A magnetic bead diluent, magnetic microparticle reagent, and instant chemiluminescence assay kit.

Abstract

This application relates to the field of immunoassay, specifically disclosing a magnetic bead diluent, magnetic microparticle reagents, and a point-of-care chemiluminescence assay kit. The magnetic bead diluent comprises component A: Tris-HCl buffer, glycerol, and preservative, with the total volume of the magnetic bead diluent being 100%. The volume percentage of glycerol is 10-40%, and the volume percentage of the preservative is 0.08-0.2%. Component B comprises sodium chloride, gelatin, and a non-reducing disaccharide. The concentration of sodium chloride in the magnetic bead diluent is 8-100 g / L, the concentration of gelatin is 3-20 g / L, and the concentration of the non-reducing disaccharide is 3-10 g / L. The magnetic bead diluent of this application can be used to dilute antigens or antibodies coupled to magnetic beads and for chemiluminescence detection, offering advantages such as minimal impact from handling and transportation and high accuracy.

Description

Technical Field

[0001] This application relates to the field of immunoassay, specifically to a magnetic bead diluent, a magnetic microparticle reagent, and a point-of-care chemiluminescence assay kit. Background Technology

[0002] Magnetic particle chemiluminescence immunoassay originated in the 1970s and has since developed into a mature, highly sensitive, and highly specific immunoassay technique. Its detection sensitivity and accuracy are significantly superior to enzyme-linked immunosorbent assays (ELISA) and fluorescence chromatography, and it can completely replace radioimmunoassay. Its advantages, including high sensitivity, strong specificity, long reagent shelf life, fast detection speed, wide detection range, simple operation, and ease of automation, have made it widely recognized by clinical laboratory personnel.

[0003] Currently, most large hospitals use large-scale, fully automated chemiluminescence immunoassay (CCI) devices for clinical immunoassay. During testing, multiple tests or multiple reagent components for the same test are involved. Sharing the same sample needle with different components can lead to incomplete cleaning and cross-contamination, affecting testing efficiency. Therefore, the demand for small-scale CCI devices, also known as point-of-care testing (POCT) CCI devices, and their matching reagent kits is increasing. Small-scale magnetic particle CCI instruments are equipped with single-use reagent strips (one strip per sample). The wells of the strips contain magnetic particles, reaction reagents, and washing solution to meet the needs of point-of-care sample testing in primary hospitals or departments.

[0004] Most single-use chemiluminescence reagents pre-package the required reagent components for immunoassay into the wells of the reagent strip, such as magnetic microparticles (conjugated antigens or antibodies), labeled antibodies or antigens, sample diluent, working wash solution, luminescent substrate solution, or luminescent trigger solution. Because magnetic microparticles tend to settle over long periods, and single-use chemiluminescence reagent strips are not suitable for agitation to fully suspend the magnetic microparticles before sample addition, reagent manufacturers often use a tip to aspirate other components (such as the sample or diluted sample, labeled antibodies or antigens) and add them to the wells of the magnetic microparticles for the immunoassay reaction. Components not involved in the immunoassay reaction are then washed away using magnetic separation.

[0005] Because the magnetic particles involved in the immune reaction are small (50-100uL), the magnetic beads may stick to the tube wall or the upper aluminum foil seal during the transportation of the test strips due to activities such as handling, loading or unloading. This can result in different numbers of magnetic particles involved in the immune reaction in the sample test, causing inconsistent test results even for the same sample. This manifests as the reagent repeatability or batch-to-batch difference failing to meet the product's technical requirements, leading to misdiagnosis and delays in early diagnosis or treatment for patients. Summary of the Invention

[0006] To improve detection precision, this application provides a magnetic bead diluent, magnetic microparticle reagent, and a point-of-care chemiluminescence reagent kit.

[0007] Firstly, this application provides a magnetic bead diluent, employing the following technical solution:

[0008] A magnetic bead diluent, comprising,

[0009] Component A: Tris-HCl buffer, glycerol, preservative.

[0010] Based on the total volume of the magnetic bead diluent as 100%, the volume percentage of glycerol is 10-40%, and the volume percentage of the preservative is 0.08-0.2%.

[0011] Component B: Sodium chloride, gelatin, non-reducing disaccharides.

[0012] The concentration of sodium chloride in the magnetic bead diluent is 8-100 g / L, the concentration of gelatin in the magnetic bead diluent is 3-20 g / L, and the concentration of non-reducing disaccharide in the magnetic bead diluent is 3-10 g / L.

