Immunoreactive trypsin (pro-trypsin) determination kit and detection method thereof

Abstract

The invention discloses an immunoreactive trypsin (pro-trypsin) determination kit and a detection method thereof, belongs to the technical field of kits, and provides the immunoreactive trypsin (pro-trypsin) determination kit and the detection method which are used for quantitative determination of neonatal immunoreactive trypsin (pro-trypsin). According to the present invention, the detection sensitivity and the specificity of the reagent are effectively improved by optimizing the antibody pairing, the IRT recombinant antigen is prepared by optimizing the IRT dominant sequence and using the dominant expression system, the reactivity and the activity stability of the IRT in the calibration product and the reaction carrier and the tracer agent are improved, and the stability of the calibration product is further improved; by optimizing the coating and sealing process of the reaction carrier, the reaction background is reduced, and the signal-to-noise ratio, the reactivity, the sensitivity, the precision and the stability are improved; by adopting a full-automatic intelligent detection method, the detection speed, efficiency, precision and accuracy are improved.

Description

Technical Field

[0001] The present invention relates to the technical field of kits, and particularly relates to a kit for determining immunoreactive trypsin (ogen) and a detection method thereof. Background Art

[0002] Cystic fibrosis (CF) is one of the common autosomal recessive genetic diseases affecting multiple systems including the respiratory, digestive, endocrine, and reproductive systems in Caucasians. The cystic fibrosis transmembrane conductance regulator (CFTR) gene is currently the only known pathogenic gene for CF. CF is relatively common in Caucasians, with an incidence of 1 / 2,000. In the past, few cases of CF were reported in China. CF was included in the first batch of rare disease catalogs in China in 2018. In recent years, CF has been gradually recognized. In the past 10 years, the number of CF patients reported in China has exceeded 2.5 times the total number reported in the previous 30 years. Early diagnosis of CF has a great impact on the prognosis of patients. The research progress on CF gene modification has brought about a revolution in treatment, significantly improving the lifespan of CF patients in European and American countries. Due to the differences in genotypes and phenotypes between Chinese CF children and Caucasians, there are problems of insufficient diagnosis and irregular treatment of this disease in China.

[0003] Newborn screening, as one of the most effective public health measures, has been carried out for nearly 50 years. Starting from the single detection of phenylketonuria in 1962, with the improvement of screening technology, it has now developed into a screening for more than 50 diseases including endocrine system diseases (congenital hypothyroidism, congenital adrenal hyperplasia), hemoglobinopathies, infectious diseases (toxoplasmosis infection, human immunodeficiency virus infection), cystic fibrosis, and inborn errors of metabolism. As the third-level preventive measure for reducing birth defects, newborn screening is an important task for improving the population quality and reducing and lowering children's mental retardation, and has become a great innovation in public health in the 20th century. Early diagnosis of CF can be carried out by measuring the blood value of immunoreactive trypsin (ogen) (IRT) in newborns. IRT is a pancreatic zymogen based on protein and plays an important role in human protein digestion. The current IRT reagents for CF screening have problems such as low clinical sensitivity, weak specificity, weak and easily inactivated IRT reaction signals in calibrators, poor stability of calibrators, low signal-to-noise ratio, poor detection precision, and low automation level. Summary of the Invention

[0004] The present invention provides a kit for determining immunoreactive trypsin (ogen) and a detection method thereof, which are used for quantitative determination of immunoreactive trypsin (ogen) in newborns.

[0005] The present invention solves its technical problems by adopting the following technical solutions: An immunoreactive trypsin (ogen) assay kit for quantitatively determining immunoreactive trypsin (ogen) in newborns, comprising the following components: filter paper dried blood spot calibrator, filter paper dried blood spot quality control product, reaction carrier, tracer, experimental buffer, washing solution and inducer.

[0006] As a preferred embodiment of the present invention, the preparation method of the filter paper dried blood spot calibrator is as follows: Dilute immunoreactive trypsin with a calibrator diluent to twice the required concentration, add an equal volume of blood cells, mix well, and drop 50 μL per point on the filter paper, spotting calibrator points A, B, C, D, E and F, and the calibrator concentrations are 0, 25, 50, 100, 250, 500 ng / mL respectively. Air-dry quickly at room temperature under dehumidification conditions and store for later use.

[0007] 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 immunoreactive trypsin with a calibrator diluent to twice the required concentration, add an equal volume of blood cells, mix well, and drop 50 μL per point on the filter paper, spotting quality control points C1, C2 and C3, and the quality control product concentrations are 50, 100, 250 ng / mL respectively. Air-dry quickly at room temperature under dehumidification conditions and store for later use.

[0008] As a preferred embodiment of the present invention, the calibrator diluent includes one of human serum, sheep serum, 50 mmol / L Tris-HCl buffer containing 5 g / L bovine serum albumin with a pH of 7.8.

[0009] As a preferred embodiment of the present invention, the calibrator diluent further includes a stabilizer, and the concentration of the stabilizer in the calibrator diluent is 5 - 20 μg / mL. The stabilizer includes at least one of dithiothreitol, β-mercaptoethanol, tris(2-carboxyethyl)phosphine hydrochloride 6-aminohexane, trypsin inhibitor (R21221), and diisopropyl fluorophosphate.

[0010] As a preferred embodiment of the present invention, the concentration of trypsin inhibitor (R21221) in the calibrator diluent is 10 μg / mL.

[0011] As a preferred embodiment of the present invention, the immunoreactive trypsin includes natural human immunoreactive trypsin or recombinant human immunoreactive trypsin; among them, natural human immunoreactive trypsin is a full-length protein and has no chymotrypsin activity against N-succinyl-alanyl-alanyl-prolyl-phenylalanine p-nitroanilide. Amino acid sequence of recombinant human immunoreactive trypsin: LNNDIMLIKLSSRAVINARVSTISLPTAPPATGTKCLISGWGNTASSGADYPDELQCLDAPVLSQAKCEASYPGKITSNMFCVGFLEGGKDSCOGDSGGPVC; or VGGYNCEENSVPYQVSLNSGYHFCGGSLINEQWVVSAGHCYKSRIQVRLGEHNIEVLEGNEQFINAAKIRHPQYDRKTLNNDIMLIKLSSRAVINARVSTISLPTAPPATGTKCLISGWGNTASSGADYPDELQCLDAPVLSQAKCEASYPGKITSNMFCVGFLEGGKDSCQGDSGGPVVCNGQLQGVVSWGDGCAQKNKPGVYTKVYNYVKWIKNTIAANS.

[0012] 5. The immunoreactive trypsin (ogen) assay kit according to claim 1, wherein the method for preparing the reaction carrier is as follows: Dilute the immunoreactive trypsin antibody with a coating buffer to the optimal working concentration, coat it in the wells of a 96-well microplate, with a coating volume of 50 - 200 μL, incubate at 2 - 8 °C for 18 - 22 h, then wash once with a washing working solution with a volume of 100 - 300 μL / well, then add 100 - 250 μL of the blocking solution to each well and incubate at 36 - 38 °C for 1 - 3 h, discard the blocking solution, drain and air-dry, vacuum-seal the film, and store for later use at 2 - 8 °C; the coating buffer is a phosphate buffer with a concentration of 20 mmol / L and a pH of 4.3; The blocking solution contains at least one of bovine serum albumin, calf serum, newborn calf serum, fetal bovine serum, casein, fish gelatin, polyvinylpyrrolidone 90, polyvinylpyrrolidone 360, polyethylene glycol 6000, polyethylene glycol 20000, Tween-20, Tween-40, sucrose, trehalose, sorbitol; the blocking buffer is a phosphate buffer with a concentration of 20 mmol / L and a pH of 4.3; The immunoreactive trypsin antibody is a rabbit or mouse monoclonal antibody, and the monoclonal antibody is one of a human immunoreactive trypsin 1 monoclonal antibody and a human immunoreactive trypsinogen 2 monoclonal antibody; among them, the monoclonal antibody is one of a human immunoreactive trypsin 1 monoclonal antibody and a human immunoreactive trypsinogen 2 monoclonal antibody. Preferably, the human immunoreactive trypsin 1 monoclonal antibody and the human immunoreactive trypsinogen 2 monoclonal antibody are mixed in a certain optimal ratio, and the preferred ratio is 1:1 to 3:1. The human immunoreactive trypsin 1 monoclonal antibody is active against synthetic substrates Boc-Phe-Ser-Arg-Mec, Boc-Leu-Thr-Arg-Mec, Boc-Gln-Ala-Arg-Mec, and Boc-Val-Pro-Arg-Mec, and has a reactivity of more than 98% with various forms of human immunoreactive trypsinogen; among them, the immunoreactive trypsinogen 2 monoclonal antibody has a reactivity of no more than 7% and 3% with human immunoreactive trypsin 1 and human immunoreactive trypsin 2, respectively; the coating human immunoreactive trypsin 1 monoclonal antibody and the human immunoreactive trypsinogen 2 monoclonal antibody and the labeled human immunoreactive trypsin 1 monoclonal antibody and the human immunoreactive trypsinogen 2 monoclonal antibody have different determinants for immunoreactive trypsin or immunoreactive trypsinogen.

[0013] As a preferred embodiment of the present invention, the blocking solution is a mixture of 0.2 - 4 g / L polyvinylpyrrolidone 360, 0.01 - 0.5 ml / L Tween-20, and 30 - 80 g / L sucrose, and more preferably a mixture of 1 g / L polyvinylpyrrolidone 360, 0.015 ml / L Tween-20, and 50 g / L sucrose.

[0014] As a preferred embodiment of the present invention, the tracer is a lanthanide-labeled immunoreactive trypsin antibody, and its preparation method is as follows: The immunoreactive trypsin antibody is first subjected to antibody storage liquid replacement treatment with 50 mmol / L carbonate buffer solution at pH 9.6. The mass ratio of the immunoreactive trypsin antibody to the lanthanide element is mixed at 1:1 to 3:1, and incubated with shaking at 2 - 8 °C for 24 - 72 h. Chromatographic purification is carried out using a gel column filled with Sephacryl S-200, and the collected tracer mother liquor is diluted with a diluent into a semi-finished product; The lanthanide element is at least one of europium chelate (sodium N- 1 -(p-isothiocyanatobenzyl)-diethylenetriamine tetraacetic acid europium) and samarium chelate; The immunoreactive trypsin antibody is a rabbit or mouse monoclonal antibody.

