Procalcitonin detection kit, preparation method and application

Abstract

The invention relates to a procalcitonin detection kit as well as a preparation method and application thereof. The kit provided by the invention comprises a first antibody coated on magnetic microspheres, a second antibody marked with a tracing marker and a calibrator, the specific binding sites of the procalcitonin identified by the first antibody and the second antibody are different; the calibrator comprises a procalcitonin antigen A and a diluent, the procalcitonin antigen A has a sequence as shown in SEQ ID NO: 1, and the diluent comprises a buffer solution and a protein stabilizer. The calibration product is high in storage stability, the kit is low in matrix effect, and a detection result can be rapidly obtained.

Description

[0001] This application is a divisional application for the original application number: CN201911304611.4, the original application date is December 17, 2019, and the invention name is A procalcitonin detection kit, preparation method and use. Technical Field

[0002] The invention belongs to the field of immunoassay, and specifically relates to a procalcitonin detection kit, a preparation method and use thereof. Background Art

[0003] Procalcitonin (PCT) is the precursor of calcitonin (CT). It is a glycoprotein composed of 116 amino acid residues with a relative molecular mass of 13,000. It has no hormonal activity and is the encoded product of the Calci gene on chromosome 11. Since Assicot et al. first reported in 1993 that PCT can be used as an early marker of bacterial infection, it has been widely used in the diagnosis and differential diagnosis of infectious diseases as a new inflammatory indicator. It is currently recognized as the most sensitive and specific indicator for the diagnosis of sepsis.

[0004] Under normal circumstances, CalcimRNA is transcribed in human thyroid parafollicular cells (C cells), translated into PCT precursor, and then glycosylated and removed by specific enzymes to generate PCT, which is then hydrolyzed by different proteases to form mature CT and katacalcin (KC). The C-terminus of mature CT is amidated to form active CT. In addition to the synthesis and secretion in C cells under physiological conditions, PCT also has ectopic secretion. Plasma PCT is still elevated in patients with severe infection after thyroidectomy. Medullary thyroid cell carcinoma and small cell lung cancer can synthesize CT and PCT at the same time, which increases plasma CT and PCT. LPS, interleukin (IL)-1, IL-2, IL-6 and tumor necrosis factor-α (TNF-α) have a positive stimulatory effect on PCT mRNA expression, which can induce a large amount of PCT in a short time. In severe bacterial infection or sepsis, plasma PCT increases significantly, and the degree of increase is significantly positively correlated with the severity of the infection; while CT does not change significantly, suggesting that the production of PCT and CT are two relatively independent processes under inflammatory conditions.

[0005] PCT is a secondary inflammatory factor that cannot initiate sepsis reaction by itself, but it can amplify and aggravate the pathological process of sepsis. The abnormal increase of plasma PCT level in sepsis suggests that PCT has certain pathophysiological functions in acute immune response, but it is not yet fully understood.

[0006] PCT products only appear in adherent monocytes and adipocytes, and are not found in circulating granulocytes. Systemic inflammation affects the adhesion of tissues and monocytes, affecting their PCT production. The degree of PCT increase depends on the scope and severity of inflammation in the body, while viral infections and autoimmune diseases cannot induce PCT production.

[0007] PCT is degraded by specific proteolytic enzymes in the body, with a half-life of about 25 to 30 hours. It is rarely excreted through the kidneys, and the renal clearance rate of plasma PCT is less than 1 mL / min. For patients with renal failure, there is no significant increase in plasma PCT levels, and the ratio of plasma PCT levels to urine PCT levels is about 4:1, which remains relatively constant. Although continuous venovenous hemofiltration or hemodialysis can remove some PCT, there is no significant change in plasma PCT levels. Therefore, the detection of plasma PCT is also suitable for patients with renal failure or receiving artificial kidney treatment.

[0008] Commonly used PCT detection methods in clinical practice mainly include quantitative method and semi-quantitative method:

[0009] The semi-quantitative method mainly includes colloidal gold colorimetry, using a specially prepared PCT-Q test card, which is simple and fast. The entire test process does not exceed 30 minutes, and the results are divided into 4 levels: normal <0.5ng / mL; mildly elevated >0.5ng / mL; significantly elevated >2ng / mL; significantly elevated >10ng / mL. This method does not rely on instruments, is simple and fast to operate, and is suitable for bedside monitoring or point-of-care testing (POCT). The disadvantage is that it cannot be accurately quantified.

[0010] Quantitative detection includes radioimmunoassay, chemiluminescence, transmission immunoturbidimetry, etc. Among them, radioimmunoassay detects a mixture of free procalcitonin, bound procalcitonin, and calcitonin gene-related peptide precursor, but cannot distinguish the above three substances. Its detection sensitivity is 4ng / L, and the linear range is 10-77ng / L. This method takes a long time to detect (19-22h), and there is contamination by radioactive elements, so its use is limited.

[0011] Double antibody sandwich immunochemiluminescence method: Use two monoclonal antibodies to bind to two sites of the procalcitonin molecule, which can eliminate cross-reactions. Its detection limit is 0.02ng / mL. This method is simple to operate, pollution-free, highly specific, and highly sensitive, and can be tested in batches.

[0012] Transmission immunoturbidimetry: This method is simple, rapid, and can be automated, making it suitable for batch testing. However, compared with the first two methods, it has a lower sensitivity.

[0013] Enzyme-linked immunosorbent assay (ELISA): Taking the double antibody sandwich method as an example, a specific antibody is coated on a solid phase carrier, and after adding the sample to be tested, another enzyme-labeled antibody is added to form a double antibody sandwich complex, and then the instrument can quantitatively detect the immune response through the color development of the luminescent substrate. Due to its shortcomings such as cumbersome operation, low sensitivity, narrow linear range and long detection cycle, in some cases it cannot help doctors to judge the patient's condition in a timely and effective manner, especially for critically ill patients, thus limiting the scope of use of this type of product.

[0014] In terms of POCT testing, colloidal gold immunochromatography, as a POCT test method, has the advantages of small sample volume, simplicity and rapidity, and is suitable for bedside testing. However, due to the poor sensitivity of this method, its application in the clinical detection of procalcitonin is further limited.

[0015] As a popular POCT detection method in the market, time-resolved fluorescence immunoassay has the basic advantages of POCT testing technology. However, due to manual operation, it has large operating errors, high precision deviations, and cannot guarantee both sensitivity and linear range at the same time, and the test results are easily affected by the external environment.

