxine assay kit, which uses the filter paper dried blood spot thyroxine assay kit and the detection system described above, wherein the detection system comprises (1) a filter paper dried blood spot quality analysis module; (2) a barcode scanning module; (3) a filter paper dried blood spot punching and cutting module; (4) a sample addition and liquid transfer module; (5) a gripping arm module; (6) an oscillation and incubation module; (7) a plate washing module; (8) a measurement module; (9) a computer data analysis module; (10) an instrument support platform; and (11) an information management system module. The testing steps include: (1) Blood sample collection: Use filter paper dried blood spots to collect blood samples, and enter the sample-related information into the information management system module; (2) Sample quality analysis: A filter paper dried blood spot quality analysis module containing a high-definition industrial camera system, image processing and analysis system is used to analyze the quality of the filter paper dried blood spot sample and determine whether the sample quality is qualified; (3) Barcode recognition: Use the barcode scanning module to read the barcode of the filter paper dried blood sample, export the carrier sample related information, and link it to the sample experimental process; (4) Cutting and collecting calibrators, quality control products, and samples to be tested: Use a filter paper dried blood smear module containing a filter paper dried blood smear sample conveying device, a high-definition industrial camera system, an image analysis and positioning system, a filter paper dried blood smear blood spot cutter, and a high-precision motion positioning device to cut a blood spot with a diameter of 3.2 mm (1 / 8 inch) and fix it on the plate needle. Use a gripper module with horizontal / vertical motion function and high-precision positioning function to move the plate needle to the temporary storage position of the plate needle holder; (5) Adding tracer working solution: Use a gripper module with lateral / longitudinal motion function and high-precision positioning function to move the 96-well microplate reaction carrier to an oscillation incubation module with bottom heating, constant temperature control system, and vortex oscillation function. Use a sample loading and liquid transfer module with lateral / longitudinal motion function and high-precision positioning function to dilute the tracer and elution release buffer at a volume ratio of 1:50 and mix them to prepare a tracer working solution. Use a sample loading and liquid transfer module with lateral / longitudinal motion function and high-precision positioning function to add 50-200 μL of tracer working solution, preferably 150 μL, into the microwell cup of the 96-well microplate reaction carrier; (6) First incubation: Use a gripper module with lateral / vertical movement function and high-precision positioning function to move the plate needle to a 96-well microplate reaction carrier containing tracer working solution. The blood spot fixed on the plate needle is immersed in the tracer working solution in the 96-well microplate reaction carrier, and then incubated in an oscillating incubation module at room temperature for 90 to 150 minutes, preferably 120 minutes; (7) Washing the plate: Dilute the cleaning agent with purified water at a volume ratio of 1:25 to form a cleaning working solution. Use a gripper module with lateral / vertical movement and high-precision positioning functions to move the plate needle to the temporary storage position of the plate needle holder. At the same time, use a gripper module with lateral / vertical movement and high-precision positioning functions to move the 96-well microplate reaction carrier to the plate washing module. The plate washing module uses the cleaning working solution to wash the 96-well microplate reaction carrier 4 to 8 times. (8) Adding inducer: Use a gripper module with lateral / vertical motion function and high-precision positioning function to move the 96-well microplate reaction carrier to an oscillation incubation module with bottom heating, constant temperature control system, and vortex oscillation function, and use a sample pipetting module with lateral / vertical motion function and high-precision positioning function to add 50-200 μL of inducer into the microwell cup of the 96-well microplate reaction carrier; (9) Second incubation: The 96-well microplate reaction carrier with the inducer was oscillated at room temperature at a motor speed of 1200 rpm for 2 to 10 minutes on the incubation position in the oscillating incubation module; (10) Detection: A gripper module with lateral / vertical movement function and high-precision positioning function is used to move the 96-well microplate reaction carrier with the inducing agent to the position of the measurement module. The measurement module has a high-precision shift and positioning device, a window opening and closing device, an automatic filter switching measurement system, and a photoelectric signal conversion measurement system. The corresponding T4 detection program is selected, and the excitation wavelength of 340 nm and the detection wavelength of 613 nm are used for detection to obtain the fluorescence count value; (11) Result analysis: The measured fluorescence count values are transmitted to a computer data analysis module that contains a microcomputer system and laboratory (testing department) information connection port. The results are analyzed using the LOG-B / Bmax axis conversion method and the SPLINE dose-response curve fitting algorithm. The test results of the samples are obtained through the test software algorithm and transmitted to the information management system module.

[0026] Among them, the instrument modules involved in the detection methods (1) to (10) are all fixed into an integrated body by the instrument support platform and contain a safety protection panel. During the operation of the detection system, the safety protection panel is always in a closed state to ensure the biosafety protection during the detection operation and the safety of the detection system operation. The detection methods (1) to (10) are also related to the information management system module. The information management system module includes system function modules (basic data setting, user management, sample management, patient self-service query, statistical report, clinical treatment), report module (traditional project report management, thalassemia report management, tandem mass spectrometry report management, urine organic acid report management, genetic test report management, allergen report management), laboratory management module (laboratory, quality control management). The information management system module is built on the network to realize cloud management of each functional module, standardization and network management of screening and diagnosis and treatment workflows, and barcoding and information management of the entire process from ordering, blood collection, logistics transportation, testing to reporting, query, tracking and follow-up.

[0027] Beneficial effects of the present invention: The filter paper dried blood smear thyroxine determination kit and detection method provided by the present invention are used for the quantitative determination of thyroxine in newborns. By inventing a complex solution of T4 antigen dry powder, the uniformity and stability of the production of filter paper dried blood smear calibration products and quality control products are achieved; by optimizing the pairing of antigens and antibodies, the detection sensitivity and precision of the reagents are effectively improved, and the linear range is widened; by optimizing the reaction carrier and tracer process, the detection sensitivity of the reagents is improved, and the working range of the curve is widened; by inventing an elution and release buffer, the elution, release and detection of TT4 in filter paper dried blood smears are achieved; and the use of a fully automatic intelligent detection method improves the detection speed, efficiency and precision. Provided is a fully automatic detection method for TT in filter paper dried blood smears with high sensitivity, a wide curve working range and good precision. 4、 kits and detection methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 2 is a diagram of the detection system of the present invention.

[0029] Figure 2 The figure is a dose-response curve. DETAILED DESCRIPTION

[0030] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention are clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0031] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0032] In the present invention, when referring to numerical ranges, unless otherwise specified, the numerical ranges are considered continuous and include the minimum and maximum values of the range, as well as every value between such minimum and maximum values. Furthermore, when a range refers to an integer, every integer between the minimum and maximum values of the range is included. In addition, when multiple ranges are provided to describe a feature or characteristic, the ranges can be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges subsumed therein.

[0033] In the present invention, there is no particular limitation on the specific dispersion and stirring treatment methods.

[0034] The reagents and instruments used in the present invention without indicating the manufacturer are all conventional products that can be purchased from the market.