[0015] Among them, the monoclonal antibody is one of a monoclonal antibody against human immunoreactive trypsin 1 and a monoclonal antibody against human immunoreactive trypsinogen 2. Preferably, a monoclonal antibody against human immunoreactive trypsin 1 labeled with a lanthanide element and a monoclonal antibody against human immunoreactive trypsinogen 2 labeled with a lanthanide element are mixed in a certain optimal ratio, and the preferred ratio is 1:1 to 3:1. The monoclonal antibody against human immunoreactive trypsin 1 is active against synthetic substrates Boc-Phe-Ser-Arg-Mec, Boc-Leu-Thr-Arg-Mec, Boc-Gln-Ala-Arg-Mec, and Boc-Val-Pro-Arg-Mec, and has a reactivity of more than 98% with various forms of human immunoreactive trypsinogen. The monoclonal antibody against immunoreactive trypsinogen 2 has a reactivity of no more than 2% and 6% with human immunoreactive trypsin 1 and human immunoreactive trypsin 2, respectively. The lanthanide element-labeled monoclonal antibody against human immunoreactive trypsin 1 and the monoclonal antibody against human immunoreactive trypsinogen 2 have different determinants against immunoreactive trypsin or immunoreactive trypsinogen from the monoclonal antibody against human immunoreactive trypsin 1 and the monoclonal antibody against human immunoreactive trypsinogen 2 used for coating.

[0016] As a preferred embodiment of the present invention, the experimental buffer is a 50 mmol / L, pH 7.8 tris(hydroxymethyl)aminomethane-hydrochloric acid buffer containing 8.5 g / L sodium chloride, 0.2 mL / L Proclin ® 300, 7.4 mg / L disodium ethylenediaminetetraacetate, 1 mL / L mouse serum, 100 mL / L newborn bovine serum, 1 mg / L casein, and 3 - 10 μg / mL trypsin inhibitor (R21221); The cleaning solution is a 50 mmol / L, pH 7.8 tris(hydroxymethyl)aminomethane-hydrochloric acid buffer containing 1.125 g / L sodium chloride, 0.2 mL / L Tween-20, and 30 ppm Proclin ® 300 preservative; The inducer is an enhancement solution containing 0.0494 g / L sodium acetate (anhydrous), 0.0043 g / L β-naphthoyltrifluoroacetone, 0.021 g / L tri-n-octylphosphine oxide, 1.143 ml / L glacial acetic acid, 0.5 ml / L absolute ethanol, 0.5 ml / L Triton X-100, and purified water.

[0017] As a preferred embodiment of the present invention, it further includes plate needles and barcodes; The barcode is a barcode containing project information.

[0018] As a preferred embodiment of the present invention, the plate needle comprises a plate needle socket body and a detachable strip-shaped plate needle fixed on the socket body. The strip-shaped plate needle is a conical columnar body with a needle tip at the front end and a card slot 3 mm behind the needle tip. The conical columnar body is fixed on a strip-shaped gelatinous carrier in an 8-needle or 12-needle pattern. The plate needle spacing is adapted to the micro-holes of a 96-well enzyme-linked immunosorbent assay microplate. The needle tip can pass through the center of a 3.2-mm blood spot and fix the blood spot on the card slot.

[0019] The present invention also provides a detection method for an immunoreactive trypsin (ogen) assay kit. Based on the above-mentioned immunoreactive trypsin (ogen) assay kit, it includes the following modules: (1) a barcode scanning module; (2) a filter paper dried blood spot quality analysis module; (3) a filter paper dried blood spot punching module; (4) a sample adding pipetting module; (5) a gripping arm module; (6) an oscillating incubation module; (7) a 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 barcode scanning module is used to identify and read the barcode information of the filter paper dried blood spot sample and the carrier barcode. The filter paper dried blood spot quality analysis module analyzes the quality of the filter paper dried blood spot sample. Qualified filter paper dried blood spot samples to be tested, calibration products, and quality control products are punched with an appropriate amount of blood spots by the filter paper dried blood spot punching module and fixed onto the plate needles, and the plate needles with the fixed blood spots are transferred to the bracket temporary storage position of the instrument support platform. The gripping arm module moves the carrier to the oscillating incubation module, and the sample adding pipetting module adds an appropriate amount of tracer working solution diluted with an experimental buffer into the carrier. The gripping arm module places the plate needles with the fixed blood spots into the carrier in a specified order and immerses the blood spots into the tracer working solution. Then the carrier is oscillated and incubated for an appropriate time on the oscillating incubation module. The gripping arm module transfers the plate needles with the fixed blood spots to the bracket temporary storage position of the instrument support platform. At the same time, the gripping arm module moves the carrier to the washing module for washing. After washing, the gripping arm module moves the carrier to the oscillating incubation module, and the sample adding pipetting module adds an appropriate amount of inducer into the carrier, and the oscillating incubation module oscillates and incubates at room temperature for an appropriate time. The gripping arm module moves the carrier to the measurement module to read the count value. The count value is analyzed by the computer data analysis module, and the result is transmitted to the information management system module.

[0020] As a preferred embodiment of the present invention, it includes the following steps: (1) Sample blood collection: Collect the sample blood with filter paper dried blood spots and input the relevant sample information into the information management system module. (2) Sample quality analysis: Use the filter paper dried blood spot quality analysis module containing a high-definition industrial camera system, an image processing and analysis system to analyze the quality of the filter paper dried blood spot sample and determine whether the sample quality is qualified. (3)Barcode recognition: Use a barcode scanning module to read the barcode of the dried blood spot sample on the filter paper, export the relevant information of the carrier sample, and associate it with the sample experimental process; (4)Cutting and collection of calibrators, quality control products, and samples to be tested: Cut a blood spot with a diameter of 3.2 mm (1 / 8 inch) from the dried blood spot filter paper of the sample to be tested with qualified quality, as well as the dried blood spot filter papers of calibrators and quality control products, using a filter paper dried blood spot punching module containing a filter paper dried blood spot sample transfer device, a high-definition industrial camera system, an image analysis and positioning system, a filter paper dried blood spot blood spot cutter, and a high-precision motion positioning device, and fix it on the plate needle. Use a gripper arm module with horizontal / vertical movement functions and high-precision positioning functions to move the plate needle to the temporary storage position of the plate needle holder; (5)Adding tracer working solution: Use a gripper arm module with horizontal / vertical movement functions and high-precision positioning functions to move the 96-well microplate reaction carrier to an oscillating incubation module with bottom heating, a constant temperature control system, and a vortex oscillation function. Use a pipetting module with horizontal / vertical movement functions and high-precision positioning functions to dilute and mix the tracer and the experimental buffer in a volume ratio of 1:50 to prepare a tracer working solution. Use a pipetting module with horizontal / vertical movement functions and high-precision positioning functions to add 50 - 200 µL of the tracer working solution to the micro-cups of the 96-well microplate reaction carrier; (6)First incubation: Use a gripper arm module with horizontal / vertical movement functions and high-precision positioning functions to move the plate needle into the 96-well microplate reaction carrier containing the 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 at room temperature with shaking in the oscillating incubation module for 90 - 150 min, preferably 120 min; (7)Washing the plate: Dilute the cleaning solution with purified water in a volume ratio of 1:25 to prepare a cleaning working solution. Use a gripper arm module with horizontal / vertical movement functions 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 arm module with horizontal / vertical movement functions 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 - 8 times; (8)Adding inducer: Use a gripper arm module with horizontal / vertical movement functions and high-precision positioning functions to move the 96-well microplate reaction carrier to an oscillating incubation module with bottom heating, a constant temperature control system, and a vortex oscillation function. Use a pipetting module with horizontal / vertical movement functions and high-precision positioning functions to add 50 - 200 µL of inducer to the micro-cups of the 96-well microplate reaction carrier; (9)Second incubation: The 96-well microplate reaction carrier with the inducer is incubated on the incubation position in the oscillating incubation module at room temperature with a motor speed of 1,200 rpm for 2 - 10 minutes; (10)Detection: The 96-well microplate reaction carrier with the inducer is moved to the measurement module position by using a gripper arm module with lateral / longitudinal movement function and high-precision positioning function. The measurement module has a high-precision displacement and positioning device, a window opening and closing device, an automatic filter switching measurement system, and a photoelectric signal conversion measurement system. The corresponding immunoreactive trypsin detection program is selected, and the fluorescence meter value is obtained by detecting with an excitation wavelength of 340 nm and a detection wavelength of 613 nm. The detection work needs to be completed within 30 minutes after adding the inducer; (11)Result analysis: The measured fluorescence meter value is transmitted to the computer data analysis module containing a microcomputer system and a laboratory (clinical laboratory) information connection port. The LOG-LOG_B axis conversion method and the SPLINE dose-response curve fitting algorithm are used for result analysis. The test result of the sample is obtained through the test software algorithm, and the result is transmitted to the information management system module.

[0021] As a preferred implementation of the present invention, the following steps are further included: (12)All the instrument modules involved in the detection methods (1) - (10) are fixed into an integrated body by an instrument support platform and are equipped with a safety protection panel. During the operation of the detection system, the safety protection panel is always in a closed state to ensure biosafety protection during the detection operation and the safe operation of the detection system. The detection methods (1) - (10) are also all associated with the information management system module. The information management system module includes a system function module (basic data setting, user management, sample management, patient self-service query, statistical report, clinical treatment), a report module (traditional item report management, thalassemia report management, tandem mass spectrometry report management, urinary organic acid report management, gene detection report management, allergen report management), and a laboratory management module (laboratory, quality control management). The information management system module is structured on a network to realize cloud management of each function module, standardized and networked management of the screening and diagnosis and treatment work processes, and realize barcoding and informatization management of the whole process from order opening, blood collection, logistics transportation, detection to report, query, and follow-up.

[0022] Advantages of the present invention: The neonatal cystic fibrosis screening kit and detection method provided by the present invention are used for the quantitative determination of immunoreactive trypsin (ogen) in newborns. By optimizing the antibody pairing, the detection sensitivity and specificity of the reagent are effectively improved. By selecting the dominant sequence of IRT and using the dominant expression system to prepare the IRT recombinant antigen, the reactivity and activity stability of IRT in the calibrator with the reaction carrier and tracer in the present invention are improved, and the stability of the calibrator (thermal stability, transportation stability, real-time stability, and experimental repeatability) is further improved. By optimizing the coating and blocking processes of the reaction carrier, the reaction background is reduced, and the signal-to-noise ratio, reactivity, sensitivity, precision, and stability are improved. Moreover, the fully automatic intelligent detection method is adopted to improve the detection speed, efficiency, precision, and accuracy. An automated detection kit with a high signal-to-noise ratio, good reactivity, high sensitivity (excellent blank limit, detection limit, and quantification limit), high precision (high within-batch precision and between-batch precision), good stability (long shelf life, good transportation stability, good thermal stability, and good stability after opening), and good accuracy is provided. Description of the Drawings

[0023] Figure 1 It is a detection result diagram of Example 1 of the present invention.

