Kit for coupling fluorescent coded microspheres with antigens and use thereof

Abstract

This invention provides a kit for conjugating fluorescently encoded microspheres to antigens and its application. The kit includes microsphere coating solution A and / or microsphere blocking solution D, or further includes at least one of microsphere activating solution, microsphere preservation solution, activator A and activator B. Microsphere coating solution A includes 0.0736–0.0744 mol / L sodium acetate solution and 0.177–0.201 mol / L acetic acid solution, with a pH of 4.4–4.6. Microsphere blocking solution D, per 100 mL, includes 1.5–1.7 g Tris, 0.43–0.53 g sucrose, 1.7–1.9 g trehalose, 0.9–1.1 g BSA, 1.9–2.1 g PEG6000, 0.12–0.18 g Triton X-100 and 0.09–0.11 mL Proclin 300, with a pH of 7.3–7.5.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a kit for conjugating fluorescently encoded microspheres with antigens and its application. Background Technology

[0002] Autoimmune liver disease (ALD) is a special type of chronic liver disease caused by immune dysfunction. It mainly includes autoimmune hepatitis (AIH), primary biliary cholangitis (PBC), and primary sclerosing cholangitis (PSC). Among them, PBC and AIH have autoantibodies with high sensitivity and specificity.

[0003] PBC is a chronic autoimmune intrahepatic cholestatic disease, whose main autoantibodies include AMA, AMA-M2, anti-gp210 antibody and anti-Sp100 antibody.

[0004] Most patients with autoimmune hemorrhage (AIH) have one or more high-titer autoantibodies in their serum, but these autoantibodies are usually disease-specific. AIH can be classified into two types based on the type of autoantibody: Type 1, which is positive for antinuclear antibodies (ANA) or anti-smooth muscle antibodies (ASMA), and Type 2, which is positive for anti-liver kidney microsome-1 (anti-LKM-1) or anti-liver cytosol-1 (anti-LC-1). Type 1 AIH accounts for approximately 90% of cases.

[0005] Studies have shown that approximately 10.2% of AIH patients are ANA and ASMA negative at onset, but some become ANA positive during follow-up. Compared to classic AIH patients, antibody-negative AIH patients have lower serum IgG levels and may be in the progressive stage of fibrosis at onset; however, there were no statistically significant differences between the two groups in terms of the degree of histological inflammation at onset and the rate of biochemical response remission within six months. Therefore, timely diagnosis and initiation of treatment can help improve the prognosis of antibody-negative AIH patients.

[0006] In recent years, various methods have been used to detect antibodies against autoimmune liver disease, such as indirect fluorescence assay, Human flow cytometry, hemagglutination assay, radioimmunoassay, or enzyme-linked immunosorbent assay. However, these methods are usually qualitative and their efficiency in detecting autoimmune liver disease cannot meet clinical needs. Summary of the Invention

[0007] Based on this, the present invention provides a kit for conjugating fluorescently encoded microspheres to antigens, fluorescently encoded microspheres conjugated to antigens, and a kit for detecting autoimmune liver diseases. The specific technical solution is as follows:

[0008] According to one aspect of the present invention, a kit for conjugating fluorescently encoded microspheres with an antigen is provided, the kit comprising microsphere coating solution A and / or microsphere blocking solution D, or further comprising one or more of microsphere activating solution, microsphere preservation solution, activator A and activator B;

[0009] The microsphere coating solution A includes a sodium acetate solution of 0.0736 mol / L to 0.0744 mol / L and an acetic acid solution of 0.177 mol / L to 0.201 mol / L, and the pH value of the microsphere coating solution A is 4.4 to 4.6.

[0010] The microsphere blocking solution D contains 5g-7g Tris, 0.43g-0.53g sucrose, 7g-9g trehalose, 0.9g-1g BSA, 9g-2.1g PEG6000, 0.12g-0.18g Triton X-100, and 0.09mL-0.11mL Proclin 300 per 100mL. The pH value of the microsphere blocking solution D is 7.3-7.5.

[0011] In one embodiment, the microsphere activation solution comprises 0.505 g to 0.555 g of 2-morpholine ethanesulfonic acid and 0.475 g to 0.525 g of sodium 2-morpholine ethanesulfonic acid per 100 mL; the pH value of the microsphere activation solution is 5.9 to 6.1.

[0012] In one embodiment, the kit further includes at least one of microsphere coating solution B, microsphere coating solution C, and microsphere coating solution D;

[0013] Optionally, per 100 mL, the microsphere coating solution B includes 1 g to 2 g of 2-morpholine ethanesulfonic acid and 0.9 g to 1.0 g of sodium 2-morpholine ethanesulfonic acid, and the pH value of the microsphere coating solution B is 5.95 to 6.15;

[0014] Optionally, per 100 mL, the microsphere coating solution C includes 0.09 g to 0.15 g of anhydrous disodium hydrogen phosphate, 0.014 g to 0.034 g of sodium dihydrogen phosphate dihydrate, and 0.08 mL to 0.12 mL of Proclin 300, and the pH value of the microsphere coating solution C is 7.3 to 7.5;

[0015] Optionally, per 100 mL, the microsphere coating solution D includes 0.09 g to 0.15 g of anhydrous sodium carbonate, 0.09 g to 0.15 g of sodium bicarbonate and 0.06 g to 0.12 g of sodium chloride, and the pH value of the microsphere coating solution D is 9.5 to 9.7.

