CN118258998B - Antigen-specific B cell detection probe and preparation method and application thereof - Google Patents
Antigen-specific B cell detection probe and preparation method and application thereof Download PDFInfo
- Publication number
- CN118258998B CN118258998B CN202410692326.9A CN202410692326A CN118258998B CN 118258998 B CN118258998 B CN 118258998B CN 202410692326 A CN202410692326 A CN 202410692326A CN 118258998 B CN118258998 B CN 118258998B
- Authority
- CN
- China
- Prior art keywords
- antigen
- specific
- probe
- polypeptide
- cells
- Prior art date
- Legal status (The legal status 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 status listed.)
- Active
Links
- 239000000427 antigen Substances 0.000 title claims abstract description 140
- 102000036639 antigens Human genes 0.000 title claims abstract description 140
- 108091007433 antigens Proteins 0.000 title claims abstract description 140
- 210000003719 b-lymphocyte Anatomy 0.000 title claims abstract description 105
- 238000001514 detection method Methods 0.000 title claims abstract description 79
- 239000000523 sample Substances 0.000 title claims abstract description 73
- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 82
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 79
- 229920001184 polypeptide Polymers 0.000 claims abstract description 78
- 239000007850 fluorescent dye Substances 0.000 claims abstract description 24
- 206010039073 rheumatoid arthritis Diseases 0.000 claims abstract description 16
- 229920001223 polyethylene glycol Polymers 0.000 claims abstract description 12
- PEEHTFAAVSWFBL-UHFFFAOYSA-N Maleimide Chemical compound O=C1NC(=O)C=C1 PEEHTFAAVSWFBL-UHFFFAOYSA-N 0.000 claims abstract description 10
- 239000002202 Polyethylene glycol Substances 0.000 claims abstract description 8
- 102000000503 Collagen Type II Human genes 0.000 claims description 30
- 108010041390 Collagen Type II Proteins 0.000 claims description 30
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 22
- 238000002965 ELISA Methods 0.000 claims description 20
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 14
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 14
- 238000006243 chemical reaction Methods 0.000 claims description 14
- 238000000034 method Methods 0.000 claims description 13
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 12
- 238000002156 mixing Methods 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- 239000012502 diagnostic product Substances 0.000 claims description 9
- 239000013067 intermediate product Substances 0.000 claims description 9
- 238000000502 dialysis Methods 0.000 claims description 8
- 229910052757 nitrogen Inorganic materials 0.000 claims description 8
- 239000003153 chemical reaction reagent Substances 0.000 claims description 7
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 7
- 239000012153 distilled water Substances 0.000 claims description 6
- 150000002148 esters Chemical class 0.000 claims description 6
- 239000002994 raw material Substances 0.000 claims description 5
- 230000010355 oscillation Effects 0.000 claims description 4
- 239000000047 product Substances 0.000 claims description 4
- 238000005886 esterification reaction Methods 0.000 claims description 3
- 125000002653 sulfanylmethyl group Chemical group [H]SC([H])([H])[*] 0.000 claims description 3
- 239000012295 chemical reaction liquid Substances 0.000 claims 1
- 125000003396 thiol group Chemical group [H]S* 0.000 claims 1
- 238000003745 diagnosis Methods 0.000 abstract description 22
- 230000008685 targeting Effects 0.000 abstract description 8
- 238000000684 flow cytometry Methods 0.000 abstract description 7
- 230000001717 pathogenic effect Effects 0.000 abstract description 7
- 239000012472 biological sample Substances 0.000 abstract description 2
- 238000003759 clinical diagnosis Methods 0.000 abstract 1
- 230000035945 sensitivity Effects 0.000 description 16
- 210000002966 serum Anatomy 0.000 description 12
- 239000000243 solution Substances 0.000 description 12
- 239000012114 Alexa Fluor 647 Substances 0.000 description 9
- 201000010099 disease Diseases 0.000 description 9
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 9
- 201000000596 systemic lupus erythematosus Diseases 0.000 description 8
- 210000004027 cell Anatomy 0.000 description 7
- 238000011161 development Methods 0.000 description 7
- 108091008875 B cell receptors Proteins 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 238000003556 assay Methods 0.000 description 5
- 235000018102 proteins Nutrition 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 238000005406 washing Methods 0.000 description 5
- 208000021386 Sjogren Syndrome Diseases 0.000 description 4
- 238000002372 labelling Methods 0.000 description 4
- 201000008482 osteoarthritis Diseases 0.000 description 4
- 210000005259 peripheral blood Anatomy 0.000 description 4
- 239000011886 peripheral blood Substances 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 description 3
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 210000005087 mononuclear cell Anatomy 0.000 description 3
- 238000012286 ELISA Assay Methods 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 208000009386 Experimental Arthritis Diseases 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 239000003054 catalyst Substances 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 239000012228 culture supernatant Substances 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000003248 secreting effect Effects 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- RWYHOIUBWBDNSP-UHFFFAOYSA-N 1-oxidanylpyrrolidine-2,5-dione Chemical compound ON1C(=O)CCC1=O.ON1C(=O)CCC1=O RWYHOIUBWBDNSP-UHFFFAOYSA-N 0.000 description 1
- 150000003923 2,5-pyrrolediones Chemical class 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 108010001857 Cell Surface Receptors Proteins 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- RHGKLRLOHDJJDR-BYPYZUCNSA-N L-citrulline Chemical compound NC(=O)NCCC[C@H]([NH3+])C([O-])=O RHGKLRLOHDJJDR-BYPYZUCNSA-N 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 1
