WO2023151281A1 - 一种羧基荧光微球表面活化方法及其应用 - Google Patents

一种羧基荧光微球表面活化方法及其应用 Download PDF

Info

Publication number
WO2023151281A1
WO2023151281A1 PCT/CN2022/121137 CN2022121137W WO2023151281A1 WO 2023151281 A1 WO2023151281 A1 WO 2023151281A1 CN 2022121137 W CN2022121137 W CN 2022121137W WO 2023151281 A1 WO2023151281 A1 WO 2023151281A1
Authority
WO
WIPO (PCT)
Prior art keywords
microsphere
labeling
fluorescent microspheres
carboxyl
carboxy
Prior art date
Application number
PCT/CN2022/121137
Other languages
English (en)
French (fr)
Inventor
张变强
彭洁
王丽娇
严俊
冯涛
盛长忠
粟艳
周泽奇
Original Assignee
丹娜(天津)生物科技股份有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 丹娜(天津)生物科技股份有限公司 filed Critical 丹娜(天津)生物科技股份有限公司
Publication of WO2023151281A1 publication Critical patent/WO2023151281A1/zh

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/02—Use of particular materials as binders, particle coatings or suspension media therefor
    • C09K11/025—Use of particular materials as binders, particle coatings or suspension media therefor non-luminescent particle coatings or suspension media
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56961—Plant cells or fungi
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
    • G01N33/587—Nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
    • G01N2333/38—Assays involving biological materials from specific organisms or of a specific nature from fungi from Aspergillus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
    • G01N2333/39—Assays involving biological materials from specific organisms or of a specific nature from fungi from yeasts
    • G01N2333/40—Assays involving biological materials from specific organisms or of a specific nature from fungi from yeasts from Candida
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
    • G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
    • G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • G01N2400/12—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar
    • G01N2400/24—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar beta-D-Glucans, i.e. having beta 1,n (n=3,4,6) linkages between saccharide units, e.g. xanthan
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
    • G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • G01N2400/38—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence, e.g. gluco- or galactomannans, Konjac gum, Locust bean gum or Guar gum
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms

Definitions

  • the invention belongs to the field of biochemical detection reagents, in particular to a method for activating the surface of carboxyl fluorescent microspheres and an application thereof.
  • Immunochromatography technology As an important branch of POCT (Point of care testing, instant detection), began in the 1980s, and was widely used in various in vitro diagnostic scenarios represented by colloidal gold labeling. In scientific comparison, it also has corresponding shortcomings, mainly in poor precision and insufficient sensitivity; therefore, in the early 20th century, in order to improve the performance of chromatography products, various manufacturers replaced colloidal gold with fluorescent substances, and the performance was greatly improved.
  • Early chromatography used fluorescein, and later gradually changed to fluorescent microspheres, because fluorescent microspheres contain more fluorescent substances, which can effectively amplify the fluorescent signal and improve detection sensitivity.
  • Fluorescence immunochromatography involves the use of microspheres to label antigens and antibodies.
  • the method of microsphere labeling antigen or antibody is mainly adsorption method and covalent coupling method.
  • the characteristics of the microspheres after reaction are detected to improve the sensitivity of the test.
  • the surface of microspheres commonly used in the covalent coupling method is coupled with functional groups - carboxyl groups.
  • one is a one-step method using carbodiimide (EDC) as a crosslinking agent
  • EDC carbodiimide
  • NHS N-hydroxysuccinimide
  • the principle of the one-step method is to first react with the carboxyl group to form an intermediate product, the intermediate product reacts with the amino group, and then remove the intermediate product to realize the coupling between the carboxyl group and the amino group.
  • the principle of the two-step method is to first react with carboxyl groups, then react with NHS to form intermediate products, react intermediate products with amino groups, and then remove intermediate products to realize the coupling of carboxyl groups and amino groups.
  • Polysaccharides are a class of macromolecular substances widely present in a variety of organisms.
  • the capsules and cell walls of bacteria and fungi are essentially composed of polysaccharide structures of specific repeating units. Whether it is a one-step method or a two-step method, the intermediate product reacts with the amino group, but most fungal and bacterial antigens are mainly composed of polysaccharides and contain polysaccharide molecules, so when the above general method is used, the effect of antigen labeling is poor , so it is urgent to find a method suitable for labeling polysaccharide antigens.
  • the present invention aims to overcome the defects in the prior art, and proposes a method for surface activation of carboxy fluorescent microspheres and its application.
  • the present invention provides a method for surface activation of carboxy fluorescent microspheres, comprising the steps of:
  • microsphere activation buffer I containing 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (DMTMM) and pimelic acid diacyl Hydrazine (PDH) microsphere activation buffer II;
  • step S3 Add the microsphere activation buffer I and microsphere activation buffer II prepared in step S2 to the carboxyl fluorescent microspheres obtained in step S1 according to the mass ratio of DMTMM and PDH at a ratio of 1:(2-4) to avoid light After 1-24 hours of rotation activation, the surface-activated carboxyl fluorescent microsphere solution can be obtained.
  • the volume ratio (V/V) of the carboxyl fluorescent microspheres in the step S1 to the MES buffer is 1:(1-20), more preferably 1:(4-10).
  • the microsphere activation buffer I and the microsphere activation buffer II are added to the carboxy fluorescent microspheres obtained in the step S1 according to the mass ratio of DMTMM and PDH at a ratio of 1:2.
  • the volume ratio (V/V) of the carboxyl fluorescent microspheres to the total amount of microsphere activation buffer I and microsphere activation buffer II is 1: (0.01-5), more preferably 1: (0.05-1 ).
  • the concentration of DMTMM in the microsphere activation buffer I is 10-500 mg/mL; more preferably 50-200 mg/mL.
  • the concentration of PDH in the microsphere activation buffer II is 10-500 mg/mL; more preferably 50-200 mg/mL.
  • the particle size range of the carboxyl fluorescent microspheres is 100-300 nm, more preferably 100-200 nm.
  • the concentration of the MES buffer is 10-200 mM, and the pH value is 4.5-9.0, more preferably, the concentration of the MES buffer is 50 mM, and the pH value is 6.0.
  • the present invention also provides a surface-activated carboxyl fluorescent microsphere obtained by the above method.
  • the present invention also provides a method for labeling polysaccharide antigens with the above-mentioned surface-activated carboxy fluorescent microspheres, the method comprising the following steps:
  • the blocking solution in the step b is 50mM MES buffer solution containing 1-10% (w/v) BSA, and the pH value is 6.0.
  • the blocking solution in step b contains 10% (w/v) BSA, and the blocking time is 1-2 hours.
  • the present invention also provides the polysaccharide antigen labeled with carboxy fluorescent microspheres obtained by the above method.
  • the present invention also provides a fluorescent immunochromatographic test strip comprising the polysaccharide antigen labeled with carboxy fluorescent microspheres.
  • the present invention has the following advantages:
  • the present invention uses DMTMM and PDH in combination to activate carboxy fluorescent microspheres, and the activated carboxy fluorescent microspheres can be efficiently coupled with polysaccharide antigens, which overcomes the one-step and two-step methods of labeling antigen coupling in the prior art. The disadvantage of poor connection effect.
  • the polysaccharide antigen labeling method of the present invention has mild reaction conditions, and the entire labeling process can be completed by using MES buffer.
  • the fluorescent immunochromatography test strip provided by the present invention uses an indirect method to detect antibodies, and has good sensitivity, specificity and stability.
  • test reagents used in the following examples are conventional biochemical reagents; the experimental methods, unless otherwise specified, are conventional methods.
  • microspheres Take 13 1.5mL EP tubes, add 100 ⁇ L carboxy fluorescent microspheres (1% solid content, hereinafter referred to as microspheres) respectively, add 400 ⁇ L of 50mM MES buffer respectively, and pipette Mix well, and then ultrasonically disperse with an ultrasonic crusher.
  • DTMM 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride
  • PDH pimelic acid dihydrazide
  • ADH adipic acid dihydrazide
  • Microsphere activation the microsphere solution was added dropwise with different activation solutions shown in Table 1 while shaking. Wrap it in tinfoil to avoid light, and activate it for 1 hour at room temperature with a rotator at 40r/min.
  • Microsphere washing centrifuge at 10°C for 20 min at 18,000 g, discard the supernatant, add 500 ⁇ L of MES buffer solution to the microsphere pellet, blow and mix with a pipette, and then ultrasonically disperse with a sonicator.
  • Microsphere labeling Add the antigen shown in the following table 2 to each tube of microsphere solution, and add the antigen concentration as shown in the table below, and mix well after adding the antigen. Rotate at room temperature at 40r/min and avoid light for overnight reaction (about 16h).
  • Microsphere sealing 55 ⁇ L of blocking solution was added to each tube of microspheres, and the reaction was carried out at room temperature with a rotator at 40 r/min in the dark for 2 hours.
  • Microsphere washing Centrifuge at 18,000 g at 10°C for 20 min, discard the supernatant, add 1 mL of PBST buffer to the microsphere pellet, and pipette to mix. Repeat the above washing operation two more times.
  • test kit includes the following components and preparation methods:
  • Microsphere-labeled Candida mannan the labeling amount is: 8 ⁇ g/mL, the labeling reaction time is 3 hours, and the blocking reaction time is 16 hours.
  • Microsphere-labeled chicken IgY antibody Labeled by NHS+EDC conventional method, labeling amount: 5 ⁇ g/mL, labeling reaction time 3h, blocking reaction time 16h.
  • Fluorescent pad preparation Chicken IgY-microspheres and Candida mannan-microspheres were mixed at a ratio of 1:1. After mixing, dilute 50 times with reconstitution solution. Microsphere working solution spray volume: 8 ⁇ L/cm. Drying method: drying at 30 ⁇ 37°C for 1-4h.
  • Detection line coating the coating diluent is 0.1M PB solution with pH 7.4, and the coating concentration is 2mg/mL goat anti-human IgG antibody.
  • the coating diluent is 0.1M PB solution with pH 7.4, and the coating concentration is 2mg/mL goat anti-chicken IgY antibody.
  • NC film drying process the drying method is 1-4h at 30-37°C.
  • sample pad treatment solution 0.1M Tris, 1% BSA, 0.5% PVP K-30, pH8.5.
  • Sample pad drying parameters 30-37 °C drying 1-4h.
  • Sample diluent formula 0.1M PB buffer containing 1% sodium caseinate, 0.05% Tween-20, 0.05% Proclin 300.
  • Reagent reading time is 15-20 minutes
  • Reagent reaction conditions are room temperature of 10-40°C and humidity ⁇ 80%.
  • Tris 100.00g of sucrose, 50.00g of trehalose, and 10.00g of BSA into a beaker, add 800mL of process water, stir and dissolve with a magnetic stirrer, add 0.1M HCl to adjust the pH to 8.5, and mix well, Dilute to 1000mL to obtain a complex solution.
  • Ultrasonic dispersion of microspheres Take 1mg of microspheres, add 50mM MES buffer, and disperse by ultrasonic.
  • Microsphere activation Add 30 ⁇ L of 10 mg/mL EDC solution and 30 ⁇ L of 10 mg/mL NHS solution dropwise to the microsphere solution while shaking, wrap it in tin foil to avoid light, and activate at room temperature for 20 minutes with a rotator at 40 r/min.
  • Microsphere washing centrifuge at 10°C at 18,000 g for 20 min, discard the supernatant, add 1000 ⁇ L of 25 mM HEPES buffer solution to the microsphere pellet, blow and mix with a pipette, and then ultrasonically disperse with a sonicator.
  • Microsphere labeling Add chicken IgY antibody solution for labeling, the labeling concentration is 5 ⁇ g/mL, and it is required to avoid light for overnight reaction.
  • Microsphere sealing Add blocking solution to the microspheres, wrap them in tin foil to avoid light, and rotate them at room temperature for 2 hours.
  • Washing of microspheres wash away chicken IgY antibodies that did not participate in the reaction with 25mM HEPES buffer, and wash twice in total.
  • test kit includes the following components and preparation methods:
  • Microsphere-labeled Aspergillus galactomannan labeling amount: 15 ⁇ g/mL, labeling reaction time 3 hours, blocking reaction time 16 hours.
  • Microsphere-labeled chicken IgY antibody Labeled by NHS+EDC conventional method, labeling amount: 5 ⁇ g/mL, labeling reaction time 3h, blocking reaction time 16h.
  • the chicken IgY antibody-microsphere complex and the Aspergillus galactomannan-microsphere complex were mixed according to 1:1 equal volume, and the microsphere complex was diluted 50 times with the reconstitution solution.
  • Microsphere working solution spray volume 8 ⁇ L/cm. Drying method: drying at 30 ⁇ 37°C for 1-4h.
  • the coating diluent is 0.1M PB solution with pH 7.4, and the coating concentration is 2mg/mL goat anti-human IgG antibody.
  • the coating diluent is 0.1M PB solution with pH 7.4, and the coating concentration is 2mg/mL goat anti-chicken IgY antibody.
  • the drying method is to dry at 30-37°C for 1-4h.
  • sample pad treatment solution 0.1M Tris, 1% BSA, 0.5% PVP K-30, pH8.5.
  • Sample pad drying parameters 30-37 °C drying 1-4h.
  • Sample diluent formula 0.1M PB buffer containing 1% BSA, 0.05% Tween-20, 0.05% Proclin300.
  • Reagent reading time is 15-20 minutes
  • Reagent reaction conditions are room temperature of 10-40°C and humidity ⁇ 80%.
  • the preparation method of the kit in this example is basically the same as that in Example 1, and will not be repeated here.
  • Candida albicans mannan-carboxyl fluorescent microspheres
  • the Candida mannan IgG antibody detection test strip was prepared, and the labeling effect was verified by testing some internal reference products of the enterprise. The test results are shown in the table below:
  • the concentration value ⁇ 135AU/mL is negative, and the concentration value ⁇ 135AU/mL is positive.
  • the concentration value ⁇ 135AU/mL is negative, and the concentration value ⁇ 135AU/mL is positive.
  • the concentration value ⁇ 135AU/mL is negative, and the concentration value ⁇ 135AU/mL is positive.
  • the Aspergillus galactomannan IgG antibody detection test strip was prepared, and the labeling effect was verified by testing some internal reference products of the enterprise. The test results are shown in the table below:
  • the concentration value ⁇ 135AU/mL is negative, and the concentration value ⁇ 135AU/mL is positive.
  • the concentration value ⁇ 135AU/mL is negative, and the concentration value ⁇ 135AU/mL is positive.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Analytical Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Biotechnology (AREA)
  • Botany (AREA)
  • Nanotechnology (AREA)
  • Mycology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Materials Engineering (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Optics & Photonics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