[0024] Figure 2 It is a dose-response curve diagram of the neonatal immunoreactive trypsin assay kit (time-resolved fluorescence immunoassay) applicable to neonatal cystic fibrosis screening of the present invention. Detailed Embodiments

[0025] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0026] In the present invention, among the technically characterized described in an open-ended manner, there are included closed technical solutions composed of the listed features, as well as open technical solutions including the listed features.

[0027] In the present invention, regarding the numerical range, unless otherwise specified, the above numerical range is considered continuous and includes the minimum and maximum values of the range, as well as each value between such minimum and maximum values. Further, when the range refers to an integer, it includes each integer between the minimum and maximum values of the range. In addition, when providing multiple ranges to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all sub-ranges subsumed therein.

[0028] In the present invention, there is no particular limitation on the specific dispersion and stirring treatment methods.

[0029] The reagents or instruments used in the present invention that are not specified by the manufacturer are all conventional products that can be obtained through commercial purchase. The raw materials used in each comparative example and the raw materials used in the parallel experiments of each example are the same commercially available products unless otherwise specified.

[0030] Example 1 Calibration diluent effects of calibration products and quality control products of filter paper dry blood spots for a neonatal immunoreactive trypsin assay kit (time-resolved fluorescence immunoassay) applicable to neonatal cystic fibrosis screening: Both the calibration product and the quality control product of the filter paper dry blood spot were prepared by diluting immunoreactive trypsin (sequence: LNNDIMLIKLSSRAVINARVSTISLPTAPPATGTKCLISGWGNTASSGADYPDELQCLDAPVLSQAKCEASYPGKITSNMFCVGFLEGGKDSCOGDSGGPVC) to twice the required concentration with the calibration diluent, and adding an equal volume of blood cells (for example, 1 mL of the calibration diluent-diluted calibration product or quality control product mother liquor was added with 1 mL of blood cells) to prepare the calibration product and the quality control product. After thorough mixing, it was spotted on S&S903 filter paper at 50 μL / drop, and calibration products A, B, C, D, E, and F spots and quality control products C1, C2, and C3 spots were spotted. The calibration product concentrations were approximately 0, 25, 50, 100, 250, and 500 ng / mL respectively, and the quality control product concentrations were 50, 100, and 250 ng / mL respectively. It was quickly dried at room temperature under dehumidification, put into a storage bag, vacuum laminated, and stored at low temperature for standby. The calibration diluent is one of human serum, sheep serum, and 50 mmol / L, pH 7.8 Tris-HCl buffer containing 5 g / L bovine serum albumin. 10 μg / mL trypsin inhibitor (R21221) was added thereto. The calibration products and quality control products prepared by diluting immunoreactive trypsin at the same theoretical concentration with different calibration diluents had the following reactivity effects under other preferred conditions as shown in the table below.

[0031] Table 1

[0032] From the above results, it can be seen that for calibration products and quality control products with the same concentration diluted with human serum and sheep serum, the background of calibration product A is relatively high, and the reactivity of calibration products B - F and quality control products C1 - C3 is more than 50% lower than that of calibration products with the same concentration diluted with 50 mmol / L, pH 7.8 Tris-HCl buffer containing 5 g / L bovine serum albumin. It is possible that substances hydrolyzing trypsin or substances activating the autolysis of trypsin itself are contained in the matrices of human serum and sheep serum. Preferably, 50 mmol / L, pH 7.8 Tris-HCl buffer containing 5 g / L bovine serum albumin is used as the matrix to dilute immunoreactive trypsin, which can significantly improve the immunoreactivity (hydrolysis stability) of immunoreactive trypsin in the matrix and its reactivity in the immunoreaction.

[0033] Example 2 Effect of stabilizers on filter paper dry blood spot calibration products and filter paper dry blood spot quality control products of a neonatal immunoreactive trypsin assay kit (time-resolved fluorescence immunoassay) suitable for neonatal cystic fibrosis screening: Both the filter paper dry blood spot calibration products and the filter paper dry blood spot quality control products are prepared by diluting immunoreactive trypsin (sequence: LNNDIMLIKLSSRAVINARVSTISLPTAPPATGTKCLISGWGNTASSGADYPDELQCLDAPVLSQAKCEASYPGKITSNMFCVGFLEGGKDSCOGDSGGPVC) with a calibration product diluent to a stock solution twice the required concentration, and adding an equal volume of blood cells (such as 1 mL of calibration product or quality control product stock solution diluted with calibration product diluent, adding 1 mL of blood cells) to prepare calibration products and quality control products. After thorough mixing, it is spotted on S&S903 filter paper at 50 μL / drop, and calibration products A, B, C, D, E, and F and quality control products C1, C2, and C3 are spotted. The concentrations of the calibration products are approximately 0, 25, 50, 100, 250, 500 ng / mL respectively, and the concentrations of the quality control products are 50, 100, 250 ng / mL respectively. It is quickly air-dried at room temperature under dehumidification, put into a storage bag, vacuum laminated, and stored at low temperature for standby. The calibration product diluent is one of 50 mmol / L, pH 7.8 Tris-HCl buffer. One or two of dithiothreitol, β-mercaptoethanol, tris(2-carboxyethyl)phosphine hydrochloride 6-aminohexane, trypsin inhibitor (R21221), and diisopropyl fluorophosphate are added, and calibration products and quality control products prepared by diluting immunoreactive trypsin at the same theoretical concentration have the following effects under other preferred conditions in the table.

[0034] Table 2

[0035] From the above results, it can be seen that using 10 μg / mL trypsin inhibitor (R21221) can effectively improve the real-time stability and precision of calibration products and quality control products.

[0036] Examples 3 - 6 Antigen effects of the use of calibration products and quality control products on filter paper dry blood spots of a neonatal immunoreactive trypsin assay kit (time-resolved fluorescence immunoassay) applicable to neonatal cystic fibrosis screening: Example 3 The full-length natural human immunoreactive trypsin protein (without chymotrypsin activity against N-succinyl-alanyl-alanyl-prolyl-phenylalanine p-nitroanilide) was diluted into a stock solution twice the required concentration with 50 mmol / L, pH 7.8 Tris-HCl buffer containing 5 g / L bovine serum albumin. Equal volume of blood cells (such as 1 mL of the calibration product or quality control product stock solution diluted from the calibration product, adding 1 mL of blood cells) was added, and after thorough mixing, it was spotted on S&S903 filter paper at 50 μL / drop. Calibration products A, B, C, D, E, and F points and quality control products C1, C2, and C3 points were spotted, with calibration product concentrations of approximately 0, 25, 50, 100, 250, 500 ng / mL respectively, and quality control product concentrations of 50, 100, 250 ng / mL respectively, among which 10 μg / mL trypsin inhibitor (R21221) was added. It was quickly air-dried at room temperature under dehumidification, packed into a storage bag, vacuum laminated, and stored at low temperature for standby.

[0037] Example 4 The recombinant human immunoreactive trypsin (sequence: LNNDIMLIKLSSRAVINARVSTISLPTAPPATGTKCLISGWGNTASSGADYPDELQCLDAPVLSQAKCEASYPGKITSNMFCVGFLEGGKDSCOGDSGGPVC) was diluted into a stock solution twice the required concentration with 50 mmol / L, pH 7.8 Tris-HCl buffer containing 5 g / L bovine serum albumin. Equal volume of blood cells (such as 1 mL of the calibration product or quality control product stock solution diluted from the calibration product, adding 1 mL of blood cells) was added, and after thorough mixing, it was spotted on S&S903 filter paper at 50 μL / drop. Calibration products A, B, C, D, E, and F points and quality control products C1, C2, and C3 points were spotted, with calibration product concentrations of approximately 0, 25, 50, 100, 250, 500 ng / mL respectively, and quality control product concentrations of 50, 100, 250 ng / mL respectively, among which 10 μg / mL trypsin inhibitor (R21221) was added. It was quickly air-dried at room temperature under dehumidification, packed into a storage bag, vacuum laminated, and stored at low temperature for standby.

[0038] Example 5 Recombinant human immunoreactive trypsin (sequence: VGGYNCEENSVPYQVSLNSGYHFCGGSLINEQWVVSAGHCYKSRIQVRLGEHNIEVLEGNEQFINAAKIRHPQYDRKTLNNDIMLIKLSSRAVINARVSTISLPTAPPATGTKCLISGWGNTASSGADYPDELQCLDAPVLSQAKCEASYPGKITSNMFCVGFLEGGKDSCQGDSGGPVVCNGQLQGVVSWGDGCAQKNKPGVYTKVYNYVKWIKNTIAANS) was diluted with 50 mmol / L, pH 7.8 Tris-HCl buffer containing 5 g / L bovine serum albumin into a stock solution twice the required concentration. An equal volume of blood cells (such as 1 mL of calibrator or control stock solution diluted with calibrator, added with 1 mL of blood cells) was added. After thorough mixing, it was spotted on S&S 903 filter paper at 50 μL / drop. Calibrators A, B, C, D, E, and F points and controls C1, C2, and C3 points were spotted, with the calibrator concentrations being approximately 0, 25, 50, 100, 250, 500 ng / mL respectively, and the control concentrations being 50, 100, 250 ng / mL respectively, and 10 μg / mL trypsin inhibitor (R21221) was added. It was quickly dried by dehumidification at room temperature, put into a storage bag, vacuum laminated, and stored at low temperature for later use.

[0039] Example 6 Recombinant human immunoreactive trypsin (sequence: LNNDIMLIKLSSRAVINARVSTISLPTAPPATGTKCLISGWGNTASSGADYPDELQCLDAPVLSQAKCEASYPGKITSNMFCVGFLEGGKDSCOGDSGGPVC) was diluted with 50 mmol / L, pH 7.8 Tris-HCl buffer containing 5 g / L bovine serum albumin into a stock solution twice the required concentration. An equal volume of blood cells (such as 1 mL of calibrator or control stock solution diluted with calibrator, added with 1 mL of blood cells) was added. After thorough mixing, it was spotted on S&S 903 filter paper at 50 μL / drop. Calibrators A, B, C, D, E, and F points and controls C1, C2, and C3 points were spotted, with the calibrator concentrations being approximately 0, 25, 50, 100, 250, 500 ng / mL respectively, and the control concentrations being 50, 100, 250 ng / mL respectively, and 10 μg / mL trypsin inhibitor (R21221) was added. It was dried overnight at room temperature, put into a storage bag, vacuum laminated, and stored at low temperature for later use.