[0013] Preferably, a magnetic bead diluent includes,

[0014] Component A: Tris-HCl buffer, glycerol, preservative.

[0015] Based on the total volume of the magnetic bead diluent as 100%, the volume percentage of glycerol is 10-30%, and the volume percentage of the preservative is 0.1-0.2%.

[0016] Component B: Sodium chloride, gelatin, non-reducing disaccharides.

[0017] The concentration of sodium chloride in the magnetic bead diluent is 9-100 g / L, the concentration of gelatin in the magnetic bead diluent is 5-20 g / L, and the concentration of the non-reducing disaccharide in the magnetic bead diluent is 5-10 g / L.

[0018] To replenish the volume to 100% using Tris-HCl buffer.

[0019] The addition of sodium chloride, glycerol, gelatin, and a non-reducing disaccharide establishes a magnetic bead diluent with high viscosity under storage conditions (2-8℃) and low viscosity at incubation temperature (37℃). Magnetic microparticle reagents prepared using this diluent have the advantage of high viscosity under storage or transportation conditions (2-8℃) and low viscosity at incubation temperature (37℃, the reaction temperature during sample detection). This reduces the risk of magnetic beads adhering to the upper tube wall or upper aluminum foil seal during transport due to handling, loading, or unloading, which could lead to reduced accuracy in sample detection due to varying numbers of magnetic microparticles participating in the immune reaction. Furthermore, this provides clinical clients with highly sensitive, specific, and precise point-of-care chemiluminescence reagents, avoiding misdiagnosis caused by low accuracy and preventing delays in early diagnosis or treatment.

[0020] Furthermore, by adjusting the amounts of sodium chloride, glycerol, gelatin, and non-reducing disaccharides, a magnetic bead diluent with a viscosity of not less than 4.6 Pa·S under storage conditions (2-8℃) and a viscosity of not more than 1.9 Pa·S under incubation temperature (37℃) was obtained.

[0021] Preferably, the concentration of sodium chloride in the magnetic bead diluent is 8-100 g / L;

[0022] In some embodiments of this application, the sodium chloride content in each 100 ml of magnetic bead diluent can be 0.8 g, 0.9 g, 1.0 g, 1.2 g, 2 g, 5 g, 8 g, or 10 g;

[0023] More preferably, the sodium chloride content is 0.9g per 100ml of magnetic bead diluent.

[0024] As the sodium chloride content increases, the viscosity of the magnetic bead diluent gradually increases, but excessively high ion concentrations may affect protein solubility and the immune response between antigens and antibodies.

[0025] Preferably, the glycerol accounts for 10-40% of the total volume of the magnetic bead diluent;

[0026] In some embodiments of this application, the glycerol content in each 100 ml of magnetic bead diluent is 10 ml, 20 ml, 30 ml, or 40 ml;

[0027] Preferably, the concentration of the gelatin in the magnetic bead diluent is 3-20 g / L;

[0028] In some embodiments of this application, the gelatin content in each 100 ml of magnetic bead diluent may be 0.3 g, 0.5 g, 1.0 g, or 2.0 g;

[0029] More preferably, the concentration of the gelatin in the magnetic bead diluent is 5 g / L.

[0030] The addition of gelatin and glycerin can increase the viscosity of the magnetic bead diluent, thus keeping the diluent at a high viscosity at low temperatures. This reduces the adhesion of magnetic beads to the upper tube wall or upper aluminum foil seal of the reagent strip during handling, allowing for more accurate acquisition of the required amount of magnetic beads during sampling, thereby improving the accuracy of the detection.

[0031] Preferably, the concentration of trehalose in the magnetic bead diluent is 3-10 g / L;

[0032] In some embodiments of this application, the trehalose content in each 100 ml of magnetic bead diluent may be 0.3 g, 0.5 g, 0.7 g, or 1.0 g.

[0033] More preferably, the concentration of trehalose in the magnetic bead diluent is 5 g / L.

[0034] Preferably, the concentration of the Tris-HCl buffer is 150-250 mM;

[0035] More preferably, the concentration of the Tris-HCl buffer is 200 mM.

[0036] Preferably, the pH of the Tris-HCl buffer solution is 7.5-8.5.

[0037] More preferably, the pH of the Tris-HCl buffer solution is 8.

