A procalcitonin determination kit and a preparation and detection method thereof

Abstract

The application discloses a procalcitonin determination kit and a preparation and detection method thereof. The kit comprises reaction adjusting reagent R1, antibody latex reagent R2 and freeze-dried calibrant which are stored separately. The R1 contains HEPES, sodium chloride, polyethylene glycol 6000, L-arginine, polyvinylpyrrolidone K30, ethylenediaminetetraacetic acid disodium, non-ionic surfactant and preservative. The R2 contains first and second sensitized latexes made of carboxylated polystyrene latex particles with the same particle size and different antibody binding amounts, trehalose, poloxamer 188, bovine serum albumin and preservative. The reference calibrant contains recombinant procalcitonin, bovine serum albumin, trehalose, mannitol and glycine. In the preparation, the R1 is prepared, the two sensitized latexes are mixed to obtain the R2, and the calibrant is freeze-dried. The kit reduces non-specific aggregation, improves detection sensitivity, widens the linear measurement interval and improves storage stability, and is used for quantitative detection of procalcitonin in serum or plasma.

Description

Technical Field

[0001] This invention relates to the field of in vitro diagnostic reagents and immunoassay detection technology, specifically to a kit for quantitative detection of procalcitonin in serum or plasma using latex-enhanced immunoturbidimetry and its preparation method. Background Technology

[0002] Procalcitonin (PCT) is a precursor to calcitonin, normally secreted by thyroid C cells. When the body is stimulated by infections such as bacteria, fungi, or parasites, especially by pro-inflammatory responses caused by bacterial infections, many different organs and tissues produce PCT, increasing its concentration in the blood. Because PCT concentration is correlated with the occurrence and severity of systemic infections and sepsis, it is frequently used as an adjunct in the diagnosis of infections and unexplained fever, for early identification and monitoring of sepsis during treatment, and as a reference for antibiotic use and discontinuation. Compared to blood cultures, PCT testing requires less time and a smaller blood sample, making it more suitable for rapid clinical testing.

[0003] Currently, common methods for PCT detection include chemiluminescence immunoassay, fluorescence immunoassay, and latex-enhanced immunoturbidimetry. Among these, latex-enhanced immunoturbidimetry can be performed on fully automated biochemical analyzers, offering convenience, speed, and instrument adaptability. Generally, anti-PCT antibodies are coupled to the surface of latex particles. PCT in the sample undergoes immunoaggregation with the sensitized latex, and the PCT concentration is determined by measuring the change in absorbance of the reaction solution.

[0004] However, most existing antibody latex reagents use a single antibody sensitization density or latex particles of different sizes. When the antibody sensitization density is too low, it cannot effectively capture low concentrations of PCT. Conversely, when the antibody sensitization density is too high, rapid particle aggregation and compression of the response in high-concentration areas can easily occur, along with an increase in nonspecific background. When using batches of latex with different particle sizes for blending, differences in the sedimentation rate, surface curvature, and aggregation kinetics of the latex particles can easily cause batch-to-batch inconsistencies and storage instability. Furthermore, while coagulant components like polyethylene glycol can accelerate the immunoaggregation reaction, inappropriate dosage or system composition can also promote nonspecific aggregation, leading to increased blank absorbance and fluctuations in low-value detection results. During liquid storage, sensitized latex may undergo antibody conformational changes, particle fusion, or sedimentation. In the case of reference calibrators, problems such as decreased PCT activity, incomplete reconstitution of lyophilized products, and insufficient value stability may also occur. Therefore, there is a need to provide a procalcitonin assay kit that can balance low-concentration detection sensitivity, wide measurement range, detection precision, and storage stability. Summary of the Invention

[0005] To address the aforementioned deficiencies, this invention provides a procalcitonin assay kit, comprising separately stored reaction conditioning reagent R1, antibody latex reagent R2, and reference calibrator; The reaction regulating reagent R1 includes a first buffer system, sodium chloride, polyethylene glycol 6000, L-arginine, polyvinylpyrrolidone K30, disodium ethylenediaminetetraacetate, a first nonionic surfactant, a first preservative, and water. The antibody latex reagent R2 comprises a second buffer system, a first sensitized latex, a second sensitized latex, trehalose, poloxamer 188, bovine serum albumin, a second preservative, and water. The first and second sensitized latexes are formed by coupling anti-human procalcitonin polyclonal antibodies to two portions of carboxylated polystyrene latex particles, respectively. These two portions of carboxylated polystyrene latex particles are obtained from latex particle dispersions from the same production batch after thorough mixing. The average particle size ratio of the first sensitized latex to the second sensitized latex is 0.90–1.10, and the antibody binding amount per unit mass of latex particles in the first sensitized latex is 1.5 to 4.0 times that of the second sensitized latex. The mass ratio of the two, based on the dry weight of the latex particles, is 1:2 to 2:1. The mass-volume percentage concentration of the latex particles in the antibody latex reagent R2, based on dry weight, is 0.25%. The reference calibrator comprises recombinant procalcitonin, a third buffer system, bovine serum albumin, trehalose, mannitol, glycine, a third preservative, and water, and is stored in lyophilized form.

[0006] Further, the reaction regulating reagent R1 comprises 20 mmol / L to 100 mmol / L HEPES, 50 mmol / L to 200 mmol / L sodium chloride, 10 g / L to 50 g / L polyethylene glycol 6000, 5 mmol / L to 30 mmol / L L-arginine, 1 g / L to 10 g / L polyvinylpyrrolidone K30, 0.5 mmol / L to 5 mmol / L disodium ethylenediaminetetraacetate, 0.1 g / L to 1.0 g / L of a first nonionic surfactant, and a first preservative with a volume fraction of 0.03% to 0.10%, with a pH value of 7.2 to 8.0.

[0007] Further, the mass ratio of polyethylene glycol 6000 to polyvinylpyrrolidone K30 is 5:1 to 10:1, the final concentration of L-arginine is 10 mmol / L to 20 mmol / L; the first nonionic surfactant is Tween-20, the first preservative is ProClin 300, and the pH value of the reaction regulating reagent R1 is 7.4 to 7.8.