[0040] The calibrators and quality control products prepared in Examples 3 to 6 were tested for their reactivity, stability and precision under other preferred conditions, and the results are compared as follows.

[0041] Table 3

[0042] The reactivity (detection response values of calibrators B to F and quality control products C1 to C3), stability and precision of the calibrators and quality control products prepared in Example 4 were better than those prepared in Examples 3, 5 and 6. This may be related to the fact that natural human immunoreactive trypsin full-length protein (without chymotrypsin activity against N-succinyl-alanyl-alanyl-prolyl-phenylalanine p-nitroanilide), recombinant human immunoreactive trypsin (sequence: VGGYNCEENSVPYQVSLNSGYHFCGGSLINEQWVVSAGHCYKSRIQVRLGEHNIEVLEGNEQFINAAKIRHPQYDRKTLNNDIMLIKLSSRAVINARVSTISLPTAPPATGTKCLISGWGNTASSGADYPDELQCLDAPVLSQAKCEASYPGKITSNMFCVGFLEGGKDSCQGDSGGPVVCNGQLQGVVSWGDGCAQKNKPGVYTKVYNYVKWIKNTIAANS) is a full-length protein. The full-length sequence trypsin may be extremely easy to destroy red blood cells during the preparation process and may interact with substances such as proteins and ions released from red blood cells, causing autolysis of trypsin; in addition, the full-length sequence trypsin may also easily act on the antibodies in the reaction carrier and tracer, decompose the antibodies, and reduce the antigen-antibody reactivity; the full-length sequence trypsin may also interact with other substances during the preparation process and reaction process of the calibrators and quality control products, and the intensity of the interaction may be non-uniform during this process, resulting in poor precision; natural human immunoreactive trypsin also has certain biosafety risks during use; therefore, recombinant human immunoreactive trypsin (sequence: LNNDIMLIKLSSRAVINARVSTISLPTAPPATGTKCLISGWGNTASSGADYPDELQCLDAPVLSQAKCEASYPGKITSNMFCVGFLEGGKDSCOGDSGGPVC) is preferably used as the raw material for calibrators and quality control products.

[0043] The reactivity (detection response values of calibrators B - F and quality control products C1 - C3), drying stability, and precision of calibrators and quality control products prepared by dehumidifying at room temperature and drying quickly are superior to those dried overnight at room temperature. Dehumidifying at room temperature and drying quickly may reduce the interaction time between trypsin and cellular substances or other protein substances, which is beneficial to improving the immunoreactivity, drying stability, and precision of trypsin. Therefore, calibrators and quality control products prepared by dehumidifying at room temperature and drying quickly are preferred.

[0044] Examples 7 - 12 The effects of coating buffer and blocking buffer of a 96 - well microplate reaction carrier for a neonatal immunoreactive trypsin assay kit (time - resolved fluorescence immunoassay) applicable to neonatal cystic fibrosis screening.

[0045] Example 7 The immunoreactive trypsin antibody for coating was diluted to 3.5 μg / mL with 20 mmol / L phosphate buffer at pH 4.3. The corresponding blocking solution was 20 mmol / L phosphate buffer at pH 4.3 containing 1 g / L polyvinylpyrrolidone 360, 0.015 mL / L Tween - 20, and 50 g / L sucrose. The coating volume was 100 μL, incubated at 2 - 8°C for 20 h, then washed once with 300 μL / well of wash working solution, and then 150 μL of blocking solution was added to each well and incubated at 37°C for 2 h. The blocking solution was discarded, dried by centrifugation, air - dried, vacuum - sealed, and stored at 2 - 8°C for later use.

[0046] Example 8 The immunoreactive trypsin antibody for coating was diluted to 3.5 μg / mL with 20 mmol / L phosphate buffer at pH 4.3. The corresponding blocking solutions were 20 mmol / L phosphate buffer at pH 7.2 containing 1 g / L polyvinylpyrrolidone 360, 0.015 mL / L Tween - 20, and 50 g / L sucrose. The coating volume was 100 μL, incubated at 2 - 8°C for 20 h, then washed once with 300 μL / well of wash working solution, and then 150 μL of blocking solution was added to each well and incubated at 37°C for 2 h. The blocking solution was discarded, dried by centrifugation, air - dried, vacuum - sealed, and stored at 2 - 8°C for later use.

[0047] Example 9 The immunoreactive trypsin antibody for coating was diluted to 3.5 μg / mL with 20 mmol / L citrate buffer at pH 6.6. The corresponding blocking solutions were 20 mmol / L phosphate buffer at pH 7.2 containing 1 g / L polyvinylpyrrolidone 360, 0.015 mL / L Tween-20, and 50 g / L sucrose. The coating volume was 100 μL, and it was incubated at 2 - 8°C for 20 h. Then it was washed once with the washing solution working fluid at a volume of 300 μL / well. Then 150 μL of the blocking solution was added to each well and incubated at 37°C for 2 h. The blocking solution was discarded, and after centrifuging to dry, it was air-dried, vacuum-sealed, and stored at 2 - 8°C for later use.

[0048] Example 10 The immunoreactive trypsin antibody for coating was diluted to 3.5 μg / mL with 20 mmol / L phosphate buffer at pH 7.2. The corresponding blocking solutions were 20 mmol / L phosphate buffer at pH 7.2 containing 1 g / L polyvinylpyrrolidone 360, 0.015 mL / L Tween-20, and 50 g / L sucrose. The coating volume was 100 μL, and it was incubated at 2 - 8°C for 20 h. Then it was washed once with the washing solution working fluid at a volume of 300 μL / well. Then 150 μL of the blocking solution was added to each well and incubated at 37°C for 2 h. The blocking solution was discarded, and after centrifuging to dry, it was air-dried, vacuum-sealed, and stored at 2 - 8°C for later use.

[0049] Example 11 The immunoreactive trypsin antibody for coating was diluted to 3.5 μg / mL with 20 mmol / L Tris-HCl buffer at pH 7.8. The corresponding blocking solutions were 20 mmol / L phosphate buffer at pH 7.2 containing 1 g / L polyvinylpyrrolidone 360, 0.015 mL / L Tween-20, and 50 g / L sucrose. The coating volume was 100 μL, and it was incubated at 2 - 8°C for 20 h. Then it was washed once with the washing solution working fluid at a volume of 300 μL / well. Then 150 μL of the blocking solution was added to each well and incubated at 37°C for 2 h. The blocking solution was discarded, and after centrifuging to dry, it was air-dried, vacuum-sealed, and stored at 2 - 8°C for later use.

[0050] Example 12 The immunoreactive trypsin antibody for coating was diluted to 3.5 μg / mL with 20 mmol / L carbonate buffer at pH 9.6. The corresponding blocking solutions were 20 mmol / L phosphate buffer at pH 7.2 containing 1 g / L polyvinylpyrrolidone 360, 0.015 mL / L Tween-20, and 50 g / L sucrose. The coating volume was 100 μL, and it was incubated at 2 - 8°C for 20 h. Then it was washed once with the washing solution working fluid at a volume of 300 μL / well. Then 150 μL of the blocking solution was added to each well and blocked and incubated at 37°C for 2 h. The blocking solution was discarded, dried by centrifugation and then air-dried, vacuum-sealed, and stored at 2 - 8°C for later use.

[0051] Using the 96-well enzyme-labeled microplate reaction carriers prepared in Examples 7 - 12, the reactivity, sensitivity, and precision of the calibrators and quality control products were detected under other preferred conditions, and the results are compared as follows.

[0052] Table 4

[0053] From the above results, it can be seen that the background of the reaction plate prepared in Example 7 is the lowest (the detection response value of calibrator A), the signal-to-noise ratio is high (calibrator B / calibrator A), the reactivity is good (the detection response values of calibrators B - F and quality control products C1 - C3), the sensitivity is good, and the precision is high. It was also found that as the pH value of the coating solution or the blocking solution increased, the background increased, and the reactivity showed a decreasing trend, indicating an increase in non-specific adsorption and a decrease in the specific adsorption amount or activity of the coating antibody. Therefore, the immunoreactive trypsin antibody for coating was diluted with 20 mmol / L phosphate buffer at pH 4.3, and the corresponding blocking solutions were 20 mmol / L phosphate buffer at pH 4.3 containing 1 g / L polyvinylpyrrolidone 360, 0.015 mL / L Tween-20, and 50 g / L sucrose.

[0054] Example 13 Effect of the blocking agent on the 96-well enzyme-labeled microplate reaction carrier of a neonatal immunoreactive trypsin assay kit (time-resolved fluorescence immunoassay) suitable for neonatal cystic fibrosis screening: The immunoreactive trypsin antibody for coating was diluted to 3.5 μg / mL with 20 mmol / L phosphate buffer at pH 4.3. The corresponding blocking solution was the buffer containing the blocking agent. The coating volume was 100 μL, and it was incubated at 2 - 8°C for 20 h. Then it was washed once with the washing solution working fluid at a volume of 300 μL / well. Then 150 μL of the blocking solution was added to each well and blocked and incubated at 37°C for 2 h. The blocking solution was discarded, dried by centrifugation and then air-dried, vacuum-sealed, and stored at 2 - 8°C for later use.