[0016] In one embodiment, the kit further includes at least one of microsphere blocking solution A, microsphere blocking solution B, and microsphere blocking solution C;

[0017] Optionally, per 100 mL, the microsphere blocking solution A comprises 0.152 g to 0.189 g of anhydrous disodium hydrogen phosphate, 0.116 g to 0.147 g of sodium dihydrogen phosphate dihydrate, 1.686 g to 1.749 g of sodium chloride, 0.297 g to 0.328 g of BSA, 0.003 g to 0.022 g of Tween 20, 1.844 g to 1.906 g of Glycine, 0.053 mL to 0.072 mL of Proclin 300, and 6.156 mL to 6.344 mL of goat serum, and the pH value of the microsphere blocking solution A is 6.4 to 6.6;

[0018] Optionally, per 100 mL, the microsphere blocking solution B comprises 4.5 g to 4.7 g Tris, 0.43 g to 0.53 g sucrose, 5.04 g to 5.34 g trehalose, 0.92 g to 1.08 g BSA, 0.03 g to 0.05 g Tween 20, 5.85 g to 6.15 g Glycine, 0.16 g to 0.2 g Triton X-405, and 0.19 mL to 0.21 mL Proclin 300, and the pH value of the microsphere blocking solution B is 7.9 to 8.1.

[0019] Optionally, per 100 mL, the microsphere blocking solution C includes 1.5 g to 1.7 g Tris, 0.43 g to 0.53 g sucrose, 0.9 g to 1.1 g BSA, 0.94 g to 1.06 g PEG2000 and 0.19 mL to 0.21 mL Proclin300, and the pH value of the microsphere blocking solution C is 7.3 to 7.5.

[0020] In one embodiment, the microsphere preservation solution comprises, per 100 mL, 5.9 g to 6.1 g HEPES buffer, 4.9 g to 5.1 g sucrose, 95 g to 2.05 g trehalose, 0.25 g to 0.35 g BSA, 1.45 g to 1.55 g mannitol, 0.54 g to 0.66 g disodium EDTA and 0.09 mL to 0.11 mL Proclin 300; the pH of the microsphere preservation solution is 7.3 to 7.5.

[0021] In one embodiment, the kit satisfies at least one of the following conditions:

[0022] (1) The activator A includes N-hydroxythiosuccinimide and the microsphere activation solution;

[0023] (2) The activator B includes 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and the microsphere activation solution.

[0024] A fluorescently encoded microsphere conjugated with an antigen was prepared using the aforementioned kit for conjugating fluorescently encoded microspheres with an antigen.

[0025] A kit for detecting autoimmune liver disease includes at least one of the above-described fluorescently encoded microspheres conjugated to an antigen; said antigen is LC-1 antigen, LKM-1 antigen, gp210 antigen, Sp100 antigen, AMA-M2 antigen, CENP-B antigen, ASMA antigen, SLA / LP antigen, or Ro52 antigen.

[0026] In one embodiment, the kit further includes a sample diluent and one or more fluorescently labeled goat anti-human IgG.

[0027] In one embodiment, the sample diluent comprises 0.006 g to 0.046 g of anhydrous disodium hydrogen phosphate, 0.332 g to 0.432 g of sodium dihydrogen phosphate dihydrate, 0.28 g to 0.38 g of sodium chloride, and 0.09 mL to 0.11 mL of Proclin 300 per 100 mL.

[0028] Compared with traditional technologies, the present invention has the following advantages:

[0029] This invention improves the microsphere coating and blocking solutions, thereby enhancing the coupling efficiency between fluorescent immunomicrospheres and antigens. This improves the signal-to-noise ratio and detection efficiency of flow cytometry, providing a reference for the screening, diagnosis, and treatment of autoimmune liver diseases in clinical practice. Attached Figure Description

[0030] Figure 1 This is a schematic diagram of the hydrophobic structure of the ASMA antigen. Detailed Implementation

[0031] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to limit the invention. Unless otherwise specifically stated, all raw materials, reagents, instruments, and equipment used in this invention are commercially available or can be prepared by existing methods.

[0033] the term

[0034] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0035] In this invention, terms such as "multiple", "various", "multiple times", and "multi-source" are used, and unless otherwise specified, they refer to a quantity greater than or equal to 2. For example, "one or more" means one or more types.

[0036] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0037] In this article, terms such as "preferred," "better," "more suitable," and "ideal" are merely used to describe implementation methods or examples that achieve better results, and should be understood not to limit the scope of protection of this invention.

[0038] In this invention, terms such as "further," "even more," and "particularly" are used for descriptive purposes and to indicate differences in content, but should not be construed as limiting the scope of protection of this invention.

[0039] In this invention, "optionally," "optionally," and "optional" mean that they are optional, that is, they are selected from either "with" or "without." If multiple "options" appear in a technical solution, unless otherwise specified and there are no contradictions or mutual constraints, each "option" is independent.

[0040] In this invention, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0041] Some embodiments of the present invention provide a kit for conjugating fluorescently encoded microspheres with antigens, the kit comprising a microsphere activation solution, a microsphere coating solution A, a microsphere blocking solution D, a microsphere preservation solution, an activator A, and an activator B;

[0042] The microsphere coating solution A includes a sodium acetate solution of 0.0736 mol / L to 0.0744 mol / L and an acetic acid solution of 0.177 mol / L to 0.201 mol / L. The pH value of the microsphere coating solution A is 4.4 to 4.6.

