S100-beta chemiluminescence detection kit and preparation method thereof

Abstract

This invention relates to the field of chemiluminescence detection technology for in vitro diagnostic reagents, specifically to an S100-β chemiluminescence detection kit and its preparation method. It provides a chemiluminescence detection kit for central nervous system-specific proteins using magnetic microparticles. The kit comprises: S100-β calibrator, S100-β monoclonal antibody conjugated with carboxyl magnetic beads, S100-β monoclonal antibody labeled with alkaline phosphatase, AMPPD chemiluminescence substrate solution acted upon by alkaline phosphatase, and concentrated washing buffer. This kit achieves the following: no sample pretreatment required, easy operation, high automation, high sensitivity, automatic result output in 16 minutes, good stability of calibrator and enzyme label, and low cost. It can meet the clinical needs for auxiliary diagnosis and treatment of acute ischemic brain injury.

Description

Technical Field

[0001] This invention relates to the field of chemiluminescence detection technology for in vitro diagnostic reagents, and particularly to an S100-β chemiluminescence detection kit and its preparation method. Background Technology

[0002] S100-β protein is the main and most active member of the S100 family. This class of proteins are acidic calcium-binding proteins, first isolated from bovine brain by Moore et al. They were named S100 proteins because they are soluble in 100% saturated ammonium sulfate solution under neutral pH conditions.

[0003] Chinese patent application CN113087782A discloses a lyophilization method for central nervous system-specific proteins. This lyophilization method can effectively preserve central nervous system-specific proteins and help maintain their maximum activity. However, manual reconstitution with liquid is required during use, which is prone to errors and batch-to-batch variations. CN109856403A discloses a time-resolved fluorescence immunochromatographic assay kit for S100-β protein. While this invention effectively eliminates interference from non-specific fluorescence, the preparation process is complex, the detection sensitivity is low, the linear range is narrow, and batch-to-batch variations are uncontrollable. CN110672849B discloses an S100 protein detection kit. This invention has a wide linear range and can achieve automated detection; however, the sample requires dilution pretreatment, results are available in 40 minutes, the detection time is long, and the initial sample dilution is cumbersome. CN113092786A discloses a buffer solution and its application in a central nervous system-specific protein detection kit. This invention has high sensitivity and can complete the detection in a short time. However, the design and development of this reagent kit uses a biotin and streptavidin system, which has a certain biotin interference effect. Moreover, the calibrator only maintains its activity for 6 months under 2-8℃ conditions, which is a short retention time. In addition, this invention is a single-use reagent, and the development cost of the reagent plus consumables is relatively high. Summary of the Invention

[0004] The purpose of this invention is to provide an S100-β chemiluminescence detection kit and its preparation method, aiming to solve the above-mentioned problems and provide a chemiluminescence detection kit and its preparation method for central nervous system specific proteins that is simple to detect, requires no sample pretreatment, is easy to operate, has a high degree of automation, high sensitivity, automatically produces results in 16 minutes, has good stability of calibrators and enzyme markers, and is low in cost. It can meet the clinical auxiliary diagnosis and treatment of acute ischemic brain injury.

[0005] To achieve the above objectives, the present invention provides an S100-β chemiluminescence detection kit and its preparation method, wherein the kit comprises: S100-β calibrator, S100-β monoclonal antibody conjugated with carboxyl magnetic beads, S100-β monoclonal antibody labeled with alkaline phosphatase, AMPPD chemiluminescence substrate solution acted upon by alkaline phosphatase, and concentrated washing buffer.

[0006] The S100-β calibrator can be prepared by the following method.

[0007] (1) Prepare S100-β calibrator buffer, comprising a buffer containing salts, animal protein, nonionic surfactant, 4-aminoantipyrine, sugars, reducing agent, polymer and preservative.

[0008] (2) Buffer solutions can, to some extent, offset and mitigate the effects of strong acids or bases on the pH of the solution, thus maintaining a relatively stable pH. Salts in the buffer solution are also crucial for maintaining the stability of the protein solution because the charge shielding of ions in the solution reduces the charge-charge repulsion between protein molecules. Animal protein plays a role in reducing the background of the kit and improving stability, while the addition of 4-aminoantipyrine and sugars further enhances stability. Nonionic surfactants mainly interact with proteins through hydrophobic forces; the interaction between their hydrophobic chains and the hydrophobic groups of the protein can have a certain impact on the structure and function of both the surfactant and the protein. Reducing agents can counteract the damage of oxidants to protein thiol groups. Polymer compounds can adsorb onto the protein surface to form a polymeric surface protective film, which can improve protein stability. Preservatives can inhibit the growth of bacteria and microorganisms and also improve protein stability.