- RHGKLRLOHDJJDR-UHFFFAOYSA-N Ndelta-carbamoyl-DL-ornithine Natural products OC(=O)C(N)CCCNC(N)=O RHGKLRLOHDJJDR-UHFFFAOYSA-N 0.000 description 1
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 1
- 241000219061 Rheum Species 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 230000006052 T cell proliferation Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 230000003460 anti-nuclear Effects 0.000 description 1
- 230000030741 antigen processing and presentation Effects 0.000 description 1
- 206010003246 arthritis Diseases 0.000 description 1
- 230000006472 autoimmune response Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000027455 binding Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 235000013477 citrulline Nutrition 0.000 description 1
- 229960002173 citrulline Drugs 0.000 description 1
- 238000006482 condensation reaction Methods 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000005206 flow analysis Methods 0.000 description 1
- 238000001215 fluorescent labelling Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 210000005104 human peripheral blood lymphocyte Anatomy 0.000 description 1
- 230000028996 humoral immune response Effects 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 230000003832 immune regulation Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000016784 immunoglobulin production Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 102000006240 membrane receptors Human genes 0.000 description 1
- 210000001806 memory b lymphocyte Anatomy 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 125000004433 nitrogen atom Chemical group N* 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000007119 pathological manifestation Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 150000003141 primary amines Chemical class 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- 239000012265 solid product Substances 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 239000012089 stop solution Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- PXQLVRUNWNTZOS-UHFFFAOYSA-N sulfanyl Chemical class [SH] PXQLVRUNWNTZOS-UHFFFAOYSA-N 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- ANRHNWWPFJCPAZ-UHFFFAOYSA-M thionine Chemical compound [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N)=CC=C3N=C21 ANRHNWWPFJCPAZ-UHFFFAOYSA-M 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
- G01N15/1434—Optical arrangements
-
- 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"
-
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising 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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56966—Animal cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/10—Musculoskeletal or connective tissue disorders
- G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
- G01N2800/102—Arthritis; Rheumatoid arthritis, i.e. inflammation of peripheral joints
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Pathology (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Microbiology (AREA)
- Dispersion Chemistry (AREA)
- Zoology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Optics & Photonics (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Rehabilitation Therapy (AREA)
- Rheumatology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses an antigen-specific B cell detection probe, a preparation method and application thereof, wherein the probe comprises an antigen polypeptide for targeting antigen-specific B cells, and a fluorescent dye and an eight-arm polyethylene glycol maleimide which are respectively and covalently connected with the antigen polypeptide. The invention directly targets pathogenic B cells by constructing the antigen-specific B cell detection probe, can detect the proportion of pathogenic antigen-specific B cells in a biological sample of a patient by flow cytometry to be used as a diagnosis index of Rheumatoid Arthritis (RA), and has remarkable clinical diagnosis value. In addition, the combined detection of antigen-specific B cells and autoantibodies corresponding to antigen polypeptides can further improve RA diagnosis efficiency.
Description
Technical Field
The invention relates to the technical field of disease diagnosis, in particular to an antigen-specific B cell detection probe, a preparation method and application thereof.
Background
Antigen-specific B cells are a specific type of B lymphocytes whose surface expresses B Cell Receptors (BCR) capable of recognizing and binding to a specific antigen. After BCR recognizes a specific antigen, antigen-specific B cells are activated and participate in immune responses, including antibody production, memory B cell formation, and antigen presentation, etc., playing a key role in the immune system, protecting the body from pathogens. However, in autoimmune diseases, antigen-specific B cells are abnormally activated, and the occurrence and development of the disease are promoted by the production of autoantibodies and participation in the pathway of immune regulation imbalance and the like. In rheumatoid arthritis (rheumatoid arthritis, RA), antigen-specific B cells are abnormally activated by RA-associated autoantigens, produce a large number of autoantibodies including rheumatoid factors (rheumatoid factor, RF) and anti-cyclic citrullinated peptide (anti-CCP) antibodies, etc., and release inflammatory mediators, promote disease development and progression （Bugatti S, Codullo V, Caporali R, Montecucco C. B cells in rheumatoid arthritis. Autoimmun Rev. 2007;7(2):137-142）. in systemic lupus erythematosus (systemic lupus erythematosus, SLE), and further form immune complexes and activate immune cells by secreting various autoantibodies such as antinuclear antibodies and anti-double-stranded DNA antibodies, etc., directly involved in the disease development and development process （Tsokos GC, Lo MS, Costa Reis P, Sullivan KE. New insights into the immunopathogenesis of systemic lupus erythematosus. Nat Rev Rheumatol. 2016;12(12):716-730）. systematic study of antigen-specific B cells is critical for the recognition and regulation of normal immune responses and autoimmune responses, and for the accurate detection and recognition of this population of cells.