[0016] At present, although there are kits for detecting procalcitonin on the market, many of them have shortcomings such as narrow linear range, insufficient sensitivity, and high cost. For example, CN101029897A has too many markers and a complicated reaction process; the CN107367620A reagent reaction system is relatively complex, and high-throughput testing is difficult to achieve. At the same time, there is still room for improvement in terms of improving stability and reducing matrix effects for calibrators. Summary of the invention

[0017] Problem that the invention aims to solve

[0018] In order to solve the problems existing in the prior art, the object of the present invention is to provide a procalcitonin detection kit, which includes a calibrator with high storage stability, has a low matrix effect and can quickly obtain detection results.

[0019] Another object of the present invention is to provide a method for preparing the aforementioned procalcitonin detection kit.

[0020] Another object of the present invention is to provide a use of the aforementioned procalcitonin detection kit.

[0021] Solutions for solving problems

[0022] Specifically, the present invention records the following technical solutions.

[0023] [1] A procalcitonin detection kit, the kit comprising: a first antibody coated on a magnetic microsphere, a second antibody labeled with a tracer marker, and a calibrator; the procalcitonin specific binding sites recognized by the first antibody and the second antibody are different; the calibrator comprises procalcitonin antigen A and a diluent, the procalcitonin antigen A is a sequence as shown in SEQ ID NO:1, and the diluent comprises a buffer and a protein stabilizer.

[0024] [2] The procalcitonin detection kit according to [1], wherein the buffer is selected from a HEPES buffer having a concentration of 0.03 to 0.06 M, and the protein stabilizer is selected from bovine serum albumin.

[0025] [3] The procalcitonin detection kit according to [1] or [2], wherein the first antibody has a sequence as shown in SEQ ID NO:4, and the second antibody has a sequence as shown in SEQ ID NO:8.

[0026] [4] A procalcitonin detection kit according to any one of the technical solutions [1] to [3], wherein the mass ratio of the magnetic microspheres to the first antibody is 100:1.5-2.5.

[0027] [5] A procalcitonin detection kit according to any one of the technical solutions [1] to [4], wherein the molar ratio of the tracer marker to the second antibody is not less than 15:1.

[0028] [6] A procalcitonin detection kit according to any one of the technical solutions [1] to [5], wherein the kit further comprises a magnetic bead cleaning solution and substrate solution A and substrate solution B. Preferably, the magnetic microsphere cleaning solution contains PBST buffer, the substrate solution A is a mixture of H2O2 and HNO3, and the substrate solution B is a mixture of Triton X-100 and NaOH.

[0029] [7] A procalcitonin detection kit according to any one of the technical solutions [1] to [6], wherein the tracer marker is selected from one or more of the group consisting of luminol, alkaline phosphatase, horseradish peroxidase, acridinium ester and adamantane; preferably, the tracer marker is acridinium ester.

[0030] [8] A method for preparing a procalcitonin detection kit according to any one of the technical solutions [1] to [7], wherein the preparation method comprises:

[0031] Magnetic microsphere coating step: coating the first antibody on the magnetic microsphere;

[0032] Tracing marker labeling step: labeling the tracing marker on the second antibody;

[0033] Steps for preparing calibrators.

[0034] [9] The preparation method according to [8], wherein the coupling time between the first antibody and the magnetic microspheres in the magnetic microsphere coating step is 1 to 1.5 hours.

[0035]

[10] Use of the procalcitonin detection kit according to any one of the technical solutions [1] to [7] in the preparation of reagents for diagnosing infectious diseases.

[0036] Effects of the Invention

[0037] By using the test kit of the present invention, the concentration of procalcitonin in the sample can be quickly detected with extremely high sensitivity. The calibrator provided by the present invention has good storage stability and low matrix effect. In conjunction with the applicant's chemiluminescent immunoassay analyzer, the concentration of PCT in the sample can be detected automatically, quickly, sensitively and quantitatively. The reagent of the present invention adopts a one-step reaction, and the reaction time only takes 6 minutes. The time for adding samples, washing, and detection is about 3.5 minutes, and the report time can be controlled within 10 minutes. The test kit of the present invention can be used to diagnose the severity of the infection as early as possible and accurately, and provide sufficient time for the treatment of the infection, as well as provide sufficient support for the prognosis detection of infected patients.

[0038] In another embodiment, the reagent is simple to operate and can be used for batch testing by the device, and the throughput is sufficient to meet the needs of most users.

[0039] In another embodiment, the calcitonin detection kit provided by the present invention can reduce nonspecific binding, improve detection sensitivity and detection stability while saving costs. In addition, the present invention has a relatively wide linear range, and the upper limit of detection can reach 100ng / mL. At a high concentration of 5000ng / mL, the anti-HOOK ability can still meet the use requirements. This shows that even when the PCT antigen content in the patient is extremely high, there is no possibility of misdiagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The graph showing the matching rate of four groups of antibodies in Example 2 of the present invention is shown;

[0041] Figure 2 An eight-point linear correlation diagram of Embodiment 10 of the present invention is shown;

[0042] Figure 3 The graph showing the compliance rate of 61 Roche samples according to the tenth embodiment of the present invention is shown. DETAILED DESCRIPTION

[0043] In order to better illustrate the present invention, numerous specific details are provided in the following specific embodiments. It should be understood by those skilled in the art that the present invention can be implemented without certain specific details. In other examples, methods, means, equipment and steps well known to those skilled in the art are not described in detail in order to highlight the subject matter of the present invention.

[0044] It should be noted that:

[0045] In this specification, the term "magnetic microspheres" and "magnetic beads" have the same meaning, referring to superparamagnetic microspheres with small particle size, generally with super strong paramagnetism. Its surface can be coated with specific antibodies, receptors, etc., and used to separate and purify targets in samples. Magnetic beads have been widely used in many fields such as immunoassay, nucleic acid separation and extraction, cell sorting, enzyme fixation, etc.

[0046] In this specification, the references to "some specific / preferred embodiments", "other specific / preferred embodiments", "embodiments", etc., mean that the specific elements (e.g., features, properties and / or characteristics) described in connection with the embodiments are included in at least one embodiment described herein, and may or may not exist in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any suitable manner.

[0047] In this specification, the meaning of "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process. In this specification, "optional" or "optionally" means that the event or situation described below may or may not occur, and the description includes the situation where the event occurs and the situation where the event does not occur.