[0009] (3) Buffer solutions can be the TRIS system, with a pH between 7 and 8 and a concentration of 0.01-0.05 mol / L. Salts can be potassium chloride or sodium chloride, with a concentration of 150 mM-300 mM. Animal proteins can be bovine serum albumin or casein, with a concentration of 10 g / L-20 g / L. Nonionic surfactants can be fatty acid monoglycerides, glyceryl monostearate, or polysorbate (Tween-type), with a volume concentration of 0.1%-0.5%. 4-Aminoantipyrine 0.01-0.05 mg / mL. Sugars can be sucrose, D-trehalose, or pullulan, with a concentration of 0.04-0.08 mg / mL, and reduced glutathione 0.5%-1%. Polymers can be PEG20000, with a concentration of 10 g / L-30 g / L. Preservatives can be ProClin950 or KV300 series, with a volume concentration of 1 ml / L-3 ml / L.

[0010] (4) Prepare S100-β calibrators with concentrations of 60 ng / ml, 40 ng / ml, 20 ng / ml, 5 ng / ml, 0.5 ng / ml and 0.1 ng / ml using calibrator dilution buffer.

[0011] 2. The detection kit prepared in this invention preferably uses magnetic beads with a particle size of 1μm-3μm; the magnetic beads have an iron oxide core and a polystyrene surface coated with a polymer containing carboxyl active groups. The S100-β monoclonal antibody conjugated to carboxyl magnetic beads can be prepared by the following method.

[0012] (1) First, prepare the buffer solution for coupling, which mainly includes: coupling solution: 100mM MES pH4.8; blocking solution: 3M ethanolamine, 1% BSA, 0.5% T-20, pH8.0; preservation solution: 0.05% proclin-300 PBS buffer.

[0013] (2) First, mix the magnetic beads with a vortex mixer. The amount of magnetic beads used depends on the required amount. Place them in an EP tube, add coupling solution to make the final concentration of the magnetic beads 5 mg / mL, mix with a vortex mixer, then place them on a magnetic separation rack to enrich the magnetic beads, remove the supernatant, and then add coupling solution to repeat the washing once.

[0014] (3) Calculate the amount of EDC and NHS used. The final concentration of EDC and NHS is 1.5 mg / mL, and the final concentration of magnetic beads is 5 mg / mL. First, add the coupling solution and vortex to mix. Then add EDC and NHS and vortex to mix again. Finally, place the mixture in a 37°C environment and rotate to mix for 30 min.

[0015] (4) After the magnetic beads are activated, place them on a magnetic separator, remove the supernatant, and calculate the required antibody volume. First, add the coupling solution and vortex to mix. Then add the antibody at a concentration of 2 mg / mL to 5 mg / mL. Vortex again to mix, and then incubate at 37°C for 1-2 hours. In this coupling reaction, the ratio of antibody to magnetic beads is 1:50 to 1:150, and the final concentration of magnetic beads is 5 mg / mL.

[0016] (5) After the coupling reaction is completed, place the EP tube on the magnetic separation rack, remove the supernatant, add the blocking solution, and at this time, the final concentration of the magnetic beads is 5 mg / mL. Also place it in a 37°C environment and rotate to mix for 1-2 hours.

[0017] (6) After the blocking is completed, the coupling solution is washed three times. Finally, the magnetic beads are stored in the preservation solution. At this time, the working concentration of S100-β monoclonal antibody coupled with carboxyl magnetic beads is 0.5-2ug / ml.

[0018] 3. Alkaline phosphatase-labeled S100-β monoclonal antibodies can be prepared by the following method.

[0019] (1) Dissolve and dilute the heteroterminated bifunctional crosslinking agent with DMF, and react it with S100-β antibody diluted with buffer A at a molar ratio of 1:10 to 1:60 at a temperature of 20 to 37°C in the dark for 0.5 to 1 h.