一种羧基荧光微球表面活化方法及其应用，表面活化方法包括如下步骤：S1：将羧基荧光微球置于MES缓冲液中超声分散；S2：分别配制含有DMTMM的微球活化缓冲液Ⅰ和含有PDH的微球活化缓冲液Ⅱ；S3：将步骤S2制备得到的微球活化缓冲液Ⅰ和微球活化缓冲液Ⅱ按照DMTMM和PDH质量比为1:(2-4)的比例加入到步骤S1得到的羧基荧光微球中避光旋转活化1-24h即得到表面活化的羧基荧光微球溶液，通过将DMTMM和PDH组合使用于活化羧基荧光微球，活化后的羧基荧光微球能够与抗原高效偶联，克服了现有技术中的一步法和两步法标记抗原偶联效果较差的缺点。

Description

一种羧基荧光微球表面活化方法及其应用 技术领域
本发明属于生化检测试剂领域，尤其是涉及一种羧基荧光微球表面活化方法及其应用。
背景技术
对某些抗原或特异性蛋白进行检测时，通常会应用到免疫标记技术。免疫层析技术作为POCT(Point of care testing，即时检测)的重要分支始于20世纪80年代，以胶体金标记作为代表大量应用于各种体外诊断场景，但是与酶联免疫/化学发光这些方法学比较其也有相应的短板，主要表现在精密度差，灵敏度不足；因此在二十世纪初各厂家为改善层析产品性能将胶体金更换为荧光物质，性能大幅提升。早期层析使用荧光素，后来逐渐转变为荧光微球，因为荧光微球含有更多的荧光物质，可有效放大荧光信号，提高检测灵敏度。荧光免疫层析技术中涉及用到微球标记抗原和抗体。微球标记抗原或抗体的方法主要是吸附法和共价偶联法，目的是将待测物质相对应的抗体或抗原包被在微球上，通过待测物质与包被了相应抗原抗体的微球反应后的特性进行检测提高试验的敏感性。共价偶联法中常用的微球表面偶联有功能基团-羧基。
目前羧基微球偶联氨基的通用方法有两种：一是以碳二亚胺(EDC)为交联剂的一步法，二是以碳二亚胺(EDC)与N-羟基琥珀酰亚胺(NHS)为交联剂的两步法。一步法的原理是首先与羧基反应形成中间产物，中间产物与氨基反应，然后脱去中间产物，实现羧基与氨基的偶联。两步法的原理是首先与羧基反应，然后与NHS反应形成中间产物，中间产物与氨基反应，然后脱去中间产物，实现羧基与氨基的偶联。
多糖是一类广泛存在于多种生物体内的高分子物质。而细菌和真菌的荚膜和细胞壁本质上是由特定重复单位的多糖结构组成的。无论是一步法还是两步法，都是中间产物与氨基进行反应，但是大部分真菌和细菌类抗原主要由多糖类物质组成，含有多糖分子，因此采用上述通用方法时，抗原标记效果较差，因此亟待需要找到一种适用于标记多糖类抗原的方法。
发明内容
有鉴于此本发明旨在克服现有技术中的缺陷，提出一种羧基荧光微球表面活化方法及其应用。
为达到上述目的，本发明的技术方案是这样实现的：
第一方面，本发明提供一种羧基荧光微球表面活化方法，包括如下步骤：
S1：将羧基荧光微球置于MES缓冲液中超声分散；
S2：分别配制含有4-(4,6-二甲氧基三嗪-2-基)-4-甲基吗啉盐酸盐(DMTMM)的微球活化缓冲液Ⅰ和含有庚二酸二酰肼(PDH)的微球活化缓冲液Ⅱ；
S3：将步骤S2制备得到的微球活化缓冲液Ⅰ和微球活化缓冲液Ⅱ按照DMTMM和PDH质量比为1:(2-4)的比例加入到步骤S1得到的羧基荧光微球中避光旋转活化1-24h即得到表面活化的羧基荧光微球溶液。
优选的，所述步骤S1的羧基荧光微球与MES缓冲液的体积比(V/V)为1：(1-20)，更优选1：(4-10)。
优选的，所述步骤S3中微球活化缓冲液Ⅰ和微球活化缓冲液Ⅱ按照DMTMM和PDH质量比为1:2的比例加入到步骤S1得到的羧基荧光微球中。
优选的，所述羧基荧光微球与微球活化缓冲液Ⅰ和微球活化缓冲液Ⅱ总量的体积比(V/V)为1：(0.01-5)，更优选1：(0.05-1)。
优选的，所述微球活化缓冲液Ⅰ中DMTMM的浓度为10-500mg/mL；更优选为50-200mg/mL。
优选的，所述微球活化缓冲液Ⅱ中PDH的浓度为10-500mg/mL；更优选为50-200mg/mL。
优选的，所述羧基荧光微球的粒径范围为100-300nm，更优选为100-200nm。
优选的，所述MES缓冲液的浓度为10-200mM，pH值为4.5-9.0，更优选的，所述MES缓冲液的浓度为50mM，pH值为6.0。
第二方面，本发明还提供一种由上述方法得到的表面活化的羧基荧光微球。
第三方面，本发明还提供了一种应用上述表面活化的羧基荧光微球标记多糖类抗原的方法，所述方法包括如下步骤：
a、向表面活化的羧基荧光微球溶液中添加浓度为5-30μg/mL的抗原溶液进行标记，避光反应1-24h；
b、加入含有1-10％(w/v)BSA的封闭液避光旋转混匀，封闭时间为1-24h；
c、用磷酸盐缓冲液洗去未参与反应的抗原，反复清洗2-5遍；
d、将微球标记物保存在微球复溶液中即得到羧基荧光微球标记的多糖类抗原。
优选的，所述步骤b中封闭液为含1-10％(w/v)BSA的50mM MES缓冲液，pH值为6.0。
更优选的，所述步骤b中封闭液中含有10％(w/v)BSA，封闭时间为1-2h。
第四方面，本发明还提供了上述方法得到的羧基荧光微球标记的多糖类抗原。
第五方面，本发明还提供了包含上述羧基荧光微球标记的多糖类抗原的荧光免疫层析试纸条。
相对于现有技术，本发明具有以下优势：
(1)本发明将DMTMM和PDH组合使用用于活化羧基荧光微球，活化后的羧基荧光微球能够与多糖类抗原高效偶联，克服了现有技术一步法和两步法标记抗原偶联效果差的缺点。
(2)本发明的多糖类抗原标记方法反应条件温和，采用MES缓冲液即可完成整个标记过程。
(3)本发明提供的荧光免疫层析试纸条，采用间接法检测抗体，具有较好的敏感性、特异性和稳定性。
具体实施方式
除有定义外，以下实施例中所用的技术术语具有与本发明创造所属领域技术人员普遍理解的相同含义。以下实施例中所用的试验试剂，如无特殊说明，均为常规生化试剂；所述实验方法，如无特殊说明，均为常规方法。
下面结合实施例来详细说明本发明创造。
实施例1：羧基荧光微球的活化效果比较
1、羧基荧光微球标记多糖抗原
(1)微球超声分散：取13个1.5mL EP管，分别加入100μL羧基荧光微球(1％固含量，下简称微球)，分别补加400μL的50mM MES缓冲液，移液器吹吸混匀，再用超声破碎机超声分散。
(2)如下表1所示分别配制4-(4,6-二甲氧基三嗪-2-基)-4-甲基吗啉盐酸盐(DMTMM)、庚二酸二酰肼(PDH)、己二酸二酰肼(ADH)三种活化溶液，这些溶液皆为现配现用。
(3)微球活化：微球溶液一边振荡一边逐滴加入如下表1所示的不同活化溶液。锡箔纸包裹避光，室温旋转仪40r/min活化1h。
(4)微球洗涤：10℃18000g离心20min，弃上清，微球沉淀分别加入500μL的MES缓冲液，移液器吹吸混匀，再用超声破碎机超声分散。
(5)微球标记：向每管微球溶液中分别加入如下表2所示抗原，加入抗原浓度如下表所示，加入抗原后混匀。室温旋转仪40r/min避光过夜反应(约16h)。
(6)微球封闭：每管微球加入55μL封闭液，室温旋转仪40r/min避光反应2h。
(7)微球洗涤：18000g 10℃离心20min，弃上清，微球沉淀加入1mL PBST缓冲液，移液器吹吸混匀。再重复两遍上述洗涤操作。
(8)微球保存：沉淀用200μL微球复溶液复溶，再用超声破碎机超声分散，然后置于4℃冰箱避光保存。