[0008] Furthermore, the average particle size of the carboxylated polystyrene latex particles is 100nm to 180nm, and the particle size variation coefficient is no greater than 10%; the antibody binding amount per unit mass of latex particles in the first sensitized latex and the second sensitized latex is 100μg / mg to 200μg / mg and 30μg / mg to 90μg / mg, respectively, and the ratio of the two is 1.5:1 to 4.0:1; the antibody binding amount is calculated as the difference between the amount of antibody added and the amount of unbound antibody in the supernatant after coupling.

[0009] Further, the antibody latex reagent R2 comprises 10 mmol / L to 50 mmol / L HEPES, 10 g / L to 50 g / L trehalose, 0.5 g / L to 5 g / L poloxamer 188, 1 g / L to 10 g / L bovine serum albumin, and a second preservative with a volume fraction of 0.03% to 0.10%; the second preservative is ProClin 300, and the pH value of the antibody latex reagent R2 is 7.0 to 7.8.

[0010] Further, the reference calibrators include six concentration levels of procalcitonin: 0 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 2 ng / mL, 10 ng / mL, and 50 ng / mL. The reference calibrators before lyophilization include 10 mmol / L to 50 mmol / L Tris-HCl, 5 g / L to 30 g / L bovine serum albumin, 20 g / L to 80 g / L trehalose, 10 g / L to 50 g / L mannitol, 5 g / L to 30 g / L glycine, and a third preservative with a volume fraction of 0.03% to 0.10%. The third preservative is ProClin 300, and the pH value before lyophilization is 7.2 to 7.6.

[0011] This application also provides a method for preparing the procalcitonin assay kit as described above, comprising the following steps: S1: Mix the first buffer system, sodium chloride, polyethylene glycol 6000, L-arginine, polyvinylpyrrolidone K30, disodium ethylenediaminetetraacetate, the first nonionic surfactant, the first preservative and water and adjust the pH value to obtain reaction regulating reagent R1; S2: Carboxylated polystyrene latex raw materials of the same particle size batch were divided into two parts after being measured by the dry weight of latex particles. Different antibody feed amounts were used to conjugate anti-human procalcitonin polyclonal antibodies to obtain the first sensitized latex and the second sensitized latex. After blocking, centrifugation to remove the supernatant and resuspending, the two were mixed in a mass ratio of 1:2 to 2:1 based on the dry weight of latex particles. The second buffer system, trehalose, poloxamer 188, bovine serum albumin and the second preservative were added to adjust the mass-volume percentage concentration of latex particles to 0.25% to obtain antibody latex reagent R2. S3: Mix recombinant procalcitonin, the third buffer system, bovine serum albumin, trehalose, mannitol, glycine, the third preservative, and water, filter, and freeze-dry to obtain the reference calibrator.

[0012] Further, in step S2, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide are used to activate carboxylated polystyrene latex particles; when preparing the first sensitized latex and the second sensitized latex, the mass ratio of anti-human procalcitonin polyclonal antibody to latex particles by dry weight is 1:(4-8) and 1:(10-30), respectively, the coupling temperature is 20℃-25℃, the coupling time is 2h-4h, and bovine serum albumin is used for blocking after coupling.

[0013] Further, in step S2, the first sensitized latex and the second sensitized latex are centrifuged at a relative centrifugal force of 10000×g to 20000×g for 20 min to 40 min, respectively, and resuspended using a resuspension containing a second buffer system, trehalose, poloxamer 188, bovine serum albumin and a second preservative. In step S3, the reference calibrator is pre-frozen at -60℃ to -40℃ for 2h to 6h, dried at a vacuum of 10Pa to 30Pa and a partition temperature of -30℃ to -10℃ for 8h to 16h, and then dried at 15℃ to 25℃ for 2h to 6h.

[0014] This application also provides a method for detecting procalcitonin using the procalcitonin assay kit described above, comprising the following steps: T1: Mix the test sample and reaction regulating reagent R1 at a volume ratio of 1:30 to 1:80 and incubate at 37°C for 1 min to 3 min. Select the detection wavelength in the range of 570 nm to 700 nm and read the initial absorbance. T2: Add antibody latex reagent R2 at a volume ratio of 3:1 to 6:1, react at 37°C for 2 to 5 minutes, and read the absorbance of the reaction at the same detection wavelength. T3: Determine the procalcitonin concentration in the sample based on the difference between the reaction absorbance and the initial absorbance, and the calibration curve established using a reference calibrator. This application has the following beneficial effects: Compared with existing technologies, this invention thoroughly mixes carboxylated polystyrene latex particle dispersions from the same production batch and then divides them into two parts. This ensures that the two parts of latex particles have the same raw material source before antibody conjugation, and that their initial average particle size, particle size distribution, surface carboxyl group state, and dispersion state are statistically consistent, thereby reducing the differences in sedimentation behavior and immunoaggregation kinetics between latex particles from different production batches. Based on this, different antibody dosages are used to form a first sensitized latex and a second sensitized latex with different antibody binding capacities. The higher sensitization density latex enhances the low-concentration PCT capture ability, while the lower sensitization density latex alleviates rapid aggregation and response compression in the high-concentration region, thus balancing detection sensitivity, linear measurement range, and repeatability. "Same production batch" refers to the two parts of carboxylated polystyrene latex particles being taken from latex particle raw materials with the same production batch number; "same production batch" does not require the two parts of latex particles to have exactly the same average particle size after sensitization, but rather indicates that the two parts of latex particles have the same raw material source and a consistent initial particle size distribution before antibody conjugation. High-sensitization-density latex enhances the capture capacity of low-concentration PCT, while low-sensitization-density latex mitigates rapid aggregation and response compression in high-concentration regions, thus balancing detection sensitivity, measurement range, and repeatability. Polyethylene glycol 6000 in reaction modifier R1 promotes antigen-antibody complex aggregation, and by eliminating volume effects, it increases the probability of effective collision between antigen and antibody, forming specific immune aggregates. L-arginine can reversibly interact with charged and aromatic residues on the antibody surface, weakening non-specific interactions between antibody molecules and between antibodies and sample impurities. Polyvinylpyrrolidone K30, as a hydrophilic nonionic polymer, increases the hydration level of the latex particle interface and forms a steric barrier, reducing the probability of direct contact and non-specific flocculation of latex particles not bridged by PCT. L-arginine and polyvinylpyrrolidone K30 together limit the non-specific aggregation induced by polyethylene glycol 6000, enabling the system to promote PCT-mediated antigen-antibody specific aggregation while inhibiting non-antigen-mediated background aggregation. This reduces absorbance changes in blank samples and minimizes detection fluctuations in low-concentration samples. Trehalose, poloxamer 188, and bovine serum albumin in antibody latex reagent R2 play roles in antibody conformation protection, steric hindrance dispersion, and blocking stabilization, respectively, mitigating particle fusion and activity reduction in sensitized latex during liquid storage. The reference calibrator uses a composite lyophilization protection system composed of bovine serum albumin, trehalose, mannitol, and glycine, which facilitates the formation of a stable lyophilized framework, reduces residual moisture, and improves reconstitution performance, thereby maintaining the stability of PCT calibrator values. This kit is compatible with fully automated biochemical analyzers, features simple operation, shortens detection time, and improves the reliability of test results. Detailed Implementation