[0020] (2) Dissolve and dilute Traut's Reagent with buffer B, and react it with ALP diluted with buffer B at a molar ratio of 1:10 to 1:60 at a temperature of 20 to 37°C in the dark for 0.5 to 1 hour.

[0021] (3) Use an ultrafiltration tube with a molecular cutoff of less than 40 kD to desalt the reaction solutions of (1) and (2) 2-3 times. After desalting, react the reaction solutions of (1) and (2) at a mass ratio of 1:0.5 to 1:2 in the dark for 1-4 hours at a temperature of 20-37°C.

[0022] (4) The ultrafiltration tube with a molecular weight cutoff of less than 40kD is used to desalt the reaction solution in (3) 2-3 times to obtain alkaline phosphatase-labeled S100-β monoclonal antibody. Finally, the antibody is stored in a preservation solution. The working concentration of alkaline phosphatase-labeled monoclonal antibody is 0.2-2ug / ml.

[0023] The labeling buffer A consists of 10 mM phosphate buffer, 9 g / L sodium chloride, and a pH value within the range of 7.40 ± 0.05; the labeling buffer B consists of 100 mM phosphate buffer, 9 g / L sodium chloride, 10 mM disodium ethylenediaminetetraacetate, and a pH value within the range of 8.00 ± 0.05.

[0024] The enzyme-labeled dilution solution consists of 10–50 mM TRIS buffer, 4.5–18 g / L sodium chloride, 5–20 g / L sugar, 10–20 g / L protein, 0.01–0.5 mM zinc ions, 0.1–10 mM magnesium ions, 5–30 g / L PEG2000, 1–10 g / L surfactant, 10 mL / L–30 mL / L glycerol, and 1–5 g / L preservative, with a pH range of 6.0–8.0.

[0025] The chemiluminescent substrate solution used by alkaline phosphatase in this invention mainly consists of AMPPD, a fluorescent agent, and a buffer solution containing a surfactant. The provided washing solution is a concentrated solution, which saves production costs and facilitates transportation. This concentrated washing solution consists of 100 mM TRIS buffer, 100 g / L sodium chloride, 10 g / L Tween-20, 1 mL / L Triton X-100, and 10 mL / L Proclin-300.

[0026] The chemiluminescence detection kit for central nervous system-specific proteins described in this invention, used with the Guilin Youlite IA260 fully automated chemiluminescence immunoassay analyzer, has a detection range of 0.02 ng / mL to 60 ng / mL and provides results in 16 minutes. The sample detection procedure is as follows: Step 1: Add 15 μL of sample, 50 μL of S100-β-coated carboxyl magnetic beads, and 50 μL of alkaline phosphatase-labeled S100-β monoclonal antibody to a reaction tube. After incubation, the S100-β in the sample binds to the S100-β antibody immobilized on the magnetic beads. Simultaneously, the S100-β antibody-alkaline phosphate conjugate binds to another site of S100-β in the sample. React at 37°C for 10 minutes, and the magnetic field absorbs the... The first step involves attaching magnetic beads and washing away unbound substances. The second step involves adding a chemiluminescent substrate to the reaction tube. The luminescent substrate (3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoryl)-phenyl-1,2-dioxane, AMPPD) is decomposed by alkaline phosphatase, losing a phosphate group to generate an unstable intermediate. This intermediate generates a methyl m-oxobenzoate anion through intramolecular electron transfer. When the methyl m-oxobenzoate anion in the excited state returns to the ground state, chemiluminescence is generated. The number of photons generated in the reaction is then measured using a photomultiplier tube. The number of photons generated is proportional to the concentration of S100-β in the sample. The amount of analyte in the sample is determined by the calibration curve. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 This is a flowchart of an S100-β chemiluminescence detection kit and its preparation method provided by the present invention. Detailed Implementation

[0029] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0030] Please see Figure 1 This invention provides an S100-β chemiluminescence detection kit and its preparation method, comprising the following steps:

[0031] S1 prepares S100-β calibrator buffer;

[0032] The specific method for preparing the S100-β calibrator buffer is as follows:

[0033] S101 was supplemented with TIRS buffer 100 mmol / L, sodium chloride concentration 150 mM, Prolin 300 0.1%, bovine serum albumin 10 g / L, Tween-20 0.2%, 4-aminoantipyrine 0.02 mg / mL, D-trehalose concentration 0.05 mg / mL, reduced glutathione 0.5%, PEG20000 concentration 15 g / L, and preservative ProClin 950 volume concentration 3 ml / L.