表1羧基荧光微球活化方案
Figure PCTCN2022121137-appb-000001
表2羧基荧光微球标记抗原名称及标记浓度
序号 抗原名称 标记浓度
1 白色念珠菌甘露聚糖 8μg/mL
2 曲霉半乳甘露聚糖 15μg/mL
3 隐球菌荚膜多糖 10μg/mL
4 1，3-β-D-葡聚糖 10μgmL
5 光滑念珠菌甘露聚糖 10μg/mL
2、微球标记后效果验证
(1)白色念珠菌甘露聚糖-羧基荧光微球
1)羧基荧光微球标记后，将鸡IgY-微球和白色念珠菌甘露聚糖-微球按1∶1混合，混合后使用复溶液稀释50倍，喷涂荧光垫。T线用2mg/mL羊抗人IgG抗体划线，待干燥后，组装，切条。将人源化白色念株菌甘露聚糖IgG抗体用样本稀释液按4000倍稀释后进行检测。比较荧光值，荧光值越高，表明微球标记效果越好。
2)羧基荧光微球标记后，将鸡IgY-微球和白色念珠菌甘露聚糖-微球按1∶1混合，混合后使用复溶液稀释50倍，喷涂荧光垫。T线用2mg/mL羊抗人IgG抗体划线，待干燥后，组装，切条。检测部分企业内部参考品，37℃放置10天，检测部分企业内部参考品。筛选 阳性样本(P1、P4、P7、P9、L2、L3)加速前后浓度值的平均相对偏差(相对偏差＝|加速前浓度值-加速后浓度值|÷加速前浓度值)较小且阴阳性参考品符合率较高的微球活化方案。
(2)曲霉半乳甘露聚糖-羧基荧光微球
1)羧基荧光微球标记后，将鸡IgY-微球和曲霉半乳甘露聚糖-微球按1:1混合，混合后使用复溶液稀释50倍，喷涂荧光垫。T线用2mg/mL羊抗人IgG抗体划线，待干燥后，组装，切条。将人源化曲霉半乳甘露聚糖IgG抗体用样本稀释液按4000倍稀释后进行检测。比较荧光值，荧光值越高，表明微球标记效果越好。
2)羧基荧光微球标记后，将鸡IgY-微球和曲霉半乳甘露聚糖-微球按1:1混合，混合后使用复溶液稀释50倍，喷涂荧光垫。T线用2mg/mL羊抗人IgG抗体划线，待干燥后，组装，切条。检测部分企业内部参考品，37℃放置10天，检测部分企业内部参考品。筛选阳性样本(P1、P4、P7、P9、L2、L3)加速前后浓度值的平均相对偏差(相对偏差＝|加速前浓度值-加速后浓度值|÷加速前浓度值)较小且阴阳性参考品符合率较高的微球活化方案。
(3)隐球菌荚膜多糖-羧基荧光微球
羧基荧光微球标记后，将鸡IgY-微球和隐球菌荚膜多糖-微球按1:1混合，混合后使用复溶液稀释50倍，喷涂荧光垫。T线用2mg/mL羊抗鼠IgG抗体划线，待干燥后，组装，切条。将隐球菌荚膜多糖抗体(鼠源)用0.1M PBS按2000倍稀释后进行检测。比较荧光值，荧光值越高，表明微球标记效果越好。
(4)1,3-β-D-葡聚糖-羧基荧光微球
羧基荧光微球标记后，将鸡IgY-微球和1,3-β-D-葡聚糖-微球按1:1混合，混合后使用复溶液稀释50倍，喷涂荧光垫。T线用2mg/mL羊抗兔IgG抗体划线，待干燥后，组装，切条。将1,3-β-D-葡聚糖抗体(兔源)用0.1M PBS按2000倍稀释后进行检测。比较荧光值，荧光值越高，表明微球标记效果越好。
(5)光滑念珠菌甘露聚糖-羧基荧光微球
羧基荧光微球标记后，将鸡IgY-微球和光滑念珠菌甘露聚糖-微球按1:1混合，混合后使用复溶液稀释50倍，喷涂荧光垫。T线用2mg/mL羊抗兔IgG抗体划线，待干燥后，组装，切条。将光滑念株菌甘露聚糖抗体(兔源)用0.1M PBS按2000倍稀释后进行检测。比较荧光值，荧光值越高，表明微球标记效果越好。
实施例2制备念珠菌甘露聚糖IgG抗体检测试剂盒(荧光免疫层析法)
该试剂盒包括以下组分和制备方法：
1、微球标记抗原抗体：
微球标记念珠菌甘露聚糖：标记量为：8μg/mL，标记反应时间3h，封闭反应时间16h。
微球标记鸡IgY抗体：采用NHS+EDC常规方法标记，标记量：5μg/mL，标记反应时间3h，封闭反应时间16h。
2、荧光垫制备：鸡IgY-微球和念珠菌甘露聚糖-微球1:1混合。混合后使用复溶液稀释50倍。微球工作液喷涂量：8μL/cm。干燥方式：30～37℃烘干1-4h。
3、检测线包被：包被稀释液为pH 7.4的0.1M PB溶液，包被浓度为2mg/mL羊抗人IgG抗体。
4、质控线包被：包被稀释液为pH 7.4的0.1M PB溶液，包被浓度为2mg/mL羊抗鸡IgY抗体。
5、NC膜干燥工艺：干燥方式为30-37℃烘干1-4h。
6、样品垫
样品垫处理液组分：0.1M Tris、1％BSA、0.5％PVP K-30，pH8.5。
样品垫干燥参数：30-37℃烘干1-4h。
7、反应体系
样本稀释液液配方：含1％酪蛋白钠、0.05％Tween-20、0.05％Proclin 300的0.1M PB的缓冲液。
样本稀释倍数：200倍；
加样量：90～100μL；
试剂读数时间为15～20min；
试剂反应条件为10～40℃的室温条件，湿度<80％。
本实施例念珠菌甘露聚糖IgG抗体检测试剂盒(荧光免疫层析法)的制备方法中，除以下试剂外，其余均按照常规方法制备。
1)配制复溶液
称取Tris 2.42g、蔗糖100.00g、海藻糖50.00g、BSA 10.00g放入烧杯中，加入800mL工艺用水，使用磁力搅拌器搅拌溶解后，加入0.1M HCl调节pH至8.5，充分混匀后，定容至1000mL，即得复溶液。
2)配制样品垫处理液
称取Tris 12.11g、BSA 10g、PVP K-30 5g放入烧杯中，加入800mL工艺用水，使用磁力搅拌器搅拌溶解后，加入0.1M HCl调节pH至8.5，充分混匀后，加入工艺用水定容至1000mL，即得样品垫处理液。
3)配制样本稀释液
称取10g酪蛋白钠，量取500μL Tween-20，500μL Proclin 300，加入800mL 0.1M PB，使用磁力搅拌器搅拌溶解后，加入0.1M PB定容至1000mL，即得样本稀释液。
4)0.01M PBS缓冲液配制
称取氯化钠8.00g，氯化钾0.20g，磷酸氢二钠1.44g，磷酸二氢钾0.24g，加入800mL工艺用水，充分混匀后，定容至1000mL即可。
5)PBST缓冲液配制
量取800mL 0.01M PBS，加入500μL Tween 20，充分混匀后，定容至1000mL，即得PBST缓冲液。
6)鸡IgY抗体微球标记方法
a.微球超声分散：取1mg微球，加入50mM MES缓冲液，超声分散。
b.微球活化：微球溶液一边振荡一边逐滴加入30μL 10mg/mL EDC溶液、30μL10mg/mL NHS溶液，锡箔纸包裹避光，室温旋转仪40r/min活化20min。
c.微球洗涤：10℃18000g离心20min，弃上清，微球沉淀加入1000μL 25mM HEPES缓冲液，移液器吹吸混匀，再用超声破碎机超声分散。
d.微球标记：添加鸡IgY抗体溶液进行标记，标记浓度为5μg/mL，要求避光过夜反应。
e.微球封闭：微球中加入封闭液，锡箔纸包裹避光，室温旋转封闭2h。
f.微球洗涤：用25mM HEPES缓冲液洗去未参与反应的鸡IgY抗体，共清洗2遍。
g.标记物保存：将微球标记物保存在微球复溶液中。
实施例3制备曲霉半乳甘露聚糖IgG抗体检测试剂盒(荧光免疫层析法)
该试剂盒包括以下组分和制备方法：
1、微球标记抗原抗体