[0043] Per 100 mL, the microsphere blocking solution D contains 1.5 g–1.7 g Tris (tris(hydroxymethyl)aminomethane), 0.43 g–0.53 g sucrose, 1.7 g–1.9 g trehalose, 0.9 g–1.1 g BSA (Bovine Serum Albumin), 1.9 g–2.1 g PEG6000, 0.12 g–0.18 g Triton X-100, and 0.09 mL–0.11 mL Proclin 300. The pH of the microsphere blocking solution D is 7.3–7.5.

[0044] Understandably, the isoelectric point of the LC-1 antigen is around pH 5.95. Therefore, preparing a microsphere coating solution with a pH of 4.4–4.6 allows the LC-1 antigen to become positively charged overall, while the carboxyl group is negatively charged. The attraction between the positive and negative charges causes the microspheres to bind to the antigen, thereby improving the coupling efficiency between the LC-1 antigen and the fluorescently encoded microspheres and increasing the detection sensitivity of this indicator. LC-1 antibody is the only detectable autoantibody in some patients with type 2 AIH, and LC-1 antibody is associated with the disease activity and progression of AIH; therefore, improving the detection efficiency of LC-1 antibody is of great significance for patients with type 2 AIH.

[0045] The hydrophobic structure of ASMA antigen is as follows Figure 1 As shown, most antigen structures have a hydrophobicity index above 0, meaning the overall antigen structure is hydrophobic. However, adding hydrophilic substances such as PEG2000 and sucrose to the microsphere blocking solution significantly increases antigen solubility and enhances the blocking effect on hydrophobic sites.

[0046] Currently, the Euromon indirect immunofluorescence assay is commonly used to determine ASMA indicators, but this method has drawbacks such as being cumbersome and time-consuming. Flow cytometry, on the other hand, is faster and more convenient. Based on this, the fluorescent immunoglobulins conjugated with antigens have been optimized and improved to enhance the efficiency of flow cytometry in detecting ASMA indicators.

[0047] Preferably, the microsphere coating solution A includes a 0.074 mol / L sodium acetate solution and a 0.189 mol / L acetic acid solution, with a pH of 4.5.

[0048] Preferably, per 100 mL, the microsphere blocking solution D contains 1.6 g Tris, 0.48 g sucrose, 1.8 g trehalose, 1.0 g BSA, 2.0 g PEG6000, 0.15 g Triton X-100 and 0.10 mL Proclin 300, with a pH of 7.4.

[0049] In some embodiments, the microsphere activation solution comprises 0.505 g to 0.555 g of 2-morpholine ethanesulfonic acid (MES) and 0.475 g to 0.525 g of sodium 2-morpholine ethanesulfonate (MES-Na) per 100 mL; the pH of the microsphere activation solution is 5.9 to 6.1.

[0050] Preferably, per 100 mL, the microsphere activation solution includes 0.53 g of 2-morpholine ethanesulfonic acid and 0.5 g of sodium 2-morpholine ethanesulfonic acid; the pH value is 6.0.

[0051] In some embodiments, the kit also includes at least one of microsphere coating solution B, microsphere coating solution C, and microsphere coating solution D.

[0052] In some specific examples, per 100 mL, the microsphere coating solution B contains 1.1 g to 1.2 g of 2-morpholine ethanesulfonic acid and 0.9 g to 1.0 g of sodium 2-morpholine ethanesulfonic acid, and the pH value of the microsphere coating solution B is 5.95 to 6.15.

[0053] Preferably, per 100 mL, the microsphere coating solution B contains 1.15 g of 2-morpholine ethanesulfonic acid and 0.95 g of sodium 2-morpholine ethanesulfonic acid, and the pH value of the microsphere coating solution B is 6.05.

[0054] In some specific examples, per 100 mL, the microsphere coating solution C includes 0.09 g to 0.15 g of anhydrous disodium hydrogen phosphate, 0.014 g to 0.034 g of sodium dihydrogen phosphate dihydrate, and 0.08 mL to 0.12 mL of Proclin 300, and the pH value of the microsphere coating solution C is 7.3 to 7.5.

[0055] Preferably, per 100 mL, the microsphere coating solution C includes 0.12 g of anhydrous disodium hydrogen phosphate, 0.024 g of sodium dihydrogen phosphate dihydrate and 0.1 mL of Proclin 300, and the pH value of the microsphere coating solution C is 7.4.

[0056] In some specific examples, per 100 mL, the microsphere coating solution D contains 0.09 g to 0.15 g of anhydrous sodium carbonate, 0.09 g to 0.15 g of sodium bicarbonate and 0.06 g to 0.12 g of sodium chloride, and the pH value of the microsphere coating solution D is 9.5 to 9.7.

[0057] Preferably, per 100 mL, the microsphere coating solution D comprises 0.12 g anhydrous sodium carbonate, 0.12 g sodium bicarbonate and 0.09 g sodium chloride, and the pH value of the microsphere coating solution D is 9.6.

[0058] In some embodiments, the kit also includes at least one of microsphere blocking solution A, microsphere blocking solution B, and microsphere blocking solution C.

[0059] In some of these examples, per 100 mL, microsphere blocking solution A contains 0.152 g–0.189 g anhydrous disodium hydrogen phosphate, 0.116 g–0.147 g sodium dihydrogen phosphate dihydrate, 1.686 g–1.749 g sodium chloride, 0.297 g–0.328 g BSA, 0.003 g–0.022 g Tween 20, 1.844 g–1.906 g Glycine, 0.053 mL–0.072 mL Proclin 300, and 6.156 mL–6.344 mL goat serum, and the pH of microsphere blocking solution A is 6.4–6.6.