[0038] In one embodiment of this application, the Tris-HCl buffer solution has a concentration of 200 mM and a pH of 8.

[0039] The preservative is Proclin 300.

[0040] The addition of preservatives extends the shelf life of the magnetic bead diluent and improves the stability of antigens or antibodies stored in the magnetic bead diluent.

[0041] A method for preparing a magnetic bead diluent includes the following steps:

[0042] Add Tris-HCl buffer, glycerol, and preservative to S1 according to the formula, and stir well.

[0043] S2 then adds sodium chloride, gelatin, and trehalose to the liquid obtained in S1, and stirs until homogeneous to obtain the magnetic bead dilution solution.

[0044] Secondly, this application provides a magnetic microparticle reagent, which adopts the following technical solution:

[0045] A magnetic microparticle reagent prepared using the above-described magnetic bead diluent, the magnetic microparticle reagent further comprising an antibody or antigen coupled with magnetic beads.

[0046] A magnetic microparticle reagent is prepared by dissolving antibodies or antigens coupled to magnetic beads in a magnetic bead diluent. The concentration of the antibodies or antigens coupled to the magnetic beads in the magnetic microparticle reagent is 0.1-10 μg / mL.

[0047] In several embodiments of this application, the concentration of the antibody or antigen coupled to the magnetic beads in the magnetic microparticle reagent can be 0.1 ug / mL, 0.2 ug / mL, 0.5 ug / mL, 1 ug / mL, 2 ug / mL, 5 ug / mL or 10 ug / mL.

[0048] After being diluted with magnetic bead diluent, the antibodies or antigens coupled with magnetic beads can be stored in the test strips. Furthermore, because the viscosity of the magnetic bead diluent is adjusted using sodium chloride, glycerin, gelatin, and trehalose, the adhesion of magnetic beads to the upper tube wall or upper aluminum foil seal of the test strip is reduced during handling, loading, and unloading. This improves the uniformity of the number of magnetic beads during sample testing and reduces the impact of random differences in the number of magnetic beads on the accuracy of the test strip results.

[0049] Preferably, the magnetic microparticle reagent has a magnetic bead concentration of 100-200 μg / mL and a MYO antibody concentration coupled to the magnetic beads of 0.2-10 μg / mL.

[0050] Thirdly, the technical solution adopted in this application for providing a point-of-care chemiluminescence assay kit is as follows:

[0051] A point-of-care chemiluminescence assay kit includes a reagent strip loaded with labeled antibody or antigen reagent and magnetic microparticle reagent;

[0052] The magnetic microparticle reagent used is the magnetic microparticle reagent described above.

[0053] Preferably, the instantaneous detection chemiluminescence kit also includes a sample diluent, a chemiluminescence substrate solution, and a working wash solution.

[0054] The sample diluent includes Tris-HCl buffer, NaCl, preservative, NP40, Tween, glycerol, and newborn calf serum.

[0055] Preferably, the sample diluent includes Tris-HCl buffer, NaCl, Proclin 300, NP40, Tween 20, glycerol, and newborn calf serum.

[0056] Based on a total sample dilution volume of 100%, use Proclin 300 0.08-0.12 v%, NP40 0.5-1.5 v%, TW-20 0.08-1.2 v%, glycerol 8-15 v%, and newborn calf serum 8-12 v%; replenish the remaining volume with Tris-HCl buffer.

[0057] The concentration of Tris-HCl buffer is 180mM-220mM, and the pH is 7.5-8.5;

[0058] The NaCl content in each 100 ml sample dilution is 0.8-100 g.

[0059] In some preferred embodiments of this application, based on a total sample diluent volume of 100%, the components are Proclin 300 0.1 v%, NP40 1 v%, TW-20 0.1 v%, glycerol 10 v%, and newborn calf serum 10 v%, with the remainder replenished using Tris-HCl buffer.

[0060] The concentration of Tris-HCl buffer is 200 mM, and the pH is 8.0;

[0061] The NaCl content in each 100 ml sample dilution is 0.9 g.

[0062] Preferably, the working wash solution, with a total volume of 100%, comprises 0.5% TW-20, 0.1% Proclin 300, and sodium chloride, and is supplemented with 200mM Tris-HCl buffer at pH 8.0 to make up the balance. The sodium chloride content in each 100 ml of working wash solution is 0.9 g.

[0063] Preferably, the labeled antibody or antigen reagent is an ALP-labeled antibody or antigen diluted proportionally using a label diluent.