[0048] In the context of describing the invention (especially in the context of the following claims), the terms "a", "an", and "the" and similar language are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0049] In this specification, the numerical range expressed using "numerical value A to numerical value B" or "numerical value A-numerical value B" means a range including the endpoints numerical values ​​A and B.

[0050] The term "comprising" and any variations thereof in the specification and claims of the present invention and the above drawings are intended to cover non-exclusive inclusions. For example, a process, method or system, product or device comprising a series of steps or units is not limited to the listed steps or units, but may optionally include steps or units not listed, or may optionally include other steps or units inherent to these processes, methods, products or devices.

[0051] <First aspect>

[0052] The invention provides a procalcitonin (PCT) detection kit, wherein the kit comprises: a first antibody coated on a magnetic microsphere, a second antibody marked with a tracer marker and a calibrator; the procalcitonin recognized by the first antibody and the second antibody has different specific binding sites; the calibrator comprises procalcitonin antigen A and a diluent, the procalcitonin antigen A is a sequence as shown in SEQ ID NO: 1, and the diluent comprises a buffer and a protein stabilizer.

[0053] The present invention adopts a double antibody sandwich method, which mainly uses two different specific antibodies, wherein the binding site of the antibody labeled with the tracer marker is different from the binding site of the antibody coated on the magnetic microsphere. The selection of these binding sites is not only conducive to the labeling of the tracer or the coating of the magnetic microsphere, but also does not hinder the antibody from binding to the antigen to form a sandwich complex, thereby improving the specificity and sensitivity of the reaction.

[0054] In some specific embodiments of the present invention, the first antibody and the second antibody of the present invention are both monoclonal antibodies against PCT. In a technical solution, in order to improve the signal value of the test sample and the conformity rate of the test sample, preferably, the sequence of the amino acid site of procalcitonin recognized by the first antibody of the present invention is a sequence as shown in SEQ ID NO:4, and the sequence of the amino acid site of procalcitonin recognized by the second antibody is a sequence as shown in SEQ ID NO:8.

[0055] The present invention does not specifically limit the types of tracer markers applicable to the present invention. For example, the tracer marker may be luminol, alkaline phosphatase, horseradish peroxidase, acridinium ester and adamantane. Preferably, the tracer marker is acridinium ester, which is relatively low in background luminescence, high in signal-to-noise ratio, fast and concentrated in light release, high in luminescence efficiency, high in luminescence intensity when applied to chemiluminescence detection, easy to bind to protein and no reduction in photon yield after binding, and the marker is stable (can be stored for several months at 2-8°C). In addition, acridinium ester has good solubility in water, is stable and not easily hydrolyzed.

[0056] The present invention does not specifically limit the type of magnetic microspheres applicable to the present invention, and the magnetic microspheres may be magnetic beads commonly used in the art. As one embodiment of the present invention, the magnetic microspheres used in the present invention are micron-sized solid-phase microspheres with paramagnetism and a maximum protein adsorption capacity formed by compounding nano-sized Fe2O3 and Fe3O4 magnetic particles with polymer materials. The magnetic microspheres can be rapidly magnetized under the action of an external magnetic field, and the remanence is zero after the magnetic field disappears. In the present invention, there is no specific limitation on the type of polymer materials used for compounding with the magnetic microspheres.

[0057] The particle size of the magnetic beads used in the present invention should be 1-5 μm. As one of the embodiments of the present invention, the magnetic beads can be surface modified to add multiple active groups. The types of active groups in the present invention include but are not limited to -OH and -COOH.

[0058] In some specific embodiments of the present invention, in order to improve the detection sample signal, the mass ratio of the magnetic microspheres to the first antibody is 100:1.5-2.5. For the stability and convenience of operation, it is further preferred that the mass ratio of the magnetic microspheres to the first antibody is 100:1.8-2.2. In other specific embodiments of the present invention, the molar ratio of the tracer marker to the second antibody is not less than 15:1. If it is lower than 15:1, the detection signal will be affected.

[0059] In some specific embodiments of the present invention, when the kit is used, the first antibody-coated magnetic microspheres are diluted to 0.03-0.08 mg / mL according to the magnetic microsphere usage concentration, and the second antibody labeled with the tracer marker is diluted to 0.5-2 μg / mL, preferably 1 μg / mL.

[0060] The calibrator of the present invention comprises procalcitonin antigen A and diluent. In order to improve the stability of the calibrator and further reduce the matrix effect, the inventors have conducted in-depth research on the antigen, diluent and protein stabilizer used in the calibrator. At present, procalcitonin antigen (PCT antigen) includes multiple products such as PCT (full length), NPCT, CT and PCT (NPCT removed), and the amino acid sequences of procalcitonin antigens from different manufacturers are also different. The inventors have found through research that in the present invention, when the procalcitonin antigen A having the sequence shown in SEQ ID NO: 1 is used, a very high antigen binding titer can be obtained. At the same test concentration, the use of procalcitonin antigen A has a higher signal than other procalcitonin antigens.

[0061] The diluent of the calibrator of the present invention includes a buffer and a protein stabilizer. The buffer of the present invention is selected from 4-hydroxyethylpiperazineethanesulfonic acid buffer (HEPES) with a concentration of 0.03 to 0.06M. Preferably, the pH value of the HEPES buffer system is 8.0. Compared with other buffer systems, such as phosphate buffered saline (PBS) or tris (hydroxymethyl) aminomethane hydrochloric acid buffer (Tris-HCl), the system using HEPES as a buffer has a small decrease in the luminescence value after accelerated destruction for seven days, and the stability of the calibrator is high. The protein stabilizer used in the present invention is selected from bovine serum albumin (BSA), which has the functions of a cryoprotectant and a dehydration protectant, and can play a good protective role on PCT antigens. Further preferably, the mass percentage of BSA in the buffer is 2 to 3wt%, which helps to further improve stability. In other embodiments of the present invention, when the concentration of bovine serum albumin in the buffer is 2.5%, the stability of the PCT freeze-dried calibrator can be maximized. The calibrators of the present invention include low-point calibrators and high-point calibrators. The low-point calibrators refer to a standard solution obtained by diluting a PCT working solution to 300 pg / mL using a HEPES solution containing 2 to 3 wt% bovine serum albumin and then freeze-drying the solution. The high-point calibrators refer to a standard solution obtained by diluting a PCT working solution to 25000 pg / mL using a HEPES solution containing 2 to 3 wt% bovine serum albumin and then freeze-drying the solution.