[0060] Preferably, per 100 mL, the microsphere blocking solution A comprises 0.171 g anhydrous disodium hydrogen phosphate, 0.131 g sodium dihydrogen phosphate dihydrate, 1.717 g sodium chloride, 0.313 g BSA, 0.013 g Tween 20, 1.875 g Glycine, 0.063 mL Proclin 300, and 6.25 mL goat serum, and the pH value of the microsphere blocking solution A is 6.5.

[0061] In some of these examples, per 100 mL, the microsphere blocking solution B contains 4.5 g–4.7 g Tris, 0.43 g–0.53 g sucrose, 5.04 g–5.34 g trehalose, 0.92 g–1.08 g BSA, 0.03–0.05 g Tween 20, 5.85 g–6.15 g Glycine, 0.16 g–0.2 g Triton X-405, and 0.19 mL–0.21 mL Proclin 300, and the pH of the microsphere blocking solution B is 7.9–8.1.

[0062] Preferably, per 100 mL, the microsphere blocking solution B contains 4.6 g Tris, 0.48 g sucrose, 5.19 g trehalose, 1.0 g BSA, 0.04 g Tween 20, 6.0 g Glycine, 0.18 g Triton X-405 and 0.20 mL Proclin 300, and the pH value of the microsphere blocking solution B is 8.0.

[0063] In some of these examples, the microsphere blocking solution C contains 1.5 g to 1.7 g Tris, 0.43 g to 0.53 g sucrose, 0.9 g to 1.1 g BSA, 0.94 g to 1.06 g PEG2000, and 0.19 mL to 0.21 mL Proclin300 per 100 mL, and the pH of the microsphere blocking solution C is 7.3 to 7.5.

[0064] Preferably, per 100 mL, the microsphere blocking solution C contains 1.6 g Tris, 0.48 g sucrose, 1.0 g BSA, 1.0 g PEG2000 and 0.20 mL Proclin300, and the pH value of the microsphere blocking solution C is 7.4.

[0065] In some embodiments, the microsphere preservation solution comprises, per 100 mL, 5.9 g to 6.1 g HEPES buffer, 4.9 g to 5.1 g sucrose, 1.95 g to 2.05 g trehalose, 0.25 g to 0.35 g BSA, 1.45 g to 1.55 g mannitol, 0.54 g to 0.66 g disodium EDTA and 0.09 mL to 0.11 mL Proclin 300; the pH of the microsphere preservation solution is 7.3 to 7.5.

[0066] Preferably, per 100 mL, the microsphere preservation solution comprises 6.0 g HEPES buffer, 5.0 g sucrose, 2.0 g trehalose, 0.30 g BSA, 1.50 g mannitol, 0.60 g disodium EDTA and 0.10 mL Proclin 300; the pH of the microsphere preservation solution is 7.4.

[0067] In some embodiments, activator A includes N-hydroxythiosuccinimide (Sulfo-NHS) and the microsphere activating solution described above.

[0068] In some specific examples, the mass ratio of N-hydroxythiosuccinimide to the volume of the microsphere activation solution is 1 mg: 20 μL.

[0069] In some embodiments, activator B includes 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and microsphere activating solution.

[0070] In some specific examples, the mass ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to the volume of the microsphere activation solution is 1 mg: 20 μL.

[0071] Using the above kit can improve the conjugation effect between fluorescent immunoglobulins and antigens, thereby improving the signal-to-noise ratio and accuracy of flow cytometry detection of autoantibodies.

[0072] Some embodiments of the present invention also provide fluorescently encoded microspheres prepared using the above-described kit for conjugating fluorescently encoded microspheres with antigens.

[0073] Some embodiments of the present invention also provide a kit for detecting autoimmune liver disease, the kit comprising at least one fluorescently encoded microsphere coupled to an antigen; the antigen is selected from one or more of LC-1 antigen, LKM-1 antigen, gp210 antigen, Sp100 antigen, AMA-M2 antigen, CENP-B antigen, ASMA antigen, SLA / LP antigen and Ro52 antigen.

[0074] In some embodiments, the kit includes nine fluorescently encoded microspheres, which are conjugated to LC-1 antigen, LKM-1 antigen, gp210 antigen, Sp100 antigen, AMA-M2 antigen, CENP-B antigen, ASMA antigen, SLA / LP antigen, and Ro52 antigen, respectively.

[0075] The kit for detecting autoimmune liver disease of the present invention includes nine antigen indicators, which can enable more comprehensive and accurate screening and diagnosis of autoimmune liver disease in clinical practice.

[0076] In some embodiments, the kit also includes a sample diluent and one or more fluorescently labeled goat anti-human IgG.

[0077] In some specific examples, the fluorescent label is phycoerythrin.

[0078] In some embodiments, the sample diluent contains 0.006 g to 0.046 g of anhydrous disodium hydrogen phosphate, 0.332 g to 0.432 g of sodium dihydrogen phosphate dihydrate, 0.28 g to 0.38 g of sodium chloride, and 0.09 mL to 0.11 mL of Proclin 300 per 100 mL.

[0079] Preferably, per 100 mL, the sample diluent contains 0.026 g anhydrous disodium hydrogen phosphate, 0.382 g sodium dihydrogen phosphate dihydrate, 0.33 g sodium chloride, and 0.10 mL Proclin 300.

[0080] In some specific examples, the pH of the sample diluent was 7.3 to 7.5.