[0015] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0016] The antibody binding amount referred to in this application means the apparent antibody binding amount per unit mass of latex particles determined by the difference in feed amount, and calculated according to the following formula: ; In the formula, Q represents the amount of antibody bound per unit mass of latex particles, expressed in μg / mg. The total mass of the anti-human procalcitonin polyclonal antibody fed during the coupling step is expressed in μg. The total mass of antibodies that did not bind to latex particles after coupling was completed, in μg. The dry weight of the latex particles involved in the coupling is in mg. The total mass of unbound antibodies is calculated based on the antibody concentration and volume of the coupling supernatant; if a pre-blocking washing step is present, the mass of unbound antibodies in the coupling supernatant and each washing buffer is combined for calculation. The antibody concentration in the coupling supernatant is determined using the micro-BCA method, and an antibody standard curve is prepared using the same coupling buffer.

[0017] Example 1

[0018] This embodiment provides a procalcitonin assay kit, which includes separately stored reaction conditioning reagent R1, antibody latex reagent R2, and reference calibrators at six concentration levels.

[0019] The reaction regulating reagent R1 consisted of 20 mmol / L HEPES, 200 mmol / L sodium chloride, 10 g / L polyethylene glycol 6000, 15 mmol / L L-arginine, 1 g / L polyvinylpyrrolidone K30, 5 mmol / L disodium ethylenediaminetetraacetate, 0.5 g / L Tween-20, and 0.03% (v / v) ProClin 300, with a pH of 7.6. The mass ratio of polyethylene glycol 6000 to polyvinylpyrrolidone K30 was 10:1.

[0020] The antibody latex reagent R2 consists of 30 mmol / L HEPES, 50 g / L trehalose, 0.5 g / L poloxamer 188, 5 g / L bovine serum albumin, 0.10% ProClin 300 (v / v), and a first sensitized latex and a second sensitized latex, with a pH of 7.4.

[0021] The first and second sensitized latexes were prepared using the same batch of carboxylated polystyrene latex particles with an average particle size of 100 nm and a particle size variation coefficient of 10%. The average particle size of the first sensitized latex was 117 nm, and the average particle size of the second sensitized latex was 130 nm, with a ratio of 0.90 between the two.

[0022] The antibody binding capacity of the first sensitized latex was 100 μg / mg latex particle, and the antibody binding capacity of the second sensitized latex was 30 μg / mg latex particle, with an antibody binding capacity ratio of 10:3. The mass ratio of the first sensitized latex to the second sensitized latex, based on the dry weight of the latex particles, was 1:1, and the total concentration of latex particles in R2 was 0.25% (w / v).

[0023] The reference calibrators consisted of 10 mmol / L Tris-HCl, 30 g / L bovine serum albumin, 50 g / L trehalose, 10 g / L mannitol, 15 g / L glycine, and 0.06% ProClin 300 (v / v), with a pH of 7.6 before lyophilization. The PCT concentrations after reconstitution of the six reference calibrators were 0 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 2 ng / mL, 10 ng / mL, and 50 ng / mL, respectively.

[0024] The reagent kit preparation method provided in this embodiment includes the following steps S1-S3.

[0025] S1: Prepare reaction regulator R1.

[0026] Add purified water equivalent to 80% of the target volume to the preparation container, then add HEPES, sodium chloride, polyethylene glycol 6000, L-arginine, polyvinylpyrrolidone K30, and disodium EDTA sequentially, stirring until completely dissolved. Add Tween-20 and ProClin 300 to adjust the pH to 7.6, and then add purified water to bring the volume to a final volume. After filtration through a 0.22 μm filter membrane, the reaction conditioning reagent R1 is obtained.

[0027] S2: Preparation of antibody latex reagent R2.

[0028] S21: Thoroughly mix the same batch of carboxylated polystyrene latex stock solution with an average particle size of 100 nm and a particle size variation coefficient of 10%. Take 1.00 mL of each solution to obtain two portions with a dry weight of 100.0 mg of latex particles. Add 2-(N-morpholine)ethanesulfonic acid buffer system and purified water to the two portions of latex stock solution and bring the volume to 10.00 mL to make the final concentration of MES 50 mmol / L, pH 6.0, and the dry weight concentration of latex particles 10 g / L. Add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl, abbreviated as EDC) and N-hydroxysuccinimide (NHS) to make their final concentrations 2 g / L and 5 g / L, respectively, and activate at 25 °C for 30 min.

[0029] S22: The two activated latex particles were centrifuged and resuspended in 20 mmol / L HEPES coupling buffer at pH 7.4. For the preparation of the first sensitized latex, the mass ratio of anti-human procalcitonin polyclonal antibody to latex particles (dry weight) was 1:8; for the preparation of the second sensitized latex, the mass ratio was 1:30. Both latex particles were coupled at 20°C for 3 h.

[0030] S23: After conjugation, samples were taken separately, latex particles and conjugation supernatant were separated, the mass of unbound antibody in the supernatant was determined, and the antibody binding amount was calculated according to the aforementioned formula. The antibody binding amount of the first sensitized latex was controlled at 100 μg / mg, and the antibody binding amount of the second sensitized latex was controlled at 30 μg / mg.

[0031] S24: Bovine serum albumin was added to both coupling solutions to achieve a final concentration of 10 g / L, and the mixture was blocked at 23°C for 1 h. Subsequently, the mixture was centrifuged at a relative centrifugal force of 20000×g for 30 min, the supernatant was removed, and the suspension was resuspended in R2 resuspension containing HEPES, trehalose, poloxamer 188, bovine serum albumin, and ProClin 300.