[0035] Example 1 Effect of dissolving solution of L-T4, raw material of filter paper dried blood spot calibrator and filter paper dried blood spot quality control material, for filter paper dried blood spot thyroxine assay kit (time-resolved fluorescence immunoassay) for neonatal hypothyroidism screening: (1) Dissolving solution ① is dimethyl sulfoxide-polyethylene glycol 300-Tween 80-normal saline solution. The preparation method is to take 1 mL of working solution as an example. Take 110 μL of 22.5 mg / mL clear dimethyl sulfoxide stock solution and add it to 450 μL of polyethylene glycol 300 and mix evenly; add 45 μL of Tween-80 to the above system and mix evenly; then continue to add 395 μL of normal saline to make the volume 1 mL, accurately weigh L-thyroxine dry powder and dissolve it into 5 mg / mL for use.

[0036] (2) Dissolving solution ② is dimethyl sulfoxide-sulfobutyl-β-cyclodextrin saline solution. The preparation method is to take 1 mL of working solution as an example. Take 110 μL of 22.5 mg / mL clear dimethyl sulfoxide stock solution and add it to 890 μL of 15% to 25% sulfobutyl-β-cyclodextrin saline solution, mix well, and accurately weigh L-thyroxine dry powder and dissolve it into 5 mg / mL for use.

[0037] (3) Dissolving solution ③ is dimethyl sulfoxide-corn oil solution. The preparation method is to take 1 mL of working solution as an example. Take 110 μL of 22.5 mg / mL clear dimethyl sulfoxide stock solution and add it to 890 μL of corn oil. Mix well. Accurately weigh L-thyroxine dry powder and dissolve it into 5 mg / mL for later use.

[0038] (4) The L-thyroxine dissolved above was diluted with 5 g / L bovine serum albumin in 50 mmol / L Tris-HCl buffer to concentrations of 0, 4, 8, 16, 32, and 64 μg / dL serum for the calibrator and 6, 14, and 24 μg / dL serum for the quality control. Then, an equal volume of blood cells was added to prepare the calibrator and quality control. After thorough mixing, 50 μL / spot was applied on S&S903 filter paper. The calibrator points A, B, C, D, E, and F and the quality control points C1, C2, and C3 were spotted. The theoretical concentrations of the calibrator were approximately 0, 2, 4, 8, 16, and 32 μg / dL serum, respectively, and the theoretical concentrations of the quality control were 3, 7, and 12 μg / dL serum, respectively. The samples were quickly dried at room temperature, placed in storage bags, vacuum-laminated, and stored at low temperature for future use. The reactivity effects under other preferred conditions are shown in the following table.

[0039] Table 1

[0040] As can be seen from Table 1, dissolving solution ① dissolves L-T4 dry powder quickly, has a good appearance and inhibition rate in the preparation of filter paper dried blood spot calibrators and quality control products, and has a long shelf life when stored in liquid form at 2-8°C. The ratio of the detected fluorescence value of calibrator B to the detected fluorescence value of calibrator A (B / A inhibition rate) reaches a minimum and is within 70%-90%, indicating higher sensitivity. The ratio of the detected fluorescence value of calibrator F to the detected fluorescence value of calibrator A (F / A inhibition rate) reaches a minimum and is within 10%-25%, indicating that there is sufficient difference between the binding rate counts at each dose point on the curve.

[0041] Example 2 A reaction carrier coated with T4 antigen for a filter paper dried blood spot thyroxine assay kit (time-resolved fluorescence immunoassay) suitable for neonatal hypothyroidism screening: T4 antigen was diluted to an optimal working concentration of 4 μg / mL with 50 mmol / L carbonate buffer at pH 9.6 and coated in the microwells of a 96-well enzyme-labeled microplate with a coating volume of 100 μL. The plates were incubated at 2–8°C for 20 h, then washed once with 200 μL / well of working wash solution. 150 μL of blocking solution (20 mmol / L phosphate buffer at pH 7.2 containing 10 g / L bovine serum albumin and 50 g / L sucrose) was added to each well and incubated at 37°C for 2 h. The blocking solution was discarded, the plates were spun dry, air-dried, vacuum-sealed, and stored at 2–8°C for later use.

[0042] Example 3 A reaction carrier-coated T4 antigen derivative for a filter paper dried blood spot thyroxine assay kit (time-resolved fluorescence immunoassay) suitable for neonatal hypothyroidism screening: T4 antigen derivatives (such as T4-BSA, T4-MSA, T4-KLH, T4-BSG, T4-OVA, T4-HSA, T4-KLH-BSA, T4-KLH-MSA, T4-KLH-BSG, T4-KLH-OVA, and T4-KLH-HSA) were diluted to an optimal working concentration of 4 μg / mL with 50 mmol / L carbonate buffer, pH 9.6, and coated in 100 μL of the wells of a 96-well enzyme-labeled microplate. The wells were incubated at 2–8°C for 20 h, then washed once with 200 μL / well of the wash buffer. 150 μL of blocking buffer (20 mmol / L phosphate buffer, pH 7.2, containing 10 g / L bovine serum albumin and 50 g / L sucrose) was added to each well and blocked and incubated at 37°C for 2 h. h. Discard the blocking solution, spin dry and air dry, vacuum seal the film, and store at 2-8℃ for later use.

[0043] Example 4 A reaction carrier of a filter paper dried blood spot thyroxine assay kit (time-resolved fluorescence immunoassay) suitable for neonatal hypothyroidism screening is coated with a ligand bound to a T4 antigen derivative: The ligand bound to the thyroxine antigen derivative (such as streptavidin, avidin) was diluted to an optimal working concentration of 4 μg / mL with 50 mmol / L carbonate buffer at pH 9.6, coated in the microwells of a 96-well enzyme-labeled microplate with a coating volume of 100 μL, incubated at 2-8°C for 20 h, then washed once with a working solution of 200 μL / well, and then 150 μL of blocking solution (20 mmol / L phosphate buffer at pH 7.2 containing 10 g / L bovine serum albumin and 50 g / L sucrose) was added to each well and incubated at 37°C for 2 h. The blocking solution was discarded, the plates were shaken dry and air-dried, and then the plates were incubated with 50 mmol / L pH 7.2 containing 5 g / L bovine serum albumin. Dilute a thyroxine antigen derivative (e.g., T4-Bio, T4-BSA-Bio, T4-MSA-Bio, T4-KLH-Bio, T4-BSG-Bio, T4-OVA-Bio, T4-HSA-Bio, T4-KLH-BSA-Bio, T4-KLH-MSA-Bio, T4-KLH-BSG-Bio, T4-KLH-OVA-Bio, T4-KLH-HSA-Bio) with 7.8% Tris-HCl buffer to prepare a working solution. Add 100 μL of this ligand-binding T4 antigen derivative working solution to each well of a reaction carrier coated with a ligand (e.g., streptavidin, avidin). Incubate at 2-8°C for 20 h. Wash the wells four times with 200 μL / well of the wash solution, spin dry, and set aside.

[0044] Table 2

[0045] Table 3

[0046] The experimental results indicate that coating with T4-KLH-BSG is the most effective. This is likely due to the directional adsorption of BSG onto the solid support in a suitable buffer system. Furthermore, the bridging effect of KLH as a linker arm allows for uniform and fully exposed T4 antigens on the outer layer, reducing steric hindrance during the antigen-antibody reaction, resulting in optimal reactivity between T4-KLH-BSG and the antibody. Coating with SA combined with T4-KLH-BSG-Bio also offers certain performance advantages, but this involves additional reagent components, increased cost, and more steps, which can introduce increased errors. Therefore, T4-KLH-BSG was chosen for coating.