[0081] Preferably, the pH value of the sample diluent is 7.4.

[0082] Using the above-mentioned kit for detecting autoimmune liver disease of the present invention, quantitative analysis of multiple antigen indicators can be achieved, providing a reference for the screening and treatment of autoimmune liver disease in clinical practice.

[0083] The present invention will be further described below with reference to specific embodiments and comparative examples, but these should not be construed as limiting the scope of protection of the present invention.

[0084] The reaction reagents used in the following embodiments of the present invention were prepared according to the following methods:

[0085] Microsphere activation solution (pH = 6.0): Dissolve 1.06 g of 2-morpholine ethanesulfonic acid and 1.00 g of sodium 2-morpholine ethanesulfonate in 200 mL of pure water.

[0086] Microsphere coating solution A (pH = 4.5): Prepared by mixing 3.7 mL of 0.2 mol / L sodium acetate solution with 6.3 mL of 0.3 mol / L acetic acid solution.

[0087] Microsphere coating solution B (pH = 6.05): Dissolve 1.15 g of 2-morpholine ethanesulfonic acid and 0.95 g of sodium 2-morpholine ethanesulfonate in 100 mL of pure water.

[0088] Microsphere coating solution C (pH = 7.4): Dissolve 0.12 g of anhydrous disodium hydrogen phosphate, 0.024 g of sodium dihydrogen phosphate dihydrate and 0.1 mL of Proclin 300 in 100 mL of pure water.

[0089] Microsphere coating solution D (pH = 9.6): Dissolve 0.12 g of anhydrous sodium carbonate, 0.12 g of sodium bicarbonate and 0.9 g of sodium chloride in 100 mL of pure water.

[0090] Microsphere blocking solution A (pH = 6.5): Dissolve 0.546 g anhydrous disodium hydrogen, 0.420 g sodium dihydrogen phosphate, 5.496 g sodium chloride, 1.0 g BSA, 0.04 g Tween 20, 6.0 g Glycine, 0.2 mL Proclin 300 and 20 mL goat serum in 300 mL pure water.

[0091] Microsphere blocking solution B (pH = 8.0): Dissolve 4.6 g of 50 mM Tris, 0.48 g of sucrose, 5.19 g of trehalose, 1.0 g of BSA, 0.04 g of Tween 20, 6.0 g of Glycine, 0.18 g of Triton X-405 and 0.2 mL of Proclin 300 in 100 mL of pure water.

[0092] Microsphere blocking solution C (pH = 7.4): Dissolve 1.6 g of 50 mM Tris, 0.48 g of sucrose, 1.0 g of BSA, 1.0 g of PEG2000 and 0.2 mL of Proclin300 in 100 mL of pure water.

[0093] Microsphere blocking solution D (pH = 7.4): Dissolve 1.6 g of 50 mM Tris, 0.48 g of sucrose, 1.8 g of trehalose, 1.0 g of BSA, 2.0 g of PEG6000, 0.15 g of Triton X-100 and 0.1 mL of Proclin 300 in 100 mL of pure water.

[0094] Microsphere preservation solution (pH = 7.4): Dissolve 6.0 g of 500 mM HEPES, 5.0 g of sucrose, 2.0 g of trehalose, 0.3 g of BSA, 1.5 g of mannitol, 0.6 g of EDTA-Na2·2H2O and 0.1 mL of Proclin 300 in 100 mL of pure water.

[0095] Sample dilution (pH = 7.4): Dissolve 0.026 g anhydrous disodium hydrogen, 0.382 g sodium dihydrogen phosphate, 0.33 g sodium chloride and 0.1 mL Proclin 300 in 100 mL pure water.

[0096] Activator A: Take 20 mg of N-hydroxythiosuccinimide (Sulfo-NHS) and mix it with 400 μL of microsphere activation solution by vortexing for 30 s.

[0097] Activator B: Take 20 mg of 1-ethyl(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and mix with 400 μL of microsphere activation solution by vortexing for 30 s.

[0098] The goat anti-human phycoerythrin solution used in the examples was purchased from Solarbio, catalog number: K0001G-PE; the goat anti-human phycoerythrin solution was diluted with the sample diluent at a ratio of 1:200.

[0099] Example 1:

[0100] (1) Treatment of fluorescently encoded microspheres

[0101] The fluorescently coded magnetic microspheres were vortexed for 30 seconds and inverted 5 times to mix. 100 μL of the fluorescently coded magnetic microspheres were added to each of the four centrifuge tubes and the centrifuge tubes were placed on a magnetic rack for magnetic adsorption for 2 minutes. The supernatant was then separated and discarded.

[0102] Add 200 μL of microsphere activation solution to each of the four centrifuge tubes, vortex for 15 s, sonicate in ultrasonic cleaner for 20 s, and magnetically adsorb for 2 min, then separate and discard the supernatant; repeat the washing once more; then add 180 μL of microsphere activation solution to each of the four test tubes, vortex for 15 s, and sonicate for 20 s; to obtain four microsphere resuspensions M.

[0103] Add 10 μL of activator A and 10 μL of activator B to each of the four centrifuge tubes, vortex for 15 s, react at 25 °C in the dark for 20 min, and then perform magnetic adsorption for 2 min. Separate and discard the supernatant.