[0064] More preferably, the label diluent includes Tris-HCl buffer, NaCl, preservative, stabilizer, NP40, glycerol, and newborn calf serum;

[0065] Based on the total volume of the marker diluent as 100%, the amount of preservative is 0.08-0.12v, stabilizer is 8-12v, NP40 is 0.5-1.5v, glycerol is 8-12v%, and newborn calf serum is 8-12v%.

[0066] The concentration of NaCl in the marker dilution solution is 0.8-1.2 g / L.

[0067] The concentration of ALP-labeled antibody or antigen in the labeled antibody or antigen reagent is 0.1-10 μg / mL.

[0068] In some embodiments of this application, the concentration of the ALP-labeled antibody or antigen may be 0.1 μg / mL, 0.2 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 5 μg / mL, or 10 μg / mL.

[0069] A point-of-care chemiluminescence assay kit includes a reagent strip with at least one sample well, at least one diluent well, at least one working wash well, at least one enzyme-labeled reagent well, at least one magnetic microparticle well, and at least one luminescent substrate solution well. The reagent strip also includes a light-shielded detection cup for detecting the chemiluminescence signal.

[0070] The sample wells are used to load test samples, the diluent reagent wells are filled with sample diluent, the working wash reagent wells are filled with working wash, the enzyme-labeled reagent wells are filled with labeled antibody or antigen reagents, the magnetic microparticle reagent wells are filled with magnetic microparticle reagents, and the luminescent substrate reagent wells are filled with chemiluminescent substrate solution.

[0071] Fourthly, this application provides a real-time chemiluminescence detection method.

[0072] A point-of-care chemiluminescence detection method: Take 50-100 μL of serum sample, and determine whether to pre-dilute the serum sample with sample diluent according to project requirements; add the sample (or pre-diluted sample), labeled antibody or antigen reagent to the reagent well containing magnetic microparticle reagent, and perform antigen-antibody reaction (incubation) to form antigen-antibody complex. After washing, add the labeled antigen-antibody complex to the reagent well, catalyze the luminescent substrate to emit light or trigger the substrate to emit light, detect the light signal RLU value, and calculate the content of the target substance in the sample to be tested according to the built-in calibration curve of the chemiluminescence detection device.

[0073] In summary, this application has the following beneficial effects:

[0074] The addition of sodium chloride, glycerol, gelatin, and trehalose established a magnetic bead diluent with high viscosity under storage conditions (2-8℃) and low viscosity at incubation temperature (37℃). Magnetic microparticle reagents prepared using this diluent have the advantages of high viscosity under storage conditions (2-8℃) and low viscosity at incubation temperature (37℃). This reduces the problem of magnetic beads sticking to the upper tube wall or upper aluminum foil seal during transport due to handling, loading, or unloading, which could lead to reduced accuracy of test results due to variations in the number of magnetic microparticles participating in the immune reaction. Attached Figure Description

[0075] Figure 1 This is a schematic diagram of the overall structure of the chemiluminescence immunoassay kit for immediate detection in this application.

[0076] Figure labeling: 1. Sample well; 2. Diluent reagent well; 3. Working wash reagent well; 4. Enzyme-labeled reagent well; 5. Magnetic microparticle reagent well; 6. Luminescent substrate solution reagent well; 7. Light-proof detection cup. Detailed Implementation

[0077] The present application will be further described in detail below with reference to the embodiments.

[0078] It should be noted that: unless otherwise specified, the following examples shall be conducted under conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the instruments, materials, reagents, etc. used in the following examples shall be commercially available.

[0079] The antibodies and antigens used in the magnetic linkage reagent and enzyme-labeled antibody were purchased from HaiTai Biotechnology (Shanghai) Co., Ltd.; the magnetic microparticles were purchased from Thermo Fisher Scientific.

[0080] Example of preparing magnetic bead diluent

[0081] A magnetic bead diluent includes component A and component B;

[0082] Component A: Tris-HCl buffer, glycerol, preservative.

[0083] Based on the total volume of the magnetic bead diluent as 100%, the volume percentage of glycerol is 10-30%, and the volume percentage of the preservative is 0.1-0.2%.

[0084] Component B: Sodium chloride, gelatin, non-reducing disaccharides.

[0085] The concentration of sodium chloride in the magnetic bead diluent is 8-100 g / L, the concentration of gelatin in the magnetic bead diluent is 3-20 g / L, and the concentration of non-reducing disaccharide in the magnetic bead diluent is 3-10 g / L.

