WO2023103464A1 - 锌转运蛋白8抗体化学发光免疫检测试剂盒及其制备方法 - Google Patents

锌转运蛋白8抗体化学发光免疫检测试剂盒及其制备方法 Download PDF

Info

Publication number
WO2023103464A1
WO2023103464A1 PCT/CN2022/115049 CN2022115049W WO2023103464A1 WO 2023103464 A1 WO2023103464 A1 WO 2023103464A1 CN 2022115049 W CN2022115049 W CN 2022115049W WO 2023103464 A1 WO2023103464 A1 WO 2023103464A1
Authority
WO
WIPO (PCT)
Prior art keywords
zinc transporter
antigen
monomer
chemiluminescent
zinc
Prior art date
Application number
PCT/CN2022/115049
Other languages
English (en)
French (fr)
Inventor
安文俊
李会平
祝亮
钱纯亘
Original Assignee
深圳市亚辉龙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 深圳市亚辉龙生物科技股份有限公司 filed Critical 深圳市亚辉龙生物科技股份有限公司
Priority to EP22902897.2A priority Critical patent/EP4375646A1/en
Publication of WO2023103464A1 publication Critical patent/WO2023103464A1/zh

Links

Classifications

    • 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
    • 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
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • 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
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • 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
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/583—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with non-fluorescent dye 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
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • 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
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the invention relates to the technical field of biomedical detection, in particular to a zinc transporter 8 antibody chemiluminescent immunodetection kit and a preparation method thereof.
  • Zinc transporter 8 (Zinc transporters member 8, ZnT8), localized in pancreatic ⁇ cells in the form of dimers, can transport cytoplasmic zinc ions into insulin storage/secretory vesicles. The reduction of the transfer function of zinc transporter 8 will affect the synthesis, storage and secretion of insulin, which can increase the risk of type 1 diabetes mellitus (T1DM). ZnT8 protein can also be used as an antigen to cause ⁇ -cell autoimmune damage and induce type 1 diabetes (T1DM).
  • Type 1 diabetes is characterized by the presence of circulating autoantibodies in the patient's serum, including islet cell antibodies (ICA), glutamic acid decarboxylase antibodies (GADA), tyrosine phosphatase antibodies (IA-2A), human insulin antibodies (IAA ) and zinc transporter 8 antibody (ZnT8A).
  • ICA islet cell antibodies
  • GADA glutamic acid decarboxylase antibodies
  • IA-2A tyrosine phosphatase antibodies
  • IAA human insulin antibodies
  • ZnT8A zinc transporter 8 antibody
  • ZnT8A is a specific antibody for the diagnosis of type 1 diabetes, and its positive rate in the initial cases of type 1 diabetes is second only to anti-GAD antibody. Detection of ZnT8A can help to assess the risk of type 1 diabetes, and has high diagnostic and prognostic value for first-degree relatives of diabetic patients.
  • Chemiluminescent immunoassay is an immunoassay method that directly labels antigens or antibodies with chemiluminescent agents.
  • Chemiluminescent substances commonly used for labeling are acridinium ester compounds—acridinium ester (AE), which is an effective luminescent marker, which emits light by initiating the action of luminescent reagents (NaOH, H 2 O 2 ), and its luminescence occurs within one second. Finished in minutes, glowing for a quick flicker.
  • the zinc transporter 8 antigen has a complex molecular structure and often exists in the form of polymers.
  • the zinc transporter 8 antigen is labeled with a chemiluminescent marker.
  • the activity of the zinc transporter 8 antigen is not high, and the repeatability is poor. The rate is low, which leads to low detection sensitivity.
  • the first object of the present invention is to provide a zinc transporter 8 antibody chemiluminescent immunoassay kit, which uses a zinc transporter 8 monomer antigen labeled with a chemiluminescence group, to detect zinc transporter 8 8 antibody for detection, since the zinc transporter 8 monomer antigen has fully exposed antigenic sites, which can bind more chemiluminescent groups, so that the zinc transporter 8 monomer antigen labeled with chemiluminescent groups has a higher detection sensitivity.
  • a zinc transporter 8 antibody chemiluminescent immunoassay kit provided by the invention comprises: a zinc transporter 8 monomer antigen labeled with a chemiluminescent group, and the zinc transporter 8 monomer antigen is polymerized by reducing the zinc transporter 8 Body antigens are obtained.
  • the second object of the present invention is to provide a method for preparing the zinc transporter 8 antibody chemiluminescent immunoassay kit, comprising the steps of preparing a zinc transporter 8 monomer antigen labeled with a chemiluminescence group:
  • the zinc transporter 8 monomeric antigen is labeled with a chemiluminescent marker to obtain the zinc transporter 8 monomeric antigen labeled with the chemiluminescent marker.
  • the zinc transporter 8 antibody chemiluminescent immunoassay kit uses the zinc transporter 8 monomer antigen labeled with a chemiluminescence group to detect the zinc transporter 8 antibody, since the zinc transporter 8 monomer antigen is composed of zinc
  • the transporter 8 multimer antigen is reduced, which reduces the influence of multimer ratio differences, has fully exposed antigenic sites, can bind more chemiluminescent groups, and improves the labeling rate of chemiluminescent markers, so that the labeling
  • the zinc transporter 8 monomer antigen with chemiluminescent groups has higher detection sensitivity.
  • the traditional zinc transporter 8 (ZnT8) antibody detection method often uses magnetic particle chemiluminescence (CLIA). 8 antigen and zinc transporter 8 antigen labeled with chemiluminescent marker react to form a double-antigen sandwich antibody complex, add pre-excitation solution and excitation solution for luminescent reaction, and calculate the content of zinc transporter 8 antibody according to the luminescence intensity and standard curve.
  • CLIA magnetic particle chemiluminescence
  • the labeling method of zinc transporter 8 antigen generally directly labels the acridinium ester on the ZnT8 antigen to be conjugated, and the ZnT8 antigen often exists in the form of a dimer.
  • This labeling method has a low labeling rate and poor stability of the labeled ZnT8 antigen. , poor activity, poor repeatability and large batch-to-batch difference.
  • the first aspect of the present invention provides a zinc transporter 8 antibody chemiluminescent immunoassay kit, comprising: a zinc transporter 8 monomer antigen labeled with a chemiluminescent group, The zinc transporter 8 monomer antigen is obtained by reducing the zinc transporter 8 multimer antigen.
  • the present invention creatively uses the zinc transporter 8 monomer antigen labeled with a chemiluminescent group to detect the zinc transporter 8 antibody, since the zinc transporter 8 monomer antigen is obtained by reducing the zinc transporter 8 multimer antigen, it has sufficient
  • the exposed antigenic site has higher activity and can bind more chemiluminescent groups, thereby increasing the labeling rate of chemiluminescent markers, making the zinc transporter 8 monomer antigen labeled with chemiluminescent groups have higher Detection sensitivity.
  • the zinc transporter 8 monomeric antigen is also modified with a blocking group, and the blocking group is connected to the zinc transporter 8 monomeric antigen to play a blocking role and prevent the zinc transporter 8 monomeric antigen from repolymerizing , affect the activity of the zinc transporter 8 monomeric antigen, and reduce the binding efficiency of the zinc transporter 8 monomeric antigen and the chemiluminescent group.
  • the blocking group is a sulfhydryl blocking group
  • the sulfhydryl blocking group is connected to the sulfhydryl group of the zinc transporter 8 monomeric antigen to play a blocking role and prevent the zinc transporter 8 monomeric antigen from repolymerizing through the sulfhydryl group , affect the activity of the zinc transporter 8 monomeric antigen, and reduce the binding efficiency of the zinc transporter 8 monomeric antigen and the chemiluminescent group.
  • the sulfhydryl blocking group is derived from at least one of N-ethylmaleimide (NEM), N-hydroxymaleimide, and iodoacetamide. It can be understood that the source of the blocking group is not limited by the range of blocking agents listed above, and any substance that can be combined with a sulfhydryl group to block the sulfhydryl group and does not affect the subsequent chemiluminescence immunoassay can be used as a blocking agent. Provide blocking groups.