However, there are still a number of difficulties and challenges in precisely targeting and recognizing antigen-specific B cells. Currently, the most common method in research is the tetramer method (tetramer). The method comprises the steps of tetramerizing antigen peptide by means of a biotin-streptavidin system, and combining the antigen peptide with a plurality of BCRs on the surface of B cells, so that the affinity and stability of the polypeptide and the BCRs are greatly improved, and the antigen-specific B cells （Kerkman PF, Fabre E, van der Voort EI, et al. Identification and characterisation of citrullinated antigen-specific B cells in peripheral blood of patients with rheumatoid arthritis. Ann Rheum Dis. 2016;75(6):1170-1176）. are identified in a targeted manner. However, the above method has the limitations of high preparation difficulty (the technology is only mastered in a few laboratories at present) and high cost, and the difficulty of researching the pathogenic B cells in the group is increased. In addition, because the proportion of antigen-specific B cells is extremely low and the antigen-specific B cells are limited by the sensitivity of the probe, the antigen-specific B cells obtained by detection only account for 0.01% -0.06% or even lower （Kristyanto H, Holborough-Kerkvliet MD, Lelieveldt L, et al. Multifunctional, Multivalent PIC Polymer Scaffolds for Targeting Antigen-Specific, Autoreactive B Cells. ACS Biomater Sci Eng. 2022;8(4):1486-1493）. of the total B cells, and therefore, development of a detection probe with high sensitivity, simple preparation and low cost is needed.
The preparation of antigen-specific B cell detection probes is expected to provide a solution to this academic problem. Autoantibodies are currently the primary indicator of RA diagnosis, including RF and anti-CCP antibodies. However, these autoantibodies have limited sensitivity and specificity, and more than one third of patients still lack diagnostic markers and are misdiagnosed by missed diagnosis. It would be worth studying whether the diagnostic efficacy of RA could be improved by detecting antigen-specific B cells upstream. The preparation of antigen-specific B cell detection probes is expected to provide a solution to this academic problem.
Collagen type II (CII) is one of the most common RA-associated autoantigens, CII antibodies that stimulate T cell proliferation and B cell production. The CII secondary immunization can cause the occurrence of arthritis in mice, and a pathological manifestation similar to RA, namely collagen-induced arthritis (CIA), occurs. In addition, CII antibodies can also be used for diagnosis of RA, but are less sensitive (only 40-50%）（Burkhardt H, Sehnert B, Bockermann R, Engström A, Kalden JR, Holmdahl R. Humoral immune response to citrullinated collagen type II determinants in early rheumatoid arthritis. Eur J Immunol. 2005;35(5):1643-1652）.
In view of this, the present invention has been made.
Disclosure of Invention
The purpose of the invention is that: aiming at the dilemma that the current method for targeting and recognizing antigen-specific B cells is difficult to prepare and has low sensitivity, the invention provides a novel antigen-specific B cell detection probe; aiming at the difficult problem of poor sensitivity of the conventional RA diagnosis method in clinic, a new diagnosis index is provided for the assistance of RA diagnosis.
Based on the above, the invention also provides a preparation method and application of the antigen-specific B cell detection probe.
The technical scheme of the invention is as follows:
In a first aspect, the invention provides an antigen-specific B cell detection probe comprising an antigen polypeptide for targeting an antigen-specific B cell, and a fluorescent dye and an eight-arm polyethylene glycol maleimide (MAL-PEG-8 arm) covalently linked to the antigen polypeptide, respectively.
MAL-PEG-8arm belongs to one of PEG materials, has the advantages of good water solubility, stability, low immunogenicity, high clinical practicability and the like, and has wide application in medicine. The invention uses PEG with good water solubility and high stability as carrier to form probe skeleton. Labeling the antigen polypeptide by using a fluorescent dye, and then connecting the antigen polypeptide labeled with the fluorescent dye with MAL-PEG-8 arm.
According to the antigen-specific B cell probe, the group of pathogenic antigen-specific B cells can be directly targeted based on the specific binding function of the antigen polypeptide, the antigen-specific B cells are detected by a flow cytometry to serve as diagnostic indexes of RA, diagnosis is carried out by calculating the ratio of the pathogenic antigen-specific B cells, the problem of poor sensitivity caused by the conventional diagnosis by using serum markers can be effectively avoided, and the accuracy of RA diagnosis is improved.
Alternatively or preferably, the antigen polypeptide is a type II collagen polypeptide, and the amino acid sequence is shown as SEQ ID NO. 1 (amino acid sequence: C-GA- (cit) -GLTG- (cit) -PGDA-GPOGPO, wherein the 4 th and 9 th positions are citrulline).
Alternatively or preferably, the fluorescent dye is AF647 or Cy5.5, both of which are commercially available. AF647, cy5.5 are more photostable than other fluorescent dyes conjugated to NHS (for conjugation to polypeptides) ester labels.
AF647 (Alexa Fluor 647) has the characteristics of large polarity, excellent water solubility and high fluorescence stability, is particularly suitable for marking protein, the marked protein is not easy to aggregate and denature, and the surface of the protein can tolerate marking a plurality of AF647 dye molecules so as to achieve the effect of signal multiplication.
Cy5.5 (Cyanine 5.5) has good photostability and chemical stability, and can form covalent bonds with proteins through its NHS esters, maleimides, or other active esters.
In a second aspect, the invention provides a method of preparing a probe as described in any one of the preceding, comprising the steps of:
(1) Uniformly mixing three raw materials of antigen polypeptide, fluorescent dye-NHS and triethylamine in anhydrous DMSO, carrying out oscillation reaction under the protection of nitrogen, and putting the mixture into a dialysis bag for dialysis in distilled water after the reaction is finished so as to remove unreacted raw materials, thereby obtaining an intermediate product;
(2) Adding the intermediate product and eight-arm polyethylene glycol maleimide (MAL-PEG-8 arm) into PBS, mixing, reacting under the protection of nitrogen, and putting into a dialysis bag to dialyze in distilled water after the reaction is finished so as to remove unreacted intermediate product and eight-arm polyethylene glycol maleimide, thereby obtaining the probe.