[0086] The Tris-HCl buffer solution has a concentration of 150-250 mM and a pH of 7.5-8.5.

[0087] A method for preparing a magnetic bead diluent: Sodium chloride, gelatin, trehalose, glycerol, and preservative are added to Tris-HCl buffer according to the formula amount, stirred evenly, and then Tris-HCl buffer is added to the target volume. After mixing evenly, the magnetic bead diluent is obtained.

[0088] Preparation Example 1

[0089] A magnetic bead diluent includes Tris-HCl buffer, glycerol, preservative, sodium chloride, gelatin, and trehalose. In this preparation example, Proclin 300 is used as the preservative.

[0090] Based on the total volume of the magnetic bead diluent being 100%, the volume percentage of glycerin is 10% and the volume percentage of the preservative is 0.1%.

[0091] The concentration of sodium chloride in the magnetic bead diluent is 9 g / L, the concentration of gelatin in the magnetic bead diluent is 5 g / L, and the concentration of trehalose in the magnetic bead diluent is 5 g / L.

[0092] The Tris-HCl buffer solution has a concentration of 200 mM and a pH of 8.

[0093] A method for preparing a magnetic bead diluent: Sodium chloride, gelatin, trehalose, glycerol, and preservative are added to Tris-HCl buffer according to the formula amount, stirred evenly, and then Tris-HCl buffer is added to the target volume. After mixing evenly, the magnetic bead diluent is obtained.

[0094] Preparation Example 2

[0095] In this preparation example, the total volume of the magnetic bead diluent is 100%, the volume percentage of glycerol is 20%, and the formulations of other components are the same as in Preparation Example 1.

[0096] Preparation Example 3

[0097] In this preparation example, the total volume of the magnetic bead diluent is 100%, the volume percentage of glycerol is 30%, and the formulations of other components are the same as in Preparation Example 1.

[0098] Preparation Example 4

[0099] In this preparation example, the total volume of the magnetic bead diluent is 100%, the volume percentage of glycerol is 40%, and the formulations of other components are the same as in Preparation Example 1.

[0100] Preparation Example 5

[0101] In this preparation example, the concentration of gelatin in the magnetic bead diluent is 3 g / L, and the formulations of other components are the same as in Preparation Example 3.

[0102] Preparation Example 6

[0103] In this preparation example, the concentration of gelatin in the magnetic bead diluent is 10 g / L, and the formulations of other components are the same as in Preparation Example 3.

[0104] Preparation Example 7

[0105] In this preparation example, the concentration of gelatin in the magnetic bead diluent is 20 g / L, and the formulations of other components are the same as in Preparation Example 3.

[0106] Preparation Example 8

[0107] In this preparation example, the concentration of trehalose in the magnetic bead diluent is 3 g / L, and the formulations of other components are the same as in Preparation Example 3.

[0108] Preparation Example 9

[0109] In this preparation example, the concentration of trehalose in the magnetic bead diluent is 7 g / L, and the formulations of other components are the same as in Preparation Example 3.

[0110] Preparation Example 10

[0111] In this preparation example, the concentration of trehalose in the magnetic bead diluent is 10 g / L, and the formulations of other components are the same as in Preparation Example 3.

[0112] Preparation Example 11

[0113] In this preparation example, the concentration of sodium chloride in the magnetic bead diluent is 8 g / L, and the formulations of other components are the same as in Preparation Example 3.

[0114] Preparation Example 12

[0115] In this preparation example, the concentration of sodium chloride in the magnetic bead diluent is 50 g / L, and the formulations of other components are the same as in Preparation Example 3.

[0116] Preparation Example 13

[0117] In this preparation example, the concentration of sodium chloride in the magnetic bead diluent is 100 g / L, and the formulations of other components are the same as in Preparation Example 3.

[0118] The partial component distributions in Preparation Examples 1-10 are shown in Table 1.

[0119] Table 1 shows the amounts of glycerol, sodium chloride, gelatin, and trehalose used in Preparation Examples 1-10.