[0034] S2 prepared S100-β recombinant protein into S100-β calibrators with concentrations of 60 ng / ml, 40 ng / ml, 20 ng / ml, 5 ng / ml, 0.5 ng / ml, and 0.1 ng / ml using calibrator dilution buffer;

[0035] S3 was used to prepare S100-β monoclonal antibodies conjugated with carboxyl magnetic beads;

[0036] The specific method for preparing the S100-β monoclonal antibody conjugated with carboxyl magnetic beads is as follows:

[0037] S301 prepared the buffer solution for coupling: Coupling solution: 100mM MES pH 4.8; Blocking solution: 3M ethanolamine, 1% BSA, 0.5% T-20, pH 8.0; Preservation solution: 0.05% proclin-300 PBS buffer.

[0038] S302 Mix 1μm magnetic beads with a vortex mixer. Take the amount of magnetic beads as needed, place them in an EP tube, add coupling solution to make the final concentration of magnetic beads 5mg / mL, mix with a vortex mixer, then place on a magnetic separation rack to enrich the magnetic beads, remove the supernatant, and then add coupling solution again to repeat the washing once.

[0039] The amounts of EDC and NHS used in the S303 calculation were both 1.5 mg / mL, and the final concentration of the magnetic beads was 5 mg / mL. First, the coupling solution was added and vortexed to mix. Then, EDC and NHS were added, and the mixture was vortexed again to mix. Finally, the mixture was placed in a 37°C environment and rotated to mix for 30 min.

[0040] After activation of the S304 magnetic beads, they were placed on a magnetic separator to remove the supernatant, and the required antibody volume was calculated. The coupling buffer was added first, and the mixture was vortexed to mix. Then, the antibody was added at a concentration of 2 mg / mL. The mixture was vortexed again to mix, and then incubated at 37°C for 1 hour. In this coupling reaction, the antibody-to-magnetic-bead ratio was 1:50, and the final concentration of the magnetic beads was 5 mg / mL.

[0041] After the S305 coupling reaction is completed, the EP tube is placed on a magnetic separation rack, the supernatant is removed, and a blocking solution is added. At this point, the final concentration of the magnetic beads is 5 mg / mL. The tube is then placed in a 37°C environment and rotated to mix for 1 hour.

[0042] After S306 blocking, the conjugation solution was added and the washing was repeated three times. Finally, the magnetic beads were stored in the preservation solution. At this time, the working concentration of S100-β monoclonal antibody conjugated with carboxyl magnetic beads was 1ug / ml.

[0043] S4 was used to prepare alkaline phosphatase-labeled S100-β monoclonal antibody;

[0044] The specific method for preparing the alkaline phosphatase-labeled S100-β monoclonal antibody is as follows:

[0045] S401 was dissolved and diluted with DMF to form an heteroterminated bifunctional cross-linking agent, and then reacted with S100-β antibody diluted with buffer A at a molar ratio of 1:20 at 20–37°C in the dark for 1 hour.

[0046] S402 was dissolved and diluted with buffer B to dissolve Traut's Reagent, and reacted with ALP diluted with buffer B at a molar ratio of 1:10 at a temperature of 20-37°C in the dark for 0.5 h.

[0047] S403 was dissolved and diluted with buffer B to dissolve Traut's Reagent, and reacted with ALP diluted with buffer B at a molar ratio of 1:10 at a temperature of 20-37°C in the dark for 0.5 h.

[0048] The S404 ultrafiltration tube with a molecular cutoff of less than 40kD desalted the reaction solution (3) three times to obtain alkaline phosphatase-labeled S100-β monoclonal antibody. Finally, the antibody was stored in a preservation solution. The working concentration of the alkaline phosphatase-labeled monoclonal antibody was 0.5ug / ml.

[0049] The S405 enzyme-labeled dilution solution consists of 50 mM TRIS buffer, 9 g / L sodium chloride, 10 g / L sucrose, 15 g / L casein, 0.01 mM zinc ions, 5 mM magnesium ions, 10 g / L PEG 20000, 2 g / L Triton X-100, 10 mL / L glycerol, 1 mL / L proclin-300, and a pH of 7.4.

[0050] S5 was used to prepare the AMPPD chemiluminescent substrate solution and concentrated wash solution for alkaline phosphatase.