[0062] In the present invention, the kit further comprises: a magnetic microsphere cleaning solution, a substrate solution A and a substrate solution B.

[0063] The magnetic microsphere cleaning solution in the present invention is phosphate-tween buffer (PBST); preferably, the magnetic microsphere cleaning solution also includes a preservative. The preservative applicable to the present invention may be a preservative commonly used in the art, such as potassium sorbate, sodium benzoate, sodium nitrite, sodium azide, proclin-300 (the main active ingredients are 2-methyl-4-isothiazoline-3-one and 5-chloro-2-methyl-4-isothiazoline-3-one) and one or more of antibiotics.

[0064] The substrate solution A in the present invention is a mixture of H2O2 and HNO3, preferably, the mass fraction of H2O2 is 0.01-5.0%, and the concentration of HNO3 is 0.01-1.0 mol / L. The substrate solution B is a mixture of polyethylene glycol octylphenyl ether (Triton X-100) and NaOH, preferably, the mass fraction of Triton X-100 is 0.01-2.0%, and the concentration of NaOH is 0.05-1 mol / L.

[0065] <Second Aspect>

[0066] The present invention also provides a method for preparing the procalcitonin PCT detection kit according to <the first aspect>, wherein the preparation method comprises:

[0067] Magnetic microsphere coating step: coating the first antibody on the magnetic microsphere;

[0068] Tracing marker labeling step: labeling the tracing marker on the second antibody;

[0069] Steps for preparing calibrators.

[0070] As one of the embodiments of the present invention, in the magnetic microsphere coating step, the coupling time between the first antibody and the magnetic microsphere is 1 to 1.5 hours.

[0071] As one of the embodiments of the present invention, 2-(N-morpholino)ethanesulfonic acid (MES) buffer is used in the first antibody coating magnetic microspheres step.

[0072] As one of the embodiments of the present invention, a blocking solution is also used in the step of coating the magnetic microspheres with the first antibody, and the blocking solution is phosphate buffered saline (PBS) containing casein. The concentration range of the blocking agent is preferably 0.01 to 0.05 M, and the amount of the blocking agent is 100 to 300 μL.

[0073] As one of the embodiments of the present invention, the antibody coupling buffer used in the tracer marker labeling step is selected from HEPES buffer or carbonate buffer (CB). In a specific embodiment of the present invention, it is selected from CB buffer (pH=9.2).

[0074] As one of the embodiments of the present invention, the labeling time of the second antibody labeled with the tracer marker (i.e. the reaction time at room temperature and away from light) is 1.5 to 3 hours; optionally, the labeling time of the second antibody labeled with the tracer marker is 2 hours.

[0075] As one of the embodiments of the present invention, the step of preparing the calibration product includes taking the working solution containing the procalcitonin antigen A of the present invention, diluting it with a HEPES solution containing 2-3% bovine serum albumin to obtain high and low point standard solutions and then freeze-drying them.

[0076] <Third Aspect>

[0077] The present invention also provides a use of the procalcitonin detection kit according to <the first aspect> in preparing a reagent for diagnosing infectious diseases.

[0078] Infectious diseases have a high incidence rate. As the disease progresses, pathogens will spread and cause patients to experience various related clinical symptoms. If symptomatic treatment is not given in time, it will affect the patient's prognosis. Bacteria and viruses are the main pathogens that cause infectious diseases. Generally speaking, the procalcitonin level in patients with infectious diseases will fluctuate abnormally. In a normal body, the procalcitonin content is relatively low, but if internal inflammation occurs, the procalcitonin level will increase. Therefore, procalcitonin can effectively reflect the severity of infectious diseases and can be used as a clinical detection indicator for monitoring. Procalcitonin has excellent diagnostic specificity, especially for bacterial infectious diseases.

[0079] The procalcitonin detection kit of the present invention can be used in conjunction with a compatible fully automatic chemiluminescence analyzer (Yao Xuanfeng series) to quickly and effectively detect the procalcitonin level in the sample to be tested, which is helpful for clinical judgment of whether the patient has an infectious disease.

[0080] The sample to be tested in the present invention is serum, plasma and whole blood obtained directly, or it can be a sample obtained by extracting human blood and separating it.

[0081] The specific detection process includes: diluting the first antibody-coated magnetic microspheres and the second antibody labeled with a tracer marker in the kit respectively. In some specific embodiments of the present invention, the first antibody-coated magnetic microspheres are diluted to 0.03-0.08 mg / mL according to the concentration of the magnetic microspheres, and the second antibody labeled with a tracer marker is diluted to 0.5-2 μg / mL; the diluted reagents and the sample to be tested or / and the calibration substance are added to the reaction cup in sequence, and the whole adding process takes about 0.5 minutes; after the solution in the reaction cup is fully mixed, it is incubated at 37°C for 6 minutes; then the reaction cup is placed under magnetic conditions and washed three times with a magnetic microsphere cleaning solution, and the whole process takes 2 minutes; chemiluminescent excitation liquid is added to the reaction cup and the photon value is detected; according to the light intensity detected in the reaction cup, the instrument automatically calculates the concentration of PCT in the sample to be tested, and the inspection calculation process takes 1 minute. It can be seen that the test result can be obtained in 10 minutes using the kit provided by the present invention.

[0082] In the present invention, the first antibody-coated magnetic microspheres, the acridinium ester labeled antibody and the antigen to be tested are fully mixed in a reaction cup to obtain a magnetic microsphere (i.e., magnetic microparticle)-antibody-antigen-acridinium ester complex. After washing, adding chemiluminescent excitation liquid, and detecting the light signal intensity, the PCT concentration in the sample is obtained.

[0083] The kit provided by the present invention can detect the PCT concentration of the sample with extremely high sensitivity. The detection limit of the present invention is no higher than 10pg / mL, and the precision within a day can be maintained within 5%, which is of great significance for the identification of infection diagnosis. The linear correlation R>0.990, the detection recovery rate should be within 85% to 115%, the accuracy is good, and it has a good correlation with the Roche PCT kit. The present invention has a wide linear range, and the upper limit of detection can reach 100ng / mL. The hook effect, also known as the HOOK effect, refers to the phenomenon of false negatives due to an inappropriate antigen-antibody ratio. The anti-HOOK ability of the present invention can still meet the use requirements at a high concentration of 5000ng / mL.