[0120] glycerin / % gelatin g / L Trehalose g / L Sodium chloride g / L Preparation Example 1 10 5 5 9 Preparation Example 2 20 5 5 9 Preparation Example 3 30 5 5 9 Preparation Example 4 40 5 5 9 Preparation Example 5 30 3 5 9 Preparation Example 6 30 10 5 9 Preparation Example 7 30 20 5 9 Preparation Example 8 30 5 3 9 Preparation Example 9 30 5 7 9 Preparation Example 10 30 5 10 9 Preparation Example 11 30 5 5 8 Preparation Example 12 30 5 5 50 Preparation Example 13 30 5 5 100

[0121] Comparative Preparation Example 1

[0122] In this comparative preparation example, the total volume of the magnetic bead diluent is 100%, the volume percentage of glycerol is 5%, and the formulations of other components are the same as in preparation example 3.

[0123] Comparative Preparation Example 2

[0124] In this comparative preparation example, the volume percentage of glycerol is 45%, and the formulations of other components are the same as in preparation example 3.

[0125] Comparative preparation example 3

[0126] In this comparative preparation example, no gelatin was added, and the formulations of other components were the same as in preparation example 3.

[0127] Comparative preparation example 4

[0128] In this comparative preparation example, no trehalose was added, and the formulations of other components were the same as in preparation example 3.

[0129] Comparative preparation example 5

[0130] In this comparative preparation example, the sodium chloride concentration was 3 g / L, and the formulations of other components were the same as in preparation example 3.

[0131] Comparative preparation example 6

[0132] In this comparative preparation example, the sodium chloride concentration was 110 g / L, and the formulations of other components were the same as in preparation example 3.

[0133] Examples of magnetic microparticle reagents

[0134] The magnetic particle reagent was prepared by conjugating MYO antibody to magnetic microparticles using the EDC method. The conjugation method is described in the technical information provided by Thermo. The magnetic beads with conjugated antibody were diluted with the magnetic bead diluent prepared in Preparation Examples 1-13 or Comparative Preparation Examples 1-6 to prepare the magnetic microparticle reagent.

[0135] The concentration of the magnetic microparticle reagent beads was 200 μg / mL, and the concentration of MYO antibody conjugated on the magnetic beads was 10 μg / mL.

[0136] Table 3 shows the correspondence between Examples 1-13 and Preparation Examples 1-13, and between Comparative Examples 1-6 and Comparative Preparation Examples 1-6.

[0137]

[0138] A point-of-care chemiluminescence assay kit, such as Figure 1 As shown, the reagent strip includes at least one sample well 1, at least one diluent well 2, at least one working wash well 3, at least one enzyme-labeled reagent well 4, at least one magnetic microparticle well 5, and at least one luminescent substrate solution well 6. The reagent strip also includes a light-shielding detection cup 7 for detecting chemiluminescent signals.

[0139] Sample well 1 is used to load test samples, diluent well 2 is loaded with sample diluent, working wash well 3 is loaded with working wash, enzyme-labeled reagent well 4 is loaded with labeled antibody or antigen reagent, magnetic microparticle reagent well 5 is loaded with magnetic microparticle reagent, and luminescent substrate reagent well 6 is loaded with chemiluminescent substrate solution.

[0140] In some embodiments of this application, the above-mentioned instantaneous chemiluminescence assay kit is a myoglobin (MYO) chemiluminescence immunoassay kit.

[0141] Preparation of a Chemiluminescence Kit for Real-Time Detection of Myoglobin (MYO)

[0142] A MYO reagent kit includes a reagent strip with at least one sample well 1, at least one diluent well 2, at least one working wash well 3, at least one enzyme-labeled reagent well 4, at least one magnetic microparticle well 5, and at least one luminescent substrate solution well 6. The reagent strip also includes a light-shielded detection cup 7 for detecting chemiluminescent signals.

[0143] The sample wells are used to load MYO in human serum or plasma. The diluent reagent well 2 is loaded with sample diluent. The working wash reagent well 3 is loaded with working wash. The enzyme-labeled reagent well 4 is loaded with labeled antibody or antigen reagent. The magnetic microparticle reagent well 5 is loaded with magnetic microparticle reagent coupled with MYO antibody. The luminescent substrate reagent well 6 is loaded with chemiluminescent substrate solution.

[0144] The working wash solution is a 200 mM pH 8.0 Tris-HCl buffer containing a final concentration of 0.9% (w / v) NaCl, 0.5% (v / v) TW-20, and 0.1% (v / v) Proclin 300 (all w / v refers to g / mL).