[0104] Add 200 μL of microsphere coating solution A, microsphere coating solution B, microsphere coating solution C, and microsphere coating solution D to four centrifuge tubes, respectively. Vortex for 15 s, sonicate for 20 s, and magnetically adsorb for 2 min. Separate and discard the supernatant. Repeat the washing process twice: add 200 μL of microsphere coating solution A, microsphere coating solution B, microsphere coating solution C, or microsphere coating solution D to four centrifuge tubes to resuspend the microspheres. Vortex for 15 s and sonicate for 20 s to obtain four microsphere resuspensions N.

[0105] Add 10 μg of LC-1 antigen to each of four centrifuge tubes, vortex to mix, and react at room temperature in the dark for 2 h; then magnetically adsorb for 2 min, and separate and discard the supernatant.

[0106] Add 300 μL of microsphere blocking solution A to each of the four centrifuge tubes, vortex for 15 seconds to mix, sonicate for 20 seconds, and react at 25°C in the dark for 60 minutes.

[0107] After sealing, add 400 μL of microsphere preservation solution to each of the four centrifuge tubes, vortex for 15 s, sonicate for 20 s, and magnetically adsorb for 20 min, then separate and remove the supernatant; repeat the washing process twice; add 400 μL of microsphere preservation solution to each of the four centrifuge tubes to resuspend the microspheres, and vortex to mix for 15 s; obtain four types of fluorescently encoded microspheres conjugated with LC-1 antigen; store at 2℃~8℃ in the dark.

[0108] The four fluorescently encoded microspheres conjugated with LC-1 antigen were mixed in a ratio of 1:1:1:1 to obtain a microsphere mixture.

[0109] (2) Signal value detection

[0110] The test samples, along with the aforementioned microsphere mixture and diluted sheep anti-human phycoerythrin solution, were tested using iMutli. The test samples included 3 LC-1 negative samples and 2 LC-1 positive samples. The signal values ​​for each sample are shown in Table 1; the signal-to-noise ratio calculation results are shown in Table 2.

[0111] Table 1. Signal values ​​of LC-1 antibody detected after treating fluorescently encoded microspheres with different coating solutions.

[0112] Background 933 4895 960 36856 SP3 (negative) 5446 7928 10663 40625 SP53 (positive) 72226 13542 18751 91073 SP108 (positive) 26058 9454 8789 55820 Blood test result -199# (negative) 13952 17992 29906 62941 Blood test result -200# (negative) 5929 13379 16080 44372

[0113] Table 2. Signal-to-noise ratio of LC-1 antibody detection after treatment with different coating solutions for fluorescently encoded microspheres.

[0114] SP3 (negative) 5.84 1.62 11.11 1.10 SP53 (positive) 77.41 2.77 19.53 2.47 SP108 (positive) 27.93 1.93 9.16 1.51 Blood test result -199# (negative) 14.95 3.68 31.15 1.71 Blood test result -200# (negative) 6.35 2.73 16.75 1.20

[0115] As shown in the table above, the fluorescently encoded microspheres treated with coating solution A have the highest signal-to-noise ratio and the lowest background signal value when used to detect LC-1 positive samples.

[0116] Example 2:

[0117] (1) Treatment of fluorescently encoded microspheres

[0118] The fluorescently coded magnetic microspheres were shaken in a vortex mixer for 30 seconds and inverted 5 times to mix. 100 μL of the fluorescently coded magnetic microspheres were added to each of the four centrifuge tubes, and the centrifuge tubes were placed on a magnetic rack for magnetic adsorption for 2 minutes. The supernatant was then separated and discarded.

[0119] Add 200 μL of microsphere activation solution to each of the four centrifuge tubes, vortex for 15 s, sonicate in ultrasonic cleaner for 20 s, and magnetically adsorb for 2 min, then separate and discard the supernatant; repeat the washing once more; then add 180 μL of microsphere activation solution to each of the four test tubes, vortex for 15 s, and sonicate for 20 s; to obtain four microsphere resuspensions M.

[0120] Add 10 μL of activator A and 10 μL of activator B to each of the four centrifuge tubes, vortex for 15 s, react at 25 °C in the dark for 20 min, and then perform magnetic adsorption for 2 min. Separate and discard the supernatant.

[0121] Add 200 μL of microsphere coating solution B to each of the four centrifuge tubes, vortex for 15 s, sonicate for 20 s, and magnetically adsorb for 2 min, then separate and discard the supernatant; repeat the washing process twice: add 200 μL of microsphere coating solution B to each of the four centrifuge tubes to resuspend the microspheres, then vortex for 15 s and sonicate for 20 s to obtain four microsphere resuspensions N.

[0122] Add 10 μg ASMA antigen to each of four centrifuge tubes, vortex to mix, and react at room temperature in the dark for 2 h; then magnetically adsorb for 2 min, and separate and discard the supernatant.

[0123] Add 300 μL of microsphere blocking solution A, microsphere blocking solution B, microsphere blocking solution C and microsphere blocking solution D to four centrifuge tubes respectively, vortex for 15 s to mix, sonicate for 20 s, and react at 25 ℃ in the dark for 60 min.

[0124] After sealing, 400 μL of microsphere preservation solution was added to each of the four centrifuge tubes. The tubes were vortexed for 15 s, sonicated for 20 s, and magnetically adsorbed for 20 min. The supernatant was then removed. The washing was repeated twice. The microspheres were resuspended in the four centrifuge tubes by adding 400 μL of microsphere preservation solution and vortexed for 15 s. Four fluorescently encoded microspheres conjugated with ASMA antigen were obtained. The microspheres were stored at 2℃~8℃ in the dark.

[0125] The four fluorescently encoded microspheres conjugated with ASMA antigen were mixed in a ratio of 1:1:1:1 to obtain a microsphere mixture.

[0126] (2) Signal value detection

[0127] The test samples, along with the microsphere mixture and diluted goat anti-human phycoerythrin solution, were tested using iMutli; the cutoff value was set to 25000. The test samples included two ASMA-negative samples and two ASMA antibody-positive samples. The signal values ​​for each sample are shown in Table 3; the signal-to-noise ratio calculation results are shown in Table 4.

[0128] Table 3. Signal values ​​of ASMA antibody detected after treatment with different blocking solutions on fluorescently encoded microspheres.

[0129] Background 3898 3955 2822 4259 3# (Positive) 28251 29801 25630 53022 5# (Positive) 69471 76188 63920 105102 146# (negative) 11612 11807 9924 10535 147# (Negative) 13975 13491 11413 12514

[0130] Table 4. Signal-to-noise ratio of ASMA antibody detection after treatment with different blocking solutions for fluorescently encoded microspheres.

[0131] 3# (Positive) 7.25 7.54 9.08 12.45 5# (Positive) 17.82 19.26 22.65 24.68 146# (negative) 2.98 2.99 3.52 2.47 147# (Negative) 3.59 3.41 4.04 2.94

[0132] As shown in the table above, treating fluorescently encoded microspheres with blocking solution D can improve the signal-to-noise ratio for detecting LC-1 positive samples.

[0133] (3) Yin-Yang Concordance Rate Analysis

[0134] Sixteen samples were collected for testing. The signal values ​​of ASMA antibodies were detected using microspheres treated with four different blocking solutions in step (1) of this embodiment, with a cutoff value of 25000. Qualitative detection of the samples was performed using the Euromon indirect immunofluorescence method. The positive and negative concordance rates between the detection results of each fluorescently coded microsphere and the detection results of the Euromon indirect immunofluorescence method were calculated. The results are shown in Table 5, where “-” indicates ASMA antibody negative, and “+”, “++”, and “+++” indicate ASMA antibody positive.

[0135] Negative concordance rate = Number of samples that tested negative by the Euromon indirect immunofluorescence assay and had a signal value less than 25,000 in qualitative detection ÷ Number of samples that tested negative by the Euromon indirect immunofluorescence assay

[0136] Positive concordance rate = Number of samples that tested positive by the Euromon indirect immunofluorescence assay and had a signal value greater than 25,000 in qualitative detection ÷ Number of samples that tested positive by the Euromon indirect immunofluorescence assay

[0137] Table 5. Concordance rates for ASMA antibody detection after treatment with different blocking solutions on fluorescently encoded microspheres.

[0138]

[0139] As shown in Table 5, compared with the results of the Euromon indirect immunofluorescence assay, the positive and negative concordance rates of the fluorescent immunoglobulins after treatment with blocking solution D were both greater than 85%, indicating higher detection efficiency.

[0140] Example 3:

[0141] (1) The processing steps of fluorescently encoded microspheres are basically the same as those in Example 1, except that the coating solution is microsphere coating solution B, the blocking solution is microsphere blocking solution A, and 10 μg of SLA / LP antigen, Ro52 antigen, AMA-M2 antigen, Sp100 antigen, Gp210 antigen, LKM-1 antigen and CENP-B antigen are added to 7 centrifuge tubes respectively.

[0142] (2) Signal value detection

[0143] The test sample, the above-mentioned microsphere mixture, and the diluted sheep anti-human phycoerythrin solution were tested using iMutli; the test results are shown in Tables 6 and 7 below.

[0144] Table 6. Signal values ​​of different antigen indicators detected by flow cytometry.

[0145] Background 3587 3564 3327 3541 3025 3301 2790 1 45571 4251 161933 4896 47874 14710 59781 2 15789 11751 4571 39450 4988 152470 4457 3 271145 15477 14470 4547 4104 49871 8495 4 7849 40057 6549 11214 12469 6764 14587 5 12487 8873 23785 18754 15771 16784 478431

[0146] Table 7. Signal-to-noise ratio of different antigen indicators detected by flow cytometry

[0147] 1 12.70 1.19 48.67 1.38 15.83 4.46 21.43 2 4.40 3.30 1.37 11.14 1.65 46.19 1.60 3 75.59 4.34 4.35 1.28 1.36 15.11 3.04 4 2.19 11.24 1.97 3.17 4.12 2.05 5.23 5 3.48 2.49 7.15 5.30 5.21 5.08 171.48

[0148] As shown in Tables 6 and 7, the microsphere coating solution and microsphere blocking solution of the present invention can be used to couple various antigens with fluorescent immunomicrospheres to achieve quantitative detection of multiple indicators.

[0149] In summary, by using the microsphere coating solution and microsphere blocking solution of this invention to process fluorescent immunoglobulins and conjugate them with antigens, it is possible to achieve rapid and accurate quantitative detection of multiple antibody indicators, providing a reference for the screening and diagnosis of autoimmune liver diseases in clinical practice and better guiding clinical medication needs.

[0150] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0151] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A kit for conjugating fluorescently encoded microspheres with antigens, characterized in that, The kit includes microsphere coating solution A and microsphere blocking solution D, and also includes one or more of microsphere activating solution, microsphere preservation solution, activator A and activator B; The microsphere coating solution A includes a sodium acetate solution of 0.0736 mol / L to 0.0744 mol / L and an acetic acid solution of 0.177 mol / L to 0.201 mol / L, and the pH value of the microsphere coating solution A is 4.4 to 4.