[0084] The kit provided by the present invention is used to detect the PCT concentration in a sample. The total detection time is short, and the test result can be obtained in only about 10 minutes. The PCT concentration of the sample can be fully automatically and quickly detected, which is convenient for early and accurate diagnosis of infectious diseases.

[0085] Example

[0086] Embodiment 1

[0087] This embodiment provides a method for preparing a PCT detection kit and a method for detecting procalcitonin in human blood.

[0088] Preparation 1: First antibody coated magnetic microspheres

[0089] (1) 10 mg of magnetic microspheres (average particle size 1.5 μm, purchased from Bangs Laboratories, solid content 2.54%) were weighed and suspended in 1 mL of 0.1 M MES buffer. After adsorption by magnet for 5 to 10 min, the supernatant was discarded, and the above washing steps were repeated 3 to 5 times. 1 mL of the above buffer (0.1 M MES buffer) was added and vortexed to mix.

[0090] (2) Add 200 μg of the first PCT monoclonal antibody (the amino acid sites of procalcitonin recognized by the first antibody are 21-40, i.e., the sequence is the sequence shown in SEQ ID NO: 4) so ​​that the mass ratio of magnetic microspheres to antibodies is 50:1, vortex mix, and incubate at 37° C. for 1 h.

[0091] (3) Add 10 μL of 10 mg / mL 1-(3-dimethylaminopropyl)-3-ethyldiimide hydrochloride (EDC), vortex to mix, and incubate at 37°C for 1.5 h.

[0092] (4) Take 200 μL of PBS solution (0.02 M) containing 1% casein for blocking, and block for 2 h.

[0093] (5) Add 1 mL of luminescence recovery solution 1 (5 g of bovine serum albumin + 400 mL of 0.025 M Tris-HCl buffer + 0.1-2‰ Tween-80 + 100 mL of newborn calf serum) to the blocked magnetic bead suspension, adsorb with a magnet, remove the supernatant, and repeat the above washing steps 3 to 5 times.

[0094] (6) Place the prepared magnetic beads in 1 mL of luminescence recovery solution 1 and store at 2-8°C.

[0095] Preparation 2: Preparation of acridinium ester-labeled PCT monoclonal antibody solution

[0096] (1) 1 mg of the second PCT monoclonal antibody (the amino acid sites of procalcitonin recognized by the second antibody are 102-111, i.e., the sequence is the sequence shown in SEQ ID NO: 8) is placed in a dialysis bag and dialyzed for 24 hours using no less than 2 L of 0.05 M CB buffer (pH = 9.2), with the solution changed four times in the middle. After the dialysis is completed, the antibody is added to a Millipore Amicon Ultra ultrafiltration centrifuge tube for concentration to a concentration of greater than 5 mg / mL.

[0097] (2) The concentrated PCT monoclonal antibody was placed in a 0.5 mL centrifuge tube, and 100 μL of 5 mg / mL acridinium ester acid-NHS ester solution (NSP-SA-NHS) dissolved in DMF was added to make the molar ratio of NSP-SA-NHS to PCT monoclonal antibody not less than 15:1. After mixing, the mixture was reacted at room temperature in the dark for 2 h, and 100 μL of 100 mg / mL lysine solution was added to react for 30 min to terminate the reaction.

[0098] (3) Place the marker in a dialysis bag and dialyze for 24 h with no less than 2 L of citric acid-sodium citrate buffer (0.01 mol / L, pH = 4.5), changing the solution four times in between to separate free NSP-SA-NHS.

[0099] (4) Collect the dialyzed marker, add 5% BSA solution to make the final BSA concentration 1%, then add an equal volume of glycerol and store at -20°C.

[0100] Preparation 3: Preparation of Calibrators

[0101] PCT working solution (procalcitonin antigen A having the sequence shown in SEQ ID NO: 1) was diluted to 300 pg / mL and 25000 pg / mL high and low point standard solutions using 0.05 M HEPES solution containing 2.5% bovine serum albumin, and then lyophilized.

[0102] The components of the kit are respectively constituted by using the magnetic microsphere suspension coated with the second anti-PCT antibody obtained in Preparation 1, the first PCT antibody labeled with acridinium ester in Preparation 2, and the high and low point calibrators in Preparation 3.

[0103] Preparation 4: Preparation of chemiluminescent substrate solution

[0104] The chemiluminescent pre-substrate solution A is a mixed solution of H2O2 and HNO3, wherein the mass fraction of H2O2 is 0.01-5.0% and the concentration of HNO3 is 0.01-1.0 mol / L.

[0105] The chemiluminescent pre-substrate solution B is a mixture of Triton X-100 and NaOH, wherein the mass fraction of Triton X-100 is 0.01-2.0%, and the concentration of NaOH is 0.05-1 mol / L.

[0106] Preparation 5: Preparation of cleaning solution

[0107] The cleaning solution of the present invention is a PBST solution with a pH value of 7.0-9.0 and a concentration of 0.02 mol / L, wherein the mass fraction of Tween-20 is 0.5%.

[0108] The first antibody obtained in Preparation 1 is used to coat the magnetic microspheres, the second antibody obtained in Preparation 2 is labeled with acridinium ester, the high and low point calibrators in Preparation 3, the chemiluminescent substrate solution in Preparation 4 and the magnetic microsphere cleaning solution in Preparation 5 are used to prepare a procalcitonin detection kit.

[0109] The PCT concentration in the sample was detected using the kit and a fully automatic chemiluminescence analyzer.

[0110] Specifically, the PCT concentration in the sample to be tested is measured using a fully automatic chemiluminescence analyzer according to the following steps:

[0111] 1. Dilute the first antibody-coated magnetic microspheres prepared in Preparation 1 to 0.05 mg / mL (reagent 1) according to the concentration of magnetic microspheres, and dilute the acridinium ester-labeled second antibody prepared in Preparation 2 to 1 μg / mL (reagent 2);

[0112] 2. Add 50 μL of reagent 1 and 50 μL of reagent 2 to the reaction cup in sequence;

[0113] 3. Add 50 μL of the sample to be tested and / or the calibrator into the reaction cup. The entire adding process takes 0.5 minutes.

[0114] 4. After the solution in the reaction cup is fully mixed, incubate at 37°C for 6 minutes;

[0115] 5. Place under magnetic conditions and wash three times with magnetic microsphere cleaning solution. The whole process takes 2 minutes.