[0032] S25: Using the R2 resuspension of this embodiment, the dry weight concentration of latex particles in the first and second sensitized latexes was adjusted to 10 g / L. The average particle size of the first sensitized latex was measured to be 117 nm and the average particle size of the second sensitized latex was 130 nm using dynamic light scattering, with a particle size ratio of 0.90. 5.00 mL of the first sensitized latex and 5.00 mL of the second sensitized latex were measured to make a 1:1 mass ratio based on the dry weight of the latex particles. The R2 resuspension was added and the volume was adjusted to 40.00 mL, making the latex particle mass-volume percentage concentration 0.25% based on dry weight. The pH was adjusted to 7.4 to obtain the antibody latex reagent R2.

[0033] S3: Prepare reference calibrators.

[0034] S31: Prepare a calibrator matrix containing Tris-HCl, bovine serum albumin, trehalose, mannitol, glycine, and ProClin 300, and adjust the pH to 7.6.

[0035] S32: Add recombinant procalcitonin to prepare six concentration levels after reconstitution: 0 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 2 ng / mL, 10 ng / mL, and 50 ng / mL. Filter each concentration calibrator through a 0.22 μm filter membrane and fill each vial with 1.0 mL.

[0036] S33: After filling, the calibrators are pre-frozen at -50℃ for 2 h, dried once at a chamber pressure of 30 Pa and a partition temperature of -20℃ for 16 h, and then dried a second time at 15℃ for 4 h. The calibrators are then sealed with stoppers to obtain lyophilized reference calibrators. When using, each bottle is reconstituted with 1.0 mL of purified water.

[0037] This embodiment also provides a method for detecting procalcitonin using the above-mentioned kit, including the following T1-T3 steps.

[0038] T1: Mix the sample to be tested with the reaction regulating reagent R1 at a volume ratio of 1:30, incubate at 37°C for 2 min, and read the initial absorbance A0 at a wavelength of 650 nm.

[0039] T2: Add R2 according to the volume ratio of reaction regulating reagent R1 to antibody latex reagent R2 of 4:1, react at 37℃ for 5 min, and read the reaction absorbance A1 at a wavelength of 650 nm.

[0040] T3: Calculate the absorbance difference ΔA=A1-A0, and determine the procalcitonin concentration in the sample to be tested based on the calibration curve established by the six reference calibrators.

[0041] Example 2

[0042] The procalcitonin assay kit provided in this embodiment includes separately stored reaction conditioning reagent R1, antibody latex reagent R2, and reference calibrators at six concentration levels.

[0043] The reaction regulating reagent R1 consisted of 60 mmol / L HEPES, 50 mmol / L sodium chloride, 50 g / L polyethylene glycol 6000, 30 mmol / L L-arginine, 10 g / L polyvinylpyrrolidone K30, 0.5 mmol / L disodium ethylenediaminetetraacetate, 1.0 g / L Tween-20, and 0.06% (v / v) ProClin 300, with a pH of 7.2. The mass ratio of polyethylene glycol 6000 to polyvinylpyrrolidone K30 was 5:1.

[0044] The antibody latex reagent R2 consists of 50 mmol / L HEPES, 10 g / L trehalose, 2.5 g / L poloxamer 188, 1 g / L bovine serum albumin, 0.06% ProClin 300 (v / v), and a first sensitized latex and a second sensitized latex, with a pH of 7.8.

[0045] The first and second sensitized latexes were prepared using the same batch of carboxylated polystyrene latex particles with an average particle size of 140 nm and a particle size variation coefficient of 7%. The average particle size of the first sensitized latex was 165 nm, and the average particle size of the second sensitized latex was 150 nm, with a ratio of 1.10 between the two.

[0046] The antibody binding capacity of the first sensitized latex was 200 μg / mg latex particle, and the antibody binding capacity of the second sensitized latex was 50 μg / mg latex particle, with an antibody binding capacity ratio of 4.0:1. The mass ratio of the first sensitized latex to the second sensitized latex, based on the dry weight of the latex particles, was 1:2, and the total concentration of latex particles in R2 was 0.25% (w / v).

[0047] The reference calibrators consisted of 30 mmol / L Tris-HCl, 5 g / L bovine serum albumin, 80 g / L trehalose, 30 g / L mannitol, 30 g / L glycine, and 0.03% ProClin 300 (v / v), with a pH of 7.4 before lyophilization. The PCT concentrations after reconstitution of the six reference calibrators were 0 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 2 ng / mL, 10 ng / mL, and 50 ng / mL, respectively.

[0048] The reagent kit preparation method provided in this embodiment includes the following steps S1-S3.

[0049] S1: According to the final concentration specified in this embodiment, HEPES, sodium chloride, polyethylene glycol 6000, L-arginine, polyvinylpyrrolidone K30 and disodium ethylenediaminetetraacetate were added to purified water in sequence and stirred until completely dissolved; Tween-20 and ProClin 300 were added to adjust the pH to 7.2, purified water was added to make up the volume, and the mixture was filtered through a 0.22 μm filter membrane to obtain the reaction conditioning reagent R1.

[0050] S2: Preparation of antibody latex reagent R2.

[0051] S21: Carboxylated polystyrene latex stock solutions from the same production batch, with an average particle size of 140 nm and a particle size variation coefficient of 7%, were thoroughly mixed. 1.00 mL of each solution was measured to obtain two portions, each with a latex particle dry weight of 100.0 mg. 2-(N-morpholine)ethanesulfonic acid buffer system and purified water were added to each of the two latex stock solutions, and the volume was adjusted to 10.00 mL to achieve a final MES concentration of 50 mmol / L, a pH of 6.0, and a latex particle dry weight concentration of 10 g / L. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl, abbreviated as EDC) and N-hydroxysuccinimide (NHS) were added to achieve final concentrations of 2 g / L and 5 g / L, respectively. The mixture was then activated at 25 °C for 30 min.

[0052] S22: The two activated latex particles were resuspended separately in 20 mmol / L HEPES coupling buffer at pH 7.4. When preparing the first sensitized latex, the mass ratio of anti-human procalcitonin polyclonal antibody to latex particles (dry weight) was 1:4; when preparing the second sensitized latex, the mass ratio was 1:18. Both latex particles were coupled at 23°C for 4 hours.

[0053] S23: Determine the mass of unbound antibody in the coupling supernatant and calculate the antibody binding amount according to the aforementioned formula. Control the antibody binding amount of the first sensitized latex to be 200 μg / mg and the antibody binding amount of the second sensitized latex to be 50 μg / mg.