[0051] The specific method for preparing the AMPPD chemiluminescent substrate solution and concentrated washing solution acted upon by the alkaline phosphatase is as follows:

[0052] The chemiluminescent substrate solution used by S501 alkaline phosphatase mainly consists of AMPPD, a fluorescent agent, and a buffer solution containing a surfactant. The provided washing solution is a concentrated washing solution, which consists of 100 mM TRIS buffer, 100 g / L sodium chloride, 10 g / L Tween-20, 1 mL / L Triton X-100, and 10 mL / L Proclin-300.

[0053] The kit of Example 1 is composed of the S100-β calibrator prepared above, the S100-β monoclonal antibody coupled with carboxyl magnetic beads, the S100-β monoclonal antibody labeled with alkaline phosphatase, the AMPPD chemiluminescent substrate solution acted on by alkaline phosphatase, and the concentrated washing solution.

[0054] S6 prepares different types of detection kits by changing the parameters of a certain type of specified substance as a comparison item, and then completes the performance test;

[0055] Specifically, in Example 2, the magnetic beads with a particle size of 2.7 μm were preferred, and the remaining steps were the same as those in Example 1.

[0056] Example 3: Alkaline phosphatase-labeled S100-β monoclonal antibody. The antibody was then stored in a preservation solution. The working concentration of the alkaline phosphatase-labeled monoclonal antibody was 1 μg / ml. The kits used in Example 1 were identical.

[0057] Comparative Example 1: Using an S100-β calibrator buffer with a different formulation than that in the kit of Example 1, the calibrator buffer formulation for Comparative Example 1 was as follows: TIRS buffer 100 mmol / L, pH 7.4, sodium chloride concentration 150 mM, Prolin 300 0.1%, bovine serum albumin 10 g / L, Tween-20 0.2%, reduced glutathione 0.5%, PEG 20000 at a concentration of 15 g / L, and ProClin 950 preservative at a volume concentration of 3 ml / L. All other steps were the same as those in the kit of Example 1.

[0058] Comparative Example 2: Using a different formulation of the S100-β calibrator buffer than that in the kit of Example 1, the calibrator buffer of Comparative Example 2 was formulated as follows: TIRS buffer 100 mmol / L, pH 7.4, sodium chloride concentration 150 mM, Prolin 300 0.1%, bovine serum albumin 10 g / L, Tween-20 0.2%, 4-aminoantipyrine 0.02 mg / mL, D-trehalose concentration 0.05 mg / mL, reduced glutathione 0.5%, and preservative ProClin 950 volume concentration 3 mL / L. The remaining steps were the same as those in the kit of Example 1.

[0059] Comparative Example 3: In the preparation of S100-β monoclonal antibody conjugated with carboxyl magnetic beads, no blocking agent was added after the conjugation reaction was completed. The remaining steps were the same as those in the kit of Example 1.

[0060] Comparative Example 4: By using an S100-β enzyme-labeled diluent with a different formulation than that in the kit of Example 1, the formulation of the S100-β enzyme-labeled diluent in Comparative Example 4 was: TRIS buffer 50 mM, sodium chloride 9 g / L, sucrose 10 g / L, casein 15 g / L, zinc ions 0.01 mM, magnesium ions 5 mM, PEG 20000 10 g / L, Triton X-100 2 g / L, proclin-300 1 mL / L, pH 7.4, and the remaining steps were the same as those in the kit of Example 1.

[0061] Finally, the performance of the S100-β magnetic microparticle chemiluminescent immunoassay kits from each example and comparative example was tested.

[0062] 1. Stability test of S100-β standard product

[0063] S100-β recombinant protein was prepared into S100-β calibrators with concentrations of 60 ng / ml, 40 ng / ml, 20 ng / ml, 5 ng / ml, 0.5 ng / ml, and 0.1 ng / ml using different calibrator buffers. Each calibrator was divided in half and incubated at 2-8℃ / 37℃ for 7 days. Detection was then performed using the Guilin Ulite Central Nervous System Specific Protein Chemiluminescence Detection Kit (Magnetic Microparticle Chemiluminescence) on the Guilin Ulite IA260 Fully Automated Chemiluminescence Immunoassay Analyzer.

[0064] Table 1: Signal retention rate of the S100-β standard prepared in Example 1 after storage at 2-8℃ / 37℃ for 7 days.