[0116] 6. After adding 100 μL of chemiluminescent excitation solution A to the reaction cup, add 100 μL of chemiluminescent excitation solution B and immediately detect the photon value;

[0117] 7. Based on the light intensity detected in the reaction cup, the instrument automatically calculates the concentration of PCT in the sample to be tested. The entire test and calculation process takes 1 minute.

[0118] Embodiment 2

[0119] Antibody panel screening for PCT detection kits

[0120] In the process of preparation 1 and preparation 2 of this embodiment, there are four PCT monoclonal antibodies: antibody 1 recognizes amino acid sites 21-40 (sequence is the sequence shown in SEQ ID NO: 4), antibody 2 recognizes amino acid sites 60-69 (sequence is the sequence shown in SEQ ID NO: 5), antibody 3 recognizes amino acid sites 72-81 (sequence is the sequence shown in SEQ ID NO: 6), antibody 4 recognizes amino acid sites 96-105 (sequence is the sequence shown in SEQ ID NO: 7), and antibody 5 recognizes amino acid sites 102-111 (sequence is the sequence shown in SEQ ID NO: 8). Antibody 1 is coated on magnetic microspheres to form an antibody 1-magnetic microsphere suspension, and antibodies 2, 3, 4, and 5 are respectively labeled on acridinium ester to obtain antibody 2-acridinium ester, antibody 3-acridinium ester, antibody 4-acridinium ester, and antibody 5-acridinium ester. The above antibodies were combined and paired to obtain group 1 (magnetic microsphere-antibody 1 and acridinium ester-antibody 2), group 2 (ie, Example 1) (magnetic microsphere-antibody 1 and acridinium ester-antibody 3), group 3 (magnetic microsphere-antibody 1 and acridinium ester-antibody 4), and group 4 (magnetic microsphere-antibody 1 and acridinium ester-antibody 5). The prepared kit was used with an automatic chemiluminescence analyzer to detect serum and EDTA plasma. The results are shown in Tables 1 and 2. Figure 1 , where signal 1, signal 2, signal 3, and signal 4 refer to light intensity.

[0121] Table 1 Results of the kit antibody pairing screening of 16 plasma samples

[0122]

[0123] Table 2 Comparison of serum and plasma samples of kit antibody combination

[0124]

[0125] Table 1 and Figure 1The results show that the consistency rate (R value) of the four groups for detecting serum samples is the highest in group 4, and the overall signal value is the highest, so group 4 is finally selected. Table 2 Use group 4 to detect 14 homologous samples, and there is no significant deviation overall, so the antibodies preferably used in the preparation process of the kit of the present invention are group 4 (magnetic microspheres-antibody 1 and acridinium ester-antibody 5).

[0126] Embodiment 3

[0127] This example provides a screening process for the coupling time between the first PCT monoclonal antibody and the magnetic microspheres during the preparation of the first antibody-coated magnetic microspheres.

[0128] Example 3 uses the same preparation 1 process as Example 1. Compared with the preparation 1 process of Example 1, Example 3 mainly changes the incubation time of step (2) in preparation 1. In step (2) of preparation 1, Example 3 uses 30 min, 1 h or 1.5 h as the incubation time. The three first antibody-coated magnetic microspheres prepared in Example 3 are respectively combined with the second antibody-labeled acridinium ester obtained in Preparation 2 of Example 1 to obtain three kits, which are then tested with a fully automatic chemiluminescence analyzer, and the test results are shown in Table 3.

[0129] Table 3 Screening results of the first PCT monoclonal antibody and magnetic microsphere coupling time

[0130]

[0131] The results in Table 3 show that, except for 30 min, the coupling time of the first PCT monoclonal antibody and the magnetic microspheres is close to that of 1 h and 1.5 h. Therefore, in the preparation process of the first antibody-coated magnetic microspheres in the kit of the present invention, 1 h and 1.5 h can be selected, but in order to retain a certain error control space, the preferred coupling time is 1 h.

[0132] Embodiment 4

[0133] This example provides a screening process for the antibody coating ratio during the preparation of the first antibody-coated magnetic microspheres.

[0134] Example 4 adopts the same preparation 1 process as Example 1. Compared with the preparation 1 process of Example 1, Example 4 mainly changes the mass ratio of magnetic microspheres to antibodies in step (2) of preparation 1. In step (2) of preparation 1, Example 4 adopts 100:2, 100:1.5 or 100:1 as the mass ratio of magnetic microspheres to antibodies. The three first antibody-coated magnetic microspheres prepared in Example 4 are respectively combined with the second antibody-labeled acridinium ester obtained in preparation 2 of Example 1 to obtain three kits, which are then tested with a fully automatic chemiluminescence analyzer, and the test results are shown in Table 4.

[0135] Table 4 Screening results of the mass ratio of magnetic microspheres to the first antibody

[0136]

[0137] The results in Table 4 show that when screening the mass ratio of magnetic microspheres to antibodies, the calibration signal detected at the ratios of 100:1.5 and 100:2 is higher, and the results are close. When the mass ratio is lower than 100:1.5, the signal is significantly reduced. For the stability and simplicity of operation, and to retain a certain error control space, the preferred mass ratio of magnetic microspheres to antibodies in the preparation process of the kit of the present invention is 100:2.

[0138] Embodiment 5

[0139] This example provides a screening process for a buffer solution during the preparation of acridinium ester labeled with a second antibody.

[0140] Example 5 uses the same preparation 2 process as Example 1. Compared with the preparation 2 process of Example 1, Example 5 mainly changes the type of buffer in step (1) of preparation 2. In step (1) of preparation 2, Example 5 uses 0.05M CB buffer (pH=9.2), 0.05M CB buffer (pH=9.6) or 0.05M HEPES buffer (pH=8.0), 0.05M HEPES buffer (pH=8.5) respectively. The four second antibodies prepared in Example 5 are labeled with acridinium ester and combined with the first antibody-coated magnetic microspheres obtained in preparation 1 of Example 1 to obtain four kits, and the calibration products are tested. The test results are shown in Table 5.

[0141] Table 5 Screening results of buffers in the preparation of the second antibody labeled acridinium ester (calibrator)

[0142]

[0143] The results in Table 5 show that when HEPES diluent or CB diluent is used as the buffer (pH 8.0-9.6), there is no significant difference in the four groups of test results. In order to retain a certain error control space, CB (pH = 9.2) is selected. Therefore, the preferred buffer for the kit of the present invention in the process of preparing acridinium ester labeled antibodies is 0.05M CB buffer (pH = 9.2).