6. The microsphere blocking solution D comprises, per 100 mL, 1.5 g to 1.7 g Tris, 0.43 g to 0.53 g sucrose, 1.7 g to 1.9 g trehalose, 0.9 g to 1.1 g BSA, 1.9 g to 2.1 g PEG6000, 0.12 g to 0.18 g Triton X-100 and 0.09 mL to 0.11 mL Proclin 300, and the pH value of the microsphere blocking solution D is 7.3 to 7.

5. The activator A includes N-hydroxythiosuccinimide and the microsphere activation solution; The activator B includes 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and the microsphere activation solution.

2. The kit for conjugating fluorescently encoded microspheres with antigens according to claim 1, characterized in that, The microsphere activation solution comprises 0.505g~0.555g of 2-morpholine ethanesulfonic acid and 0.475g~0.525g of sodium 2-morpholine ethanesulfonic acid per 100mL; the pH value of the microsphere activation solution is 5.9~6.

1.

3. The kit for conjugating fluorescently encoded microspheres with antigens according to any one of claims 1 to 2, characterized in that, The kit also includes at least one of microsphere coating solution B, microsphere coating solution C and microsphere coating solution D; The microsphere coating solution B contains 1.1g~1.2g of 2-morpholine ethanesulfonic acid and 0.9g~1.0g of sodium 2-morpholine ethanesulfonate per 100mL, and the pH value of the microsphere coating solution B is 5.95~6.15; The microsphere coating solution C contains 0.09 g to 0.15 g of anhydrous disodium hydrogen phosphate, 0.014 g to 0.034 g of sodium dihydrogen phosphate dihydrate, and 0.08 mL to 0.12 mL of Proclin 300 per 100 mL, and the pH value of the microsphere coating solution C is 7.3 to 7.

5. The microsphere coating solution D contains 0.09 g to 0.15 g of anhydrous sodium carbonate, 0.09 g to 0.15 g of sodium bicarbonate and 0.06 g to 0.12 g of sodium chloride per 100 mL, and the pH value of the microsphere coating solution D is 9.5 to 9.

7.

4. The kit for conjugating fluorescently encoded microspheres with antigens according to any one of claims 1 to 2, characterized in that, The kit also includes at least one of microsphere blocking solution A, microsphere blocking solution B and microsphere blocking solution C; Per 100 mL, the microsphere blocking solution A comprises 0.152 g to 0.189 g of anhydrous disodium hydrogen phosphate, 0.116 g to 0.147 g of sodium dihydrogen phosphate dihydrate, 1.686 g to 1.749 g of sodium chloride, 0.297 g to 0.328 g of BSA, 0.003 g to 0.022 g of Tween 20, 1.844 g to 1.906 g of Glycine, 0.053 mL to 0.072 mL of Proclin 300, and 6.156 mL to 6.344 mL of goat serum, and the pH value of the microsphere blocking solution A is 6.4 to 6.

6. Per 100 mL, the microsphere blocking solution B comprises 4.5 g to 4.7 g Tris, 0.43 g to 0.53 g sucrose, 5.04 g to 5.34 g trehalose, 0.92 g to 1.08 g BSA, 0.03 g to 0.05 g Tween 20, 5.85 g to 6.15 g Glycine, 0.16 g to 0.2 g Triton X-405, and 0.19 mL to 0.21 mL Proclin 300, and the pH value of the microsphere blocking solution B is 7.9 to 8.

1. The microsphere blocking solution C contains 1.5g~1.7g Tris, 0.43g~0.53g sucrose, 0.9g~1.1g BSA, 0.94g~1.06g PEG2000 and 0.19mL~0.21mL Proclin300 per 100mL, and the pH value of the microsphere blocking solution C is 7.3~7.

5.

5. The kit for conjugating fluorescently encoded microspheres with antigens according to any one of claims 1 to 2, characterized in that, The microsphere preservation solution comprises, per 100 mL, 5.9 g to 6.1 g HEPES buffer, 4.9 g to 5.1 g sucrose, 1.95 g to 2.05 g trehalose, 0.25 g to 0.35 g BSA, 1.45 g to 1.55 g mannitol, 0.54 g to 0.66 g disodium EDTA and 0.09 mL to 0.11 mL Proclin 300; the pH of the microsphere preservation solution is 7.3 to 7.

5.

6. A fluorescently encoded microsphere conjugated to an antigen, characterized in that, Prepared using the kit for conjugating fluorescently encoded microspheres with antigens as described in any one of claims 1 to 5.

7. A kit for detecting autoimmune liver disease, characterized in that, It includes at least one fluorescently encoded microsphere conjugated with an antigen as described in claim 6; the antigen is LC-1 antigen, LKM-1 antigen, gp210 antigen, Sp100 antigen, AMA-M2 antigen, CENP-B antigen, ASMA antigen, SLA / LP antigen, or Ro52 antigen.

8. The kit for detecting autoimmune liver disease according to claim 7, characterized in that, The kit also includes sample diluent and one or more fluorescently labeled goat anti-human IgG.

9. The kit for detecting autoimmune liver disease according to claim 8, characterized in that, The sample diluent contains 0.006 g to 0.046 g of anhydrous disodium hydrogen phosphate, 0.332 g to 0.432 g of sodium dihydrogen phosphate dihydrate, 0.28 g to 0.38 g of sodium chloride, and 0.09 mL to 0.11 mL of Proclin 300 per 100 mL.

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