[0055] Among them, the closing agents are: (1) 50 g / L bovine serum albumin, (2) 5 g / L bovine serum albumin, (3) 0.5 g / L bovine serum albumin, (4) 100 mL / L calf serum, (5) 10 mL / L calf serum, (6) 1 mL / L calf serum, (7) 100 mL / L newborn bovine serum, (8) 10 mL / L newborn bovine serum, (9) 1 mL / L newborn bovine serum, (10) 100 mL / L fetal bovine serum, (11) 10 mL / L fetal bovine serum, (12) 1 mL / L fetal bovine serum, (13) 10 g / L casein, (14) 1 g / L casein, (15) 0.1 g / L casein, (16) 10 g / L fish gelatin, (17) 1 g / L fish gelatin, (18) 0.1 g / L fish gelatin, (19) 8 g / L polyvinylpyrrolidone 90, (20) 4 g / L polyvinylpyrrolidone 90, (21) 1 g / L polyvinylpyrrolidone 90, (22) 0.2 g / L polyvinylpyrrolidone 90, (23) 0.1 g / L polyvinylpyrrolidone 90, (24) 8 g / L polyvinylpyrrolidone 360, (25) 4 g / L polyvinylpyrrolidone 360, (26) 4 g / L polyvinylpyrrolidone 360, (27) 1 g / L polyvinylpyrrolidone 360, (28) 1 g / L polyvinylpyrrolidone 360, (29) 0.1 g / L polyvinylpyrrolidone 360, (30) 0.1 g / L polyvinylpyrrolidone 360, (31) 10 g / L polyethylene glycol 6000, (32) 1 g / L polyethylene glycol 6000, (33) 0.1 g / L polyethylene glycol 6000, (34) 10 g / L polyethylene glycol 20 000, (35) 1 g / L polyethylene glycol 20 000, (36) 0.1 g / L polyethylene glycol 20 000, (37) 1 g / L polyvinylpyrrolidone 360 and 0.12 ml / L Tween-20, (38) 1 g / L polyvinylpyrrolidone 360 and 0.06 ml / L Tween-20, (39) 1 g / L polyvinylpyrrolidone 360 and 0.03 ml / L Tween-20, (40) 1 g / L polyvinylpyrrolidone 360 and 0.015 ml / L Tween-20, (41) 1 g / L polyvinylpyrrolidone 360 and 0.0075 ml / L Tween-20, (42) 1 g / L polyvinylpyrrolidone 360 and 0.12 ml / L Tween-40, (43) 1 g / L polyvinylpyrrolidone 360 and 0.015 ml / L Tween-40, (44) 1 g / L polyvinylpyrrolidone 360 and 0.0075 ml / L Tween-40, (45) 1 g / L polyvinylpyrrolidone 360, 0.015 ml / L Tween-20 and 100 g / L sucrose, (46) 1 g / L polyvinylpyrrolidone 360, 0.015 ml / L Tween-20 and 50 g / L sucrose, (47) 1 g / L polyvinylpyrrolidone 360, 0.015 ml / L Tween-20 and 25 g / L sucrose, (48) 1 g / L polyvinylpyrrolidone 360, 0.015 ml / L Tween-20 and 50 g / L trehalose, (49) 1 g / L polyvinylpyrrolidone 360, 0.015 ml / L Tween-20 and 5 g / L trehalose, (50) 1 g / L polyvinylpyrrolidone 360 and 5 g / L trehalose, (51) 1 g / L polyvinylpyrrolidone 360, 0.015 ml / L Tween-20 and 50 g / L sorbitol, (52) 1 g / L polyvinylpyrrolidone 360, 0.1 g / L polyethylene glycol 20000, 0.015 ml / L Tween-20 and 50 g / L sucrose, (53) 1 g / L polyvinylpyrrolidone 360, 0.1 g / L polyethylene glycol 20000 and 50 g / L sucrose, (54) 1 g / L polyvinylpyrrolidone 360, 0.01 g / L casein and 50 g / L sucrose, (55) 1 g / L polyvinylpyrrolidone 360, 1 mL / L fetal bovine serum and 50 g / L sucrose, (56) 1 g / L polyvinylpyrrolidone 360, 1 mL / L newborn bovine serum and 50 g / L sucrose, (57) 1 g / L polyvinylpyrrolidone 360, 1 mL / L calf serum and 50 g / L sucrose, (58) 1 g / L polyvinylpyrrolidone 360, 0.5 g / L bovine serum albumin and 50 g / L sucrose, (59) 1 g / L polyvinylpyrrolidone 360 and 50 g / L sucrose. Among them, casein will denature under acidic conditions. The blocking buffer containing casein component blocker is 20 mmol / L phosphate buffer with pH 7.2, and other blockers are 20 mmol / L phosphate buffer with pH 4.3..

[0056] The test results are as Figure 1 shown. It can be seen from Figure 1 that the background of the reaction plate prepared under the blocking condition (46) in Example 13 is the lowest (detection response value of calibrator A), the signal-to-noise ratio is high (calibrator B / calibrator A), the reactivity is good (detection response values of calibrators B - F), the sensitivity is good, the precision is high, and the real-time stability is long. Therefore, it is preferably to mix 1 g / L polyvinylpyrrolidone 360, 0.015 ml / L Tween-20 and 50 g / L sucrose as the blocker.

[0057] Examples 14 - 19 Coating and blocking effects of a 96-well enzyme-labeled microplate reaction carrier for a neonatal immunoreactive trypsin assay kit (time-resolved fluorescence immunoassay) applicable to neonatal cystic fibrosis screening.

[0058] Example 14 The monoclonal antibody against human immunoreactive trypsin 1 for coating was diluted to 3.5 μg / mL with 20 mmol / L phosphate buffer at pH 4.3. The corresponding blocking solution was 20 mmol / L phosphate buffer at pH 4.3 containing 10 g / L bovine serum albumin. The coating volume was 100 μL, and it was incubated at 2 - 8°C for 20 h. Then it was washed once with 300 μL / well of the washing solution working fluid. Then 150 μL of the blocking solution was added to each well and incubated at 37°C for 2 h. The blocking solution was discarded, and after centrifuging to dry and air-drying, it was vacuum-sealed and stored at 2 - 8°C for standby.

[0059] Example 15 The monoclonal antibody against human immunoreactive trypsinogen 2 for coating was diluted to 3.5 μg / mL with 20 mmol / L phosphate buffer at pH 4.3. The corresponding blocking solution was 20 mmol / L phosphate buffer at pH 4.3 containing 1 g / L polyvinylpyrrolidone 360, 0.015 ml / L Tween-20, and 50 g / L sucrose. The coating volume was 100 μL, and it was incubated at 2 - 8°C for 20 h. Then it was washed once with 300 μL / well of the washing solution working fluid. Then 150 μL of the blocking solution was added to each well and incubated at 37°C for 2 h. The blocking solution was discarded, and after centrifuging to dry and air-drying, it was vacuum-sealed and stored at 2 - 8°C for standby.

[0060] Example 16 3.5 μg / mL of the monoclonal antibody against human immunoreactive trypsin 1 for coating and 2.0 μg / mL of the monoclonal antibody against human immunoreactive trypsinogen 2 for coating were added simultaneously to 20 mmol / L phosphate buffer at pH 4.3. The corresponding blocking solution was 20 mmol / L phosphate buffer at pH 4.3 containing 1 g / L polyvinylpyrrolidone 360, 0.015 ml / L Tween-20, and 50 g / L sucrose. The coating volume was 100 μL, and it was incubated at 2 - 8°C for 20 h. Then it was washed once with 300 μL / well of the washing solution working fluid. Then 150 μL of the blocking solution was added to each well and incubated at 37°C for 2 h. The blocking solution was discarded, and after centrifuging to dry and air-drying, it was vacuum-sealed and stored at 2 - 8°C for standby.

[0061] Example 17 In a phosphate buffer solution with a concentration of 20 mmol / L and a pH of 4.3, 3.5 μg / mL of monoclonal antibody coated with human immunoreactive trypsin 1 and 2.0 μg / mL of monoclonal antibody coated with human immunoreactive trypsinogen 2 were added simultaneously. The corresponding blocking solution was a phosphate buffer solution with a concentration of 20 mmol / L and a pH of 4.3 containing 1 g / L of polyvinylpyrrolidone 360, 0.015 mL / L of Tween-20, and 50 g / L of sucrose. The coating volume was 50 μL, and incubation was carried out at 2 - 8°C for 20 h. Then, it was washed once with a washing solution working fluid with a volume of 200 μL / well. Then, 100 μL of the blocking solution was added to each well and incubated at 37°C for 2 h. The blocking solution was discarded, and after centrifuging to dry and air-drying, it was vacuum-sealed and stored at 2 - 8°C for later use.

[0062] Example 18 In a phosphate buffer solution with a concentration of 20 mmol / L and a pH of 4.3, 3.5 μg / mL of monoclonal antibody coated with human immunoreactive trypsin 1 and 2.0 μg / mL of monoclonal antibody coated with human immunoreactive trypsinogen 2 were added simultaneously. The corresponding blocking solution was a phosphate buffer solution with a concentration of 20 mmol / L and a pH of 4.3 containing 1 g / L of polyvinylpyrrolidone 360, 0.015 mL / L of Tween-20, and 50 g / L of sucrose. The coating volume was 150 μL, and incubation was carried out at 2 - 8°C for 20 h. Then, it was washed once with a washing solution working fluid with a volume of 300 μL / well. Then, 200 μL of the blocking solution was added to each well and incubated overnight at room temperature for blocking. The blocking solution was discarded, and after centrifuging to dry and air-drying, it was vacuum-sealed and stored at 2 - 8°C for later use.

[0063] Example 19 In a phosphate buffer solution with a concentration of 20 mmol / L and a pH of 4.3, 3.5 μg / mL of monoclonal antibody coated with human immunoreactive trypsin 1 and 2.0 μg / mL of monoclonal antibody coated with human immunoreactive trypsinogen 2 were added simultaneously. The corresponding blocking solution was a phosphate buffer solution with a concentration of 20 mmol / L and a pH of 4.3 containing 1 g / L of polyvinylpyrrolidone 360, 0.015 mL / L of Tween-20, and 50 g / L of sucrose. The coating volume was 200 μL, and incubation was carried out at 2 - 8°C for 20 h. Then, it was washed once with a washing solution working fluid with a volume of 300 μL / well. Then, 250 μL of the blocking solution was added to each well and incubated at 37°C for 2 h. The blocking solution was discarded, and after centrifuging to dry and air-drying, it was vacuum-sealed and stored at 2 - 8°C for later use.

[0064] Using the 96-well enzyme-labeled microplate reaction carrier prepared in Examples 14 - 16, the reactivity of the calibrator, the coincidence rates of positive samples (P1 - P10) and negative samples (N1 - N10) were detected under other preferred conditions, and the results are compared as follows.

[0065] Table 5

[0066] From the above results, it can be seen that the reaction plate prepared in Example 15 has good reactivity with the calibrator, and the positive sample coincidence rate and negative coincidence rate are both 10 / 10. Therefore, a mixture of monoclonal antibody against human immunoreactive trypsin 1 and monoclonal antibody against human immunoreactive trypsinogen 2 is selected for coating.