[0054] S24: Bovine serum albumin with a final concentration of 10 g / L was used for blocking at 23°C for 1 h. After blocking, the mixture was centrifuged at a relative centrifugal force of 10000×g for 40 min, the supernatant was removed, and the two sensitized latexes were resuspended using the R2 resuspension solution of this embodiment.

[0055] S25: Using the R2 resuspension of this embodiment, the dry weight concentration of latex particles in the first and second sensitized latexes was adjusted to 10 g / L. The average particle size of the first sensitized latex was measured to be 165 nm and the average particle size of the second sensitized latex was 150 nm using dynamic light scattering, with a particle size ratio of 1.10. 5.00 mL of the first sensitized latex and 10.00 mL of the second sensitized latex were measured to make a mass ratio of 1:2 based on the dry weight of the latex particles. The R2 resuspension was added and the volume was adjusted to 60.00 mL, making the latex particle mass-volume percentage concentration 0.25% based on dry weight. The pH was adjusted to 7.8 to obtain antibody latex reagent R2.

[0056] S3: Prepare reference calibrators.

[0057] S31: Prepare the calibrator matrix according to the final concentration specified in this embodiment, and adjust the pH value to 7.4.

[0058] S32: Prepare six concentration levels of PCT after reconstitution: 0 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 2 ng / mL, 10 ng / mL and 50 ng / mL. Filter the solutions through a 0.22 μm filter membrane and fill each bottle with 1.0 mL.

[0059] S33: Pre-freeze at -40℃ for 4 h, dry once at a chamber pressure of 10 Pa and a partition temperature of -30℃ for 12 h, then dry a second time at a partition temperature of 25℃ for 2 h, and then seal with a stopper. When using, add 1.0 mL of purified water to each bottle to reconstitute.

[0060] This embodiment also provides a method for detecting procalcitonin using the above-mentioned kit, including the following T1-T3 steps.

[0061] T1: Mix the sample to be tested with the reaction regulating reagent R1 at a volume ratio of 1:50, incubate at 37°C for 3 min, and read the initial absorbance A0 at a wavelength of 570 nm.

[0062] T2: Add R2 according to the volume ratio of reaction regulating reagent R1 to antibody latex reagent R2 of 6:1, react at 37℃ for 2 min, and read the reaction absorbance A1 at a wavelength of 570 nm.

[0063] T3: Calculate ΔA = A1 - A0, and determine the procalcitonin concentration in the sample to be tested based on the calibration curve established by the six reference calibrators.

[0064] Example 3

[0065] The procalcitonin assay kit provided in this embodiment includes separately stored reaction conditioning reagent R1, antibody latex reagent R2, and reference calibrators at six concentration levels.

[0066] The reaction regulating reagent R1 consisted of 100 mmol / L HEPES, 125 mmol / L sodium chloride, 30 g / L polyethylene glycol 6000, 5 mmol / L L-arginine, 5 g / L polyvinylpyrrolidone K30, 2.5 mmol / L disodium ethylenediaminetetraacetate, 0.1 g / L Tween-20, and 0.10% (v / v) ProClin 300, with a pH of 8.0. The mass ratio of polyethylene glycol 6000 to polyvinylpyrrolidone K30 was 6:1.

[0067] The antibody latex reagent R2 consists of 10 mmol / L HEPES, 30 g / L trehalose, 5 g / L poloxamer 188, 10 g / L bovine serum albumin, 0.03% ProClin 300 (v / v), and a first sensitized latex and a second sensitized latex, with a pH of 7.0.

[0068] The first and second sensitized latexes were prepared using the same batch of carboxylated polystyrene latex particles with an average particle size of 180 nm and a particle size variation coefficient of 8%. The average particle size of both the first and second sensitized latexes was 190 nm, and the ratio of their average particle sizes was 1.00.

[0069] The antibody binding amount of the first sensitized latex was 135 μg / mg latex particle, and the antibody binding amount of the second sensitized latex was 90 μg / mg latex particle, with an antibody binding ratio of 1.5:1. The mass ratio of the first sensitized latex to the second sensitized latex, based on the dry weight of the latex particles, was 2:1, and the total concentration of latex particles in R2 was 0.25% (w / v).

[0070] The reference calibrators consisted of 50 mmol / L Tris-HCl, 15 g / L bovine serum albumin, 20 g / L trehalose, 50 g / L mannitol, 5 g / L glycine, and 0.10% ProClin 300 (v / v), with a pH of 7.2 before lyophilization. The PCT concentrations after reconstitution of the six reference calibrators were 0 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 2 ng / mL, 10 ng / mL, and 50 ng / mL, respectively.

[0071] The reagent kit preparation method provided in this embodiment includes the following steps S1-S3.

[0072] S1: According to the final concentration specified in this embodiment, HEPES, sodium chloride, polyethylene glycol 6000, L-arginine, polyvinylpyrrolidone K30 and disodium ethylenediaminetetraacetate were added to purified water and stirred until completely dissolved; Tween-20 and ProClin 300 were added to adjust the pH to 8.0, purified water was added to make up the volume, and the mixture was filtered through a 0.22 μm filter membrane to obtain reaction conditioning reagent R1.

[0073] S2: Preparation of antibody latex reagent R2.

[0074] S21: Carboxylated polystyrene latex stock solutions from the same production batch, with an average particle size of 180 nm and a particle size variation coefficient of 8%, were thoroughly mixed. 1.00 mL of each solution was measured to obtain two portions, each with a latex particle dry weight of 100.0 mg. 2-(N-morpholine)ethanesulfonic acid buffer system and purified water were added to each of the two latex stock solutions, and the volume was adjusted to 10.00 mL to achieve a final MES concentration of 50 mmol / L, a pH of 6.0, and a latex particle dry weight concentration of 10 g / L. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl, abbreviated as EDC) and N-hydroxysuccinimide (NHS) were added to achieve final concentrations of 2 g / L and 5 g / L, respectively. The mixture was then activated at 25 °C for 30 min.

[0075] S22: The two activated latex particles were resuspended separately in 20 mmol / L HEPES coupling buffer at pH 7.4. When preparing the first sensitized latex, the mass ratio of anti-human procalcitonin polyclonal antibody to latex particles (dry weight) was 1:6; when preparing the second sensitized latex, the mass ratio was 1:10. Both latex particles were coupled at 25°C for 2 hours.