  • the chemiluminescent group is derived from acridinium ester (AE-NHS, DMAE-NHS), acridinic acid (9-acridinecarboxylic acid), acridine amide or acridine sulfonamide (NSP-SA-NHS) at least one of the Among them, the chemiluminescent group is connected to the amino group on the zinc transporter 8 monomer antigen, and can emit light rapidly under the excitation of the excitation solution or the pre-excitation solution, and calculate the content of the zinc transporter 8 antibody according to the luminescence intensity and the standard curve .
  • AE-NHS acridinium ester
  • acridinic acid (9-acridinecarboxylic acid
  • NSP-SA-NHS acridine amide or acridine sulfonamide
  • the second aspect of the present invention provides a method for preparing the zinc transporter 8 antibody chemiluminescent immunoassay kit, comprising the steps of preparing a zinc transporter 8 monomer antigen labeled with a chemiluminescent group:
  • the zinc transporter 8 monomeric antigen is labeled with a chemiluminescent marker to obtain the zinc transporter 8 monomeric antigen labeled with a chemiluminescent group.
  • a chemical marker dissolved in DMSO solvent was added to the ZnT8 monomer antigen solution for a period of labeling reaction, and the reaction product was desalted with a molecular weight cut-off column, using TRIS (pH 7.4) buffer as the buffer for changing the liquid, and passed column to remove free chemical markers and reaction by-products to obtain a ZnT8 monomer antigen solution labeled with chemiluminescent groups.
  • the activity of the ZnT8 antigen can be improved, so that the ZnT8 antigen exposes more active sites
  • Labeling the ZnT8 monomeric antigen with a chemiluminescent marker can increase the labeling rate of the chemiluminescent marker, and then use the chemiluminescent marker-labeled ZnT8 monomeric antigen to detect antibodies, which can improve the sensitivity of detecting ZnT8 monomeric antibodies.
  • the number of moles of the chemiluminescent marker is 1-5000 times the number of moles of the zinc transporter 8 monomer antigen, for example, it can be 5-4000 times, 10-3000 times, 20-2000 times, and further can be It is 30-1000 times, 50-400 times, 100-300 times, it can also be 200 times, 1500 times, 2500 times, 3500 times, 4500 times.
  • the reducing agent includes at least one of dithiothreitol, mercaptoethylamine, mercaptoethanol, and tricarboxyethylphosphine.
  • the number of moles of the reducing agent is 1-2500 times the number of moles of the zinc transporter 8 multimer antigen, for example, it can be 5-2000 times, 10-1500 times, 20-1000 times, and further can be 30 times. -600 times, 40-300 times, can also be 50 times, 100 times, 200 times.
  • the zinc transporter 8 monomeric antigen before or after labeling the zinc transporter 8 monomeric antigen with a chemiluminescent marker, it further includes: using a blocking agent to block the zinc transporter 8 monomeric antigen to obtain a sulfhydryl-blocked acridinium ester-labeled Zinc transporter 8 monomeric antigen, such that the zinc transporter 8 monomeric antigen is linked to the blocking group through a chemical reaction.
  • a blocking agent is added to the ZnT8 monomer antigen solution, and after reaching the working concentration of the blocking agent, it is reacted for a period of time at room temperature.
  • a PBS equilibrium desalting column is used to desalt and remove impurities from the reaction product to obtain a purified thiol-blocked The ZnT8 monomer antigen solution.
  • the step of blocking the sulfhydryl group can be performed before labeling the ZnT8 monomeric antigen, or after labeling the ZnT8 monomeric antigen.
  • the molar amount of the blocking agent is 1-2000 times, preferably 20-200 times, that of the zinc transporter 8 multimer antigen.
  • This embodiment provides a zinc transporter 8 antibody chemiluminescent immunoassay kit and its preparation method.
  • the kit includes a sulfhydryl-blocked acridinium ester-labeled zinc transporter 8 monomer antigen.
  • the preparation method of the kit includes:
  • step (2) Add dithiothreitol (DTT) to the polymer antigen solution in step (1) to make the final concentration 1.5mg/mL, mix gently, and react at room temperature (25°C) for 60min ; using a PBS equilibrated desalting column, and desalting and removing impurities from the product after the reaction, to prepare a purified zinc transporter 8 monomer antigen solution;
  • DTT dithiothreitol
  • NAM N-ethylmaleimide
  • Use standard buffer 40mM Tris-HCl, 0.5% BSA, 1% NaCl, pH 8.0 to configure the zinc transporter 8 antibody to a concentration of 5AU/mL, 20AU/mL, 80AU/mL, 200AU/mL, 800AU/mL mL, 2000AU/mL, 0.5mL per bottle, freeze-dried, and stored at 4°C for later use.
  • This embodiment provides a zinc transporter 8 antibody chemiluminescent immunoassay kit and its preparation method.
  • the kit includes a sulfhydryl-blocked acridinium ester-labeled zinc transporter 8 monomer antigen.
  • the preparation method of the kit includes:
  • step (2) Add dithiothreitol (DTT) to the zinc transporter 8 antigen solution in step (1) to make the final concentration 1.5mg/mL, mix gently, and react at room temperature (25°C) 60min; use PBS to balance the desalting column, and desalt and remove impurities from the product after the reaction to prepare the purified zinc transporter 8 monomer antigen solution;
  • DTT dithiothreitol
  • NAM N-ethylmaleimide
  • Use standard buffer 40mM Tris-HCl, 0.5% BSA, 1% NaCl, pH 8.0 to configure the zinc transporter 8 antibody to a concentration of 5AU/mL, 20AU/mL, 80AU/mL, 200AU/mL, 800AU/mL mL, 2000AU/mL, 0.5mL per bottle, freeze-dried, and stored at 4°C for later use.
  • This embodiment provides a zinc transporter 8 antibody chemiluminescent immunoassay kit and a preparation method thereof, the kit includes azidinium ester-labeled zinc transporter 8 multimer antigen, and the preparation method of the kit includes:
  • Use standard buffer 40mM Tris-HCl, 0.5% BSA, 1% NaCl, pH 8.0 to configure the zinc transporter 8 antibody to a concentration of 5AU/mL, 20AU/mL, 80AU/mL, 200AU/mL, 800AU/mL mL, 2000AU/mL, 0.5mL per bottle, freeze-dried, and stored at 4°C for later use.
  • a fully automatic chemiluminescence immunoassay analyzer is used as a detection tool.
  • the detection method in this embodiment is the double-antigen sandwich method, that is, the instrument sequentially adds 25 ⁇ L of samples, 50 ⁇ L of zinc transporter 8-coated magnetic particles, and 50 ⁇ L of acridine Acridine ester-labeled zinc transporter 8 or sulfhydryl-blocked acridinium ester-labeled zinc transporter 8 monomer antigen was reacted for 10 minutes, then magnetically separated, and the instrument sent the reaction mixture into the dark room, followed by adding 50 ⁇ L chemiluminescence pre-excitation solution, 50 ⁇ L The chemiluminescence excitation solution was used for luminescence reaction, and finally the luminescence intensity was recorded, and the zinc transporter 8 antibody content of the tested sample was calculated from the standard curve.
  • the kits in Examples 1, 2 and Comparative Example 1 were respectively used for zinc transporter 8 antibody chemiluminescent immunoassay, and the sensitivity was characterized by the detection signal value. Under the same sample concentration, the higher the signal value, it means The higher the sensitivity, the specific detection results are shown in Table 1. It can be known from Table 1 that within the concentration range of 6.83-285.93 AU/mL, the sensitivity of the kits of Examples 1 and 2 is significantly greater than that of the kit of Comparative Example 1.
  • kits in Examples 1, 2 and Comparative Example 1 were used to detect samples containing ZnT8A or samples not containing ZnT8A, and judge whether the samples contained ZnT8A according to the test results.
  • the specificity is indicated by the percentage of the actual disease-free people who are correctly judged as negative, that is, the specificity is characterized by the correct rate of testing negative samples. The higher the correct rate, the higher the accuracy and the better the specificity.
  • Table 2 shows. According to Table 2, it can be seen that the specificity of the kits of Examples 1 and 2 is significantly higher than that of the kit of Comparative Example 1.
  • Example 1 Example 2
  • Example 3 Positive 115 116 116 Negative 3 2 10 total 118 118 126 specificity 96.9% 98.0% 89.8%