[0067] Using the 96-well enzyme-labeled microplate reaction carrier prepared in Examples 16-19, the calibrator, linear range, sensitivity and precision were detected under other preferred conditions, and the results are compared as follows.

[0068] Table 6

[0069] From the above results, it can be seen that the fluorescence value of the reaction plate prepared in Example 16 reacted with the calibrator tends to be stable; the sensitivity of Example 16 is better than that of the reaction plate prepared in Example 17, and Example 16 is comparable to the reaction plates prepared in Examples 18 and 19; the precision of Example 16 is better than that of the reaction plates prepared in Examples 18 and 19.

[0070] Examples 20-28 Preparation of a tracer for a neonatal immunoreactive trypsin assay kit (time-resolved fluorescence immunoassay) suitable for neonatal cystic fibrosis screening.

[0071] Example 20 The lanthanide element-labeled immunoreactive trypsin antibody (monoclonal antibody against human immunoreactive trypsin 1 and monoclonal antibody against human immunoreactive trypsinogen 2) was first subjected to antibody storage liquid replacement treatment with 50 mmol / L carbonate buffer at pH 9.6. The immunoreactive trypsin antibody (monoclonal antibody against human immunoreactive trypsin 1 and monoclonal antibody against human immunoreactive trypsinogen 2) was mixed with N 1 - (p-isothiocyanatobenzyl)-diethylenetriamine tetraacetic acid europium sodium in a mass ratio of 1:1, and incubated with shaking at 2-8 °C for 24 h. Chromatographic purification was carried out using a gel column packed with Sephadex G-200, and the tracer mother liquor was collected. The mother liquor of the lanthanide element-labeled monoclonal antibody against human immunoreactive trypsin 1 and the mother liquor of the lanthanide element-labeled monoclonal antibody against human immunoreactive trypsinogen 2 were mixed in a volume ratio of 2:1, and then diluted into a semi-finished product with a diluent.

[0072] Example 21 The immunoreactive trypsin antibodies for lanthanide element labeling (human immunoreactive trypsin 1 monoclonal antibody and human immunoreactive trypsinogen 2 monoclonal antibody) were first subjected to antibody storage liquid replacement treatment with 50 mmol / L carbonate buffer at pH 9.6. The immunoreactive trypsin antibodies (human immunoreactive trypsin 1 monoclonal antibody and human immunoreactive trypsinogen 2 monoclonal antibody) were mixed with N 1 -sodium (p-isothiocyanatobenzyl)-diethylenetriamine tetraacetic acid europium in a mass ratio of 1:1, and incubated with shaking at 2-8 °C for 48 h. Chromatographic purification was carried out using a gel column packed with Sephacryl S-200 to collect the tracer mother liquor. The mother liquor of lanthanide element-labeled human immunoreactive trypsin 1 monoclonal antibody and the mother liquor of lanthanide element-labeled human immunoreactive trypsinogen 2 monoclonal antibody were mixed at a volume ratio of 2:1, and then diluted with a diluent into a semi-finished product.

[0073] Example 22 The immunoreactive trypsin antibodies for lanthanide element labeling (human immunoreactive trypsin 1 monoclonal antibody and human immunoreactive trypsinogen 2 monoclonal antibody) were first subjected to antibody storage liquid replacement treatment with 50 mmol / L carbonate buffer at pH 9.6. The immunoreactive trypsin antibodies (human immunoreactive trypsin 1 monoclonal antibody and human immunoreactive trypsinogen 2 monoclonal antibody) were mixed with N 1 -sodium (p-isothiocyanatobenzyl)-diethylenetriamine tetraacetic acid europium in a mass ratio of 1:1, and incubated with shaking at 2-8 °C for 72 h. Chromatographic purification was carried out using a gel column packed with Sephacryl S-200 to collect the tracer mother liquor. The mother liquor of lanthanide element-labeled human immunoreactive trypsin 1 monoclonal antibody and the mother liquor of lanthanide element-labeled human immunoreactive trypsinogen 2 monoclonal antibody were mixed at a volume ratio of 2:1, and then diluted with a diluent into a semi-finished product.

[0074] Example 23 The immunoreactive trypsin antibodies for lanthanide element labeling (human immunoreactive trypsin 1 monoclonal antibody and human immunoreactive trypsinogen 2 monoclonal antibody) were first subjected to antibody storage liquid replacement treatment with 50 mmol / L carbonate buffer at pH 9.6. The immunoreactive trypsin antibodies (human immunoreactive trypsin 1 monoclonal antibody and human immunoreactive trypsinogen 2 monoclonal antibody) were mixed with N 1-Sodium (P-isothiocyanatobenzyl)-diethylenetriaminepentaacetatoeuropate(III) were mixed at a mass ratio of 2:1, incubated with shaking at 2 - 8 °C for 48 h, purified by chromatography using a gel column packed with Sephacryl S-200, the tracer stock solution, the stock solutions of lanthanide-labeled human immunoreactive trypsin 1 monoclonal antibody and lanthanide-labeled human immunoreactive trypsinogen 2 monoclonal antibody were mixed at a volume ratio of 2:1, and then diluted with a diluent to form a semi-finished product.

[0075] Example 24 For the lanthanide-labeled immunoreactive trypsin antibodies (human immunoreactive trypsin 1 monoclonal antibody and human immunoreactive trypsinogen 2 monoclonal antibody), the antibody storage liquid was first replaced with 50 mmol / L carbonate buffer at pH 9.6. The immunoreactive trypsin antibodies (human immunoreactive trypsin 1 monoclonal antibody and human immunoreactive trypsinogen 2 monoclonal antibody) and N 1 -Sodium (P-isothiocyanatobenzyl)-diethylenetriaminepentaacetatoeuropate(III) were mixed at a mass ratio of 1:2, incubated with shaking at 2 - 8 °C for 48 h, purified by chromatography using a gel column packed with Sephacryl S-200, the tracer stock solution, the stock solutions of lanthanide-labeled human immunoreactive trypsin 1 monoclonal antibody and lanthanide-labeled human immunoreactive trypsinogen 2 monoclonal antibody were mixed at a volume ratio of 2:1, and then diluted with a diluent to form a semi-finished product.

[0076] Example 25 For both the lanthanide-labeled human immunoreactive trypsin 1 monoclonal antibody and the lanthanide-labeled human immunoreactive trypsinogen 2 monoclonal antibody, the antibody storage liquid was first replaced with 50 mmol / L carbonate buffer at pH 9.6. Both the human immunoreactive trypsin 1 monoclonal antibody and the human immunoreactive trypsinogen 2 monoclonal antibody were respectively mixed with N 1 -Sodium (P-isothiocyanatobenzyl)-diethylenetriaminepentaacetatosamarium(III) were mixed at a mass ratio of 1:1, incubated with shaking at 2 - 8 °C for 48 h, purified by chromatography using a gel column packed with Sephacryl S-200, the tracer stock solution, the stock solutions of lanthanide-labeled human immunoreactive trypsin 1 monoclonal antibody and lanthanide-labeled human immunoreactive trypsinogen 2 monoclonal antibody were mixed at a volume ratio of 1:1, and then diluted with a diluent to form a semi-finished product.

[0077] Example 26 Both the monoclonal antibody of human immunoreactive trypsin 1 labeled with lanthanide elements and the monoclonal antibody of human immunoreactive trypsinogen 2 labeled with lanthanide elements were first subjected to antibody storage liquid replacement treatment with 50 mmol / L carbonate buffer at pH 9.6. Both the monoclonal antibody of human immunoreactive trypsin 1 and the monoclonal antibody of human immunoreactive trypsinogen 2 were respectively mixed with N 1 sodium samarium diethylenetriaminepentaacetic acid-(p-isothiocyanatobenzyl) in a mass ratio of 1:1, incubated with shaking at 2 - 8 °C for 48 h, chromatographically purified using a gel column packed with Sephadex G-200, the tracer mother liquor was collected, and the mother liquor of the monoclonal antibody of human immunoreactive trypsin 1 labeled with lanthanide elements and the mother liquor of the monoclonal antibody of human immunoreactive trypsinogen 2 labeled with lanthanide elements were diluted into semi-finished products according to a volume ratio of 3:1 and then diluted with a diluent.

[0078] Example 27 The monoclonal antibody of human immunoreactive trypsin 1 labeled with lanthanide elements was first subjected to antibody storage liquid replacement treatment with 50 mmol / L carbonate buffer at pH 9.6. The monoclonal antibody of human immunoreactive trypsin 1 was mixed with N 1 sodium europium diethylenetriaminepentaacetic acid-(p-isothiocyanatobenzyl) in a mass ratio of 1:1, incubated with shaking at 2 - 8 °C for 48 h, chromatographically purified using a gel column packed with Sephadex G-200 to collect the tracer mother liquor, and the monoclonal antibody of human immunoreactive trypsin 1 labeled with lanthanide elements was then diluted into semi-finished products with a diluent respectively.

[0079] Example 28 The monoclonal antibody of human immunoreactive trypsinogen 2 labeled with lanthanide elements was first subjected to antibody storage liquid replacement treatment with 50 mmol / L carbonate buffer at pH 9.6. The monoclonal antibody of human immunoreactive trypsinogen 2 was mixed with N 1 sodium europium diethylenetriaminepentaacetic acid-(p-isothiocyanatobenzyl) in a mass ratio of 1:1, incubated with shaking at 2 - 8 °C for 48 h, chromatographically purified using a gel column packed with Sephadex G-200 to collect the tracer mother liquor, and the monoclonal antibody of human immunoreactive trypsinogen 2 labeled with lanthanide elements was then diluted into semi-finished products with a diluent respectively.

[0080] Using the tracers prepared in Examples 20 - 28, the background, linear range, sensitivity, precision, compliance rates of positive samples (P1 - P10) and negative samples (N1 - N10) were detected under other preferred conditions, and the results are compared as follows.

[0081] Table 7

[0082] From the above results, it can be seen that the tracer prepared in Example 21 has a low detection background, a wide linear range, high sensitivity, good precision, and the coincidence rates of positive and negative samples are both 10 / 10. The tracer prepared in Example 19 has low detection sensitivity and a low dilution ratio; the tracer prepared in Example 22 has a relatively high detection background and low sensitivity; the dilution ratio of Example 23 is the same as that of Example 19, and more antibodies are lost; the tracer prepared in Example 24 has a high detection background and low sensitivity; false positives occur in Example 25; false negatives occur in the tracers prepared in Example 26, Example 27, and Example 28.