Wherein, the fluorescent dye-NHS refers to a fluorescent dye connected with NHS. DMSO is dimethyl sulfoxide, PBS is phosphate buffer salt solution, and the main components are Na 2HPO4、KH2PO4, naCl and KCl, so that the product can be directly purchased from market or prepared by self. Preferably, the pH value is 6.8, and the reaction is more favorable for being carried out.
NHS is N-hydroxysuccinimide (N-Hydroxysuccinimide), which is a common primary amine activating reagent and is commonly used for synthesizing peptides, chemically modifying proteins, preparing medicaments and the like. The equation for reacting NHS with amino is as follows: NHS+H265N-R→NHS ester-R+H2525O wherein NHS represents N-hydroxysuccinimide, H 2 N-R represents an amino group-containing compound, NHS ester-R represents an activated NHS ester, and H 2 O represents water. The reaction of NHS and amino occurs mainly under alkaline conditions, and the mechanism is that the esterification reaction connection is carried out between NHS in fluorescent dye-NHS and amino (-NH 2) of antigen polypeptide.
The free amino (-NH 2) in the antigen polypeptide will react with NHS ester carried by the fluorescent dye to make the fluorescent dye covalently linked to the antigen polypeptide.
The invention adopts the fluorescent dye connected with NHS to label the antigen polypeptide, and compared with other labeling modes, other functional groups (the structure and the reaction complexity are easy to increase) are not introduced. The antigen polypeptide and the fluorescent dye are combined by the condensation reaction of amino and carboxyl, and the-NHS is in a form of carboxyl-COOH after activation, so that the synthesis process based on the reaction is simple, the reaction condition is milder, and the method is suitable for mass production and application.
Commercial fluorochromes with NHS, such as AF647-NHS, cy5.5-NHS, can be purchased directly when carrying out the preparation method described above.
Triethylamine is an alkaline catalyst, and a lone pair electron of a nitrogen atom in the triethylamine can be combined with a proton to form protonated triethylamine, so that the triethylamine has stronger alkalinity and can promote esterification reaction.
In the step (2), the intermediate product, namely the antigen polypeptide (the amino (-NH 2) of the cysteine side chain of the antigen polypeptide is connected with the NHS of the fluorescent dye) which is subjected to fluorescent labeling, is connected with maleimide (Mal) in PEG through the mercapto (-SH) of the cysteine side chain of the antigen polypeptide, so as to complete the probe construction.
In a third aspect, the invention provides the use of any of the probes described above in the preparation of a diagnostic product for rheumatoid arthritis. The probe is prepared into a flow detection reagent, flow cytometry detection is carried out, and the proportion of antigen-specific B cells is counted, so that diagnosis of RA can be carried out.
Alternatively or preferably, the diagnostic product comprises a probe-flow detection reagent. Experimental results show that the sensitivity performance reaches 70% and the specificity is 90% when the probe is used for flow cytometry detection.
Alternatively or preferably, the diagnostic product further comprises an ELISA reagent comprising a capture antigen having the same amino acid sequence as the antigen polypeptide of the probe. The combination of flow detection of antigen-specific B cells with an autoantibody ELISA detection of the corresponding antigen polypeptide can further enhance RA diagnostic efficiency.
In a fourth aspect, the invention provides a rheumatoid arthritis diagnostic product comprising a probe as described in any one of the preceding.
Alternatively or preferably, the diagnostic product further comprises a capture antigen for detection by ELISA methods, said capture antigen having an amino acid sequence identical to the amino acid sequence of the antigen polypeptide of the probe.
Compared with the prior art, the invention has the following beneficial effects:
The invention directly targets and recognizes the antigen-specific B cells by constructing the antigen-specific B cell detection probe; the proportion of antigen-specific B cells obtained by flow detection can be used as a diagnostic index for RA; the combination of probe-flow detection of antigen-specific B cells with an autoantibody ELISA detection of the same amino acid sequence as the capture polypeptide can further improve RA diagnostic efficiency.
Drawings
FIG. 1 is a diagram of a dual fluorescent probe targeting antigen-specific B cell pattern, wherein the probe is formed by connecting an antigen polypeptide with a fluorescent dye and then with a carrier skeleton of eight-arm polyethylene glycol maleimide. The antigen polypeptide is CII polypeptide, plays a role in targeting antigen-specific B cells, combines with antigen-specific B cell surface receptors (BCR), and simultaneously carries fluorescent dye (AF 647 or Cy5.5) on a probe so as to facilitate detection for identifying antigen-specific B cells.
FIG. 2 is a schematic flow chart of detection of antigen-specific B cells by CII polypeptide probes, wherein the horizontal axis represents MAL-PEG-8arm: CII-AF647 (CII polypeptide probe carries AF647 fluorescence), the vertical axis represents MAL-PEG-8arm: CII-Cy5.5 (CII polypeptide probe carries Cy5.5 fluorescence), in which the loop gate is antigen-specific B cells and the number is the proportion of antigen-specific B cells in total B cells.