[0065]

[0066] Table 2: Signal retention rate of the S100-β standard prepared in Comparative Example 1 after storage at 2-8℃ / 37℃ for 7 days.

[0067]

[0068] Table 3: Signal retention rate of the S100-β standard prepared in Comparative Example 2 after storage at 2-8℃ / 37℃ for 7 days.

[0069]

[0070]

[0071] S100-β recombinant protein was prepared into S100-β calibrators with concentrations of 60 ng / ml, 40 ng / ml, 20 ng / ml, 5 ng / ml, 0.5 ng / ml, and 0.1 ng / ml using different calibrator buffers. These calibrators were stored at 2℃~8℃ for 12 months and then detected using the Guilin Ulite Central Nervous System Specific Protein Chemiluminescence Detection Kit (Magnetic Microparticle Chemiluminescence) on the Guilin Ulite IA260 Fully Automated Chemiluminescence Immunoassay Analyzer.

[0072] Table 4-1: Long-term stability monitoring of Example 1

[0073]

[0074] Table 4-2: Stability in Example 1

[0075]

[0076] Table 5-1: Long-term stable monitoring of Comparative Example 1

[0077]

[0078] Table 5-2: Stability in Comparative Example 1

[0079]

[0080] Table 6-1: Long-term stable monitoring of Comparative Example 2

[0081]

[0082] Table 6-2: Stability in Comparative Example 2

[0083]

[0084]

[0085] 2. S100-β valence test

[0086] A chemiluminescence assay kit for central nervous system-specific proteins (magnetic microparticle chemiluminescence) was prepared using different methods for preparing S100-β monoclonal antibodies conjugated with carboxyl magnetic beads, and the potency of S100-β samples was tested.

[0087] Table 7: S100-β valence test

[0088]

[0089] 3. Clinical comparison with benchmark companies: The S100-β magnetic particle chemiluminescence detection kits prepared in Examples 1 and 3 were used to test 40 clinical S100-β samples, and the correlation coefficient r with Roche's clinical results was calculated.

[0090] Table 8: Clinical Testing (Unit: ng / ml)

[0091]

[0092]

[0093] 4. Accelerated stability test of S100-β-ALP at 37℃ for 7 days

[0094] The S100-β-ALP obtained from the alkaline phosphatase-labeled S100-β monoclonal antibody provided in this invention was diluted to a working solution of 0.5 μg / mL using different enzyme-labeled dilution buffers and placed in a 37°C oven for accelerated testing for 7 days. A control was set at 4°C. The same group of samples was analyzed using a kit containing the S100-β monoclonal antibody working solution conjugated with carboxyl magnetic beads produced by our company, and the analysis was performed on a Guilin Unitech IA260 fully automated chemiluminescence immunoassay analyzer.

[0095] Table 9: Signal retention rate of S100-β-ALP after storage at 2-8℃ / 37℃ for 7 days in Example 1

[0096]

[0097] Table 10: Signal retention rate of S100-β-ALP in Comparative Example 4 after storage at 2-8℃ / 37℃ for 7 days

[0098]

[0099] Comparison of test results:

[0100] In the stability test comparison of S100-β standard:

[0101] As shown in Tables 1-3, the signal retention rate of the S100-β standard prepared in Example 1 after storage at 2-8℃ / 37℃ for 7 days is >90%, while the signal retention rate of the S100-β standard prepared in Comparative Examples 1 and 3 after storage at 2-8℃ / 37℃ for 7 days is <90%. Therefore, Example 1 is the optimal solution.

[0102] As shown in Table 4-1-6-2, the S100-β standard prepared in Example 1 maintained a signal retention rate >90% after being stored at 2℃~8℃ for 12 months, while the S100-β standards prepared in Comparative Examples 1 and 2 maintained a signal retention rate <90% after being stored at 2℃~8℃ for 12 months. The S100-β standard does not require lyophilization; its stability remains good in liquid form, and Example 1 is the optimal solution.