[0083] Examples 29 - 30 A test method for a neonatal immunoreactive trypsin assay kit (time-resolved fluorescence immunoassay) suitable for neonatal cystic fibrosis screening: Example 29 A full-automatic test method for a neonatal immunoreactive trypsin assay kit (time-resolved fluorescence immunoassay) suitable for neonatal cystic fibrosis screening is as follows: (1) Sample blood collection: Collect sample blood using filter paper dry blood spots, and enter the relevant sample information into the information management system module; (2) Sample quality analysis: Use the filter paper dry blood spot quality analysis module with a high-definition industrial camera system, image processing and analysis system to analyze the quality of the filter paper dry blood spot samples and determine whether the sample quality is qualified; (3) Barcode identification: Use the barcode scanning module to read the barcodes of the filter paper dry blood spot samples, export the relevant information of the carrier samples, and associate them with the sample experimental process; (4) Cutting and collection of calibrators, quality control products, and samples to be tested: Use the filter paper dry blood spot punching module with a filter paper dry blood spot sample transfer device, high-definition industrial camera system, image analysis and positioning system, filter paper dry blood spot blood spot cutter, and high-precision motion positioning device to cut out a blood spot with a diameter of 3.2 mm (1 / 8 inch) from the filter paper dry blood spot of the sample to be tested with qualified quality and the filter paper dry blood spots of calibrators and quality control products, and fix it on the plate needle. Use the gripper arm module with horizontal / vertical movement 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 the gripper arm module with horizontal / vertical movement function and high-precision positioning function to move the 96-well enzyme-linked microplate reaction carrier to the oscillation incubation module with bottom heating, constant temperature control system, and vortex oscillation function. Use the pipetting module with horizontal / vertical movement function and high-precision positioning function to dilute and mix the tracer and experimental buffer in a volume ratio of 1:50 to prepare the tracer working solution. Use the pipetting module with horizontal / vertical movement function and high-precision positioning function to add 50 - 200 µL of the tracer working solution, preferably 150 µL, to the micro-cups of the 96-well enzyme-linked microplate reaction carrier; (6) First incubation: Use the gripper arm module with horizontal / vertical movement function and high-precision positioning function to move the plate needle into the 96-well enzyme-linked microplate reaction carrier with the tracer working solution added. The blood spot fixed on the plate needle is immersed in the tracer working solution in the 96-well enzyme-linked microplate reaction carrier, and then incubated at room temperature with oscillation in the oscillation incubation module for 90 - 150 min, preferably 120 min; (7) Plate washing: Dilute the cleaning solution with purified water in a volume ratio of 1:25 to prepare the cleaning working solution. Use the gripper arm module with horizontal / vertical 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 the gripper arm module with horizontal / vertical movement function and high-precision positioning function to move the 96-well enzyme-linked microplate reaction carrier to the plate washing module. The plate washing module uses the cleaning working solution to wash the 96-well enzyme-linked microplate reaction carrier 4 - 8 times, preferably 6 times.(8) Adding an inducer: Use a gripper arm module with lateral / longitudinal movement function and high-precision positioning function to move the 96-well enzyme-labeled microplate reaction carrier to an oscillating incubation module with bottom heating, constant temperature control system, and vortex oscillation function. Use a sample addition pipetting module with lateral / longitudinal movement function and high-precision positioning function to add 50 - 200 µL of inducer, preferably 150 µL, to the micro-cups of the 96-well enzyme-labeled microplate reaction carrier. (9) Second incubation: The 96-well enzyme-labeled microplate reaction carrier with the added inducer is incubated on the incubation position in the oscillating incubation module at room temperature with a motor speed of 1200 rpm for 2 - 10 min, preferably oscillated for 5 min. (10) Detection: Use a gripper arm module with lateral / longitudinal movement function and high-precision positioning function to move the 96-well enzyme-labeled microplate reaction carrier with the added inducer to the measurement module position. The measurement module has a high-precision displacement and positioning device, window opening and closing device, filter automatic switching measurement system, and photoelectric signal conversion measurement system. Select the corresponding immunoreactive trypsin detection program and perform detection using an excitation wavelength of 340 nm and a detection wavelength of 613 nm to obtain the fluorometer value. The detection work needs to be completed within 30 min after adding the inducer. (11) Result analysis: The measured fluorometer value is transmitted to a computer data analysis module containing a microcomputer system and a laboratory (clinical laboratory) information connection port. Use the LOG-LOG_B axis conversion method and SPLINE dose-response curve fitting algorithm for result analysis. Obtain the test result of the sample through the test software algorithm and transmit the result to the information management system module. All the instrument modules involved in the detection method are fixed into an integrated body by an instrument support platform and are equipped with a safety protection panel. During the operation of the detection system, the safety protection panel is always in a closed state to ensure biosafety protection during the detection operation and the safe operation of the detection system. The detection methods are also all associated with the information management system module. The information management system module includes a system function module (basic data setting, user management, sample management, patient self-service query, statistical report, clinical treatment), a report module (traditional item report management, thalassemia report management, tandem mass spectrometry report management, urinary organic acid report management, gene detection report management, allergen report management), and a laboratory management module (laboratory, quality control management). The information management system module is architected on a network to achieve cloud management of each function module, standardize and network the screening and diagnosis and treatment work processes, and achieve barcoding and informatization management of the entire process from order placement, blood collection, logistics transportation, detection to report, query, and follow-up.

[0084] Example 30 A semi-automatic test method for a neonatal immunoreactive trypsin assay kit (time-resolved fluorescence immunoassay) suitable for neonatal cystic fibrosis screening is as follows: (1)Sample blood sample collection: Collect the sample blood sample using filter paper dry blood spots, and manually enter the sample-related information into the information management system; (2) Sample quality analysis: Visually analyze the quality of the filter paper dry blood spot sample and determine whether the sample quality is qualified; Cutting and collection of calibrator, quality control product, and sample to be tested: Use a punching pliers to cut out a blood spot with a diameter of 3.2 mm (1 / 8 inch) from the filter paper dry blood spots of the calibrator, quality control product, and sample to be tested and place it into the reaction carrier of a 96-well enzyme-linked microplate; (3) Add tracer working solution: Manually dilute the tracer and experimental buffer in a volume ratio of 1:50 using a pipette and mix well to prepare the tracer working solution. Manually add 50 - 200 µL of the tracer working solution to the micro-cups of the reaction carrier of the 96-well enzyme-linked microplate using a pipette, preferably 150 µL; (4) First incubation: Incubate at room temperature on an oscillating incubator for 90 - 150 min, preferably 120 min; (5) Wash the plate: Dilute the cleaning solution with purified water in a volume ratio of 1:25 to form the cleaning working solution. Place the reaction carrier of the 96-well enzyme-linked microplate on a semi-automatic plate washer and wash the reaction carrier of the 96-well enzyme-linked microplate 4 - 8 times with the cleaning working solution, preferably 6 times. (6) Add inducer: Manually add 50 - 200 µL of inducer to the micro-cups of the reaction carrier of the 96-well enzyme-linked microplate using a pipette, preferably 150 µL. (7) Second incubation: The reaction carrier of the 96-well enzyme-linked microplate with the inducer added is oscillated at a motor speed of 1200 rpm for 2 - 10 min on an oscillating incubator at room temperature, preferably oscillated for 5 min.

[0085] (8)Detection: Transfer the reaction carrier of the 96-well enzyme-linked microplate with the inducer added to a semi-automatic fluorescence analyzer, select the corresponding immunoreactive trypsin detection program, and perform detection using an excitation wavelength of 340 nm and a detection wavelength of 613 nm to obtain the fluorometer value. The detection work needs to be completed within 30 min after adding the inducer. (9) Result analysis: Transmit the measured fluorometer value to the computer data analysis module containing a microcomputer system and a laboratory (clinical laboratory) information connection port, and perform result analysis using the LOG-LOG_B axis conversion method and the SPLINE dose-response curve fitting algorithm. Obtain the test result of the sample through the test software algorithm and transmit the result to the information management system module.

[0086] Using the test methods made in Examples 27 - 28, detect the background, linear range, sensitivity, precision, compliance rate of positive samples (P1 - P10), and compliance rate of negative samples (N1 - N10) under other preferred conditions, and the results are compared as follows.

[0087] Table 8

[0088] From the above results, it can be seen that the precision details of the test method in Example 29 are significantly better than those in Example 30.

[0089] Among them, the dose-response curve of the neonatal immunoreactive trypsin assay kit (time-resolved fluorescence immunoassay) applicable to neonatal cystic fibrosis screening according to the present invention is as Figure 2 shown.

[0090] Inspired by the ideal embodiments of the present invention described above, through the above description, relevant staff can completely make various changes and modifications without departing from the technical idea of the present invention. The technical scope of the present invention is not limited to the content in the specification, and its technical scope must be determined according to the scope of the claims.

Claims

1. An immunoreactive trypsin (ogen) assay kit, characterized in that, For the quantitative determination of immunoreactive trypsin (ogen) in newborns, it includes the following components: filter paper dried blood spot calibrator, filter paper dried blood spot quality control product, reaction carrier, tracer, experimental buffer, washing solution and inducer.

2. The immunoreactive trypsin(ogen) assay kit according to claim 1, characterized in that, The preparation method of the filter paper dried blood spot calibrator is as follows: Dilute immunoreactive trypsin with calibrator diluent to twice the required concentration, add an equal volume of blood cells, mix well, and then spot 50 μL per drop on the filter paper. Spot calibrator A, B, C, D, E and F points, and the calibrator concentrations are 0, 25, 50, 100, 250, 500 ng / mL respectively. Dry quickly at room temperature under dehumidification and store for later use; The preparation method of the filter paper dried blood spot quality control product is as follows: Dilute immunoreactive trypsin with calibrator diluent to twice the required concentration, add an equal volume of blood cells, mix well, and then spot 50 μL per drop on the filter paper. Spot quality control product C1, C2 and C3 points, and the quality control product concentrations are 50, 100, 250 ng / mL respectively. Dry quickly at room temperature under dehumidification and store for later use.

3. The immunoreactive trypsin (ogen) assay kit according to claim 2, characterized in that The calibrator diluent includes one of human serum, sheep serum, 50 mmol / L Tris-HCl buffer with 5 g / L bovine serum albumin, pH 7.8; The calibrator diluent further includes a stabilizer, and the concentration of the stabilizer in the calibrator diluent is 5 - 20 μg / mL. The stabilizer includes at least one of dithiothreitol, β-mercaptoethanol, tris(2-carboxyethyl)phosphine hydrochloride 6-aminohexane, trypsin inhibitor (R21221), diisopropyl fluorophosphate.