[0047] Example 5 A coating buffer and blocking solution for a filter paper dried blood spot thyroxine assay kit (time-resolved fluorescence immunoassay) suitable for neonatal hypothyroidism screening: The T4 antigen derivative T4-KLH-BSG was diluted with coating buffer to an optimal working concentration of 4 μg / mL and coated in the microwells of a 96-well enzyme-labeled microplate with a coating volume of 100 μL. The plates were incubated at 2-8°C for 20 h, then washed once with 200 μL / well of washing solution. 150 μL of blocking solution (blocking buffer containing 10 g / L bovine serum albumin and 50 g / L sucrose) was added to each well and incubated at 37°C for 2 h. The blocking solution was discarded, the plates were spun dry, air-dried, vacuum-sealed, and stored at 2-8°C for later use.

[0048] Coating buffer and blocking solution combination: (1) Combination ①: 50 mmol / L carbonate coating buffer at pH 9.5-9.7 combined with 50 mmol / L phosphate blocking buffer at pH 7.1-7.3; (2) Combination ②: 50 mmol / L Tris-HCl coating buffer at pH 7.7-7.9 combined with 50 mmol / L phosphate blocking buffer at pH 7.1-7.3; (3) Combination ③: 20 mmol / L phosphate coating buffer at pH 7.1-7.3 combined with 50 mmol / L phosphate blocking buffer at pH 7.1-7.3; (4) Combination ④: 10 mmol / L citrate coating buffer at pH 6.5-6.7 combined with 50 mmol / L phosphate coating buffer at pH 7.1-7.3; (5) Combination ⑤: 20 mmol / L phosphate coating buffer at pH 4.2-4.4 combined with 20 mmol / L phosphate blocking buffer at pH 4.2-4.4.

[0049] Table 4

[0050] Note: A~F are the fluorescence values of calibrators A~F, the unit is CPs.

[0051] From the experimental results, it can be seen that the combination of coating buffer and blocking buffer ① is the best combination formula. The detection fluorescence value of calibrator B / the detection fluorescence value of calibrator A (B / A inhibition rate) reaches the minimum and is within 70% to 90%, indicating that the sensitivity is higher. The detection fluorescence value of calibrator F / the detection fluorescence value of calibrator A (F / A inhibition rate) reaches the minimum and is within 10% to 25%, indicating that there is a sufficient difference between the binding rate counts at each dose point on the curve; the detection fluorescence values of calibrators A to F are the highest, indicating the best reactivity.

[0052] Example 6 Effect of coating volume on a filter paper dried blood spot thyroxine assay kit (time-resolved fluorescence immunoassay) for neonatal hypothyroidism screening: The T4 antigen derivative, T4-KLH-BSG, was diluted to an optimal working concentration of 4 μg / mL in 50 mmol / L carbonate coating buffer (pH 9.5-9.7). The plates were then coated in appropriate volumes and incubated at 2-8°C for 20 h. The plates were then washed once with 200 μL / well of working wash buffer. Each well was then blocked with 150 μL of blocking buffer (50 mmol / L phosphate blocking buffer (pH 7.1-7.3) containing 10 g / L bovine serum albumin and 50 g / L sucrose) and incubated at 37°C for 2 h. The blocking buffer was discarded, the plates were spun to dry, air-dried, vacuum-sealed, and stored at 2-8°C until further use. Coating volumes ranged from 50, 100, 150, and 200 μL.

[0053] Table 5

[0054] Note: A~F are the fluorescence values of calibrators A~F, the unit is CPs.

[0055] The experimental results show that as the coating volume increases, the reactivity becomes stronger, but the cost increases. When the coating volume is 100 μL, the detection fluorescence value of calibrator B / the detection fluorescence value of calibrator A (B / A inhibition rate) reaches a minimum and is within 70% to 90%, indicating higher sensitivity. The detection fluorescence value of calibrator F / the detection fluorescence value of calibrator A (F / A inhibition rate) reaches a minimum and is within 10% to 25%, indicating that there is a sufficient difference between the binding rate counts at each dose point on the curve. Therefore, choosing a coating volume of 100 μL is the most appropriate.

[0056] Example 7 A tracer for a filter paper dried blood spot thyroxine assay kit (time-resolved fluorescence immunoassay) suitable for neonatal hypothyroidism screening: (1) Tracer ①: T4 antibody was first treated with 50 mmol / L, pH 9.6 carbonate buffer to replace the antibody storage liquid. 1 The mixture was mixed with sodium europium-(P-isothiocyanatobenzyl)-diethylenetriaminetetraacetic acid in a mass ratio of 1:1, incubated with shaking at 2-8°C for 48 h, and purified by chromatography using a gel column filled with polyacrylamide gel S-200. The collected tracer mother solution was diluted with diluent to form a semi-finished product.

[0057] (2) Tracer ②: T4 antibody was first treated with 50 mmol / L, pH 9.6 carbonate buffer to replace the antibody storage liquid.1 The mixture was mixed with sodium europium-(P-isothiocyanatobenzyl)-diethylenetriaminetetraacetic acid in a mass ratio of 2:1, incubated with shaking at 2-8°C for 48 h, and purified by chromatography using a gel column filled with polyacrylamide gel S-200. The collected tracer mother solution was diluted with diluent to form a semi-finished product.

[0058] (3) Tracer ③: T4 antibody was first treated with 50 mmol / L, pH 9.6 carbonate buffer to replace the antibody storage liquid. 1 The mixture was mixed with sodium europium-(P-isothiocyanatobenzyl)-diethylenetriaminetetraacetic acid in a mass ratio of 3:1, incubated with shaking at 2-8°C for 48 h, and purified by chromatography using a gel column filled with polyacrylamide gel S-200. The collected tracer mother solution was diluted with diluent to form a semi-finished product.

[0059] (4) Tracer ④: T4 antibody was first treated with 50 mmol / L, pH 9.6 carbonate buffer to replace the antibody storage liquid. 1 The mixture was mixed with sodium europium-(P-isothiocyanatobenzyl)-diethylenetriaminetetraacetic acid in a mass ratio of 1:1, incubated with shaking at 2-8°C for 24 h, and purified by chromatography using a gel column filled with propylene glycol dextran gel S-200. The collected tracer mother solution was diluted with a diluent to form a semi-finished product.

[0060] (5) Tracer ⑤: T4 antibody was first treated with 50 mmol / L, pH 9.6 carbonate buffer to replace the antibody storage liquid. 1 The mixture was mixed with sodium europium-(P-isothiocyanatobenzyl)-diethylenetriaminetetraacetic acid in a mass ratio of 1:1, incubated with shaking at 2-8°C for 72 h, and purified by chromatography using a gel column filled with polyacrylamide gel S-200. The collected tracer mother solution was diluted with diluent to form a semi-finished product.

[0061] Table 6

[0062] The experimental results show that the process of tracer ① is optimal, the resulting dilution ratio is appropriate, the cost is low, and the detection fluorescence value of calibrator B / calibrator A (B / A inhibition rate) reaches a minimum and is within 70% to 90%, indicating higher sensitivity. The detection fluorescence value of calibrator F / calibrator A (F / A inhibition rate) reaches a minimum and is within 10% to 25%, indicating that there is a sufficient difference between the binding rate counts at each dose point on the curve.