微球标记曲霉半乳甘露聚糖：标记量为：15μg/mL，标记反应时间3h，封闭反应时间16h。
微球标记鸡IgY抗体：采用NHS+EDC常规方法标记，标记量：5μg/mL，标记反应时间3h，封闭反应时间16h。
2、荧光垫制备
鸡IgY抗体-微球复合物和曲霉半乳甘露聚糖-微球复合物按照1:1等体积混合，用复溶液将微球复合物50倍稀释。微球工作液喷涂量：8μL/cm。干燥方式：30～37℃烘干1-4h。
3、检测线包被
包被稀释液为pH 7.4的0.1M PB溶液，包被浓度为2mg/mL羊抗人IgG抗体。
4、质控线包被
包被稀释液为pH 7.4的0.1M PB溶液，包被浓度为2mg/mL羊抗鸡IgY抗体。
5、NC膜干燥工艺
干燥方式为30-37℃烘干1-4h。
6、样品垫
样品垫处理液组分：0.1M Tris、1％BSA、0.5％PVP K-30，pH8.5。
样品垫干燥参数：30-37℃烘干1-4h。
7、反应体系
样本稀释液液配方：含1％BSA、0.05％Tween-20、0.05％Proclin300的0.1M PB的缓冲液。
样本稀释倍数：200倍。
加样量：90～100μL。
试剂读数时间为15～20min；
试剂反应条件为10～40℃的室温条件，湿度<80％。
本实施例试剂盒的制备方法与实施例1中的基本一致，不再赘述。
检测结果如下：
1、白色念珠菌甘露聚糖-羧基荧光微球
(1)白色念珠菌甘露聚糖-羧基荧光微球标记检测结果
按照前述的方法检测荧光值，结果如下：
表3白色念珠菌甘露聚糖-羧基荧光微球标记荧光值检测结果
Figure PCTCN2022121137-appb-000002
从上表的数据可以看出，组合4-7的荧光值相对于组合1-3和8-13较高，其中组合5的荧光值最高。
(2)念珠菌甘露聚糖IgG抗体检测试剂盒(荧光免疫层析法)
1)念珠菌甘露聚糖IgG抗体检测试剂盒(荧光免疫层析法)抗原标记效果验证
按照上述研究资料所述的方案标记念珠菌甘露聚糖抗原后，制备念珠菌甘露聚糖IgG抗体检测试纸条，通过检测部分企业内部参考品，验证标记效果。检测结果如下表所示：
表4不同方法标记念珠菌甘露聚糖检测结果
Figure PCTCN2022121137-appb-000003
备注：浓度值＜135AU/mL为阴性，浓度值≥135AU/mL为阳性。
表5不同方法标记念珠菌甘露聚糖检测结果(37℃放置10天)
Figure PCTCN2022121137-appb-000004
备注：浓度值＜135AU/mL为阴性，浓度值≥135AU/mL为阳性。
表6不同方法标记念珠菌甘露聚糖检测结果(加速前后浓度值的变化)
Figure PCTCN2022121137-appb-000005
备注：上述数据为加速前后浓度值的相对偏差，相对偏差＝|加速前浓度值-加速后浓度值|÷加速前浓度值。相对偏差越小表明试纸条稳定性越好。
从上表可知，组合5加速前后浓度值的相对偏差最小，稳定性最好，且加速前后阴阳性参考品的符合率均为100％。
2)使用组合5的方案，进行念珠菌甘露聚糖IgG抗体检测试剂盒(荧光免疫层析法)性能验证，验证结果见下表。
表7念珠菌甘露聚糖IgG抗体检测试剂盒验证结果
Figure PCTCN2022121137-appb-000006
备注：浓度值＜135AU/mL为阴性，浓度值≥135AU/mL为阳性。
从上述测定结果可知，按照上述研究确定生产工艺及反应体系制备的试剂，检测企业内部参考品全部合格，符合预期结果。
2、曲霉半乳甘露聚糖-羧基荧光微球
(1)曲霉半乳甘露聚糖-羧基荧光微球标记检测结果
按照前述的方法检测荧光值，结果见下表：
表8曲霉半乳甘露聚糖-羧基荧光微球标记荧光值检测结果
Figure PCTCN2022121137-appb-000007
从上表的数据可以看出，组合4-7的荧光值相对于组合1-3和8-13较高，其中组合5的荧光值最高。
(2)曲霉半乳甘露聚糖IgG抗体检测试剂盒(荧光免疫层析法)
1)曲霉半乳甘露聚糖IgG抗体检测试剂盒(荧光免疫层析法)抗原标记效果验证
按照上述研究资料所述的方案标记曲霉半乳甘露聚糖抗原后，制备曲霉半乳甘露聚糖IgG抗体检测试纸条，通过检测部分企业内部参考品，验证标记效果。检测结果如下表所示：
表9不同方法标记曲霉半乳甘露聚糖检测结果
Figure PCTCN2022121137-appb-000008
备注：浓度值＜135AU/mL为阴性，浓度值≥135AU/mL为阳性。
表10不同方法标记曲霉半乳甘露聚糖检测结果(37℃放置10天)
Figure PCTCN2022121137-appb-000009
备注：浓度值＜135AU/mL为阴性，浓度值≥135AU/mL为阳性。
表11不同方法标记曲霉半乳甘露聚糖检测结果(加速前后浓度值的变化)
Figure PCTCN2022121137-appb-000010
备注：上述数据为加速前后浓度值的相对偏差，相对偏差＝|加速前浓度值-加速后浓度值|÷加速前浓度值。相对偏差越小表明试纸条稳定性越好。
从上表可知，组合5加速前后浓度值的相对偏差最小，稳定性最好，且加速前后阴阳性参考品的符合率均为100％。因此选择DMTMM和PDH质量为1：2的组合。
2)使用组合5的方案，进行曲霉半乳甘露聚糖IgG抗体检测试剂盒(荧光免疫层析法)性能验证，验证结果见下表：
表12曲霉半乳甘露聚糖IgG抗体检测试剂盒验证结果
Figure PCTCN2022121137-appb-000011
从上述测定结果可知，按照上述研究确定生产工艺及反应体系制备的试剂，检测企业内部参考品全部合格，符合预期结果。
(3)隐球菌荚膜多糖-羧基荧光微球
按照前面的方法检测荧光值，结果如下：
表13隐球菌荚膜多糖-羧基荧光微球荧光值检测结果
Figure PCTCN2022121137-appb-000012
从上表的数据可以看出，组合4-6的荧光值相对于组合1-3和9-13较高，其中组合5的荧光值最高。
(4)1,3-β-D-葡聚糖-羧基荧光微球
按照前面的方法检测荧光值，结果如下：
表14 1,3-β-D-葡聚糖-羧基荧光微球荧光值检测结果
Figure PCTCN2022121137-appb-000013
从上表的数据可以看出，组合4-6的荧光值相对于组合1-3和9-13较高，其中组合5的荧光值最高。
(5)光滑念珠菌甘露聚糖-羧基荧光微球
按照前面的方法检测荧光值，结果如下：
表15光滑念珠菌甘露聚糖-羧基荧光微球荧光值检测结果
Figure PCTCN2022121137-appb-000014
从上表的数据可以看出，组合5-7的荧光值相对于组合1-4和8-13较高，其中组合5的荧光值最高。
试验结论：从表1-15的数据结果可以看出DMTMM与PDH复配进行羧基荧光微球的活化，活化后的羧基荧光微球标记多糖类抗原效果优于单一组分和DMTMM与ADH复配，可见DMTMM与PDH具有协同作用，同时还可以看出，DMTMM和PDH质量比为1：2时，标记的多糖类抗原荧光值最高，且标记的多糖类抗原制得的IgG抗体检测试剂盒(荧光免疫层析法)具有较好的敏感性、特异性和稳定性。
以上所述仅为本发明创造的较佳实施例而已，并不用以限制本发明创造，凡在本发明创造的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明创造的保护范围之内。