[0076] S23: Determine the mass of unbound antibody in the coupling supernatant and calculate the antibody binding amount according to the aforementioned formula. Control the antibody binding amount of the first sensitized latex to be 135 μg / mg and the antibody binding amount of the second sensitized latex to be 90 μg / mg.

[0077] S24: Bovine serum albumin with a final concentration of 10 g / L was used for blocking at 23°C for 1 h. After blocking, the mixture was centrifuged at a relative centrifugal force of 15000×g for 20 min, the supernatant was removed, and the two sensitized latexes were resuspended using the R2 resuspension solution of this embodiment.

[0078] S25: Using the R2 resuspension of this embodiment, the dry weight concentration of the latex particles of the first sensitized latex and the second sensitized latex was adjusted to 10 g / L. The average particle size of the first sensitized latex and the second sensitized latex was measured to be 190 nm using dynamic light scattering, and the ratio of their average particle sizes was 1.00. 10.00 mL of the first sensitized latex and 5.00 mL of the second sensitized latex were measured to make a mass ratio of 2:1 based on the dry weight of the latex particles. The R2 resuspension was added and the volume was adjusted to 60.00 mL, making the mass-volume percentage concentration of the latex particles 0.25% based on dry weight. The pH was adjusted to 7.0 to obtain the antibody latex reagent R2.

[0079] S3: Prepare reference calibrators.

[0080] S31: Prepare the calibrator matrix according to the final concentration specified in this embodiment, and adjust the pH value to 7.2.

[0081] S32: Prepare six concentration levels of PCT after reconstitution: 0 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 2 ng / mL, 10 ng / mL and 50 ng / mL. Filter the solutions through a 0.22 μm filter membrane and fill each bottle with 1.0 mL.

[0082] S33: Pre-freeze at -60℃ for 6 h, dry once at a chamber pressure of 20 Pa and a partition temperature of -10℃ for 8 h, then raise the partition temperature to 20℃ for a second drying for 6 h, and then seal with a stopper. When using, add 1.0 mL of purified water to reconstitute each bottle.

[0083] This embodiment also provides a method for detecting procalcitonin using the above-mentioned kit, including the following T1-T3 steps.

[0084] T1: Mix the sample to be tested with the reaction regulating reagent R1 at a volume ratio of 1:80, incubate at 37°C for 1 min, and read the initial absorbance A0 at a wavelength of 700 nm.

[0085] T2: Add R2 according to the volume ratio of reaction regulating reagent R1 to antibody latex reagent R2 of 3:1, react at 37℃ for 3.5 min, and read the reaction absorbance A1 at a wavelength of 700 nm.

[0086] T3: Calculate ΔA = A1 - A0, and determine the procalcitonin concentration in the sample to be tested based on the calibration curve established by the six reference calibrators.

[0087] Comparative Example 1 The difference between this comparative example and Example 2 is that the mixture of the first sensitized latex and the second sensitized latex is replaced with the first sensitized latex, and the total concentration of latex particles in R2 remains 0.25% (w / v); the remaining reagent composition, preparation method and detection method are the same as in Example 2.

[0088] Comparative Example 2 The difference between this comparative example and Example 2 is that the antibody binding amount of both the first and second sensitized latexes in this comparative example is adjusted to 100 μg / mg latex particles, and the two sensitized latexes are still mixed according to the dry weight ratio specified in Example 2; the other conditions are the same as in Example 2.

[0089] Comparative Example 3 The difference between this comparative example and Example 2 is that the kit in this comparative example replaces the latex particles used in the first sensitized latex with first batch carboxylated polystyrene latex particles with an average particle size of 100 nm, and replaces the latex particles used in the second sensitized latex with second batch carboxylated polystyrene latex particles with an average particle size of 180 nm; the other conditions are the same as in Example 2.

[0090] Comparative Example 4 The difference between this comparative example and Example 2 is that L-arginine is not added to the reaction regulating reagent R1 in this comparative example, and purified water is used to make up to the original volume; the composition, preparation method and detection method of the other reagents are the same as those in Example 2.

[0091] Comparative Example 5 The difference between this comparative example and Example 2 is that polyvinylpyrrolidone K30 is not added to the reaction regulating reagent R1 in this comparative example, and purified water is used to make up to the original volume; the other conditions are the same as in Example 2.

[0092] Comparative Example 6 The difference between this comparative example and Example 2 is that trehalose is not added to the antibody latex reagent R2 in this comparative example, and purified water is used to make up to the original volume; the other conditions are the same as in Example 2.

[0093] Comparative Example 7 The difference between this comparative example and Example 2 is that poloxamer 188 is not added to the antibody latex reagent R2 in this comparative example, and purified water is used to make up to the original volume; the other conditions are the same as in Example 2.

[0094] Comparative Example 8 The difference between this comparative example and Example 2 is that mannitol and glycine are not added to the reference calibrator in this comparative example, and purified water is used to make up to the original volume; the other reagent composition, freeze-drying process and detection method are the same as in Example 2.

[0095] It should be noted that Examples 1-3 and Comparative Examples 1-8 of this application used the same batch of recombinant procalcitonin, anti-human procalcitonin polyclonal antibody, and test samples for evaluation. Except for the differences explicitly stated, the comparative examples are consistent with their corresponding examples in terms of raw material batches, preparation equipment, testing instruments, and testing procedures.

[0096] Test Example 1: Sensitivity, Precision, and Measurement Range Test To evaluate the effects of polyethylene glycol 6000, L-arginine, and polyvinylpyrrolidone K30 in R1 on the PCT detection performance of dual-sensitized density latex of the same particle size, a Hitachi 7180 biochemical analyzer was used. A1: Five blank samples were prepared from PCT-negative human serum matrix, and low-concentration samples of 0.03 ng / mL, 0.05 ng / mL, 0.08 ng / mL, 0.10 ng / mL, and 0.15 ng / mL were prepared, with each sample tested 12 times; A2: The sample was mixed with R1 at a volume ratio of 1:50, incubated at 37℃ for 2 min, and then R2 was added at a volume ratio of R1:R2 of 4:1, reacted at 37℃ for 3 min, and the absorbance before and after the reaction was read at 600 nm; A3: ... The blank limit was calculated based on the 95th percentile of the blank results, and the detection limit was calculated based on the standard deviation of the low-concentration samples; A4: The 0.20 ng / mL and 50 ng / mL quality control samples were tested 20 times each, and the recovery rate and the coefficient of variation for low-value repeatability were calculated; A5: Linear samples of 0.1 ng / mL, 0.5 ng / mL, 2 ng / mL, 10 ng / mL, 25 ng / mL and 50 ng / mL were tested, with each concentration tested 3 times, and linear regression was performed between the theoretical concentration and the measured concentration. The results are shown in Table 1.