[0063] Example 8 An elution and release buffer for a filter paper dried blood spot thyroxine assay kit (time-resolved fluorescence immunoassay) suitable for neonatal hypothyroidism screening: (1) Elution release buffer 1: 0.01% Tween 40, 0.074% disodium ethylenediaminetetraacetic acid, 0.85% sodium chloride, 0.0375% 8-anilino-1-naphthalenesulfonic acid, 0.1% Proclin ® 300, 0.05% sodium azide, 1% bovine serum albumin in 50 mmol / L Tris-HCl buffer, pH 7.8; (2) Elution release buffer 2: 0.01% Tween 40, 0.074% disodium ethylenediaminetetraacetic acid, 0.85% sodium chloride, 0.075% 8-anilino-1-naphthalenesulfonic acid, 0.1% Proclin ® 300, 0.05% sodium azide, 1% bovine serum albumin in 50 mmol / L Tris-HCl buffer, pH 7.8; (3) Elution release buffer 3: 0.01% Tween 40, 0.074% disodium ethylenediaminetetraacetic acid, 0.85% sodium chloride, 0.15% 8-anilino-1-naphthalenesulfonic acid, 0.1% Proclin ® 300, 0.05% sodium azide, 1% bovine serum albumin in 50 mmol / L Tris-HCl buffer, pH 7.8; (4) Elution release buffer 4: 0.01% Tween 40, 0.074% disodium ethylenediaminetetraacetic acid, 0.85% sodium chloride, 0.30% 8-anilino-1-naphthalenesulfonic acid, 0.1% Proclin ® 300, 0.05% sodium azide, 1% bovine serum albumin in 50 mmol / L Tris-HCl buffer, pH 7.8; (5) Elution release buffer 5: 0.01% Tween 40, 0.074% disodium EDTA, 0.85% sodium chloride, 0.025% sodium salicylate, 0.1% Proclin ® 300, 0.05% sodium azide, 1% bovine serum albumin in 50 mmol / L Tris-HCl buffer, pH 7.8; (6) Elution release buffer 6: 0.01% Tween 40, 0.074% disodium EDTA, 0.85% sodium chloride, 0.05% sodium salicylate, 0.1% Proclin ® 300, 0.05% sodium azide, 1% bovine serum albumin in 50 mmol / L Tris-HCl buffer, pH 7.8; (7) Elution release buffer 7: 0.01% Tween 40, 0.074% disodium edetate, 0.85% sodium chloride, 0.10% sodium salicylate, 0.1% Proclin ® 300, 0.05% sodium azide, 1% bovine serum albumin in 50 mmol / L Tris-HCl buffer, pH 7.8; (8) Elution release buffer 8: 0.01% Tween 40, 0.074% disodium edetate, 0.85% sodium chloride, 0.20% sodium salicylate, 0.1% Proclin ® 300, 0.05% sodium azide, 1% bovine serum albumin in 50 mmol / L Tris-HCl buffer, pH 7.8; (9) Elution release buffer 9: 0.01% Tween 40, 0.074% disodium edetate, 0.85% sodium chloride, 0.40% sodium salicylate, 0.1% Proclin ® 300, 0.05% sodium azide, 1% bovine serum albumin in 50 mmol / L Tris-HCl buffer, pH 7.8; (10) Elution release buffer 10: 0.01% Tween 40, 0.074% disodium edetate, 0.85% sodium chloride, 0.030% sodium thiomersal, 0.1% Proclin ® 300, 0.05% sodium azide, 1% bovine serum albumin in 50 mmol / L Tris-HCl buffer, pH 7.8; (11) Elution release buffer 11: 0.01% Tween 40, 0.074% disodium edetate, 0.85% sodium chloride, 0.060% sodium thiomersal, 0.1% Proclin ® 300, 0.05% sodium azide, 1% bovine serum albumin in 50 mmol / L Tris-HCl buffer, pH 7.8; (12) Elution release buffer 12: 0.01% Tween 40, 0.074% disodium edetate, 0.85% sodium chloride, 0.12% sodium thiomersal, 0.1% Proclin ® 300, 0.05% sodium azide, 1% bovine serum albumin in 50 mmol / L Tris-HCl buffer, pH 7.8; (13) Elution release buffer 13: 0.01% Tween 40, 0.074% disodium ethylenediaminetetraacetic acid, 0.85% sodium chloride, 10% trichloroacetic acid, 0.1% Proclin ®300, 0.05% sodium azide, 1% bovine serum albumin in 50 mmol / L Tris-HCl buffer, pH 7.8; (14) Elution release buffer 14: 0.01% Tween 40, 0.074% disodium edetate, 0.85% sodium chloride, 10 mg / L phenytoin sodium, 0.1% Proclin ® 300, 0.05% sodium azide, 1% bovine serum albumin in 50 mmol / L Tris-HCl buffer (pH 7.8); (15) Elution release buffer 15: 0.01% Tween 40, 0.074% disodium edetate, 0.85% sodium chloride, 20 mg / L phenytoin sodium, 0.1% Proclin ® 300, 0.05% sodium azide, 1% bovine serum albumin in 50 mmol / L Tris-HCl buffer (pH 7.8); (16) Elution release buffer 16: 0.01% Tween 40, 0.074% disodium ethylenediaminetetraacetic acid, 0.85% sodium chloride, 0.05% 5-bromo-2-methylpyridine, 0.1% Proclin ® 300, 0.05% sodium azide, 1% bovine serum albumin in 50 mmol / L Tris-HCl buffer, pH 7.8; (17) Elution release buffer 17: 0.01% Tween 40, 0.074% disodium ethylenediaminetetraacetic acid, 0.85% sodium chloride, 0.1% 5-bromo-2-methylpyridine, 0.1% Proclin ® 300, 0.05% sodium azide, 1% bovine serum albumin in 50 mmol / L Tris-HCl buffer, pH 7.8; (18) Elution release buffer 18: 0.01% Tween 40, 0.074% disodium ethylenediaminetetraacetic acid, 0.85% sodium chloride, 0.20% 5-bromo-2-methylpyridine, 0.1% Proclin ® 300, 0.05% sodium azide, 1% bovine serum albumin in 50 mmol / L Tris-HCl buffer, pH 7.8; (19) Elution release buffer 19: 0.01% Tween 40, 0.074% disodium ethylenediaminetetraacetic acid, 0.85% sodium chloride, 0.50% 5-bromo-2-methylpyridine, 0.1% Proclin ® 300, 0.05% sodium azide, 1% bovine serum albumin in 50 mmol / L Tris-HCl buffer, pH 7.8; (20) Elution release buffer 20: 0.01% Tween 40, 0.074% disodium ethylenediaminetetraacetic acid, 0.85% sodium chloride, 0.05% 3-bromo-5-hydroxypyridine, 