Claims (9)

  1. 一种羧基荧光微球标记多糖类抗原的方法，其特征在于：包括羧基荧光微球活化步骤以及标记多糖类抗原步骤；
    其中，羧基荧光微球活化步骤具体包括如下步骤：
    S1：将羧基荧光微球置于MES缓冲液中超声分散；
    S2：分别配制含有DMTMM的微球活化缓冲液Ⅰ和含有PDH的微球活化缓冲液Ⅱ；
    S3：将步骤S2制备得到的微球活化缓冲液Ⅰ和微球活化缓冲液Ⅱ按照DMTMM和PDH质量比为1:(2-4)的比例加入到步骤S1得到的羧基荧光微球中避光旋转活化1-24h即得到表面活化的羧基荧光微球溶液；
    标记多糖类抗原步骤具体包括如下步骤：
    a、向表面活化的羧基荧光微球溶液中添加浓度为5-30μg/mL的抗原溶液进行标记，避光反应1-24h；
    b、加入含有1-10％(w/v)BSA的封闭液避光旋转混匀，封闭时间为1-24h；
    c、用磷酸盐缓冲液洗去未参与反应的抗原，反复清洗2-5遍；
    d、将微球标记物保存在微球复溶液中即得到羧基荧光微球标记的多糖类抗原。
  2. 根据权利要求1所述的羧基荧光微球标记多糖类抗原的方法，其特征在于：所述步骤S1中所述羧基荧光微球与MES缓冲液的体积比(V/V)为1：(1-20)。
  3. 根据权利要求1所述的羧基荧光微球标记多糖类抗原的方法，其特征在于：所述步骤S3中微球活化缓冲液Ⅰ和微球活化缓冲液Ⅱ按照DMTMM和PDH质量比为1:2的比例加入到步骤S1得到的羧基荧光微球中；所述羧基荧光微球与微球活化缓冲液Ⅰ和微球活化缓冲液Ⅱ总量的体积比(V/V)为1：(0.01-5)。
  4. 根据权利要求1所述的羧基荧光微球标记多糖类抗原的方法，其特征在于：所述步骤S2中所述微球活化缓冲液Ⅰ中DMTMM的浓度为10-500mg/mL；所述微球活化缓冲液Ⅱ中PDH的浓度为10-500mg/mL。
  5. 根据权利要求1所述的羧基荧光微球标记多糖类抗原的方法，其特征在于：所述羧基荧光微球的粒径范围为100-300nm。
  6. 根据权利要求1所述的羧基荧光微球标记多糖类抗原的方法，其特征在于：所述MES缓冲液的浓度为10-200mM，pH值为4.5-9.0。
  7. 根据权利要求1所述的羧基荧光微球标记多糖类抗原的方法，其特征在于：所述步骤b中封闭液为含10％(w/v)BSA的50mM MES缓冲液，pH值为6.0封闭时间为1-2h。
  8. 权利要求1-7任一所述的方法制备得到的羧基荧光微球标记的多糖类抗原。
  9. 一种包含权利要求8所述的羧基荧光微球标记的多糖类抗原的荧光免疫层析试纸条。
PCT/CN2022/121137 2022-02-08 2022-09-24 一种羧基荧光微球表面活化方法及其应用 WO2023151281A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202210119536.X 2022-02-08
CN202210119536.XA CN114166812B (zh) 2022-02-08 2022-02-08 一种羧基荧光微球表面活化方法及其应用

Publications (1)

Publication Number Publication Date
WO2023151281A1 true WO2023151281A1 (zh) 2023-08-17

Family

ID=80489530

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/121137 WO2023151281A1 (zh) 2022-02-08 2022-09-24 一种羧基荧光微球表面活化方法及其应用

Country Status (2)

Country Link
CN (1) CN114166812B (zh)
WO (1) WO2023151281A1 (zh)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114166812B (zh) * 2022-02-08 2022-05-17 丹娜(天津)生物科技股份有限公司 一种羧基荧光微球表面活化方法及其应用
CN117214443B (zh) * 2023-11-09 2024-01-23 深圳市迈科龙生物技术有限公司 一种荧光微球的活化方法、定量检测血清淀粉样蛋白a的试剂盒及其制备方法
CN117471110B (zh) * 2023-12-27 2024-03-19 天津德祥生物技术股份有限公司 一种用于封闭抗原-微球指示系统的封闭液及封闭方法