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Analytical Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Nanotechnology (AREA)
  • Plasma & Fusion (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)

Abstract

一种锌转运蛋白8抗体化学发光免疫检测试剂盒，包括：标记有化学发光基团的锌转运蛋白8单体抗原，锌转运蛋白8单体抗原通过还原锌转运蛋白8多聚体抗原得到。该试剂盒采用标记有化学发光基团的锌转运蛋白8单体抗原，对锌转运蛋白8抗体进行检测，由于锌转运蛋白8单体抗原由锌转运蛋白8多聚体抗原还原得到，具有充分暴露的抗原位点，能够结合更多的化学发光基团，从而使得标记有化学发光基团的锌转运蛋白8单体抗原具有更高的检测灵敏度。

Description

锌转运蛋白8抗体化学发光免疫检测试剂盒及其制备方法 技术领域
本发明涉及生物医学检测技术领域，具体而言，涉及一种锌转运蛋白8抗体化学发光免疫检测试剂盒及其制备方法。
背景技术
锌转运蛋白8(Zinc transporters member 8，ZnT8)，以二聚体形式定位于胰岛β细胞，能将胞浆锌离子转运至胰岛素储存/分泌性囊泡内。锌转运蛋白8转运功能降低会影响胰岛素合成、储存和分泌，能增加1型糖尿病(type 1 1diabetes mellitus，T1DM)的发病风险，ZnT8蛋白也可作为抗原引起β细胞自身免疫损伤，诱发1型糖尿病(T1DM)。1型糖尿病的特征是患者血清中存在循环性自身抗体，包括胰岛细胞抗体(ICA)、谷氨酸脱羧酶抗体(GADA)、酪氨酸磷酸酶抗体(IA-2A)、人胰岛素抗体(IAA)和锌转运蛋白8抗体(ZnT8A)。
ZnT8A是诊断1型糖尿病的特异性抗体，在1型糖尿病初发病例中阳性率仅次于抗GAD抗体。检测ZnT8A可有助于评估1型糖尿病的发病危险，对糖尿病患者的一级亲属具有很高的诊断及预后评估价值。
目前，临床上检测锌转运蛋白8抗体的常用方法是磁微粒化学发光法，利用双抗原夹心法原理进行检测，测定相关发光值RLU。化学发光免疫分析(CLIA)，是用化学发光剂直接标记抗原或抗体的免疫分析方法。常用于标记的化学发光物质有吖啶酯类化合物——acridinium ester(AE)，是有效的发光标记物，其通过启动发光试剂(NaOH、H 2O 2)作用而发光，其发光在一秒钟内完成，为快速的闪烁发光。
然而，锌转运蛋白8抗原分子结构复杂，常以多聚体形式存在，采用化学发光标记物对锌转运蛋白8抗原进行标记，锌转运蛋白8抗原活性不高，重复性差批间差大，标记率低，进而导致检测灵敏度较低。
发明内容
为了解决上述问题，本发明的第一目的在于提供一种锌转运蛋白8抗体化学发光免疫检测试剂盒，该试剂盒采用标记有化学发光基团的锌转运蛋白8单体抗原，对锌转运蛋白8抗体进行检测，由于锌转运蛋白8单体抗原具有充分暴露的抗原位点，能够结合更多的化学发光基团，从而使得标记有化学发光基团的锌转运蛋白8单体抗原具有更高的检测灵敏度。
本发明提供的一种锌转运蛋白8抗体化学发光免疫检测试剂盒，包括：标记有化学发光基团的锌转运蛋白8单体抗原，锌转运蛋白8单体抗原通过还原锌转运蛋白8多聚体抗原得到。
本发明的第二目的在于提供一种上述锌转运蛋白8抗体化学发光免疫检测试剂盒的制备方法，包括制备标记有化学发光基团的锌转运蛋白8单体抗原的步骤：
利用还原剂对锌转运蛋白8多聚体抗原进行还原处理，得到锌转运蛋白8单体抗原；
采用化学发光标记物对锌转运蛋白8单体抗原进行标记反应，得到化学发光标记物标记的锌转运蛋白8单体抗原。
本发明提供的锌转运蛋白8抗体化学发光免疫检测试剂盒，采用标记有化学发光基团的锌转运蛋白8单体抗原对锌转运蛋白8抗体进行检测，由于锌转运蛋白8单体抗原由锌转运蛋白8多聚体抗原还原得到，降低多聚体比例差异 的影响，具有充分暴露的抗原位点，能够结合更多的化学发光基团，提高了化学发光标记物的标记率，从而使得标记有化学发光基团的锌转运蛋白8单体抗原具有更高的检测灵敏度。
具体实施方式
现将详细地提供本发明实施方式的参考，其一个或多个实例描述于下文。提供每一实例作为解释而非限制本发明。实际上，对本领域技术人员而言，显而易见的是，可以对本发明进行多种修改和变化而不背离本发明的范围或精神。例如，作为一个实施方式的部分而说明或描述的特征可以用于另一实施方式中，来产生更进一步的实施方式。
因此，旨在本发明覆盖落入所附权利要求的范围及其等同范围中的此类修改和变化。本发明的其它对象、特征和方面公开于以下详细描述中或从中是显而易见的。本领域普通技术人员应理解本讨论仅是示例性实施方式的描述，而非意在限制本发明更广阔的方面。
传统锌转运蛋白8(ZnT8)抗体的检测方法常采用磁微粒化学发光法(CLIA)，其技术原理为：向锌转运蛋白8抗体待测样本依次加入包被于固相载体上的锌转运蛋白8抗原、化学发光标记物标记的锌转运蛋白8抗原，反应形成双抗原夹心抗体复合物，加入预激发液和激发液进行发光反应，根据发光强度和标准曲线计算锌转运蛋白8抗体的含量。
目前，锌转运蛋白8抗原的标记方法一般将吖啶酯直接标记在待结合物ZnT8抗原上，ZnT8抗原常以二聚体形式存在，这种标记方法存在标记率低，标记的ZnT8抗原稳定性差、活性不好、重复性差批间差大等问题。
为了至少部分解决上述技术问题的至少一个，本发明的第一方面提供了一 种锌转运蛋白8抗体化学发光免疫检测试剂盒，包括：标记有化学发光基团的锌转运蛋白8单体抗原，锌转运蛋白8单体抗原通过还原锌转运蛋白8多聚体抗原得到。
本发明创造性地采用标记有化学发光基团的锌转运蛋白8单体抗原对锌转运蛋白8抗体进行检测，由于锌转运蛋白8单体抗原由锌转运蛋白8多聚体抗原还原得到，具有充分暴露的抗原位点，活性更高，能够结合更多的化学发光基团，从而提高了化学发光标记物的标记率，使得标记有化学发光基团的锌转运蛋白8单体抗原具有更高的检测灵敏度。
一些实施方案中，锌转运蛋白8单体抗原还修饰有封闭基团，封闭基团和锌转运蛋白8单体抗原相连接，用以起到封闭作用，防止锌转运蛋白8单体抗原再次聚合，影响锌转运蛋白8单体抗原的活性，降低锌转运蛋白8单体抗原和化学发光基团的结合效率。
一些实施方案中，封闭基团为巯基封闭基团，巯基封闭基团和锌转运蛋白8单体抗原的巯基相连接，用以起到封闭作用，防止锌转运蛋白8单体抗原通过巯基再次聚合，影响锌转运蛋白8单体抗原的活性，降低锌转运蛋白8单体抗原和化学发光基团的结合效率。
一些实施方案中，巯基封闭基团来自于N-乙基马来酰亚胺(NEM)、N-羟基马来酰亚胺和碘乙酰胺中的至少一种。可以理解的是，封闭基团的来源不受上述列举的封闭剂范围的限制，任何可以和巯基结合以起到封闭巯基的作用且不影响后续化学发光免疫检测的物质，均可以作为封闭剂以提供封闭基团。