[0144] Embodiment 6

[0145] This example provides a screening process for PCT antigen during the preparation of calibrators.

[0146] Example 6: Using 0.01M PBS solution with 1% bovine serum albumin, three different PCT antigens, PCT antigen A (sequence as shown in SEQ ID NO: 1), PCT antigen B (sequence as shown in SEQ ID NO: 2), and PCT antigen C (sequence as shown in SEQ ID NO: 3), were diluted into antigen diluents of different concentrations in the same proportion. The rest was the same as in Example 1. The test results are shown in Table 6.

[0147] Table 6 Comparison of PCT antigen titers from different sources

[0148] Concentration (ng / ml) Antigen A Antigen B Antigen C 500 3772444 491898 40115 250 2081787 243170 19993 50 513161 50867 4788 10 108216 11555 1513 1 11863 1583 455 0.1 1652 509 372 0 262 259 371

[0149] From the results in Table 6, it can be seen that the titer of antigen A is much higher than that of the other two groups, so antigen A is preferably used in the preparation of the calibrator of this kit.

[0150] Embodiment 7

[0151] This example provides a screening process for buffer solutions during the preparation of calibrator dilutions.

[0152] Example 7 Conventional buffers 0.02M PBS (pH=7.1), 0.05M HEPES (PH=8.0), and 0.05M Tris-HCl (pH=9.0) were used as diluents to dilute PCT antigen A into antigen diluents of different concentrations, and a comparative test was performed at 37°C for seven days. The test results are shown in Table 7.

[0153] Table 7 Comparison of antigen stability under different buffer systems

[0154]

[0155] From the results in Table 7, it can be seen that without adding any protein or sugar, the antigen was accelerated to be destroyed for seven days using 0.05M HEPES, and the luminescence value decreased the least. Therefore, this kit preferably uses 0.05M HEPES as the antigen diluent.

[0156] Embodiment 8

[0157] This example provides a process for screening protein stabilizers during the preparation of calibrator dilutions.

[0158] Example 8 Using 0.05M HEPES buffer, 2.5% bovine serum albumin (BSA), 20% newborn calf serum, and 2.5% casein were added to the buffer system, and the PCT antigen dilution solution was diluted into antigen dilution solutions of different concentrations in the same proportion, and a comparative test was performed at 37°C for seven days. The test results are shown in Table 8.

[0159] The results in Table 8 show that the use of bovine serum albumin as a protein stabilizer has an overall better stability than the other two groups. After seven days of destruction, the luminescence value decreased slightly. Therefore, this kit preferably uses bovine serum albumin as a protein stabilizer for the antigen diluent.

[0160] Table 8 Antigen accelerated stability performance after adding different protein stabilizers

[0161]

[0162] Embodiment 9

[0163] This example provides the evaluation results of the anti-HOOK ability of this kit. Example 9 uses a calibration diluent (0.05M HEPES+2.5% BSA) to prepare five high concentration points of antigen at concentrations of 5000, 3000, 2000, 1000, and 500 ng / mL, and prepares calibration products at concentrations of 100, 50, 10, 1, and 0.1 ng / mL. The rest is the same as Example 1, and the test results are shown in Table 9.

[0164] Table 9 Anti-HOOK ability test results

[0165]

[0166] It can be seen from the results in Table 9 that when the antigen concentration is as high as 5000 ng / mL, the luminescence value of the test result is still higher than the linear upper limit, indicating that the anti-HOOK ability of this kit meets the use requirements.

[0167] Embodiment 10

[0168] Performance test of the detection kit of the present invention

[0169] (1) Detection limit

[0170] The detection method was tested with reference to the test method of YY / T 1588-2018 "Procalcitonin Assay Kit" issued by the State Food and Drug Administration. The test results are shown in Tables 10-12.

[0171] Table 10 Detection results of initial calibration curve

[0172] RLU Concentration (pg / mL) S0 0.00 241 S1 0.14 2817 S2 0.90 17346 S3 8.00 175630 S4 55.00 1343276 S5 120.00 2836233

[0173] Table 11 Test results of blank limit

[0174]

[0175] Table 12 Low value sample test results

[0176]

[0177] Blank limit test: Use blank (zero value corporate calibrator) as sample to test the kit, repeat the test 20 times, calculate the mean X1 of blank response and the standard deviation (SD) of blank response, X1 (mean of blank response) + 2SD (standard deviation of blank response) is the blank limit.

[0178] Use the company's calibrator to dilute 5 samples with a concentration close to the detection limit (0.02ng / mL) for testing. Each sample is tested 5 times. The test results are sorted by size. The results should meet the following requirements:

[0179] The number of test results less than the detection limit (0.02g / mL) should be less than or equal to three. Based on the Mean+2SD obtained from the 20-well blank sample (S0) test, 5 samples with a concentration close to the detection limit (0.02ng / mL) were diluted with the company's calibrator for testing. Each sample was tested 5 times, and none of the test results was greater than the blank limit result, proving that the detection limit of this product is no higher than 10pg / mL, which is of great significance for the diagnosis of infection.

[0180] (2) Precision testing within one day

[0181] The high and low standards were tested 10 times each, and the CV was calculated based on the test results. The results are shown in Table 13.

[0182] Table 13 Precision test results within 13 days

[0183] Number of tests QCL Signal QCH Signal QCL concentration QCH concentration 1 9996 199228 0.526124 9.338542 2 10613 206696 0.560239 9.658259 3 10754 212027 0.568043 9.886137 4 10601 203002 0.559575 9.500186 5 10103 216091 0.532037 10.059662 6 10686 217772 0.564279 10.131389 7 10733 215145 0.56688 10.019284 8 10959 211064 0.579392 9.844994 9 10415 198914 0.549286 9.325086 10 10722 199281 0.566271 9.340813 Mean 10558.2 207922 0.5572126 9.7104352 SD 302.0878459 7451.741303 0.016708459 0.318655586 CV 2.86％ 3.58％ 3.00％ 3.28％

[0184] The intra-day precision of the present invention can be maintained within 5%, which is of great significance for the diagnostic accuracy of infection.

[0185] (3) Detection of linear correlation

[0186] The detection method was tested with reference to the test method of YY / T 1588-2018 "Procalcitonin Assay Kit" issued by the State Food and Drug Administration.