[0145] The sample diluent includes Tris-HCl buffer, NaCl, Proclin 300, NP40, TW-20, glycerol, and newborn calf serum; wherein, based on a total sample diluent volume of 100%, Proclin 300 0.1v%, NP40 1v%, TW-20 0.1v%, glycerol 10v%, and newborn calf serum 10v%; the remainder is replenished with Tris-HCl buffer.

[0146] The concentration of Tris-HCl buffer is 200 mM, and the pH is 8.0;

[0147] The NaCl content in each 100 ml sample dilution is 0.9 g.

[0148] The enzyme-labeled antibody reagent was ALP-labeled mouse anti-human MYO antibody diluted with enzyme-labeled diluent at a volume ratio of 1:2000; the enzyme-labeled diluent was 200mM Tris-HCl buffer at pH 8.0, containing a final concentration of 0.9% (w / v) NaCl, 3% (w / v) BSA, 10% (v / v) stabilizer, and 0.1% (v / v) Proclin 300 (all w / v refers to g / mL).

[0149] The chemiluminescent substrate solution was a 0.2 mol / L pH 9.0 Tris-HCl buffer solution containing 0.5 g / L 9-(4-chlorophenylthiobenzoyloxymethylene)-10-methyl-9,10-dihydroacrylidine-disodium salt, 0.3 mg / L 5-(tetradecanoic acid)fluorescein, 0.5 g / L aryl acylhydrazone-9,10-acrylidine derivative, 1.0 mM MgCl2, 1.0 mM ZnCl2, 5 mg / L hexadecyltrimethylammonium chloride, 0.5 g / L Proclin 300, and 0.5 wt% Tween 20.

[0150] The calibrator is a MYO antigen diluted with a calibrator diluent at different ratios. The calibrator diluent is a 200mM pH 8.0 Tris-HCl buffer containing a final concentration of 0.9% (w / v) NaCl, 2% (w / v) bovine serum albumin, 5% (w / v) trehalose, and 0.1% (v / v) Proclin 300 (all w / v refers to g / mL).

[0151] The main principle of the chemiluminescence kit for point detection of myoglobin (MYO) is as follows: the antibody or antigen coupled with the magnetic beads binds to MYO in human serum or plasma, and then forms a double antibody sandwich with ALP-labeled antibody or antigen. In the presence of the substrate, alkaline phosphatase catalyzes the substrate to emit light, and quantitative detection is performed by the chemiluminescence detection system.

[0152] During testing, the calibrator is first added to the sample well, and the reagent strip is placed in the detection instrument to measure the chemiluminescence intensity value. A calibration curve is created with the different concentrations of MYO antigen in the calibrator as the x-axis and the chemiluminescence intensity value as the y-axis. Then, the sample is added to the sample well, and the reagent strip is placed in the detection instrument to measure the chemiluminescence intensity value. The concentration of MYO in the sample is calculated based on the linear relationship.

[0153] Performance testing

[0154] 1. The viscosity (Pa·S) of the magnetic bead diluents obtained in Preparation Examples 1-13 was measured using a rotational viscometer at storage temperature (4±1℃) and incubation temperature (37±1℃) for comparison with that obtained in Preparation Examples 1-6.

[0155] 2. Pre-transport testing: The concentration of MYO in serum samples was measured using the point-of-care chemiluminescence assay kits prepared in Examples 1-13 and Comparative Examples 1-6. Each example or comparative example was measured 10 times, and the repeatability (coefficient of variation, CV) of the measurement results was compared.

[0156] Post-transport testing: The same batch of point-of-care chemiluminescence assay kits prepared by Examples 1-13 and Comparative Examples 1-6 were airlifted from Beijing to Haikou and then back to Beijing. The concentration of MYO in serum samples was measured using this batch of point-of-care chemiluminescence assay kits. Each example or comparative example was measured 10 times, and the repeatability (coefficient of variation CV) of the measurement results was compared.

[0157] The experimental results are shown in the table below.

[0158] Table 4 shows the viscosity (Pa·S) of the magnetic bead diluents obtained in Preparation Examples 1.1-1.10 and Comparative Preparation Examples 1.1-1.5 at storage temperature (4±1℃) and incubation temperature (37±1℃).