0.1% Proclin ® 300, 0.05% sodium azide, 1% bovine serum albumin in 50 mmol / L Tris-HCl buffer, pH 7.8; (21) Elution release buffer 21: 0.01% Tween 40, 0.074% disodium ethylenediaminetetraacetic acid, 0.85% sodium chloride, 0.10% 3-bromo-5-hydroxypyridine, 0.1% Proclin ® 300, 0.05% sodium azide, 1% bovine serum albumin in 50 mmol / L Tris-HCl buffer, pH 7.8; (22) Elution release buffer 22: 0.01% Tween 40, 0.074% disodium ethylenediaminetetraacetic acid, 0.85% sodium chloride, 0.20% 3-bromo-5-hydroxypyridine, 0.1% Proclin ® 300, 0.05% sodium azide, 1% bovine serum albumin in 50 mmol / L Tris-HCl buffer, pH 7.8; (23) Elution release buffer 23: 0.01% Tween 40, 0.074% disodium ethylenediaminetetraacetic acid, 0.85% sodium chloride, 0.50% 3-bromo-5-hydroxypyridine, 0.1% Proclin ® 300, 0.05% sodium azide, 1% bovine serum albumin in 50 mmol / L Tris-HCl buffer, pH 7.8; (24) Elution release buffer 24: 0.01% Tween 40, 0.074% disodium EDTA, 0.85% sodium chloride, 0.50% pepsin, 0.1% Proclin ® 300, 0.05% sodium azide, 1% bovine serum albumin in 50 mmol / L Tris-HCl buffer, pH 7.8; (25) Elution release buffer 25: 0.01% Tween 40, 0.074% disodium EDTA, 0.85% sodium chloride, 1.0% pepsin, 0.1% Proclin ® 300, 0.05% sodium azide, 1% bovine serum albumin in 50 mmol / L Tris-HCl buffer, pH 7.8; (26) Elution release buffer 26: 0.01% Tween 40, 0.074% disodium ethylenediaminetetraacetic acid, 0.85% sodium chloride, 0.075% 8-anilino-1-naphthalenesulfonic acid, 0.05% sodium salicylate, 0.1% Proclin ® 300, 0.05% sodium azide, 1% bovine serum albumin in 50 mmol / L Tris-HCl buffer, pH 7.8; (27) Elution release buffer 27: 0.074% disodium ethylenediaminetetraacetic acid, 0.85% sodium chloride, 0.075% 8-anilino-1-naphthalenesulfonic acid, 0.05% sodium salicylate, 0.1% Proclin ® 300, 0.05% sodium azide, 1% bovine serum albumin in 50 mmol / L Tris-HCl buffer, pH 7.8; (28) Elution release buffer 28: 0.01% Tween 40, 0.85% sodium chloride, 0.075% 8-anilino-1-naphthalenesulfonic acid, 0.05% sodium salicylate, 0.1% Proclin ® 300, 0.05% sodium azide, 1% bovine serum albumin in 50 mmol / L Tris-HCl buffer, pH 7.8; (29) Elution release buffer 29: 0.01% Tween 40, 0.074% disodium ethylenediaminetetraacetic acid, 0.075% 8-anilino-1-naphthalenesulfonic acid, 0.05% sodium salicylate, 0.1% Proclin ® 300, 0.05% sodium azide, 1% bovine serum albumin in 50 mmol / L Tris-HCl buffer, pH 7.8; (30) Elution release buffer 30: 0.01% Tween 40, 0.074% disodium ethylenediaminetetraacetic acid, 0.85% sodium chloride, 0.075% 8-anilino-1-naphthalenesulfonic acid, 0.05% sodium salicylate, 0.1% Proclin ® 300, 1% bovine serum albumin in 50 mmol / L Tris-HCl buffer (pH 7.8); (31) Elution release buffer 31: 0.01% Tween 40, 0.074% disodium ethylenediaminetetraacetic acid, 0.85% sodium chloride, 0.075% 8-anilino-1-naphthalenesulfonic acid, 0.05% sodium salicylate, 0.05% sodium azide, 1% bovine serum albumin in 50 mmol / L Tris-HCl buffer at pH 7.8; (32) Elution release buffer 32: 0.01% Tween 40, 0.074% disodium ethylenediaminetetraacetic acid, 0.85% sodium chloride, 0.075% 8-anilino-1-naphthalenesulfonic acid, 0.05% sodium salicylate, 0.1% Proclin ® 300, 0.05% sodium azide in 50 mmol / L Tris-HCl buffer (pH 7.8); (33) Elution release buffer 33: 0.01% Tween 40, 0.074% disodium ethylenediaminetetraacetic acid, 0.85% sodium chloride, 0.075% 8-anilino-1-naphthalenesulfonic acid, 0.05% sodium salicylate, 0.1% Proclin ® 300, 0.05% sodium azide, 10% calf serum in 50 mmol / L Tris-HCl buffer, pH 7.8; (34) Elution release buffer 34: 0.01% Tween 40, 0.074% disodium ethylenediaminetetraacetic acid, 0.85% sodium chloride, 0.075% 8-anilino-1-naphthalenesulfonic acid, 0.05% sodium salicylate, 0.1% Proclin ® 300, 0.05% sodium azide, 10% newborn calf serum in 50 mmol / L Tris-HCl buffer, pH 7.8; (35) Elution release buffer 35: 0.01% Tween 40, 0.074% disodium ethylenediaminetetraacetic acid, 0.85% sodium chloride, 0.075% 8-anilino-1-naphthalenesulfonic acid, 0.05% sodium salicylate, 0.1% Proclin ® 300, 0.05% sodium azide, 10% fetal bovine serum in 50 mmol / L Tris-HCl buffer (pH 7.8); (36) Elution release buffer 36: 0.01% Tween 40, 0.074% disodium ethylenediaminetetraacetic acid, 0.85% sodium chloride, 0.075% 8-anilino-1-naphthalenesulfonic acid, 0.05% sodium salicylate, 0.1% Proclin ® 300, 0.05% sodium azide, 1% fish gelatin in 50 mmol / L Tris-HCl buffer, pH 7.8; (37) Elution release buffer 37: 0.01% Tween 40, 0.074% disodium ethylenediaminetetraacetic acid, 0.85% sodium chloride, 0.075% 8-anilino-1-naphthalenesulfonic acid, 0.05% sodium salicylate, 0.1% Proclin ® 300, 0.05% sodium azide, 1% casein in 50 mmol / L Tris-HCl buffer, pH 7.8; (38) Elution release buffer 38: 0.01% Tween 40, 0.074% disodium ethylenediaminetetraacetic acid, 0.85% sodium chloride, 0.075% 8-anilino-1-naphthalenesulfonic acid, 0.05% sodium salicylate, 0.1% Proclin ® 300, 0.05% sodium azide, 0.1% polyvinylpyrrolidone in 50 mmol / L Tris-HCl buffer, pH 7.8.