[0103] In the S100-β valence test:

[0104] As shown in the table above, the signal-to-noise ratio (SNR) of Example 1 is 10.82 and that of Example 2 is 10.91, both of which are greater than that of Comparative Example 3. The photon quantity gradient ratios of Examples 1 and 2 are both greater than the concentration gradient ratios, while the photon quantity gradient ratio of Comparative Example 3 is less than the concentration gradient ratio. This indicates that in the kit prepared by this invention, the magnetic bead particle size can preferably be selected from 1μm to 3μm. In the preparation of S100-β monoclonal antibody conjugated with carboxyl magnetic beads, blocking treatment after the conjugation reaction can reduce the background and improve the low-end sensitivity.

[0105] 3. In clinical comparative testing with benchmark companies:

[0106] As shown in Table 8, in the central nervous system-specific protein chemiluminescence detection kit (magnetic microparticle chemiluminescence) prepared in this invention, the working concentrations of alkaline phosphatase-labeled monoclonal antibodies of 0.5 ug / ml and 1 ug / ml do not affect clinical relevance. The results of the two detection samples are basically consistent with those of Roche's products, showing good correlation, indicating that the detection kit of this invention has high detection accuracy.

[0107] 4. S100-β-ALP's stability in a 7-day accelerated test at 37℃:

[0108] As shown in Tables 9-10, in Example 1, the S100-β enzyme-labeled diluent provided by the present invention improves the 7-day accelerated stability of ALP-labeled antibodies to over 95%.

[0109] This invention provides a preservation solution for S100-β recombinant protein, which is stable for 7 days at 37℃ and 12 months at 2-8℃. It also provides a carboxyl magnetic bead-conjugated S100-β monoclonal antibody, avoiding interference from biotin in the sample and exhibiting high sensitivity. Furthermore, it provides a method for labeling S100-β antibodies and a formulation for enzyme-labeled dilution, improving reaction titer and reducing production costs. The invention increases the 7-day accelerated stability of ALP-labeled S100-β antibodies to over 95%, and the correlation coefficient R² with clinical samples from benchmark company Roche is greater than 0.975, indicating that this reagent has a good correlation with similar detection reagents in clinical test results.

[0110] The above description is merely a preferred embodiment of the S100-β chemiluminescence detection kit and its preparation method according to the present invention. It should not be construed as limiting the scope of the present invention. Those skilled in the art can understand that implementing all or part of the above embodiments and making equivalent changes in accordance with the claims of the present invention are still within the scope of the invention.

Claims

1. A method for preparing an S100-β chemiluminescence detection kit, characterized in that, Includes the following steps: Prepare S100-β calibrator buffer; Prepare S100-β calibrators with concentrations of 60 ng / ml, 40 ng / ml, 20 ng / ml, 5 ng / ml, 0.5 ng / ml, and 0.1 ng / ml using calibrator buffer. Preparation of S100-β monoclonal antibody conjugated with carboxyl magnetic beads; Preparation of alkaline phosphatase-labeled S100-β monoclonal antibody; Preparation of AMPPD chemiluminescent substrate solution and concentrated washing solution for alkaline phosphatase action; Different types of test kits were prepared by changing the parameters of a specific type of substance as a comparison, and then performance testing was performed. The specific method for preparing the S100-β calibrator buffer is as follows: Add 100 mmol / L TIRS buffer, 150 mM sodium chloride, 0.1% Prolin 300, 10 g / L bovine serum albumin, 0.2% Tween-20, 0.02 mg / mL 4-aminoantipyrine, 0.05 mg / mL D-trehalose, 0.5% reduced glutathione, 15 g / L PEG20000, and 3 mL / L ProClin 950 preservative.