[0097] Table 1

[0098] Table 1 shows that the limits of detection (LODs) for Examples 1-3 ranged from 0.035 ng / mL to 0.048 ng / mL, the recoveries for 0.20 ng / mL samples ranged from 96.8% to 101.5%, and the coefficients of variation for repeatability ranged from 3.8% to 4.9%. Furthermore, the coefficients of determination for the ranges from 0.1 ng / mL to 50 ng / mL were all not less than 0.9980, indicating that all three formulations balanced low-value response and wide-range quantification. Comparative Example 1, using only a high-sensitization-density latex, still had a low LOD, but the recoveries for 50 ng / mL samples decreased to 78.4%, indicating that a single high-density surface easily leads to response compression in high-concentration regions. Comparative Example 2 did not exhibit a sensitization-density difference, while Comparative Example 3 used batches with different particle sizes. Both showed a decrease in LOD, low-value precision, and linearity, indicating that the effectiveness of this application is not simply obtained by mixing two latexes. Comparative Examples 4 and 5, lacking L-arginine or polyvinylpyrrolidone K30 respectively, showed larger blank limits and low-value coefficients of variation. Comparative Example 7, lacking poloxamer 188, also exhibited a higher background. Comparative Examples 6 and 8 showed minimal changes in initial detection performance, with their effects only becoming apparent in stability tests. Therefore, it can be concluded that the density design of the same particle size dual-sensitization is responsible for coordinating low-concentration capture and high-concentration response, while the coagulation-dispersion equilibrium system of R1 reduces non-specific aggregation; both improve sensitivity, precision, and measurement range.

[0099] Test Example 2: Accelerated Stability Test of the Reagent Kit To evaluate the liquid stability system composed of trehalose, poloxamer 188, and bovine serum albumin in R2, and the freeze-drying protection system composed of trehalose, mannitol, and glycine in the reference calibrator, an accelerated stability test at 37°C was conducted. B1: Three independent batches were prepared for Examples 1-3 and Comparative Examples 1-8. Five vials of R2 and calibrator from each batch were stored at 37°C for 14 days, and another five vials were stored at 2°C-8°C as controls. B2: R2 was diluted 10-fold with its own buffer and measured three times at 25°C using a Zetasizer Nano ZS particle size analyzer. The average particle size increase rate was calculated. B3: 0.20 ng / mL and 2 ng / mL quality control samples were tested three times per vial using a Hitachi 7180 fully automated biochemical analyzer. The recovery rate after accelerated storage was calculated. B4: 1.0 mL of purified water was added to each vial of lyophilized calibrator, and the mixture was mixed at a rate of inverting once per second. The time for complete solid disappearance was recorded. B5: Residual moisture was determined using a coulometric Karl Fischer moisture analyzer. The PCT recovery rate of the reconstituted 2 ng / mL calibrator was determined using a commercially assigned PCT detection system. The results were taken as the arithmetic mean of the three batches. The test results are shown in Table 2.

[0100] Table 2

[0101] After storage at 37°C for 14 days, the average particle size increase rate of R2 in Examples 1-3 was only 2.9%–4.5%, and the recoveries of 0.20 ng / mL and 2 ng / mL samples were both 94.8%–99.1%. The PCT recovery rate of the lyophilized calibrator was 96.9%–98.6%, the reconstitution time was less than or equal to 48 s, and the residual moisture was less than or equal to 1.6%, indicating that the three formulations had good accelerated stability. In Comparative Examples 1-3, changing the bis-sensitization density or maintaining the same particle size resulted in a larger increase in latex particle size and greater measurement deviation, further demonstrating that a uniform particle size contributes to latex storage stability. In Comparative Examples 4 and 5, the R2 particle size did not change significantly, but the detection recovery rate after acceleration was low, further indicating that L-arginine and polyvinylpyrrolidone K30 mainly inhibit non-specific aggregation during the detection reaction stage. In Comparative Example 6, the particle size increase rate reached 15.8% after the removal of trehalose; in Comparative Example 7, the recovery rate increased to 28.6% after removing poloxamer 188, and the low-value recovery rate was only 72.4%, demonstrating that the two substances respectively played the roles of antibody conformation protection and steric hindrance dispersion. In Comparative Example 8, the calibrator recovery rate decreased to 82.7% after removing mannitol and glycine, the reconstitution time reached 135 s, and the residual moisture increased to 3.8%. Therefore, the liquid latex stabilization system and the lyophilized calibrator protection system can work together to simultaneously ensure the dispersion state of sensitized particles and the stability of PCT values.

[0102] This document describes preferred embodiments of the invention, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments will become apparent to those skilled in the art after reading the foregoing description. The inventors expect those skilled in the art to appropriately employ such variations, and the inventors intend to practice the invention in ways different from those specifically described herein. Therefore, the invention includes all modifications and equivalents to the subject matter referenced in the appended claims, where permitted by applicable law. Furthermore, the invention covers any combination of the elements described above with all possible variations, unless otherwise indicated herein or otherwise expressly contradicted by the context.

Claims

1. A procalcitonin assay kit, characterized in that, This includes separately stored reaction conditioning reagent R1, antibody latex reagent R2, and reference calibrators; The reaction regulating reagent R1 includes a first buffer system, sodium chloride, polyethylene glycol 6000, L-arginine, polyvinylpyrrolidone K30, disodium ethylenediaminetetraacetate, a first nonionic surfactant, a first preservative, and water. The antibody latex reagent R2 comprises a second buffer system, a first sensitized latex, a second sensitized latex, trehalose, poloxamer 188, bovine serum albumin, a second preservative, and water. The first and second sensitized latexes are formed by coupling anti-human procalcitonin polyclonal antibodies to two portions of carboxylated polystyrene latex particles, respectively. These two portions of carboxylated polystyrene latex particles are obtained from latex particle dispersions from the same production batch after thorough mixing. The average particle size ratio of the first to the second sensitized latex is 0.90–1.10, and the antibody binding amount per unit mass of latex particles in the first sensitized latex is 1.5 to 4.0 times that of the second sensitized latex. The mass ratio of the two latex particles based on dry weight is 1:2 to 2:1, and the mass-volume percentage concentration of latex particles in the antibody latex reagent R2 based on dry weight is 0.25%. The reference calibrator comprises recombinant procalcitonin, a third buffer system, bovine serum albumin, trehalose, mannitol, glycine, a third preservative, and water, and is stored in lyophilized form.