[0064] (39) Elution release buffer 39: 0.5% Tween 40, 0.074% disodium ethylenediaminetetraacetic acid, 0.85% sodium chloride, 0.075% 8-anilino-1-naphthalenesulfonic acid, 0.05% sodium salicylate, 0.1% Proclin ® 300, 0.05% sodium azide, 1% bovine serum albumin in 50 mmol / L Tris-HCl buffer, pH 7.8.

[0065] (40) Elution release buffer 40: 0.01% Tween 40, 0.074% disodium ethylenediaminetetraacetic acid, 0.85% sodium chloride, 0.05% 3-bromo-5-hydroxypyridine, 0.05% 5-bromo-2-methylpyridine, 0.1% Proclin ® 300, 0.05% sodium azide, 1% bovine serum albumin in 50 mmol / L Tris-HCl buffer, pH 7.8.

[0066] (41) Elution release buffer 41: 0.01% Tween 40, 0.074% disodium ethylenediaminetetraacetic acid, 0.85% sodium chloride, 0.5% 3-bromo-5-hydroxypyridine, 0.5% 5-bromo-2-methylpyridine, 0.1% Proclin ® 300, 0.05% sodium azide, 1% bovine serum albumin in 50 mmol / L Tris-HCl buffer, pH 7.8.

[0067] (42) Elution release buffer 42: 0.01% Tween 40, 0.074% disodium ethylenediaminetetraacetic acid, 0.85% sodium chloride, 0.075% 8-anilino-1-naphthalenesulfonic acid, 0.05% sodium salicylate, 0.05% 3-bromo-5-hydroxypyridine, 0.1% Proclin ® 300, 0.05% sodium azide, 1% bovine serum albumin in 50 mmol / L Tris-HCl buffer, pH 7.8.

[0068] (43) Elution release buffer 43: 0.01% Tween 20, 0.074% disodium ethylenediaminetetraacetic acid, 0.85% sodium chloride, 0.075% 8-anilino-1-naphthalenesulfonic acid, 0.05% sodium salicylate, 0.1% Proclin ® 300, 0.05% sodium azide, 1% bovine serum albumin in 50 mmol / L Tris-HCl buffer, pH 7.8.

[0069] (44) Elution release buffer 44: 0.01% Tween 60, 0.074% disodium ethylenediaminetetraacetic acid, 0.85% sodium chloride, 0.075% 8-anilino-1-naphthalenesulfonic acid, 0.05% sodium salicylate, 0.1% Proclin ® 300, 0.05% sodium azide, 1% bovine serum albumin in 50 mmol / L Tris-HCl buffer, pH 7.8.

[0070] (45) Elution release buffer 45: 0.01% Tween 80, 0.074% disodium ethylenediaminetetraacetic acid, 0.85% sodium chloride, 0.075% 8-anilino-1-naphthalenesulfonic acid, 0.05% sodium salicylate, 0.1% Proclin ® 300, 0.05% sodium azide, 1% bovine serum albumin in 50 mmol / L Tris-HCl buffer, pH 7.8.

[0071] (46) Elution release buffer 46: 0.01% Tween 40, 0.074% disodium EDTA, 0.85% sodium chloride, 0.1% Proclin ® 300, 0.05% sodium azide, 1% bovine serum albumin in 50 mmol / L Tris-HCl buffer, pH 7.8.

[0072] Table 7

[0073] From the experimental results, it can be seen that elution release buffer 26 is the optimal formula. The detection fluorescence value of calibrator B / detection fluorescence value of calibrator A (B / A inhibition rate) reaches the minimum and is within 70% to 90%, indicating higher sensitivity. The detection fluorescence value of calibrator F / detection fluorescence value of calibrator A (F / A inhibition rate) reaches the minimum and is within 10% to 25%, indicating that there is a sufficient difference between the binding rate counts at each dose point on the curve. Taking elution release buffer 46 without releasing agent as the control, the fluorescence value of calibrator A detected by elution release buffer 26 is 49.00% of the fluorescence value of calibrator A detected by elution release buffer 46, which is within 33% to 50%. At this time, it has high sensitivity (B / A) and a wide dose-response curve working range (F / A). The present invention also found that the release agents 8-anilino-1-naphthalenesulfonic acid, sodium salicylate, trichloroacetic acid, and 3-bromo-5-hydroxypyridine have a good T4 release effect; however, trichloroacetic acid is strongly acidic and a protein precipitant, which has a significant inhibitory effect on the immune response, and the fluorescence values detected by calibrators A to F are low; 8-anilino-1-naphthalenesulfonic acid and sodium salicylate have a synergistic effect and can achieve a satisfactory T4 release effect; the introduction of 3-bromo-5-hydroxypyridine did not show a synergistic effect.

[0074] Example 9 A test method for a filter paper dried blood spot thyroxine assay kit (time-resolved fluorescence immunoassay) suitable for neonatal hypothyroidism screening, using Figure 1 System shown: (1) Fully automatic inspection method: 1) Sample blood sample collection: Use filter paper dry blood spots to collect sample blood samples, and enter the sample related information into the information management system module; 2) Sample quality analysis: Use a filter paper dry blood spot quality analysis module containing a high-definition industrial camera system, image processing and analysis system to analyze the quality of the filter paper dry blood spot sample and determine whether the sample quality is qualified; 3) Barcode recognition: Use a barcode scanning module to read the filter paper dry blood spot sample barcode, export the carrier sample related information, and associate it with the sample experimental process; 4) Cutting and collection of calibration products, quality control products, and test samples: Use a filter paper dry blood spot punching module containing a filter paper dry blood spot sample conveying device, a high-definition industrial camera system, an image analysis and positioning system, a filter paper dry blood spot cutter, and a high-precision motion positioning device to cut out a 3.2 mm (1 / 8) diameter filter paper dry blood spot. inch) of blood spots and fix them on the plate needle, and use the gripper module with lateral / longitudinal movement function and high-precision positioning function to move the plate needle to the temporary storage position of the plate needle holder; 5) Add tracer working solution: Use the gripper module with lateral / longitudinal movement function and high-precision positioning function to move the 96-well microplate reaction carrier to the oscillation incubation module with bottom heating, constant temperature control system, and vortex oscillation function, and use the sample pipetting module with lateral / longitudinal movement function and high-precision positioning function to dilute the tracer and elution release buffer at a volume ratio of 1:50 and mix them to prepare the tracer working solution, and use the gripper module with lateral / longitudinal movement function to transfer the tracer to the elution release buffer at a volume ratio of 1:50 and mix them evenly. and a sample-adding pipetting module with high-precision positioning function to add 50-200µL of tracer working solution, preferably 150µL, into the microwell cup of the 96-well microplate reaction carrier; 6) First incubation: Use a gripper arm module with lateral / vertical movement function and high-precision positioning function to move the plate needle to the 96-well microplate reaction carrier with tracer working solution, soak the blood spot fixed on the plate needle in the tracer working solution in the 96-well microplate reaction carrier, and then incubate with shaking at room temperature in the shaking incubation module for 90-150 minutes, preferably 120 minutes; 7) Plate washing: Dilute the detergent with purified water at a volume ratio of 1:25 to form a cleaning working solution. Use a gripping arm module with lateral / longitudinal movement function and high-precision positioning function to move the plate needle to the temporary storage position of the plate needle holder. At the same time, use a gripping arm module with lateral / longitudinal movement function and high-precision positioning function to move the 96-well microplate reaction carrier to the plate washing module. The plate washing module uses a cleaning working fluid to clean the 96-well microplate reaction carrier 4 to 8 times, preferably 6 times.8) Adding Inducer: Using a gripper module with lateral / vertical motion and high-precision positioning, move the 96-well microplate reaction carrier to the shaking incubation module equipped with bottom heating, a constant temperature control system, and vortex shaking. Using a pipetting module with lateral / vertical motion and high-precision positioning, add 50–200 µL (preferably 150 µL) of inducer to the microwell cup of the 96-well microplate reaction carrier. 9) Second Incubation: Oscillate the 96-well microplate reaction carrier with inducer at 1200 rpm in the incubation position of the shaking incubation module at room temperature for 2–10 minutes, preferably 5 minutes. 10) Detection: A gripper module with lateral and longitudinal motion and high-precision positioning capabilities moves the 96-well microplate reaction carrier containing the inducer to the measurement module. The measurement module features high-precision displacement and positioning mechanisms, a window opening and closing mechanism, an automatic filter switching measurement system, and a photoelectric signal conversion measurement system. Fluorescence counts are determined using the corresponding T4 detection program, an excitation wavelength of 340 nm, and a detection wavelength of 613 nm. Detection must be completed within 30 minutes after the addition of the inducer. 11) Analysis: Fluorescence counts are transmitted to a computer data analysis module equipped with a microcomputer system and laboratory (laboratory) information connection port. Results are analyzed using the LOG-B / Bmax axis conversion method and the SPLINE dose-response curve fitting algorithm. Sample test results are generated using the test software algorithm and transmitted to the information management system module. All instrument modules involved in the test method are fixed to the instrument support platform as an integrated unit and include a safety protection panel. During operation, the safety protection panel remains closed to ensure biosafety during the test and safe operation of the test system. The detection methods are also related to the information management system module, which includes system function modules (basic data settings, user management, sample management, patient self-service query, statistical reports, clinical treatment), report modules (traditional project report management, thalassemia report management, tandem mass spectrometry report management, urine organic acid report management, genetic testing report management, allergen report management), laboratory management module (laboratory, quality control management). The information management system module is built on the network to realize cloud management of various functional modules, standardization and network management of screening and diagnosis and treatment work processes, and barcoding and information management of the entire process from ordering, blood collection, logistics transportation, testing to reporting, query, tracking and follow-up.