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050118199A1 (en) * 2003-10-07 2005-06-02 Esser Mark T. Process for covalently conjugating polysaccharides to microspheres or biomolecules
US20070134334A1 (en) * 2003-11-14 2007-06-14 Chugai Seiyaku Kabushiki Kaisha Crosslinked polysaccharide microparticles and method for their preparation
CN104897904A (zh) * 2015-05-29 2015-09-09 中生北控生物科技股份有限公司 一种羧基微球标记蛋白的方法
US20180235897A1 (en) * 2017-02-08 2018-08-23 Wisconsin Alumni Research Foundation Unimolecular nanoparticles for efficient delivery of therapeutic cationic peptides
CN112143484A (zh) * 2020-09-24 2020-12-29 武汉生之源生物科技股份有限公司 一种荧光微球活化剂复溶液及其应用
WO2021100041A1 (en) * 2019-11-20 2021-05-27 Yeda Research And Development Co. Ltd. Treating alzheimer's disease
CN114166812A (zh) * 2022-02-08 2022-03-11 丹娜(天津)生物科技股份有限公司 一种羧基荧光微球表面活化方法及其应用

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105646703B (zh) * 2016-02-29 2020-05-26 丹娜（天津）生物科技有限公司 新型隐球菌荚膜多糖gxm多克隆抗体及其制备方法
CN110927384B (zh) * 2019-11-29 2023-08-08 丹娜(天津)生物科技股份有限公司 一种荧光免疫层析试纸条及其制备方法和应用
CN111253629B (zh) * 2020-03-17 2021-06-15 江苏地韵医疗科技有限公司 一种凝胶、其成套原料及应用
CN113671171A (zh) * 2021-07-06 2021-11-19 安徽惠邦生物工程有限公司 信号放大量子点荧光免疫探针及其制备方法和应用

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050118199A1 (en) * 2003-10-07 2005-06-02 Esser Mark T. Process for covalently conjugating polysaccharides to microspheres or biomolecules
US20070134334A1 (en) * 2003-11-14 2007-06-14 Chugai Seiyaku Kabushiki Kaisha Crosslinked polysaccharide microparticles and method for their preparation
CN104897904A (zh) * 2015-05-29 2015-09-09 中生北控生物科技股份有限公司 一种羧基微球标记蛋白的方法
US20180235897A1 (en) * 2017-02-08 2018-08-23 Wisconsin Alumni Research Foundation Unimolecular nanoparticles for efficient delivery of therapeutic cationic peptides
WO2021100041A1 (en) * 2019-11-20 2021-05-27 Yeda Research And Development Co. Ltd. Treating alzheimer's disease
CN112143484A (zh) * 2020-09-24 2020-12-29 武汉生之源生物科技股份有限公司 一种荧光微球活化剂复溶液及其应用
CN114166812A (zh) * 2022-02-08 2022-03-11 丹娜(天津)生物科技股份有限公司 一种羧基荧光微球表面活化方法及其应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DAO-FENG LIU, DENG SHENG-LIANG,LAI WEI-HUA, XIA JUN: "Development of lateral flow assay of ractopamine based on fluorescent microspheres", FOOD & MACHINERY, vol. 28, no. 1, 18 January 2012 (2012-01-18), pages 73 - 77, XP093082891 *
SCHLOTTMANN, S.A. ; JAIN, N. ; CHIRMULE, N. ; ESSER, M.T.: "A novel chemistry for conjugating pneumococcal polysaccharides to Luminex microspheres", JOURNAL OF IMMUNOLOGICAL METHODS, ELSEVIER SCIENCE PUBLISHERS B.V.,AMSTERDAM., NL, vol. 309, no. 1-2, 20 February 2006 (2006-02-20), NL , pages 75 - 85, XP028017444, ISSN: 0022-1759, DOI: 10.1016/j.jim.2005.11.019 *

Also Published As

Publication number Publication date
CN114166812B (zh) 2022-05-17
CN114166812A (zh) 2022-03-11

Similar Documents

Publication Publication Date Title
WO2023151281A1 (zh) 一种羧基荧光微球表面活化方法及其应用
WO2018133038A1 (zh) 标记复合物及其制备方法、试剂盒、应用和检测系统
CN112730839B (zh) 一种磁微粒化学发光法测定细胞角蛋白19片段含量的试剂盒
CN111308067B (zh) 一种定量检测氟喹诺酮类药物的量子点微球免疫层析试纸条
CN114200125B (zh) 一种不含蛋白的碱性磷酸酶-抗体偶联物稀释液及其制备方法
CN109725160B (zh) 一种降钙素原(pct)检测试剂盒
US4711841A (en) Method for determining one or more antigens in a sample
WO2021088730A1 (zh) 游离前列腺特异性抗原检测试剂盒及其制备方法
CN110954691B (zh) 一种样本前处理试剂及其应用
CN113311154B (zh) 一种免疫磁微粒的偶联方法、稀释液和应用
CN116840460A (zh) 一种用于检测黄曲霉毒素b1的侧流免疫分析试剂盒及方法
JP3650765B2 (ja) イムノアッセイ用の粒子およびその処理方法
CN112143484B (zh) 一种荧光微球活化剂复溶液及其应用
CN104483492A (zh) 抗链球菌溶血素o抗体的检测试剂盒
CN112098639B (zh) 以氧化石墨烯为载体的二抗的合成及应用
US6777246B2 (en) Tertiary amine compounds for use in immunoassays
WO2022233106A1 (zh) 碱性磷酸酶标记的甲状腺素的制备方法
CN116298267B (zh) 一种抗原包被反应板的封闭液及其应用、试剂盒
CN117647644B (zh) 一种封闭剂及其在免疫检测中的应用
CN113030484B (zh) 降钙素原致敏胶乳、试剂盒及制备方法、应用
JP2020076692A (ja) 抗ストレプトリジンｏ測定用ラテックス粒子の製造方法
CN114113591B (zh) 一种灵敏检测大肠杆菌o157:h7的酶联免疫方法
CN117517681A (zh) C肽检测试剂盒及其制备方法
CN117031035A (zh) 抗链球菌脱氧核糖核酸酶b抗体的检测试剂盒及方法
CN116256517A (zh) 一种抗ngal单克隆抗体复合荧光微球复合物及其制备方法和ngal检测试剂盒

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22925639

Country of ref document: EP

Kind code of ref document: A1