4. The immunoreactive trypsin(ogen) assay kit according to claim 3, characterized in that, The immunoreactive trypsin includes natural human immunoreactive trypsin or recombinant human immunoreactive trypsin; among them, natural human immunoreactive trypsin is a full-length protein and has no chymotrypsin activity against N-succinyl-alanyl-alanyl-prolyl-phenylalanine p-nitroanilide; The amino acid sequence of recombinant human immunoreactive trypsin: LNNDIMLIKLSSRAVINARVSTISLPTAPPATGTKCLISGWGNTASSGADYPDELQCLDAPVLSQAKCEASYPGKITSNMFCVGFLEGGKDSCOGDSGGPVC; Or VGGYNCEENSVPYQVSLNSGYHFCGGSLINEQWVVSAGHCYKSRIQVRLGEHNIEVLEGNEQFINAAKIRHPQYDRKTLNNDIMLIKLSSRAVINARVSTISLPTAPPATGTKCLISGWGNTASSGADYPDELQCLDAPVLSQAKCEASYPGKITSNMFCVGFLEGGKDSCQGDSGGPVVCNGQLQGVVSWGDGCAQKNKPGVYTKVYNYVKWIKNTIAANS.

5. The immunoreactive trypsin(ogen) assay kit according to claim 1, characterized in that, The preparation method of the reaction carrier is as follows: Dilute the immunoreactive trypsin antibody with a coating buffer to the optimal working concentration, coat it in the wells of a 96-well microplate, with a coating volume of 50 - 200 μL, incubate at 2 - 8 °C for 18 - 22 h, then wash once with a washing solution working fluid with a volume of 100 - 300 μL / well, and then add 100 - 250 μL of blocking solution to each well and incubate at 36 - 38 °C for 1 - 3 h. Discard the blocking solution, drain and air-dry, vacuum seal the film, and store at 2 - 8 °C for later use; the coating buffer is a phosphate buffer with a concentration of 20 mmol / L and a pH of 4.

3. The blocking solution contains at least one of bovine serum albumin, calf serum, newborn calf serum, fetal bovine serum, casein, fish gelatin, polyvinylpyrrolidone 90, polyvinylpyrrolidone 360, polyethylene glycol 6000, polyethylene glycol 20000, Tween-20, Tween-40, sucrose, trehalose, sorbitol; the blocking buffer is a phosphate buffer with a concentration of 20 mmol / L and a pH of 4.

3. The immunoreactive trypsin antibody is a rabbit or mouse monoclonal antibody, and the monoclonal antibody is one of human immunoreactive trypsin 1 monoclonal antibody and human immunoreactive trypsinogen 2 monoclonal antibody.

6. The immunoreactive trypsin(ogen) assay kit according to claim 1, wherein The tracer is a lanthanide element-labeled immunoreactive trypsin antibody, and its preparation method is as follows: First, perform antibody storage liquid replacement treatment on the immunoreactive trypsin antibody with a carbonate buffer with a concentration of 50 mmol / L and a pH of 9.

6. Mix the immunoreactive trypsin antibody and the lanthanide element at a mass ratio of 1:1 - 3:1, oscillate and incubate at 2 - 8 °C for 24 - 72 h, perform chromatography purification using a gel column filled with Sephadex G-200, and dilute the collected tracer mother liquor with a Tris-HCl buffer with a concentration of 50 mmol / L and a pH of 7.8 containing 2 g / L bovine serum albumin into a semi-finished product. The lanthanide element is at least one of europium chelate and samarium chelate. The immunoreactive trypsin antibody is a rabbit or mouse monoclonal antibody, and the monoclonal antibody is one of human immunoreactive trypsin 1 monoclonal antibody and human immunoreactive trypsinogen 2 monoclonal antibody.

7. The immunoreactive trypsin(ogen) assay kit according to claim 1, characterized in that, The experimental buffer is 50 mmol / L Tris-HCl buffer with a pH of 7.8 containing 8.5 g / L sodium chloride, 0.2 mL / L Proclin ® 300, 7.4 mg / L disodium ethylenediaminetetraacetate, 1 mL / L mouse serum, 100 mL / L newborn bovine serum, 1 mg / L casein, and 3 - 10 μg / mL trypsin inhibitor (R21221). The cleaning solution is a 50 mmol / L tris(hydroxymethyl)aminomethane-hydrochloric acid buffer solution with a pH of 7.8 containing 1.125 g / L of sodium chloride, 0.2 mL / L of Tween-20, and 30 ppm of Proclin ® 300 preservative; The inducer is an enhancement solution containing 0.0494 g / L sodium acetate (anhydrous), 0.0043 g / L β-naphthoyltrifluoroacetone, 0.021 g / L tri-n-octylphosphine oxide, 1.143 ml / L glacial acetic acid, 0.5 ml / L absolute ethanol, 0.5 ml / L Triton X-100, and purified water.

8. The immunoreactive trypsin(ogen) assay kit according to claim 1, characterized in that, It also includes plate needles and barcodes. The barcode is a barcode containing project information.

9. A detection method for an immunoreactive trypsin(ogen) assay kit, characterized in that, The immunoreactive trypsin (ogen) assay kit according to any one of claims 1 to 8 includes the following modules: (1) a barcode scanning module; (2) a filter paper dried blood spot quality analysis module; (3) a filter paper dried blood spot punching module; (4) a sample addition pipetting module; (5) a gripper arm module; (6) an oscillation incubation module; (7) a 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 barcode scanning module is used to identify and read the barcode information of the filter paper dried blood spot sample and the carrier barcode. The filter paper dried blood spot quality analysis module is used to analyze the quality of the filter paper dried blood spot sample. For the filter paper dried blood spot samples, calibration products, and quality control products with qualified quality, an appropriate amount of blood spots are punched out by the filter paper dried blood spot punching module and fixed on the plate needles, and the plate needles with fixed blood spots are transferred to the temporary storage position of the bracket on the instrument support platform. The gripper arm module moves the carrier to the oscillation incubation module, and the sample addition pipetting module adds an appropriate amount of tracer working solution diluted with the experimental buffer to the carrier. The gripper arm module places the plate needles with fixed blood spots into the carrier in a specified order and immerses the blood spots in the tracer working solution. Then, the carrier is oscillated and incubated on the oscillation incubation module for an appropriate time. The gripper arm module transfers the plate needles with fixed blood spots to the temporary storage position of the bracket on the instrument support platform. At the same time, the gripper arm module moves the carrier to the washing module for washing. After washing, the gripper arm module moves the carrier to the oscillation incubation module, and the sample addition pipetting module adds an appropriate amount of inducer to the carrier, and the oscillation incubation module is used to oscillate and incubate at room temperature for an appropriate time. The gripper arm module moves the carrier to the measurement module to read the count value, and the count value is analyzed by the computer data analysis module, and the result is transmitted to the information management system module.

10. The detection method of the immunoreactive trypsin (ogen) assay kit according to claim 9, characterized in that, It includes the following steps: (1) Sample blood collection: Collect the sample blood using a filter paper dried blood spot and enter the sample-related information into the information management system module; (2) Sample quality analysis: Use the filter paper dried blood spot quality analysis module containing a high-definition industrial camera system, an image processing and analysis system to analyze the quality of the filter paper dried blood spot sample and determine whether the sample quality is qualified; (3) Barcode identification: Use the barcode scanning module to read the barcode of the filter paper dried blood spot sample, 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 the filter paper dried blood spot punching module containing a filter paper dried blood spot sample transfer device, a high-definition industrial camera system, an image analysis and positioning system, a filter paper dried blood spot blood spot cutter, and a high-precision motion positioning device to cut out a blood spot with a diameter of 3.2 mm (1 / 8 inch) from the filter paper dried blood spots of the qualified test samples, calibration products, and quality control products and fix it on the plate needles, and use the gripper arm module with horizontal / vertical movement functions and high-precision positioning functions to move the plate needles to the temporary storage position of the plate needle bracket; (5)Add tracer working solution: Use a gripper arm module with lateral / longitudinal movement function and high-precision positioning function to move the 96-well microplate reaction carrier to the oscillating incubation module with bottom heating, constant temperature control system, and vortex oscillation function. Use a pipetting module with lateral / longitudinal movement function and high-precision positioning function to dilute and mix the tracer and experimental buffer at a volume ratio of 1:50 to prepare the tracer working solution. Use a pipetting module with lateral / longitudinal movement function and high-precision positioning function to add 50 - 200 µL of the tracer working solution to the micro-cups of the 96-well microplate reaction carrier; (6)First incubation: Use a gripper arm module with lateral / longitudinal movement function and high-precision positioning function to move the plate needle to the 96-well microplate reaction carrier containing the 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 at room temperature with shaking in the oscillating incubation module for 90 - 150 min, preferably 120 min; (7)Wash the plate: Dilute the cleaning solution with purified water at a volume ratio of 1:25 to form the cleaning working solution and store it in the cleaning solution bucket of the washing module. Use a gripper 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 gripper 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 the cleaning working solution to wash the 96-well microplate reaction carrier 4 - 8 times; (8)Add inducer: Use a gripper arm module with lateral / longitudinal movement function and high-precision positioning function to move the 96-well microplate reaction carrier to the oscillating incubation module with bottom heating, constant temperature control system, and vortex oscillation function. Use a pipetting module with lateral / longitudinal movement function and high-precision positioning function to add 50 - 200 µL of inducer to the micro-cups of the 96-well microplate reaction carrier; (9)Second incubation: The 96-well microplate reaction carrier with inducer added is incubated at room temperature on the incubation position in the oscillating incubation module with a motor rotation speed of 1200 rpm for 2 - 10 min; (10)Detection: Use a gripper arm module with lateral / longitudinal movement function and high-precision positioning function to move the 96-well microplate reaction carrier with inducer added to the measurement module position. The measurement module has a high-precision displacement and positioning device, window opening and closing device, filter automatic switching measurement system, and photoelectric signal conversion measurement system. Select the corresponding immunoreactive trypsin detection program and use an excitation wavelength of 340 nm and a detection wavelength of 613 nm for detection to obtain the fluorometer value. The detection work needs to be completed within 30 min after adding the inducer; (11) Result analysis: The measured fluorometer values are transmitted to the computer data analysis module with a microcomputer system and an information connection port for the laboratory (clinical laboratory). The result analysis is carried out using the LOG-LOG_B 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 the results are transmitted to the information management system module.

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