[0075] (2) Semi-automatic test method: 1) Blood sample collection: Use filter paper blood spots to collect blood samples, and manually enter the sample related information into the information management system; 2) Sample quality analysis: Analyze the quality of the filter paper blood spots with the naked eye to determine whether the sample quality is qualified; Cutting and collection of calibrators, quality control materials, and test samples: Use punching forceps to cut a blood spot with a diameter of 3.2 mm (1 / 8 inch) on the filter paper blood spots of calibrators, quality control materials, and test samples and place it in a 96-well microplate reaction carrier; 3) Add tracer working solution: Use a pipette to manually dilute the tracer and elution release buffer at a volume ratio of 1:50 and mix them to prepare the tracer working solution. Use a pipette to manually add 50-200µL of tracer working solution, preferably 150µL, to the microwell cup of the 96-well microplate reaction carrier; 4) First incubation: Incubate at room temperature for 90-150 minutes on a shaking incubator. 5) Washing: Dilute the detergent with purified water at a volume ratio of 1:25 to make a working cleaning solution. Wash the 96-well microplate reaction carrier 4-8 times, preferably 6 times, with the working cleaning solution on a semi-automated plate washer. 6) Adding the inducer: Manually add 50-200 µL of the inducer, preferably 150 µL, to the microwell cup of the 96-well microplate reaction carrier using a pipette. 7) Second incubation: Oscillate the 96-well microplate reaction carrier with the inducer at 1200 rpm on a shaking incubator at room temperature for 2-10 minutes, preferably 5 minutes. 8) Detection: Transfer the 96-well microplate reaction carrier with the inducer to a semi-automated fluorescence analyzer. Detect fluorescence using the appropriate T4 detection program, using an excitation wavelength of 340 nm and a detection wavelength of 613 nm. Detection should be completed within 30 minutes of adding the inducer. 9) Result analysis: The measured fluorescence count values are transmitted to the computer data analysis module containing the microcomputer system and laboratory (testing department) information connection port, using LOG-B / B max The results are analyzed by using the axis conversion method and the SPLINE dose-response curve fitting algorithm, and the test results of the samples are obtained through the test software algorithm and transmitted to the information management system module, such as Figure 2 shown.

[0076] The test method of Example 9 was used to test the background, linear range, sensitivity, and precision under other preferred conditions. The results are compared as follows.

[0077]

[0078] From the above results, it can be seen that the precision of the fully automatic inspection method is better than that of the semi-automatic inspection method.

[0079] The above description is intended to serve as a guide for the preferred embodiments of the present invention. Based on the above description, relevant personnel are fully capable of making various changes and modifications without departing from the technical scope of the present invention. The technical scope of the present invention is not limited to the contents of the specification and must be determined according to the scope of the claims.

Claims

1. A filter paper dried blood spot thyroxine assay kit, characterized in that: include: Filter paper dried blood spot calibrators, filter paper dried blood spot quality controls, reaction carriers, tracers, elution and release buffers, cleaning agents, and inducing agents; The elution and release buffer is a 20-100 mmol / L tris-hydrochloric acid buffer containing an eluent, a release agent, a preservative and a blocking agent.

2. The filter paper dried blood spot thyroxine assay kit according to claim 1, characterized in that: The concentration of the eluent in the elution release buffer is 0.01-1 wt %, and the eluent is at least one of Tween 20, Tween 40, Tween 60, Tween 80, disodium edetate, and sodium chloride; The concentration of the release agent in the elution release buffer is 0.01-1 wt %, and the release agent is at least one of 8-anilino-1-naphthalenesulfonic acid, sodium salicylate, sodium thimerosal, trichloroacetic acid, phenytoin sodium, 5-bromo-2-methylpyridine, 3-bromo-5-hydroxypyridine and pepsin; The concentration of the preservative in the elution release buffer is 0.01-1 wt %, and the preservative is at least one of Proclin® 300, sodium azide, and gentamicin; The concentration of the blocking agent in the elution release buffer is 0.5-2 wt %, and the blocking agent is at least one of bovine serum albumin, calf serum, newborn calf serum, fetal bovine serum, fish gelatin, casein and polyvinyl pyrrolidone.

3. The filter paper dried blood spot thyroxine assay kit according to claim 1, characterized in that: The preparation method of the tracer comprises: first replacing the antibody storage liquid of T4 antibody with 50 mmol / L carbonate buffer at pH 9.6, uniformly mixing the T4 antibody and lanthanum salt at a mass ratio of 1:1 to 3:1, incubating with shaking at 2 to 8° C. for 24 to 72 hours, and chromatographically purifying the mixture using a gel column filled with polyacrylamide gel S-200. The collected tracer mother liquor is diluted with 20 mmol / L 3-(N-morpholino)propanesulfonic acid-triethanolamine buffer at a pH 7.8 containing 2 g / L bovine serum albumin at a dilution ratio of 1:(1000 to 6000) to form a semi-finished product; The lanthanum salt includes at least one of a europium salt and a samarium salt; preferably, sodium europium N1-(P-isothiocyanatobenzyl)-diethylenetriaminetetraacetate.

4. The filter paper dried blood spot thyroxine assay kit according to claim 1, characterized in that: The reaction carrier is a 96-well polystyrene enzyme-labeled microplate, and the reaction carrier contains a coating, which is T4 antigen, T4 antigen derivative or a ligand that binds to a T4 antigen derivative; The coating material in the reaction carrier is diluted with a coating buffer to a concentration of 1-10 μg / mL, coated in the reaction carrier with a coating volume of 50-200 μL, incubated at 2-8°C for 20±2 hours, then washed once with a washing solution with a volume of 100-300 μL / well, and then 100-250 μL of blocking solution is added to each well and incubated at 37°C for 1-3 hours. The blocking solution is discarded, the wells are spun and dried, vacuum-sealed, and stored at 2-8°C for later use; The coating buffer is 50 mmol / L carbonate buffer at pH 9.5-9.7, 50 mmol / L Tris-HCl buffer at pH 7.7-7.9, 50 mmol / L phosphate buffer at pH 7.1-7.3, and 20 mmol / L phosphate buffer at pH 4.2-4.