FIG. 3 shows the specificity verification of CII polypeptide probes. And (3) separating to obtain antigen-specific B cells and non-antigen-specific B cells by using CII polypeptide probes, and then culturing in vitro. And then collecting cell culture supernatants for detection of anti-CII antibodies, wherein the horizontal axis represents two B cell types, the vertical axis represents AU value (Arbitrary units) of anti-CII antibodies secreted by cells [ AU value= (OD value of serum to be detected/OD value of positive serum) ×100, OD value is 450nm detection value of an enzyme labeling instrument-570 nm detection value ], and p is less than 0.05 in the figure.
Fig. 4 shows the detection of antigen-specific B cells using CII polypeptide probes and evaluation of their diagnostic value for rheumatoid arthritis, where p <0.05, p <0.01, p <0.001 (using Kruskal-Wallis assay).
Fig. 5 shows the diagnostic value of anti-CII polypeptide antibodies against rheumatoid arthritis, where p <0.05, p <0.01, p <0.001 (using Kruskal-Wallis assay).
FIG. 6 shows the diagnostic value of CII antigen specific B cells in combination with anti-CII polypeptide antibodies for rheumatoid arthritis.
Detailed Description
For a better understanding of the present application, reference will now be made in detail to the present embodiments, examples of which are illustrated in the accompanying drawings, wherein the present application is illustrated in the accompanying drawings. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present application without making any inventive effort, shall fall within the scope of the present application. The instruments and reagents used in the examples were commercially available without any particular description.
Example 1 preparation of antigen-specific B cell detection probes
The antigen-specific B cell detection probes include MAL-PEG-8arm: CII-AF647 and MAL-PEG-8arm: CII-Cy5.5. With MAL-PEG-8arm: CII-AF647 is an example to describe the preparation process in detail.
Raw material compound: the antigen polypeptide is a type II collagen polypeptide (CII polypeptide for short), and the amino acid sequence is as follows: C-GA- (cit) -GLTG- (cit) -PGDA-GPOGPO (SEQ ID NO: 1); fluorescent dye: AF647-NHS; catalyst: and triethylamine.
The preparation process comprises the following steps:
(1) CII polypeptide (0.01 mmol), AF647-NHS (0.02 mmol) and triethylamine (2 mu L) are uniformly mixed in anhydrous DMSO, reacted under the protection of nitrogen at room temperature, and subjected to oscillation reaction. After the completion of the reaction, the whole solution was packed into a dialysis bag (MWCO 1000 Da) and dialyzed in distilled water for 2 days to remove unreacted starting compounds. The dialyzed solution was lyophilized to give an intermediate product.
(2) The intermediate product (0.01 mmol) obtained in step (1) and MAL-PEG-8arm (0.002 mmol) were mixed in PBS (ph=6.8) and reacted overnight under nitrogen at room temperature. After the completion of the reaction, the whole solution was filled into dialysis bags (MWCO 2000 Da), and then dialyzed in distilled water for 2 days to remove unreacted intermediate and MAL-PEG-8arm. Freeze-drying the dialyzed solution to obtain an antigen-specific B cell detection probe MAL-PEG-8arm: CII-AF647 solid product.
The nitrogen protection can prevent the interference of external oxidation environment, and the freeze-drying operation in the steps (1) and (2) is beneficial to the longer preservation of the product.
The product was detected with an ultraviolet spectrophotometer.
The antigen-specific B cell detection probe is MAL-PEG-8arm: when CII-Cy5.5, the fluorescent dye is changed into Cy5.5-NHS (0.02 mmol), and the rest steps are the same.
Referring to FIG. 1 for preparation and recognition modes of an antigen-specific B cell detection probe with CII polypeptide, the antigen polypeptide of the probe is respectively connected with MAL-PEG-8arm and fluorescent dye (AF 647 or Cy5.5), and after the antigen polypeptide is specifically combined with BCR on the surface of the antigen-specific B cell in a biological sample of a patient, the antigen-specific B cell is fluorescently labeled, so that the observation and statistics can be carried out.
Example 2 evaluation of Performance of antigen-specific B cell detection probes
2.1 Probe availability
Obtaining human peripheral blood mononuclear cells by lymphocyte separation liquid, and performing flow staining on CD3 +percp-Cy5.5、CD19+APC、FVD- BV510 and an antigen specific B cell detection probe MAL-PEG-8arm: CII-AF647, MAL-PEG-8arm: CII-Cy5.5, flow cytometry was performed. The gate (FSC/SSC) was placed on peripheral mononuclear cells, FVD - and CD3 -/CD19+ B cells, and the antigen-specific B cells of CII-AF647 +/ CII-Cy5.5+ could be labeled. The percentage of CII polypeptide antigen specific B cells of AF647 +/Cy5.5+ in total B cells was calculated.
As shown in fig. 2, two fluorescent double positives, AF647 and cy5.5, are circled in total B cells, targeting CII polypeptides to antigen-specific B cells; AF647 and cy5.5 were both fluorescent double negative, non-antigen specific B cells. The figure shows that the antigen-specific B cells are significantly higher in RA patients than in healthy controls, with a high recognition capacity.
2.2 Probe specificity
And (3) separating to obtain antigen-specific B cells (AF 647 +/Cy5.5+) and non-antigen-specific B cells (AF 647 -/Cy5.5-) recognized by the CII polypeptide by adopting a flow separation technology (figure 2), respectively culturing the cells, and collecting culture supernatants after 7 days for ELISA detection of the CII antibody.