[0159]

[0160] As shown in Table 4, gelatin, glycerin, trehalose, and sodium chloride all affect the viscosity of the magnetic bead diluent. The addition of these components increases the viscosity, but it decreases with increasing temperature. By selecting gelatin, glycerin, trehalose, and sodium chloride and adjusting their amounts, the viscosity of the magnetic bead diluent can be controlled to be no less than 4.6 Pa·s at the storage temperature (4±1℃) and no more than 1.9 Pa·s at the incubation temperature (37±1℃). Adjusting the viscosity of the magnetic bead diluent using these four components prevents it from flowing easily at storage temperatures, thus reducing the likelihood of magnetic beads adhering to the upper tube wall or aluminum foil seal of the reagent strip during handling and transportation.

[0161] The addition of gelatin and trehalose not only increases the viscosity of the magnetic bead diluent but also protects the protein and adjusts the liquid density, thereby allowing the magnetic beads to be more evenly dispersed in the magnetic microparticle reagent.

[0162] In the magnetic bead diluent prepared in Example 3 of this application, the viscosity at the storage temperature reached 5.4 Pa·s, while the viscosity at the incubation temperature reached 1.3 Pa·s.

[0163] Table 5 compares the precision of the instant detection chemiluminescence kits using magnetic microparticle reagents from Examples 1-13 and Comparative Examples 1-6 before and after transportation.

[0164]

[0165] Note: M represents the average concentration of MYO after 10 replicate experiments;

[0166] S represents the standard deviation of 10 repeated experiments;

[0167] CV stands for coefficient of variation, and CV = S / M.

[0168] As shown in Table 5, for Sample 2, before transportation, the repeatability (CV) of the examples was in the range of 2.78%-3.89%, and the repeatability (CV) of the comparative examples was in the range of 3.92%-4.32%. After being transported from Beijing to Haikou and then tested upon return, the repeatability (CV) of the kit 1 of this invention for detecting three different concentration levels of quality control samples was in the range of 3.12%-3.93%, while the repeatability (CV) of the comparative reagent was in the range of 8.80%-9.42%.

[0169] This demonstrates that the magnetic microparticle reagents obtained in Examples 1-13 of this application can significantly improve the accuracy of detection results after the reagent kit is transported.

[0170] The magnetic microparticle reagents prepared in Examples 1-13 of this application are less affected by transportation and handling, and can maintain extremely high accuracy during detection.

[0171] It is understood that the above embodiments are merely exemplary implementations used to illustrate the principles of the present invention, and the present invention is not limited thereto. For those skilled in the art, various modifications and improvements can be made without departing from the spirit and essence of the present invention, and these modifications and improvements are also considered to be within the scope of protection of the present invention.

Claims

1. Use of a magnetic bead diluent in the preparation of a magnetic microparticle reagent, the magnetic microparticle reagent being a combination of a magnetic bead conjugated antibody or antigen and the magnetic bead diluent, characterised in that, The magnetic bead diluent is composed of A component and B component The A component is composed of Tris-HCl buffer, glycerol and Proclin 300, the concentration of Tris-HCl buffer is 200 mM, and the pH is 7.5-8.5; The volume percentage of glycerol is 30%, and the volume percentage of Proclin 300 is 0.1% in the total volume of the magnetic bead diluent; The B component is composed of sodium chloride, gelatin and trehalose; The concentration of sodium chloride in the magnetic bead diluent is 9 g / L, the concentration of gelatin in the magnetic bead diluent is 5 g / L, and the concentration of trehalose in the magnetic bead diluent is 5 g / L. The magnetic bead diluent is composed of antibody or antigen coupled to magnetic beads and the magnetic bead diluent of claim 1.

2. A magnetic microparticle reagent configured using the magnetic bead diluent solution of claim 1, characterized by, The concentration of magnetic beads in the magnetic particle reagent is 100-200 μg / mL, and the concentration of MYO antibody coupled to the magnetic beads is 0.2-10 μg / mL.

3. The magnetic microparticle reagent according to claim 2, wherein The reagent strip is loaded with labeled antibody or antigen reagent and magnetic particle reagent.

4. A kit for the instant detection of chemiluminescence, characterized in that, The magnetic particle reagent is the magnetic particle reagent of claim 2. The sample diluent and chemiluminescent substrate solution are also included.

5. The instant detection chemiluminescent kit according to claim 4, wherein The sample diluent includes Tris-HCl buffer, NaCl, preservative, NP40, Tween, glycerol and newborn calf serum. The labeled antibody or antigen reagent is ALP-labeled antibody or antigen diluted by the marker diluent in proportion.

6. The instant detection chemiluminescent kit according to claim 4, wherein ​

Images