4.

5. The filter paper dried blood spot thyroxine assay kit according to claim 1, characterized in that: The cleaning agent is a tris-hydrochloric acid buffer containing 1.125 g / L sodium chloride, 0.2 mL / L Tween-20, and 30 ppm Proclin® 300 preservative.

6. The filter paper dried blood spot thyroxine assay kit according to claim 1, characterized in that: The inducer is an aqueous solution containing 0.0494 g / L anhydrous sodium acetate, 0.0043 g / L β-naphthylcarbonyltrifluoroacetone, 0.021 g / L tri-n-octylphosphine oxide, 1.143 ml / L glacial acetic acid, 0.5 ml / L anhydrous ethanol, and 0.5 ml / L Triton X-100.

7. The filter paper dried blood spot thyroxine assay kit according to claim 1, characterized in that: The preparation method of the filter paper dried blood spot calibrator is as follows: diluting thyroxine with a dissolving solution to a concentration of 2-30 mg / mL, then diluting it with a calibrator diluent to a concentration of 0-64 μg / dL, adding an equal volume of blood cells, and mixing to obtain a filter paper dried blood spot calibrator with a concentration of 0-32 μg / dL.

8. The filter paper dried blood spot thyroxine assay kit according to claim 1, characterized in that: The filter paper dried blood spot quality control product is prepared by diluting thyroxine with a dissolving solution to a concentration of 2 to 30 mg / mL, then diluting it with a calibrator diluent to a concentration of 6 to 24 μg / dL, adding an equal volume of blood cells, and mixing to obtain a filter paper dried blood spot calibrator with a concentration of 3 to 12 μg / dL.

9. The filter paper dried blood spot thyroxine assay kit according to any one of claims 7 to 8, characterized in that: The calibrator diluent is 50 mmol / L Tris-HCl buffer containing 5 g / L bovine serum albumin; The preparation method of the dissolving solution is as follows: 80-120 μL of 20-25 mg / mL clear dimethyl sulfoxide stock solution is added to 300-500 μL of polyethylene glycol 300 and mixed evenly; 40-60 μL of Tween-80 is added to the above system and mixed evenly; then physiological saline is added to make the volume to 1 mL and mixed evenly to obtain the dissolving solution; or The dissolving solution is prepared by adding 80 to 120 μL of a 20 to 25 mg / mL clear dimethyl sulfoxide stock solution to 880 to 920 μL of a 15% to 25% sulfobutyl-β-cyclodextrin saline solution, and mixing the mixture to obtain a dissolving solution; or The dissolving solution is prepared by adding 80-120 μL of a 20-25 mg / mL clear dimethyl sulfoxide stock solution to 880-920 μL of corn oil, and mixing the mixture to obtain a dissolving solution.

10. A method for detecting thyroxine using a filter paper dried blood spot assay kit, characterized in that: A filter paper dried blood spot thyroxine assay kit and detection system according to any one of claims 1 to 9 is used, wherein the detection system comprises (1) a filter paper dried blood spot quality analysis module; (2) a barcode scanning module; (3) a filter paper dried blood spot punching and cutting module; (4) a sample addition and liquid transfer module; (5) an arm grasping module; (6) an oscillation and incubation module; (7) a plate washing module; and (8) a measurement module. (9) Computer data analysis module; (10) Instrument support platform; (11) Information management system module; The testing steps include: (1) Blood sample collection: Use filter paper dried blood spots to collect blood samples, and enter the sample-related information into the information management system module; (2) Sample quality analysis: A filter paper dried blood spot quality analysis module containing a high-definition industrial camera system, image processing and analysis system is used to analyze the quality of the filter paper dried blood spot sample and determine whether the sample quality is qualified; (3) Barcode recognition: Use the barcode scanning module to read the barcode of the filter paper dried blood sample, export the carrier sample related information, and link it to the sample experimental process; (4) Cutting and collecting calibrators, quality control products, and samples to be tested: Use a filter paper dried blood smear module containing a filter paper dried blood smear sample conveying device, a high-definition industrial camera system, an image analysis and positioning system, a filter paper dried blood smear blood spot cutter, and a high-precision motion positioning device to cut a blood spot with a diameter of 3.2 mm (1 / 8 inch) and fix it on the plate needle. Use a gripper module with horizontal / vertical motion function and high-precision positioning function to move the plate needle to the temporary storage position of the plate needle holder; (5) Adding tracer working solution: Use a gripper module with lateral / longitudinal motion function and high-precision positioning function to move the reaction carrier to an oscillation incubation module with bottom heating, constant temperature control system, and vortex oscillation function. Use a sample loading and liquid transfer module with lateral / longitudinal motion function and high-precision positioning function to dilute the tracer and elution release buffer at a volume ratio of 1:50 and mix them to prepare a tracer working solution. Use a sample loading and liquid transfer module with lateral / longitudinal motion function and high-precision positioning function to add 50-200 μL of tracer working solution into the microporous cup of the reaction carrier; (6) First incubation: Use a gripper module with lateral / vertical movement and high-precision positioning functions to move the plate needle to a reaction carrier containing tracer working solution. The blood spot fixed on the plate needle is immersed in the tracer working solution in the reaction carrier, and then incubated in an oscillating incubation module at room temperature for 90-150 min. (7) Plate washing: Dilute the cleaning agent with purified water at a volume ratio of 1:25 to form a cleaning working solution. Use a gripper module with lateral / vertical movement and high-precision positioning functions to move the plate needle to the temporary storage position of the plate needle bracket. At the same time, use a gripper module with lateral / vertical movement and high-precision positioning functions to move the reaction carrier to the plate washing module. The plate washing module uses the cleaning working solution to wash the reaction carrier 4 to 8 times. (8) Adding inducer: Use a gripper module with lateral / vertical motion function and high-precision positioning function to move the reaction carrier to an oscillation incubation module with bottom heating, constant temperature control system, and vortex oscillation function, and use a sample pipetting module with lateral / vertical motion function and high-precision positioning function to add 50-200 μL of inducer into the microporous cup of the reaction carrier; (9) Second incubation: The reaction carrier with the inducer is oscillated at room temperature at a motor speed of 1200 rpm for 2 to 10 minutes on the incubation position in the oscillating incubation module; (10) Detection: A gripper module with lateral / vertical movement function and high-precision positioning function is used to move the reaction carrier with the inducing agent to the position of the measurement module. The measurement module has a high-precision shift and positioning device, a window opening and closing device, an automatic filter switching measurement system, and a photoelectric signal conversion measurement system. The corresponding T4 detection program is selected, and the excitation wavelength of 340 nm and the detection wavelength of 613 nm are used for detection to obtain the fluorescence count value; (11) Result analysis: The measured fluorescence count values are transmitted to a computer data analysis module containing a microcomputer system and a laboratory information connection port. The results are analyzed using the LOG-B / Bmax axis conversion method and the SPLINE dose-response curve fitting algorithm. The test results of the samples are obtained through the test software algorithm and transmitted to the information management system module.

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