一些实施方案中，化学发光基团来自于吖啶酯(AE-NHS、DMAE-NHS)、吖啶酸(9-吖啶甲酸)、吖啶酰胺或吖啶磺酰胺(NSP-SA-NHS)中的至少一种。其中，化学发光基团和锌转运蛋白8单体抗原上的氨基相连接，并且能够在激 发液或者预激发液的激发下，快速发光，根据发光强度和标准曲线计算锌转运蛋白8抗体的含量。
本发明的第二方面提供了一种上述锌转运蛋白8抗体化学发光免疫检测试剂盒的制备方法，包括制备标记有化学发光基团的锌转运蛋白8单体抗原的步骤：
利用还原剂对锌转运蛋白8多聚体抗原进行还原处理，得到锌转运蛋白8单体抗原；
采用化学发光标记物对锌转运蛋白8单体抗原进行标记反应，得到标记有化学发光基团的锌转运蛋白8单体抗原。
具体地，将锌转运蛋白8多聚体抗原用PBS缓冲液稀释制成锌转运蛋白8多聚体抗原溶液，向ZnT8多聚体抗原溶液加入还原剂，达到还原剂的工作浓度后，进行一段时间的还原反应，反应结束后采用PBS平衡脱盐柱，对反应产物脱盐除杂，制得纯化后的ZnT8单体抗原溶液；
进一步，向ZnT8单体抗原溶液中加入溶解于DMSO溶剂的化学标记物进行一段时间的标记反应，将反应产物用截留分子量的脱盐柱，以TRIS(pH 7.4)缓冲液作为换液缓冲液，过柱，除去游离的化学标记物及反应副产物，得到标记有化学发光基团的ZnT8单体抗原溶液。
可以理解的是，通过本发明的标记方法，在对锌转运蛋白8多聚体抗原进行还原处理得到ZnT8单体抗原后，可以提高ZnT8抗原的活性，使得ZnT8抗原暴露更多的活性位点，对ZnT8单体抗原进行化学发光标记物的标记，能够增加化学发光标记物的标记率，进而采用化学发光标记物标记的ZnT8单体抗原检测抗体时，能够提高检测ZnT8单体抗体的灵敏度。
一些实施方案中，化学发光标记物的摩尔数是锌转运蛋白8单体抗原的摩 尔数的1-5000倍，例如，可以是5-4000倍，10-3000倍，20-2000倍，进一步可以是30-1000倍，50-400倍，100-300倍，还可以是200倍，1500倍，2500倍，3500倍，4500倍。
一些实施方案中，还原剂包括二硫苏糖醇、巯基乙胺、巯基乙醇、三羧基乙基膦中的至少一种。
一些实施方案中，还原剂的摩尔数是锌转运蛋白8多聚体抗原摩尔数的1-2500倍，例如，可以是5-2000倍，10-1500倍，20-1000倍，进一步可以是30-600倍，40-300倍，还可以是50倍，100倍，200倍。
一些实施方案中，采用化学发光标记物对锌转运蛋白8单体抗原进行标记之前或之后还包括：采用封闭剂对锌转运蛋白8单体抗原进行封闭处理，得到巯基封闭的吖啶酯标记的锌转运蛋白8单体抗原，以使得锌转运蛋白8单体抗原经化学反应连接于封闭基团。
具体地，向ZnT8单体抗原溶液加入封闭剂，达到封闭剂工作浓度后，在室温下反应一段时间，反应结束后采用PBS平衡脱盐柱，对反应产物脱盐除杂，制得纯化后的巯基封闭的ZnT8单体抗原溶液。可以理解的是，封闭巯基的步骤可以在标记ZnT8单体抗原之前进行，也可以在标记ZnT8单体抗原之后进行。
一些实施方案中，封闭剂的摩尔数是锌转运蛋白8多聚体抗原摩尔数的1-2000倍，优选为20-200倍。
下面将结合实施例对本发明的实施方案进行详细描述。
实施例1
本实施例提供了一种锌转运蛋白8抗体化学发光免疫检测试剂盒及其制备方法，试剂盒包括巯基封闭的吖啶酯标记的锌转运蛋白8单体抗原，试剂盒的制备方法包括：
1、吖啶酯标记的锌转运蛋白8单体抗原溶液的制备
(1)将锌转运蛋白8用0.05M PBS缓冲液稀释至0.5mg/mL，制得多聚体抗原溶液；
(2)在步骤(1)的多聚体抗原溶液中加入二硫苏糖醇(DTT)，使其终浓度为1.5mg/mL，温和混匀后，在室温(25℃)条件下反应60min；采用PBS平衡脱盐柱，并对反应结束后的产物脱盐除杂，制得纯化后的锌转运蛋白8单体抗原溶液；
(3)在步骤(2)纯化后的锌转运蛋白8单体抗原溶液中加入N-乙基马来酰亚胺(NEM)，使其终浓度为0.5mg/mL，温和混匀后，在室温(25℃)条件下反应60min。采用PBS平衡脱盐柱，并对反应结束后的产物脱盐除杂，制得纯化后的巯基封闭的锌转运蛋白8单体抗原溶液；
(4)向锌转运蛋白单体抗原溶液中加入10μL溶解于DMSO溶剂的6.0mg/mL吖啶酯，于25℃反应60min，用2mL 7KD截留分子量的脱盐柱(Thermo fisher公司)以50mM TRIS(pH 7.4)缓冲液作为换液缓冲液，过柱3遍，除去游离的吖啶酯及反应副产物，得到巯基封闭的吖啶酯标记的锌转运蛋白8单体抗原。
2、锌转运蛋白8单体抗原包被的纳米磁珠的制备
取50mg羧基化的磁微粒(粒径为0.05-1um)悬浮液，磁分离去上清，用0.02M，pH为5.5MES缓冲液重悬，加入0.5-2mL新配置的10mg/mL的EDC水溶液，活化磁珠表面羧基，加入3-5mg锌转运蛋白8，室温下混悬2-10h，磁分离，去除上清，用含2％BSA的0.1M pH为8.0的Tris缓冲液重悬到1mg/mL，得到锌转运蛋白8包被的磁微粒，每瓶5mL分装保存于4℃备用。
3、锌转运蛋白8抗体定标品的制备
用标准品缓冲液(40mM Tris-HCl，0.5％BSA，1％NaCl，pH 8.0)将锌转运蛋白8抗体配置成浓度为5AU/mL、20AU/mL、80AU/mL、200AU/mL、800AU/mL、2000AU/mL，每瓶0.5mL分装冻干，4℃保存备用。
4、化学发光预激发液的配制
量取1.0升纯化水，依次加入80μL质量分数为20％的双氧水(H2O2)、1.0克叠氮化钠、1.5克吐温20，摇匀后避光存放。
5、化学发光激发液的配制
量取1.0升纯化水，依次加入0.6克氢氧化钠、0.5克PC300、0.5g叠氮化钠、1.5克Triton 405，摇匀后避光存放。
实施例2
本实施例提供了一种锌转运蛋白8抗体化学发光免疫检测试剂盒及其制备方法，试剂盒包括巯基封闭的吖啶酯标记的锌转运蛋白8单体抗原，试剂盒的制备方法包括：
1、吖啶酯标记的锌转运蛋白8单体抗原溶液的制备
(1)将锌转运蛋白8抗原用0.05M PBS缓冲液稀释至0.5mg/mL，制得锌转运蛋白8多聚体抗原溶液；
(2)在步骤(1)的锌转运蛋白8抗原溶液中加入二硫苏糖醇(DTT)，使其终浓度为1.5mg/mL，温和混匀后，在室温(25℃)条件下反应60min；采用PBS平衡脱盐柱，并对反应结束后的产物脱盐除杂，制得纯化后的锌转运蛋白8单体抗原溶液；
(3)向抗原溶液中加入10μL溶解于DMSO溶剂的6.0mg/mL吖啶酯，于25℃反应60min，用2mL 7KD截留分子量的脱盐柱(Thermo fisher公司)以50mM TRIS(pH 7.4)缓冲液作为换液缓冲液，过柱3遍，除去游离的吖啶酯及反 应副产物，得到吖啶酯标记的锌转运蛋白8单体抗原溶液；
(4)在步骤(3)的结合产物吖啶酯-抗原溶液中加入N-乙基马来酰亚胺(NEM)，使其终浓度为0.5mg/mL，温和混匀后，在室温(25℃)条件下反应60min，采用PBS平衡脱盐柱，并对反应结束后的产物脱盐除杂，制得纯化后的巯基封闭的吖啶酯标记的锌转运蛋白8单体抗原溶液。
2、锌转运蛋白8单体抗原包被的纳米磁珠的制备
取50mg羧基化的磁微粒(粒径为0.05-1um)悬浮液，磁分离去上清，用0.02M，pH为5.5MES缓冲液重悬，加入0.5-2mL新配置的10mg/mL的EDC水溶液，活化磁珠表面羧基，加入3-5mg锌转运蛋白8单体抗原，室温下混悬2-10h，磁分离，去除上清，用含2％BSA的0.1M pH为8.0的Tris缓冲液重悬到1mg/mL，得到巯基封闭的吖啶酯标记的锌转运蛋白8单体抗原包被的磁微粒，每瓶5mL分装保存于4℃备用。
3、锌转运蛋白8抗体定标品的制备
用标准品缓冲液(40mM Tris-HCl，0.5％BSA，1％NaCl，pH 8.0)将锌转运蛋白8抗体配置成浓度为5AU/mL、20AU/mL、80AU/mL、200AU/mL、800AU/mL、2000AU/mL，每瓶0.5mL分装冻干，4℃保存备用。
4、化学发光预激发液的配制
量取1.0升纯化水，依次加入80μL质量分数为20％的双氧水(H2O2)、1.0克叠氮化钠、1.5克吐温20，摇匀后避光存放。
5、化学发光激发液的配制
量取1.0升纯化水，依次加入0.6克氢氧化钠、0.5克PC300、0.5g叠氮化钠、1.5克Triton 405，摇匀后避光存放。
对比例1
本实施例提供了一种锌转运蛋白8抗体化学发光免疫检测试剂盒及其制备方法，试剂盒包括吖啶酯标记的锌转运蛋白8多聚体抗原，试剂盒的制备方法包括：
1、吖啶酯标记的锌转运蛋白8抗原溶液的制备
(1)将锌转运蛋白8抗原用0.05M PBS缓冲液稀释至0.5mg/mL，制得抗原溶液；
(2)向抗原溶液中加入10μL溶解于DMSO溶剂的6.0mg/mL吖啶酯，于25℃反应60min，用2mL 7KD截留分子量的脱盐柱(Thermo fisher公司)以50mM TRIS(pH 7.4)缓冲液作为换液缓冲液，过柱3遍，除去游离的吖啶酯及反应副产物，得到吖啶酯标记的锌转运蛋白8抗原溶液。
2、吖啶酯标记的锌转运蛋白8抗原包被的纳米磁珠的制备
取50mg羧基化的磁微粒(粒径为0.05-1um)悬浮液，磁分离去上清，用0.02M，pH为5.5MES缓冲液重悬，加入0.5-2mL新配置的10mg/mL的EDC水溶液，活化磁珠表面羧基，加入3-5mg锌转运蛋白8单体抗原，室温下混悬2-10h，磁分离，去除上清，用含2％BSA的0.1M pH为8.0的Tris缓冲液重悬到1mg/mL，得到锌转运蛋白8抗原包被的磁微粒，每瓶5mL分装保存于4℃备用。
3、锌转运蛋白8抗体定标品的制备
用标准品缓冲液(40mM Tris-HCl，0.5％BSA，1％NaCl，pH 8.0)将锌转运蛋白8抗体配置成浓度为5AU/mL、20AU/mL、80AU/mL、200AU/mL、800AU/mL、2000AU/mL，每瓶0.5mL分装冻干，4℃保存备用。
4、化学发光预激发液的配制
量取1.0升纯化水，依次加入80μL质量分数为20％的双氧水(H2O2)、1.0克叠氮化钠、1.5克吐温20，摇匀后避光存放。
5、化学发光激发液的配制
量取1.0升纯化水，依次加入0.6克氢氧化钠、0.5克PC300、0.5g叠氮化钠、1.5克Triton 405，摇匀后避光存放。
二、锌转运蛋白8抗体化学发光免疫检测
本实施例以全自动化学发光免疫分析仪为检测工具，本实施例的检测方法为双抗原夹心法，即仪器依次加入25μL的样品、50μL的锌转运蛋白8包被的磁微粒以及50μL的吖啶酯标记锌转运蛋白8或者巯基封闭的吖啶酯标记的锌转运蛋白8单体抗原，反应10min后，进行磁分离，仪器将反应混合物送入暗室，依次加入50μL化学发光预激发液、50μL化学发光激发液进行发光反应，最后记录发光强度，从标准曲线计算出被测样品的锌转运蛋白8抗体含量。
三、锌转运蛋白8抗体化学发光免疫检测试剂盒的性能评价
1、灵敏度的检测
对相同浓度的样本分别采用实施例1、2及对比例1中的试剂盒进行锌转运蛋白8抗体化学发光免疫检测，灵敏度用检测信号值表征，在相同样本浓度下，信号值越高，说明灵敏度越高，具体检测结果如表1所示。根据表1可知，在6.83～285.93AU/mL的浓度范围内，实施例1和实施例2的试剂盒灵敏度明显大于对比例1的试剂盒。
表1
Figure PCTCN2022115049-appb-000001
2、特异性检测
分别采用实施例1、2及对比例1中的试剂盒检测含ZnT8A的样本或不含ZnT8A的样本，根据检测结果判断样本是否含ZnT8A。特异性以将实际无病者正确地判断为阴性的百分率标示，即通过检测阴性样本的正确率来表征特异性，正确率越高，说明准确度越高，特异性越好，具体检测结果如表2所示。根据表2可知，实施例1和实施例2的试剂盒的特异性明显高于比对例1的试剂盒。
表2
  实施例1 实施例2 实施例3
阳性 115 116 116
阴性 3 2 10
总计 118 118 126
特异性 96.9％ 98.0％ 89.8％
3、稳定性检测
将实施例1、2及对比例1制备的试剂盒中抗原溶液分别放至4℃和37℃恒温箱7天，检测不同浓度样本的化学发光信号值，以4℃抗原溶液为对照，计算抗原溶液37℃加速7天稳定性。稳定性偏差绝对数值越小，说明工作液稳定性越好，具体检测结果如表3、表4和表5所示。根据表3、表4和表5可知，实施例1和实施例2的试剂盒中的抗原溶液的稳定性明显高于对比例1的试剂盒中的抗原溶液。
表3
Figure PCTCN2022115049-appb-000002
Figure PCTCN2022115049-appb-000003
表4
Figure PCTCN2022115049-appb-000004
表5
Figure PCTCN2022115049-appb-000005
4、批间差
使用三批不同批次抗原分别进行吖啶标记制备实施例1和实施例2以及对比例1的试剂盒，测试不同浓度样本的化学发光信号值，检测不同批次的原料 制备的试剂盒差异，以第一批试剂盒为对照，计算第二批、第三批相对偏差，相对偏差越小说明批间差越小，结果如下表6、表7和表8所示。根据表6、表7和表8的数据可知，实施例1和实施例2制备的试剂盒的批间差异远小于对比例1的试剂盒的批间差异。
表6
Figure PCTCN2022115049-appb-000006
表7
Figure PCTCN2022115049-appb-000007
表8
Figure PCTCN2022115049-appb-000008
以上实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (10)

  1. 一种锌转运蛋白8抗体化学发光免疫检测试剂盒，其特征在于，包括：标记有化学发光基团的锌转运蛋白8单体抗原，所述锌转运蛋白8单体抗原通过还原锌转运蛋白8多聚体抗原得到。
  2. 根据权利要求1所述的试剂盒，其特征在于，所述锌转运蛋白8单体抗原还修饰有封闭基团，所述封闭基团和所述锌转运蛋白8单体抗原经化学键相连接。
  3. 根据权利要求2所述的试剂盒，其特征在于，所述封闭基团为巯基封闭基团，所述封闭基团和所述锌转运蛋白8单体抗原的巯基经化学键相连接。
  4. 根据权利要求3所述的试剂盒，其特征在于，所述化学发光基团来自于吖啶酯、吖啶酸、吖啶酰胺和吖啶磺酰胺中的至少一种；
    所述巯基封闭基团来自于N-乙基马来酰亚胺、N-羟基马来酰亚胺和碘乙酰胺中的至少一种。
  5. 一种如权利要求1～4任一项所述的试剂盒的制备方法，其特征在于，包括如下步骤：
    利用还原剂对锌转运蛋白8多聚体抗原进行还原处理，得到锌转运蛋白8单体抗原；
    采用化学发光标记物对锌转运蛋白8单体抗原进行标记反应，得到标记有化学发光基团的锌转运蛋白8单体抗原。
  6. 根据权利要求5所述的制备方法，其特征在于，所述化学发光标记物的摩尔数是锌转运蛋白8单体抗原的摩尔数的1-5000倍。
  7. 根据权利要求5所述的制备方法，其特征在于，所述还原剂包括二硫苏糖醇、巯基乙胺、巯基乙醇以及三羧基乙基膦中的至少一种。
  8. 根据权利要求7所述的制备方法，其特征在于，所述还原剂的摩尔数是所 述锌转运蛋白8多聚体抗原摩尔数的1-2500倍。
  9. 根据权利要求6所述的制备方法，其特征在于，所述采用化学发光标记物对锌转运蛋白8单体抗原进行标记之前或之后还包括：
    采用封闭剂对锌转运蛋白8单体抗原进行封闭处理，得到巯基封闭的吖啶酯标记的锌转运蛋白8单体抗原，以使得锌转运蛋白8单体抗原经化学反应连接于封闭基团。
  10. 根据权利要求9所述的制备方法，其特征在于，所述封闭剂的摩尔数是所述锌转运蛋白8多聚体抗原摩尔数的1-2000倍。
PCT/CN2022/115049 2021-12-09 2022-08-26 锌转运蛋白8抗体化学发光免疫检测试剂盒及其制备方法 WO2023103464A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP22902897.2A EP4375646A1 (en) 2021-12-09 2022-08-26 Zinc transporter 8 antibody chemiluminescence immunoassay kit and preparation method thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202111498554.5A CN114184793B (zh) 2021-12-09 2021-12-09 锌转运蛋白8抗体化学发光免疫检测试剂盒及其制备方法
CN202111498554.5 2021-12-09