2. The procalcitonin assay kit according to claim 1, characterized in that, The reaction regulating reagent R1 comprises 20 mmol / L to 100 mmol / L HEPES, 50 mmol / L to 200 mmol / L sodium chloride, 10 g / L to 50 g / L polyethylene glycol 6000, 5 mmol / L to 30 mmol / L L-arginine, 1 g / L to 10 g / L polyvinylpyrrolidone K30, 0.5 mmol / L to 5 mmol / L disodium ethylenediaminetetraacetate, 0.1 g / L to 1.0 g / L of a first nonionic surfactant, and a first preservative with a volume fraction of 0.03% to 0.10%, with a pH value of 7.2 to 8.

0.

3. The procalcitonin assay kit according to claim 2, characterized in that, The mass ratio of polyethylene glycol 6000 to polyvinylpyrrolidone K30 is 5:1 to 10:1, the final concentration of L-arginine is 10 mmol / L to 20 mmol / L, the first nonionic surfactant is Tween-20, the first preservative is ProClin 300, and the pH value of the reaction regulating reagent R1 is 7.4 to 7.

8.

4. The procalcitonin assay kit according to claim 1, characterized in that, The average particle size of the carboxylated polystyrene latex particles is 100nm to 180nm, and the particle size variation coefficient is no greater than 10%; the antibody binding amount per unit mass of latex particles in the first sensitized latex and the second sensitized latex is 100μg / mg to 200μg / mg and 30μg / mg to 90μg / mg, respectively, and the ratio of the two is 1.5:1 to 4.0:1; the antibody binding amount is calculated as the difference between the amount of antibody added and the amount of unbound antibody in the supernatant after coupling.

5. The procalcitonin assay kit according to claim 1, characterized in that, The antibody latex reagent R2 comprises 10 mmol / L to 50 mmol / L HEPES, 10 g / L to 50 g / L trehalose, 0.5 g / L to 5 g / L poloxamer 188, 1 g / L to 10 g / L bovine serum albumin, and a second preservative with a volume fraction of 0.03% to 0.10%; the second preservative is ProClin 300, and the pH value of the antibody latex reagent R2 is 7.0 to 7.

8.

6. The procalcitonin assay kit according to claim 1, characterized in that, The reference calibrators include six concentration levels of procalcitonin: 0 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 2 ng / mL, 10 ng / mL, and 50 ng / mL. The reference calibrators before lyophilization include 10 mmol / L to 50 mmol / L Tris-HCl, 5 g / L to 30 g / L bovine serum albumin, 20 g / L to 80 g / L trehalose, 10 g / L to 50 g / L mannitol, 5 g / L to 30 g / L glycine, and a third preservative with a volume fraction of 0.03% to 0.10%. The third preservative is ProClin 300, and the pH value before lyophilization is 7.2 to 7.

6.

7. A method for preparing a procalcitonin assay kit as described in any one of claims 1 to 6, characterized in that, Includes the following steps: S1: Mix the first buffer system, sodium chloride, polyethylene glycol 6000, L-arginine, polyvinylpyrrolidone K30, disodium ethylenediaminetetraacetate, the first nonionic surfactant, the first preservative and water and adjust the pH value to obtain reaction regulating reagent R1; S2: Carboxylated polystyrene latex particles of the same particle size batch were divided into two parts after being weighed by dry weight of latex particles. Different antibody dosages were used to conjugate anti-human procalcitonin polyclonal antibodies to obtain the first sensitized latex and the second sensitized latex. After blocking, centrifugation to remove the supernatant and resuspending, the two were mixed in a mass ratio of 1:2 to 2:1 based on the dry weight of latex particles. The second buffer system, trehalose, poloxamer 188, bovine serum albumin and the second preservative were added to adjust the mass-volume percentage concentration of latex particles to 0.25% to obtain antibody latex reagent R2. S3: Mix recombinant procalcitonin, the third buffer system, bovine serum albumin, trehalose, mannitol, glycine, the third preservative, and water, filter, and freeze-dry to obtain the reference calibrator.

8. The preparation method according to claim 7, characterized in that, In step S2, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide are used to activate carboxylated polystyrene latex particles. When preparing the first sensitized latex and the second sensitized latex, the mass ratio of anti-human procalcitonin polyclonal antibody to latex particles by dry weight is 1:(4-8) and 1:(10-30), respectively. The coupling temperature is 20℃-25℃, the coupling time is 2h-4h, and bovine serum albumin is used for blocking after coupling.

9. The preparation method according to claim 7, characterized in that, In step S2, the first sensitized latex and the second sensitized latex are centrifuged at a relative centrifugal force of 10000×g to 20000×g for 20 min to 40 min, respectively, and resuspended in a resuspension containing a second buffer system, trehalose, poloxamer 188, bovine serum albumin and a second preservative. In step S3, the reference calibrator is pre-frozen at -60℃ to -40℃ for 2h to 6h, dried at a vacuum of 10Pa to 30Pa and a partition temperature of -30℃ to -10℃ for 8h to 16h, and then dried at 15℃ to 25℃ for 2h to 6h.

10. A method for detecting procalcitonin using the procalcitonin assay kit as described in any one of claims 1 to 6, characterized in that, Includes the following steps: T1: Mix the test sample and reaction regulating reagent R1 at a volume ratio of 1:30 to 1:80 and incubate at 37°C for 1 min to 3 min. Select the detection wavelength in the range of 570 nm to 700 nm and read the initial absorbance. T2: Add antibody latex reagent R2 at a volume ratio of 3:1 to 6:1, react at 37°C for 2 to 5 minutes, and read the absorbance of the reaction at the same detection wavelength. T3: Determine the procalcitonin concentration in the sample to be tested based on the difference between the reaction absorbance and the initial absorbance and the calibration curve established by the reference calibrator.

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