As shown in fig. 3, the results show: the anti-CII antibody secreted by the antigen-specific B cells obtained by sorting is obviously higher than that of the non-antigen-specific B cells, and the non-antigen-specific B cells do not substantially secrete the anti-CII antibody. Thus, MAL-PEG-8arm was verified: CII-AF647, MAL-PEG-8arm: the CII-Cy5.5 probe has specificity, can effectively mark antigen-specific B cells, and also shows that the antigen-specific B cells are main pathogenic cell subgroups secreting pathogenic antibodies.
Example 3 antigen-specific B cell detection probes detection of antigen-specific B cells, diagnostic value analysis for Rheumatoid Arthritis (RA)
3.1 Diagnostic value analysis of RA by antigen-specific B cell detection probe flow detection
And selecting 20 persons of RA patients, and 10 persons of disease control Systemic Lupus Erythematosus (SLE), sjogren Syndrome (SS), osteoarthritis (OA) and health control respectively, obtaining peripheral blood of the patients, separating single nuclear cells, performing flow detection, and verifying the effectiveness of antigen-specific B cell detection probes.
Flow cytometry detection techniques:
1) Taking fresh EDTA anticoagulated whole blood 3.5 mL (1 deep ultraviolet), and uniformly mixing with PBS liquid 1:1; adding 7mL of human peripheral blood lymphocyte separating medium into a 15mL centrifuge tube, slowly adding diluted whole blood into the upper layer of the separating medium, and avoiding oscillation; centrifuging at 1800rpm for 20min at room temperature; the upper diluted plasma was aspirated off, the middle cell layer was slowly aspirated to a new 15mL centrifuge tube with a 1mL pipette, PBS (about 10 mL) was added, mixed well, and centrifuged at 1800rpm for 5min; the supernatant was discarded to obtain PBMC.
2) Adding 12mL of PBS into a centrifuge tube for extracting mononuclear cells, uniformly mixing, putting into a centrifuge at 1800rpm for 5min, discarding the supernatant, and repeatedly cleaning for one time;
3) Adding 1 mL PBS, mixing, transferring to a new EP tube, centrifuging at 1800rpm for 5min, and discarding supernatant;
4) 200 mu L of PBS is added for uniform mixing, and the Percp-Cy5.5 marked CD3 antibodies are respectively added under the dark condition; APC-tagged CD19; MAL-PEG-8arm: CII-AF 647, MAL-PEG-8arm: the CII-Cy5.5 probe labeled antigen specific B cells were incubated for 30min at room temperature;
5) Adding 1 mL PBS parts of the mixture into each tube, uniformly mixing and cleaning, centrifuging at 1800rpm for 5min, and discarding the supernatant; repeating the washing once again;
6) Adding 300 mu L of PBS into each tube, uniformly mixing, and waiting for loading;
7) Filtering the sample into a detection tube by using a 200-mesh filter screen before the machine is started, so as to avoid blocking the machine due to overlarge molecular weight;
8) 500,000 cells were collected on-board using a flow cytometer, and analyzed by Beckmann software after completion.
As shown in FIG. 4, the A graph shows the result of flow analysis statistics of the ratio of antigen-specific B cells in total B cells after staining the mononuclear cells separated from different populations by using an antigen-specific B cell detection probe, and shows that the ratio of antigen-specific B cells in RA patients is significantly higher than that of normal people and related disease control groups, and has statistical significance. Panel B shows the covariate-adjusted receiver operating characteristic curve (ROC) curve of antigen-specific B cell detection probes against RA patients, reflecting the correlation between sensitivity and specificity of probe diagnosis, with an AUC of 0.908. The ROC curve in the figure has an abscissa False Positive Rate (FPR), i.e. a medically false positive; the ordinate is True Positive Rate (TPR), a medically true positive. AUC: the area under the ROC curve, which can quantitatively reflect the model performance measured based on the ROC curve.
3.2 Diagnostic value analysis of RA for anti-CII polypeptide antibody ELISA detection
The above-mentioned RA patients were selected for 20 persons, and 10 persons for disease control Systemic Lupus Erythematosus (SLE), sjogren's Syndrome (SS), osteoarthritis (OA) and healthy control, and serum isolated after obtaining peripheral blood thereof was subjected to anti-CII antibody ELISA assay, and sensitivity and specificity of the ELISA assay to RA diagnosis were analyzed and compared with antigen-specific B cell assay probes.