Publications (1)

Publication Number Publication Date
WO2023103464A1 true WO2023103464A1 (zh) 2023-06-15

Family

ID=80603987

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/115049 WO2023103464A1 (zh) 2021-12-09 2022-08-26 锌转运蛋白8抗体化学发光免疫检测试剂盒及其制备方法

Country Status (3)

Country Link
EP (1) EP4375646A1 (zh)
CN (1) CN114184793B (zh)
WO (1) WO2023103464A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114184793B (zh) * 2021-12-09 2023-08-11 深圳市亚辉龙生物科技股份有限公司 锌转运蛋白8抗体化学发光免疫检测试剂盒及其制备方法

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101100485A (zh) * 2006-07-07 2008-01-09 曾位森 一种促血管生成素-2单体型抗体的制备及应用技术
CN101709091A (zh) * 2009-03-02 2010-05-19 广东虹业抗体科技有限公司 抗促血管生成素2等聚合体蛋白的单体型抗体的制备及在免疫检测方法中的应用
WO2013071055A1 (en) * 2011-11-10 2013-05-16 Wellstat Diagnostics, Llc Assay for diabetes-associated autoantibodies
CN106226526A (zh) * 2016-06-30 2016-12-14 深圳市亚辉龙生物科技股份有限公司 一种锌转运蛋白8抗体化学发光免疫检测试剂盒及其制备方法
CN111579791A (zh) * 2020-04-30 2020-08-25 江苏省人民医院（南京医科大学第一附属医院） 一种锌转运体8胰岛自身抗体的电化学发光检测试剂盒
CN112485419A (zh) * 2020-11-25 2021-03-12 广州市进德生物科技有限公司 一种锌转运体8抗体检测试剂盒
CN114184793A (zh) * 2021-12-09 2022-03-15 深圳市亚辉龙生物科技股份有限公司 锌转运蛋白8抗体化学发光免疫检测试剂盒及其制备方法