[0187] Use high concentration samples close to the upper limit of the linear range (100ng / mL) and low concentration samples close to the lower limit of the linear range (0.02ng / mL), mix at least 5 concentrations in a certain ratio, repeat the test at least 2 times for each concentration, and calculate the mean value (yi) of the test results. Use the least squares method to fit the average value of the measured concentration and the theoretical concentration or dilution ratio, and calculate the correlation coefficient γ of the linear regression. The correlation coefficient γ is ≥ 0.990. The results are shown in Table 14 and Figure 2 .

[0188] The linear correlation R of the present invention is greater than 0.990. The theoretical concentration is the concentration calculated by the high and low sample concentration values, and the measured value is the concentration calculated by using the calibration curve. Figure 2 This indicates that the linear correlation is good.

[0189] Table 14 Detection results of linear correlation

[0190] Concentration 1 Concentration 2 Measured value Theoretical value Sample 1 0.00 0.00 0.00 0.00 Sample 2 0.03 0.03 0.03 0.02 Sample 3 0.16 0.15 0.15 0.10 Sample 4 0.68 0.71 0.70 0.48 Sample 5 3.07 3.38 3.22 2.38 Sample 6 11.82 13.30 12.56 11.88 Sample 7 55.13 58.91 57.02 59.38 Sample 8 111.27 119.02 115.14 118.76 Linear Dependence 0.9999

[0191] (4) Accuracy testing

[0192] The detection method was tested with reference to the test method of YY / T 1588-2018 "Procalcitonin Assay Kit" issued by the State Food and Drug Administration.

[0193] A high-level analyte (A) with a known concentration is added to a low-concentration serum (or other body fluid component) B. The volume ratio of the added analyte A to the serum (or other body fluid component) B is not greater than 1:9. The test is repeated 3 times and the average value is taken according to the formula:

[0194]

[0195] Calculate the recovery rate (where: R-recovery rate; C-average concentration after adding solution A to solution B; V0-volume of solution B; V S -A liquid volume; C0-the average value of B liquid concentration; C S -A solution concentration), the recovery rate should be in the range of 85% to 115%. The test results are shown in Table 15.

[0196] Table 15 Recovery test results

[0197]

[0198] The detection recovery results were within 85% to 115%, with good accuracy.

[0199] (5) Methodological comparison

[0200] After using the procalcitonin detection kit (electrochemiluminescence method) produced by Roche Diagnostics to detect part of the serum or plasma samples, the kit of the present invention was used to detect the above samples. The test results are shown in Figure 3 , Table 16. The kit of the present invention was used to detect 61 Roche samples, and the concentration values ​​(ng / mL) detected by the Roche kit were plotted against the concentration values ​​detected by the kit of the present invention. It was found that the coincidence rate was good, and the overall coincidence rate R value could reach 0.9943. It can be seen that the kit of the present invention has a good correlation with the Roche PCT kit recognized at home and abroad, and can more accurately screen out diseased individuals from healthy people.

[0201] Table 16 Roche test value compliance rate

[0202]

[0203]

[0204] The above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the embodiments here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A procalcitonin detection kit, characterized in that: The kit comprises: a first antibody coated on a magnetic microsphere, a second antibody marked with a tracer marker, and a calibrator; the specific binding sites of procalcitonin recognized by the first antibody and the second antibody are different; the calibrator comprises procalcitonin antigen A and a diluent, the procalcitonin antigen A is a sequence as shown in SEQ ID NO: 1, and the diluent comprises a buffer and a protein stabilizer; Optionally, the sequence of the amino acid site of procalcitonin recognized by the first antibody is the sequence shown in SEQ ID NO:4, and the sequence of the amino acid site of procalcitonin recognized by the second antibody is the sequence shown in SEQ ID NO:8; Optionally, the buffer is selected from HEPES buffer with a concentration of 0.03 to 0.06 M, and the protein stabilizer is selected from bovine serum albumin.

2. The procalcitonin detection kit according to claim 1, characterized in that The mass ratio of the magnetic microspheres to the first antibody is 100:1.5-2.

5.

3. The procalcitonin detection kit according to claim 1 or 2, characterized in that: The molar ratio of the tracer marker to the second antibody is not less than 15:

1.

4. The procalcitonin detection kit according to any one of claims 1 to 3, characterized in that The kit also includes a magnetic bead cleaning solution and substrate solution A and substrate solution B. Preferably, the magnetic microsphere cleaning solution contains PBST buffer, the substrate solution A is a mixture of H2O2 and HNO3, and the substrate solution B is a mixture of Triton X-100 and NaOH.

5. The procalcitonin detection kit according to any one of claims 1 to 4, characterized in that The tracer marker is selected from one or more of the group consisting of luminol, alkaline phosphatase, horseradish peroxidase, acridinium ester and adamantane; preferably, the tracer marker is acridinium ester.

6. The method for preparing the procalcitonin detection kit according to any one of claims 1 to 5, characterized in that: The preparation method comprises: Magnetic microsphere coating step: coating the first antibody on the magnetic microsphere; Tracing marker labeling step: labeling the tracing marker on the second antibody; Steps for preparing calibrators.

7. The preparation method according to claim 6, characterized in that: In the magnetic microsphere coating step, the coupling time between the first antibody and the magnetic microsphere is 1 to 1.5 hours; Optionally, the antibody coupling buffer used in the tracer marker labeling step is selected from HEPES buffer or carbonate buffer; preferably, the antibody coupling buffer is selected from CB buffer; Optionally, the labeling time of the second antibody labeled with the tracer marker is 1.5 to 3 hours; optionally, the labeling time of the second antibody labeled with the tracer marker is 2 hours.

8. The method according to claim 6 or 7, characterized in that: The step of preparing the calibration product comprises taking the working solution containing the procalcitonin antigen A of the present invention, diluting it with a HEPES solution containing 2-3% bovine serum albumin to obtain high and low point standard solutions, and then freeze-drying them.

9. The method according to any one of claims 6 to 8, characterized in that: 2-(N-morpholino)ethanesulfonic acid buffer was used in the first antibody coating magnetic microspheres step; Optionally, a blocking solution is also used in the step of coating the magnetic microspheres with the first antibody, and the blocking solution is a phosphate buffer containing casein; the concentration range of the blocking reagent is preferably 0.01 to 0.05 M, and the amount of the blocking reagent is 100 to 300 μL.

10. Use of the procalcitonin detection kit according to any one of claims 1 to 5 in the preparation of reagents for diagnosing infectious diseases.

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