ELISA detection technology:
1) Taking out the detection plate coated with CII polypeptide (amino acid sequence shown as SEQ ID NO: 1) from refrigerator, standing at room temperature, and unpacking;
2) Incubation of serum: sample serum (1:100) was diluted with 1% BSA-PBST and incubated at 37℃for 1h at 100. Mu.L per well;
3) Washing the plate: washing the plate 3 times with wash solution (0.05% PBST), leaving 300 μl of wash solution per well for 2min, forcibly removing the wash solution, and drying;
4) Secondary antibody incubation: adding goat anti-human IgG marked by horseradish peroxidase (1% BSA-PBST 1:10000 dilution, 100. Mu.L/well), and incubating at 37℃for 30min;
5) Washing the plate: washing the plate 3 times with wash solution (0.05% PBST), leaving 300 μl of wash solution per well for 2min, forcibly removing the wash solution, and drying;
6) Color development: 100 mu L of TMB color development liquid is sucked and added into each microwell, and the microwells are incubated for 15min in dark place;
7) And (3) terminating: 100. Mu.L of stop solution (2 mol/L of dilute sulfuric acid) was added to each microwell, and the optical density values at 450 nm and 570nm were read using a microplate reader and the results were calculated. The color produced remains stable for at least 30 minutes, during which time the optical density value is read;
8) And (3) calculating results: positive control standard serum is arranged on each plate, and is obtained by selecting 10 RA serum with OD value more than 1, and mixing the two serum uniformly in equal volume. The final result is represented by AU values. AU value= (OD value of serum to be measured/OD value of positive serum) ×100, OD value is 450nm detection value-570 nm detection value of enzyme labeling instrument)
As shown in FIG. 5, the A graph shows the statistical result of the detection of the anti-CII polypeptide antibody content of the peripheral blood serum of different people by using the anti-CII polypeptide antibody ELISA, and the result shows that the anti-CII polypeptide antibody in RA patients is obviously higher than that of normal people and related disease control groups, and has statistical significance. Panel B shows the ROC curve of the anti-CII polypeptide antibody ELISA detection on RA patients, reflecting the correlation between sensitivity and specificity of ELISA diagnosis, and AUC is 0.889.
In summary, flow cytometry and ELISA detection results showed that: both the CII polypeptide-targeted antigen-specific B cells and the anti-CII polypeptide antibodies were significantly increased in RA patients compared to normal humans and other disease controls. Thus, both detection techniques are of significant interest for diagnosis of RA.
The sensitivity and specificity of the two detection methods to RA diagnosis were further analyzed and the results are given in table 1 below:
TABLE 1 diagnostic value analysis of RA by antigen-specific B cells and anti-CII polypeptide antibodies and combinations thereof
The result shows that the sensitivity of the CII polypeptide serving as an antigen specificity B cell detection probe of the antigen polypeptide in the diagnosis of RA in the flow detection is 70%, and the specificity is 90%; the sensitivity of the anti-CII polypeptide antibody ELISA detection for diagnosing RA is 60 percent, and the specificity is 92.5 percent. Therefore, the sensitivity of the antigen-specific B cell detection probe is better than that of the ELISA detection of the anti-CII polypeptide antibody under the condition of ensuring the specificity. Meanwhile, the AUC of the probe detection is higher than that of ELISA detection, which shows that the probe has important significance for diagnosing RA.
3.3 Combined detection of CII antigen specific B cell detection probe and anti-CII polypeptide antibody, thereby further improving RA diagnosis efficiency
When the combined analysis and diagnosis are carried out on the flow detection of the antigen-specific B cell detection probe (the antigen polypeptide is CII polypeptide) and the ELISA detection result of the anti-CII polypeptide antibody, the sensitivity reaches 90% and the specificity reaches 90% with reference to the table 1. ROC results of the probe and ELISA combination detection showed: the AUC of the antigen-specific B cell detection probe (the antigen polypeptide is CII polypeptide) after the flow combined anti-CII polypeptide antibody ELISA reaches 0.938, which is higher than the AUC (0.908 and 0.889) of the antigen-specific B cell detection probe and the anti-CII polypeptide antibody ELISA (see figure 6), which shows that the combined diagnosis accuracy of the antigen-specific B cell detection probe and the anti-CII polypeptide antibody is higher.
FIG. 6 reflects the sensitivity and specificity of antigen-specific B cell detection probe flow assays, ELISA kit assays, and combinations thereof, respectively.
Specific examples are set forth herein to illustrate the invention in detail, and the description of the above examples is only for the purpose of aiding in understanding the core concept of the invention. It should be noted that any obvious modifications, equivalents, or other improvements to those skilled in the art without departing from the inventive concept are intended to be included in the scope of the present invention.
Claims (8)
1. An antigen-specific B cell detection probe, comprising an antigen polypeptide for recognizing an antigen-specific B cell, and a fluorescent dye and an eight-arm polyethylene glycol maleimide, which are covalently linked to the antigen polypeptide, respectively; the covalent connection is that free amino in the antigen polypeptide and NHS ester carried by fluorescent dye undergo esterification reaction, so that the fluorescent dye is covalently connected to the antigen polypeptide, and then is connected with maleimide in PEG through sulfhydryl of cysteine side chain of the antigen polypeptide;
the antigen polypeptide is a type II collagen polypeptide, and the amino acid sequence is shown as SEQ ID NO. 1.
2. The probe of claim 1, wherein the fluorescent dye is AF647 or cy5.5.
3. The method for preparing a probe according to claim 1 or 2, comprising the steps of:
(1) Uniformly mixing three raw materials of antigen polypeptide, fluorescent dye-NHS and triethylamine in anhydrous DMSO, carrying out oscillation reaction under the protection of nitrogen, after the reaction is finished, filling the reaction liquid into a dialysis bag, and dialyzing in distilled water to remove unreacted raw materials, thereby obtaining an intermediate product of the antigen polypeptide connected with the fluorescent dye;
(2) Adding the intermediate product and the eight-arm polyethylene glycol maleimide into PBS, mixing, reacting under the protection of nitrogen, and filling into a dialysis bag for dialysis in distilled water after the reaction is finished so as to remove unreacted intermediate product and the eight-arm polyethylene glycol maleimide, thereby obtaining the probe.