2. The method for preparing an S100-β chemiluminescence detection kit as described in claim 1, characterized in that, The specific method for preparing the S100-β monoclonal antibody conjugated with carboxyl magnetic beads is as follows: Prepare the buffer solution for coupling: 100mM MES pH 4.8; Blocking solution: 3M ethanolamine, 1% BSA, 0.5% T-20, pH 8.0; Preservation solution: 0.05% proclin-300 in PBS buffer. Mix the 1μm magnetic beads with a vortex mixer. Take the amount of magnetic beads as needed and place them in an EP tube. Add the coupling solution to make the final concentration of the magnetic beads 5mg / mL. Mix with a vortex mixer and then place the tube on a magnetic separator to enrich the magnetic beads. Remove the supernatant and then add the coupling solution again to repeat the washing process. Calculate the amount of EDC and NHS used. The final concentration of EDC and NHS is 1.5 mg / mL, and the final concentration of magnetic beads is 5 mg / mL. First, add the coupling solution and vortex to mix. Then add EDC and NHS and vortex to mix again. Finally, place the mixture in a 37°C environment and rotate to mix for 30 min. After the magnetic beads are activated, they are placed on a magnetic separator, the supernatant is removed, and the required volume of antibody is calculated. First, the coupling solution is added and vortexed to mix. Then, the antibody is added at a concentration of 2 mg / mL. The mixture is vortexed again and then placed in a 37°C environment for 1 hour to rotate and mix. In this coupling reaction, the ratio of antibody to magnetic beads is 1:50, and the final concentration of magnetic beads is 5 mg / mL. After the coupling reaction is complete, place the EP tube on a magnetic separation rack, remove the supernatant, add the blocking solution, and at this time, the final concentration of the magnetic beads is 5 mg / mL. Also place it in a 37°C environment and rotate to mix for 1 h. After blocking, the conjugation solution was added and the washing was repeated three times. Finally, the magnetic beads were stored in the preservation solution. At this time, the working concentration of the S100-β monoclonal antibody conjugated with the carboxyl magnetic beads was 1 ug / ml.

3. The method for preparing an S100-β chemiluminescence detection kit as described in claim 2, characterized in that, The specific method for preparing the alkaline phosphatase-labeled S100-β monoclonal antibody is as follows: (1) Dissolve and dilute the heteroterminated bifunctional crosslinking agent with DMF, and react it with S100-β antibody diluted with buffer A at a molar ratio of 1:20 at a temperature of 20-37℃ in the dark for 1 h. Buffer A consists of 10 mM phosphate buffer and 9 g / L sodium chloride, with a pH value in the range of 7.40±0.

05. (2) Dissolve and dilute Traut's Reagent with buffer B, and react it with ALP diluted with buffer B at a molar ratio of 1:10 at a temperature of 20-37℃ in the dark for 0.5h. Buffer B consists of 100mM phosphate buffer, 9g / L sodium chloride, and 10mM disodium ethylenediaminetetraacetate, with a pH value in the range of 8.00±0.

05. (3) Use an ultrafiltration tube with a molecular cutoff of less than 40kD to desalt the reaction solution of steps (1) and (2) 2-3 times. Then, react the desalted reaction solution of steps (1) and (2) at a mass ratio of 1:0.5 to 1:2 in the dark for 1-4 hours at a temperature of 20-37℃. (4) The reaction solution in step (3) is desalted three times using an ultrafiltration tube with a molecular weight cutoff of less than 40 kD to obtain alkaline phosphatase-labeled S100-β monoclonal antibody. Finally, the antibody is stored in a preservation solution. The working concentration of the alkaline phosphatase-labeled monoclonal antibody is 0.5 ug / ml. The alkaline phosphatase-labeled monoclonal antibody is diluted to a working solution of 0.5 ug / ml using enzyme-labeled dilution buffer. The enzyme-labeled dilution solution consisted of 50 mM TRIS buffer, 9 g / L sodium chloride, 10 g / L sucrose, 15 g / L casein, 0.01 mM zinc ions, 5 mM magnesium ions, 10 g / L PEG20000, 2 g / L Triton X-100, 10 mL / L glycerol, and 1 mL / L proclin-300, with a pH of 7.

4.

4. The method for preparing an S100-β chemiluminescence detection kit as described in claim 3, characterized in that, The specific method for preparing the AMPPD chemiluminescent substrate solution and concentrated washing solution acted upon by the alkaline phosphatase is as follows: The chemiluminescent substrate solution used by alkaline phosphatase mainly consists of AMPPD, a fluorescent agent, and a buffer solution containing a surfactant. The provided washing solution is a concentrated washing solution, which consists of 100 mM TRIS buffer, 100 g / L sodium chloride, 10 g / L Tween-20, 1 mL / L Triton X-100, and 10 mL / L Proclin-300.

5. An S100-β chemiluminescence detection kit, prepared by the method for preparing the S100-β chemiluminescence detection kit according to any one of claims 1-4, characterized in that, The kit comprises: the S100-β calibrator, the S100-β monoclonal antibody conjugated with carboxyl magnetic beads, the S100-β monoclonal antibody labeled with alkaline phosphatase, the AMPPD chemiluminescent substrate solution acted upon by alkaline phosphatase, and concentrated wash buffer.

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