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050009955A1 (en) * 2003-06-25 2005-01-13 Cohen Martin Paul Composite of silica reacted with blocked mercaptoalkoxysilane and alkyl silane, rubber composition with said composite, and article having component thereof
CN106084051B (zh) * 2016-02-17 2018-03-02 张梅 一种ZnT8特异性单链抗体scFv‑C27及其应用
US11016085B2 (en) * 2016-04-25 2021-05-25 The Johns Hopkins University ZNT8 assays for drug development and pharmaceutical compositions
US11892457B2 (en) * 2017-07-12 2024-02-06 The Johns Hopkins University Proteoliposome-based ZnT8 self-antigen for type 1 diabetes diagnosis
CN107449904A (zh) * 2017-09-06 2017-12-08 深圳市亚辉龙生物科技股份有限公司 一种自身免疫性糖尿病检测用反应膜条、制备和使用方法
CN113166241A (zh) * 2018-08-16 2021-07-23 约翰霍普金斯大学 人类znt8抗体
CN109734793B (zh) * 2019-03-14 2022-12-02 深圳市药品检验研究院（深圳市医疗器械检测中心） 一种ZnT8重组蛋白及其制备方法和应用
US20210302413A1 (en) * 2019-10-01 2021-09-30 The Johns Hopkins University Cell-based znt8 assay

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101100485A (zh) * 2006-07-07 2008-01-09 曾位森 一种促血管生成素-2单体型抗体的制备及应用技术
CN101709091A (zh) * 2009-03-02 2010-05-19 广东虹业抗体科技有限公司 抗促血管生成素2等聚合体蛋白的单体型抗体的制备及在免疫检测方法中的应用
WO2013071055A1 (en) * 2011-11-10 2013-05-16 Wellstat Diagnostics, Llc Assay for diabetes-associated autoantibodies
CN106226526A (zh) * 2016-06-30 2016-12-14 深圳市亚辉龙生物科技股份有限公司 一种锌转运蛋白8抗体化学发光免疫检测试剂盒及其制备方法
CN111579791A (zh) * 2020-04-30 2020-08-25 江苏省人民医院（南京医科大学第一附属医院） 一种锌转运体8胰岛自身抗体的电化学发光检测试剂盒
CN112485419A (zh) * 2020-11-25 2021-03-12 广州市进德生物科技有限公司 一种锌转运体8抗体检测试剂盒
CN114184793A (zh) * 2021-12-09 2022-03-15 深圳市亚辉龙生物科技股份有限公司 锌转运蛋白8抗体化学发光免疫检测试剂盒及其制备方法

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
"Biochemistry", 31 January 2017, CHINA LIGHT INDUSTRY PRESS, CN, ISBN: 978-7-5184-1040-8, article WANG, MIAO: "Passage; Biochemistry", pages: 111 - 112, XP009546818 *
"Food Chemistry", 30 April 2003, CHINA LIGHT INDUSTRY PRESS, CN, ISBN: 7-5019-3839-3, article FENNEMA, OWEN.R; WANG ZHANG; XU SHIYING; JIANG BO; YANG RUIJIN; ZHONG FANG: "Passage; Food Chemistry", pages: 346 - 347, XP009546817 *
"Medical Immunology Experimental Principles and Techniques", 30 April 2020, FUDAN UNIVERSITY PRESS, CN, ISBN: 978-7-309-14843-5, article CHU, YIWEI ET AL.: "Passage; Medical Immunology Experimental Principles and Techniques", pages: 100 - 101, XP009546819 *
"Modern Nutrition and Food Safety", 28 February 2013, THE SECOND MILITARY MEDICAL UNIVERSITY PRESS, CN, ISBN: 978-7-5481-0574-9, article LI, MIN: "Passage; Modern Nutrition and Food Safety", pages: 156 - 157, XP009546820 *

Also Published As

Publication number Publication date
EP4375646A1 (en) 2024-05-29
CN114184793B (zh) 2023-08-11
CN114184793A (zh) 2022-03-15

Similar Documents

Publication Publication Date Title
US10472400B2 (en) Cardiac troponin I ultra-sensitive detection reagent kit, and ultra-sensitive detection method therefor
CN108362688B (zh) 一种25羟基维生素d磁微粒化学发光检测试剂盒
WO2018000447A1 (zh) 脂联素化学发光免疫检测试剂盒及其制备方法和应用
CN107907690B (zh) 一种超敏c反应蛋白检测试剂盒及其使用方法
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
CN111323577B (zh) 一种抗心磷脂抗体IgG磁微粒化学发光免疫检测试剂盒
WO2016127301A1 (zh) rT3化学发光免疫检测试剂盒及其检测方法和应用
US10259969B2 (en) Paramagnetic supports for use as assay reagents
WO2016127320A1 (zh) 一种用于检测胃泌素-17的试剂盒及其制备方法和应用
CN112782156A (zh) 一种壳多糖酶3样蛋白1试剂盒及其制备方法
CN106918708A (zh) 一种用于检测胰岛素的竞争法胶乳增强免疫透射比浊试剂盒
WO2023124154A1 (zh) 磁珠包被物及其制备方法和检测试剂盒
WO2023103464A1 (zh) 锌转运蛋白8抗体化学发光免疫检测试剂盒及其制备方法
CN110907641B (zh) 一种透明质酸检测试剂盒及检测方法
WO2002095407A1 (fr) Procede de dosage immunologique
CN111175494A (zh) 一种甲状腺球蛋白抗体检测试剂盒及其使用方法
CN110988362B (zh) 抗组蛋白抗体测定试剂、试剂盒及其使用方法
CN112485418B (zh) 一种用于人体维生素b12含量检测的释放剂及其应用
CN113933521A (zh) 磁微粒化学发光检测试剂盒及其制备方法和应用
CN110672836B (zh) 磁珠包被物及其制备方法和应用、检测试剂盒
JPS59116548A (ja) 親水性ラテツクス粒子を含有する診断薬
CN116047087B (zh) 一种判断样本中游离生物素的方法
JP2011257243A (ja) 試料中のｃ反応性蛋白質の測定試薬、測定方法及び測定範囲の拡大方法
CN111521832A (zh) 一种用于测定25-羟基-维生素d的试剂盒
CN113238055A (zh) 空间邻近化学发光法检测降钙素原的试剂盒及其检测方法和应用

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: 22902897

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 2022902897

Country of ref document: EP

ENP Entry into the national phase

Ref document number: 2022902897

Country of ref document: EP

Effective date: 20240219

WWE Wipo information: entry into national phase

Ref document number: 2024105889

Country of ref document: RU

NENP Non-entry into the national phase

Ref country code: DE