4. Use of a probe according to claim 1 or 2 for the preparation of a diagnostic product for rheumatoid arthritis.
5. The use of claim 4, wherein the diagnostic product comprises a probe-flow detection reagent.
6. The use according to claim 5, wherein the diagnostic product further comprises an ELISA reagent comprising a capture antigen having the same amino acid sequence as the antigen polypeptide of the probe.
7. A rheumatoid arthritis diagnostic product comprising the probe of claim 1 or 2.
8. The product of claim 7, further comprising a capture antigen for detection by an ELISA method, the capture antigen having an amino acid sequence identical to the antigen polypeptide amino acid sequence of the probe.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410692326.9A CN118258998B (en) | 2024-05-31 | 2024-05-31 | Antigen-specific B cell detection probe and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410692326.9A CN118258998B (en) | 2024-05-31 | 2024-05-31 | Antigen-specific B cell detection probe and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN118258998A CN118258998A (en) | 2024-06-28 |
| CN118258998B true CN118258998B (en) | 2024-08-09 |
Family
ID=91611418
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410692326.9A Active CN118258998B (en) | 2024-05-31 | 2024-05-31 | Antigen-specific B cell detection probe and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN118258998B (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009103753A1 (en) * | 2008-02-20 | 2009-08-27 | Ablynx Nv | Methods for identifying and/or sorting cells by secreted molecule and kits for performing such methods |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130039861A1 (en) * | 2005-04-06 | 2013-02-14 | Immunomedics, Inc. | Dye Conjugated Peptides for Fluorescent Imaging |
| CA2623841C (en) * | 2005-09-27 | 2019-03-12 | National Research Council Of Canada | Methods of identifying agents capable of tmem30a mediated transmigration across the blood brain barrier (bbb) |
| TWI388334B (en) * | 2007-12-11 | 2013-03-11 | Glaxo Group Ltd | Antigen binding proteins |
| EP3310387A1 (en) * | 2015-06-16 | 2018-04-25 | Fraunhofer Gesellschaft zur Förderung der Angewand | Specific photoimmuno-theranostics for detection and elimination of skin cancer cells |
| CN111450264B (en) * | 2020-05-03 | 2021-03-02 | 四川大学华西医院 | Bimodal nanoprobe targeting glioblastoma multiforme and preparation method thereof |
| CN114377017A (en) * | 2020-10-21 | 2022-04-22 | 复旦大学 | Use of folate and folate modification to induce B cell immune tolerance and target mIgM positive expression B cell lymphoma |
| WO2023107985A1 (en) * | 2021-12-07 | 2023-06-15 | Genentech, Inc. | Cellular assays and methods to assess mhc-peptide-tcr interactions and kinetics |
-
2024
- 2024-05-31 CN CN202410692326.9A patent/CN118258998B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009103753A1 (en) * | 2008-02-20 | 2009-08-27 | Ablynx Nv | Methods for identifying and/or sorting cells by secreted molecule and kits for performing such methods |
Also Published As
| Publication number | Publication date |
|---|---|
| CN118258998A (en) | 2024-06-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP1195606A1 (en) | Allergen-microarray assay | |
| JP3105579B2 (en) | Biologically active reagents and analytical elements and methods of using the reagents | |
| EP1969373A2 (en) | Multiplexed detection of anti-red cell alloantibodies | |
| CN1866013B (en) | Liquid phase chip for parallel detection of autoantibodies, preparation method and application thereof | |
| CN113447659B (en) | Kit for detecting anti-proteasome subunit alpha 1-IgG antibody | |
| CN106459198A (en) | Method for the serological diagnosis of rheumatoid arthritis | |
| CN109342718A (en) | A kind of magnetic microparticle chemiluminescence detection method | |
| CN110018156A (en) | Application of the anti-CarP antibody as systemic lupus erythematosus diagnosis marker | |
| WO1987004794A1 (en) | Latex agglutination using avidin/biotin system | |
| CN118258998B (en) | Antigen-specific B cell detection probe and preparation method and application thereof | |
| CN109725158A (en) | Application of the polypeptide SLE2018-V001 in diagnostic system lupus erythematosus kit | |
| CN117470819A (en) | Autoimmune myositis disease detection kit and detection method thereof | |
| CN112480244A (en) | Anti-allergic nano antibody composition, antibody determination method and spray | |
| CN108535493B (en) | Method for detecting specific allergen IgE | |
| CN118240057B (en) | Antigenic peptide and application thereof in preparation of rheumatoid arthritis diagnostic product | |
| KR20010025027A (en) | Immunoassay reagents and immunoassay method | |
| CA1231047A (en) | Diagnostic test for rheumatological diseases | |
| CN108948174B (en) | Citrulline modified peptide and application thereof | |
| CN113447656A (en) | Kit for detecting anti-filamentous actin cap-forming protein beta-IgG antibody | |
| CN109212181A (en) | A kind of neutrophil gelatinase-associated lipocalin detection kit and preparation method thereof and application method | |
| CN109725156A (en) | Application of the polypeptide SLE2018-V002 in diagnostic system lupus erythematosus kit | |
| CN113447648B (en) | Kit for detecting anti-serine/arginine-rich splicing factor 9-IgG antibody | |
| CN116162134B (en) | Polypeptide or polypeptide composition | |
| CN114814203B (en) | Antinuclear antibody joint detection kit | |
| CN107090024A (en) | Detection device, detection kit and diagnostic method available for diagnostic system lupus erythematosus |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |