CN117466980A - Supported metal conjugate based on polypeptide chain skeleton and applied to metal antibody labeling and application thereof - Google Patents
Supported metal conjugate based on polypeptide chain skeleton and applied to metal antibody labeling and application thereof Download PDFInfo
- Publication number
- CN117466980A CN117466980A CN202311450244.5A CN202311450244A CN117466980A CN 117466980 A CN117466980 A CN 117466980A CN 202311450244 A CN202311450244 A CN 202311450244A CN 117466980 A CN117466980 A CN 117466980A
- Authority
- CN
- China
- Prior art keywords
- amino acid
- metal
- polypeptide chain
- antibody
- group
- 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.)
- Pending
Links
- 229910052751 metal Inorganic materials 0.000 title claims abstract description 145
- 239000002184 metal Substances 0.000 title claims abstract description 145
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 105
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 104
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 104
- 238000002372 labelling Methods 0.000 title claims abstract description 60
- 238000001514 detection method Methods 0.000 claims abstract description 49
- 229910021645 metal ion Inorganic materials 0.000 claims abstract description 38
- 230000000694 effects Effects 0.000 claims abstract description 19
- 238000004949 mass spectrometry Methods 0.000 claims abstract description 16
- 235000001014 amino acid Nutrition 0.000 claims description 60
- 229940024606 amino acid Drugs 0.000 claims description 60
- 150000001413 amino acids Chemical class 0.000 claims description 60
- 150000003862 amino acid derivatives Chemical class 0.000 claims description 47
- 150000003141 primary amines Chemical group 0.000 claims description 29
- 125000003396 thiol group Chemical group [H]S* 0.000 claims description 24
- 102000004169 proteins and genes Human genes 0.000 claims description 21
- 108090000623 proteins and genes Proteins 0.000 claims description 21
- 150000002500 ions Chemical group 0.000 claims description 20
- QPCDCPDFJACHGM-UHFFFAOYSA-N N,N-bis{2-[bis(carboxymethyl)amino]ethyl}glycine Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(=O)O)CCN(CC(O)=O)CC(O)=O QPCDCPDFJACHGM-UHFFFAOYSA-N 0.000 claims description 9
- XQUPVDVFXZDTLT-UHFFFAOYSA-N 1-[4-[[4-(2,5-dioxopyrrol-1-yl)phenyl]methyl]phenyl]pyrrole-2,5-dione Chemical compound O=C1C=CC(=O)N1C(C=C1)=CC=C1CC1=CC=C(N2C(C=CC2=O)=O)C=C1 XQUPVDVFXZDTLT-UHFFFAOYSA-N 0.000 claims description 8
- 238000012986 modification Methods 0.000 claims description 8
- 230000004048 modification Effects 0.000 claims description 8
- 229920003192 poly(bis maleimide) Polymers 0.000 claims description 7
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 6
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 claims description 6
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 claims description 6
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 claims description 6
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims description 6
- 235000018417 cysteine Nutrition 0.000 claims description 6
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 claims description 6
- 235000018102 proteins Nutrition 0.000 claims description 6
- 239000004475 Arginine Substances 0.000 claims description 5
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 claims description 5
- 239000004472 Lysine Substances 0.000 claims description 5
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims description 5
- 235000009582 asparagine Nutrition 0.000 claims description 5
- 229960001230 asparagine Drugs 0.000 claims description 5
- 239000012472 biological sample Substances 0.000 claims description 5
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 claims description 5
- 239000004471 Glycine Substances 0.000 claims description 3
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 claims description 3
- 235000004279 alanine Nutrition 0.000 claims description 3
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 3
- 229960003330 pentetic acid Drugs 0.000 claims description 3
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 2
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 claims description 2
- WDLRUFUQRNWCPK-UHFFFAOYSA-N Tetraxetan Chemical compound OC(=O)CN1CCN(CC(O)=O)CCN(CC(O)=O)CCN(CC(O)=O)CC1 WDLRUFUQRNWCPK-UHFFFAOYSA-N 0.000 claims description 2
- 229920000642 polymer Polymers 0.000 abstract description 23
- 238000000034 method Methods 0.000 abstract description 12
- 238000005516 engineering process Methods 0.000 abstract description 10
- 230000008569 process Effects 0.000 abstract description 4
- 230000015572 biosynthetic process Effects 0.000 abstract description 3
- 238000003786 synthesis reaction Methods 0.000 abstract description 3
- 238000010959 commercial synthesis reaction Methods 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 35
- 239000000243 solution Substances 0.000 description 29
- 239000000523 sample Substances 0.000 description 27
- 238000000108 ultra-filtration Methods 0.000 description 23
- 239000000872 buffer Substances 0.000 description 15
- 239000006228 supernatant Substances 0.000 description 14
- 102000017420 CD3 protein, epsilon/gamma/delta subunit Human genes 0.000 description 13
- 101000917858 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-A Proteins 0.000 description 11
- 101000917839 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-B Proteins 0.000 description 11
- 102100029185 Low affinity immunoglobulin gamma Fc region receptor III-B Human genes 0.000 description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 9
- 229940098773 bovine serum albumin Drugs 0.000 description 9
- 239000007788 liquid Substances 0.000 description 7
- 239000012528 membrane Substances 0.000 description 7
- 239000000203 mixture Substances 0.000 description 7
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 6
- 239000008055 phosphate buffer solution Substances 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 239000000047 product Substances 0.000 description 5
- 108060003951 Immunoglobulin Proteins 0.000 description 4
- 108010052285 Membrane Proteins Proteins 0.000 description 4
- 102000018697 Membrane Proteins Human genes 0.000 description 4
- 210000001744 T-lymphocyte Anatomy 0.000 description 4
- PZBFGYYEXUXCOF-UHFFFAOYSA-N TCEP Chemical compound OC(=O)CCP(CCC(O)=O)CCC(O)=O PZBFGYYEXUXCOF-UHFFFAOYSA-N 0.000 description 4
- 102000018358 immunoglobulin Human genes 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 210000005259 peripheral blood Anatomy 0.000 description 4
- 239000011886 peripheral blood Substances 0.000 description 4
- 239000008363 phosphate buffer Substances 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 238000005481 NMR spectroscopy Methods 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 3
- 239000012467 final product Substances 0.000 description 3
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 239000008351 acetate buffer Substances 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000007795 chemical reaction product Substances 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000007877 drug screening Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000000834 fixative Substances 0.000 description 2
- 238000001095 inductively coupled plasma mass spectrometry Methods 0.000 description 2
- 210000000822 natural killer cell Anatomy 0.000 description 2
- 238000004445 quantitative analysis Methods 0.000 description 2
- 238000006722 reduction reaction Methods 0.000 description 2
- 238000012712 reversible addition−fragmentation chain-transfer polymerization Methods 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 238000009010 Bradford assay Methods 0.000 description 1
- 241000699800 Cricetinae Species 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 239000012625 DNA intercalator Substances 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 229910052691 Erbium Inorganic materials 0.000 description 1
- 229910052765 Lutetium Inorganic materials 0.000 description 1
- 229910052775 Thulium Inorganic materials 0.000 description 1
- 125000003295 alanine group Chemical group N[C@@H](C)C(=O)* 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000003596 drug target Substances 0.000 description 1
- UYAHIZSMUZPPFV-UHFFFAOYSA-N erbium Chemical compound [Er] UYAHIZSMUZPPFV-UHFFFAOYSA-N 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 210000003714 granulocyte Anatomy 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- OHSVLFRHMCKCQY-UHFFFAOYSA-N lutetium atom Chemical compound [Lu] OHSVLFRHMCKCQY-UHFFFAOYSA-N 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000004005 microsphere Substances 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000010526 radical polymerization reaction Methods 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 238000003127 radioimmunoassay Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 108010027322 single cell proteins Proteins 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 239000012192 staining solution Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- FRNOGLGSGLTDKL-UHFFFAOYSA-N thulium atom Chemical compound [Tm] FRNOGLGSGLTDKL-UHFFFAOYSA-N 0.000 description 1
- 230000025366 tissue development Effects 0.000 description 1
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
-
- 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
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6848—Methods of protein analysis involving mass spectrometry
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Bioinformatics & Computational Biology (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Organic Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Food Science & Technology (AREA)
- Biophysics (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Gastroenterology & Hepatology (AREA)
- General Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Electrochemistry (AREA)
- Dispersion Chemistry (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a metal-loaded conjugate based on application of a polypeptide chain skeleton to metal antibody labeling, which comprises a polypeptide chain skeleton, wherein the polypeptide chain skeleton comprises a side chain group for modifying a metal ion group, a side chain group for modifying an antibody bridging structure, and a connecting structure which can exist among polypeptide chain skeletons, among polypeptide chain skeletons and the metal ion group capable of being combined, and among the polypeptide chain skeletons and the antibody bridging structure and avoid steric hindrance from affecting the labeling effect. The invention also discloses application of the metal antibody in detection of the metal antibody mark based on a mass spectrometry detection technology. The performance of the metal antibody labeled loaded conjugate based on the polypeptide chain skeleton and applied to mass spectrometry detection is equivalent to that of the commercial conjugate. And compared with the commercial synthesis process of the high molecular polymer framework capable of being grafted with the metal ion group, the non-polymeric conjugate disclosed by the invention has a simpler synthesis process.
Description
Technical Field
The invention relates to the technical field of mass spectrometry detection, in particular to a supported metal conjugate based on a polypeptide chain skeleton and applied to metal antibody labeling and application thereof.
Background
The technology capable of accurately detecting the expression level of the sample cell target protein plays an important role and has an application prospect in the fields of scientific research, clinical transformation diagnosis and test application, drug screening and the like. The change of the expression quantity of the protein in the cells is researched, the signal path change of organism tissue development and disease pathology occurrence can be explored, the functional cell constitution of specific tissues and organs can be accurately described, the disease characteristic monitoring index is screened out, and the clinical diagnosis test and the drug target effect detection are served. Currently, widely used techniques for qualitative and quantitative detection of target proteins in sample cells include: dye assay by Bradford method/Lowry method/BCA method, western blot, ultraviolet spectrometry, organic mass spectrometry, high performance liquid chromatography, flow cytometry, cytokine flow microsphere technique, enzyme-linked immunosorbent assay, radioimmunoassay, etc. However, these techniques do not meet the ever-increasing high-throughput, high-sensitivity, high-accuracy detection needs, serving the goal of clinically accurate medical treatment. And the quantitative analysis of the target protein provides a remarkable advantage for single-cell protein quantification by taking isotope elements with different mass numbers as signal tags. The mass flow detection technology established based on immunodetection of inductively coupled plasma mass spectrometry (ICP-MS) can realize high-sensitivity and high-accuracy quantitative analysis of more than forty target proteins at the single cell level in a sample of up to million-level cells by selecting non-naturally occurring non-radioactive metal isotopes in a biological sample as detection labels. The polypeptide chain skeleton-based metal conjugate can be applied to the metal antibody labeled load, and can be used for labeling non-naturally occurring nonradioactive metal isotopes in a biological sample on an antibody of a target protein, so that the increasingly severe detection requirements of scientific research and accurate medical service are met.
Disclosure of Invention
The invention aims to provide a metal-antibody-labeled supported metal conjugate based on a polypeptide chain skeleton and application thereof, which can realize the labeling of non-naturally-occurring nonradioactive metal isotopes in a biological sample on an antibody of a target protein.
The invention adopts the following technical scheme:
a metal-loaded conjugate based on the application of a polypeptide chain skeleton to metal antibody labeling, comprising a polypeptide chain skeleton comprising a side chain group for modifying a bindable metal ion group, a side chain group for modifying an antibody bridging structure, and a possibly existing connection structure between polypeptide chain skeletons, between polypeptide chain skeletons and bindable metal ion groups, between polypeptide chain skeletons and antibody bridging structures, which avoids steric hindrance from affecting the labeling effect.
Further, the polypeptide chain skeleton includes amino acids or/and amino acid derivatives having a side chain containing a group for modifying a metal ion-binding group, amino acids or/and amino acid derivatives having a side chain containing a group for modifying an antibody bridging structure, and amino acids or/and amino acid derivatives that may be present to avoid steric hindrance from affecting the labeling effect.
Further, the polypeptide chain skeleton includes at least 1 amino acid or/and amino acid derivative having a side chain containing a group for modifying a metal ion-binding group, 1 amino acid or amino acid derivative having a side chain containing a group for modifying an antibody bridging structure, and possibly an amino acid or/and amino acid derivative for avoiding steric hindrance from affecting the labeling effect.
Further, the polypeptide chain skeleton comprises at least 1 amino acid or/and amino acid derivative with a side chain containing primary amine groups, 1 amino acid or amino acid derivative with a side chain containing sulfhydryl groups, and possibly existing amino acid or/and amino acid derivative with steric hindrance avoiding influence on the labeling effect;
amino acid with a side chain containing primary amine groups or/and amino acid derivative with a side chain modified by primary amine groups can be combined with metal ion groups to provide a binding site of a polypeptide chain skeleton and metal ions, and amino acid with a side chain containing sulfhydryl groups or sulfhydryl groups on the side chain of the amino acid derivative modify an antibody bridging structure to provide a binding site of the polypeptide chain skeleton and an antibody to be marked, and amino acid or/and amino acid derivative with steric hindrance effect is/are prevented from extending the chain length of the polypeptide chain skeleton to avoid steric hindrance effect.
Still further, the amino acid with a side chain containing a primary amine group is one or more of asparagine, glutamine, lysine and arginine, the amino acid with a side chain containing a sulfhydryl group is cysteine, and the amino acid avoiding the influence of steric hindrance is alanine or glycine or other amino acids with side chains not containing a sulfhydryl group nor a primary amine group.
Furthermore, various amino acids and amino acid derivatives are synthesized into a polypeptide chain skeleton in a straight-chain manner, and other amino acids and amino acid derivatives are not required to be arranged in a special order except that the amino acid or amino acid derivative with a sulfhydryl group in a side chain is designed at the amino terminal or the carboxyl terminal of the polypeptide chain skeleton.
Further, the modification of the primary amine group on the side chain of the amino acid or/and amino acid derivative having a primary amine group in the side chain may be combined with a metal ion group, and the combined metal ion group may include ethylenediamine tetraacetic acid, diethylenetriamine pentaacetic acid, 1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid.
Further, the thiol-modified antibody bridge structure on the side chain of the amino acid or amino acid derivative having a thiol-group in the side chain includes bismaleimide.
Further, the modification of the primary amine group on the side chain of the amino acid or/and amino acid derivative having a primary amine group in the side chain may bind to a metal ion group, and the metal ion to which the metal ion group may bind refers to an element having one of the following atomic numbers: 3. 4, 11-13, 19-32, 37-42, 44-51, 55-60, 62-83.
The application of the supported metal conjugate based on the polypeptide chain skeleton in metal antibody labeling in detection of metal antibody labeling based on mass spectrometry detection technology.
Further, the quantitative detection of the relative expression amount of the target protein including the single cell level or the quantitative detection of the average expression level of the target protein of the total protein extract of the biological sample is detected based on the mass spectrometry detection technology.
The beneficial effects of the invention are as follows:
the method has the advantages of high throughput, high detection parameters, high sensitivity and high accuracy in single cell level detection of the target protein expression level in the sample cells, realization of a technology for accurately detecting the target protein expression level of the sample cells, and great significance and application prospect in the fields of scientific research, clinical transformation diagnostic test application, drug screening and the like. The mass spectrometry detection technology is a high-new detection technology meeting the application requirement, but the detection of the expression level of the target protein expressed by the cell to be detected depends on taking metal ions with specific metal mass numbers as detection labels, marking the metal ions onto antibodies capable of specifically recognizing the target protein to be detected, and quantifying the expression level of the target protein in the single cell by detecting the signal intensity of the metal ions with the specific metal mass numbers. Thus, conjugates that can achieve labeling of metal ions to antibodies are core technologies for achieving mass spectrometry detection applications.
The current commercial conjugate of the labeled metal antibody applied to mass spectrometry detection is a product which is prepared by performing controllable free radical polymerization reaction based on reversible addition-fragmentation chain transfer polymerization (Reversible Addition-Fragmentation Chain Transfer Polymerization, RAFT) reaction to form a high polymer framework capable of being grafted with metal ion groups, grafting the metal ion groups on the side chains of the framework and modifying the bound antibody groups at one end, and is applied to mass spectrometry detection to realize the labeling of the metal antibody. Currently, only one mass spectrometry service provider abroad (united states) provides commercial RAFT-based metal antibody labeling kits for mass spectrometry detection technology. The invention discloses a metal antibody-labeled supported metal conjugate based on a polypeptide chain skeleton, which creatively uses the polypeptide chain skeleton as a metal-supported and antibody-bound skeleton, utilizes primary amine groups on amino acid/amino acid derivative side chains with primary amine groups on side chains on polypeptide chains to modify and can be combined with metal ion groups, and mercapto-modified antibody bridging structures on amino acid/amino acid derivative side chains with mercapto groups on side chains, and is applied to mass spectrometry flow detection to realize the labeling of metal antibodies. The performance of the metal antibody labeled loaded conjugate based on the polypeptide chain skeleton and applied to the metal antibody labeling in mass spectrometry detection is equivalent to that of a commercial conjugate (commercial high molecular polymer). Compared with the commercial synthesis process of the high molecular polymer framework capable of being grafted and combined with the metal ion group, the non-polymeric conjugate disclosed by the invention has a simpler synthesis process and is more beneficial to the industrial and amplified production of products.
Drawings
FIG. 1 shows the results of nuclear magnetic resonance detection of the polypeptide chain backbone of example 1, a metal-loaded conjugate (a conjugate of the polypeptide chain backbone) based on the application of the polypeptide chain backbone to metal antibody labeling, and a commercial high molecular polymer.
FIG. 2 shows the dye specificity of a T cell population in a peripheral blood sample based on the application of a polypeptide chain backbone to a metal antibody-labeled, metal-loaded conjugate (conjugate of a polypeptide chain backbone) labeled CD3 metal antibody and a commercial high molecular polymer-labeled CD3 metal antibody.
FIG. 3 is a comparison of the detection signal intensity of a T cell population in a peripheral blood sample based on the application of a polypeptide chain backbone to a metal antibody-labeled, metal-loaded conjugate (conjugate of a polypeptide chain backbone) labeled CD3 metal antibody and a commercial high molecular polymer labeled CD3 metal antibody.
FIG. 4 is a comparison of detection signal intensity of a population of natural killer cells and monocytes in a peripheral blood sample based on a polypeptide chain backbone applied to metal antibody-labeled, metal-loaded conjugate (conjugate of polypeptide chain backbone) labeled CD16 metal antibody and commercial high molecular polymer labeled CD16 metal antibody.
Detailed Description
The present invention will be described in further detail with reference to the following specific examples and drawings, but the present invention is not limited to the following specific examples.
The following examples are provided not to limit the scope of the invention nor the order of execution of the steps described. The present invention is obviously improved and the synthesis system is amplified by the person skilled in the art in combination with the prior common knowledge, and the present invention is also within the scope of protection claimed by the present invention.
Example 1
Preparation of supported metal conjugates based on polypeptide chain backbone for metal antibody labeling
Step one, obtaining a polypeptide chain skeleton
The design principle is that the polypeptide chain skeleton comprises at least 1 amino acid or/and amino acid derivative with a side chain containing primary amine groups, 1 amino acid or amino acid derivative with a side chain containing sulfhydryl groups, and possibly 1 amino acid or/and amino acid derivative with a side chain containing no sulfhydryl groups and no primary amine groups, wherein the amino acid with a side chain containing primary amine groups is one or more of asparagine, glutamine, lysine and arginine, the amino acid with a side chain containing sulfhydryl groups is cysteine, and the amino acid with a side chain containing no sulfhydryl groups and no primary amine groups is alanine or glycine or other amino acids with a side chain containing no sulfhydryl groups and no primary amine groups. Amino acids or/and amino acid derivatives with side chains containing primary amine groups are modified with primary amine groups on the side chains to bind metal ion groups to provide binding sites for polypeptide chain skeletons and metal ions, and amino acids or/and amino acid derivatives with side chains containing sulfhydryl groups are modified with sulfhydryl groups to modify antibody bridging structures to provide binding sites for polypeptide chain skeletons and antibodies to be labeled, and amino acids or/and amino acid derivatives with side chains free of sulfhydryl groups and free of primary amine groups extend polypeptide chain skeleton chain lengths to avoid steric hindrance from affecting the labeling effect. All kinds of amino acids and amino acid derivatives are synthesized into polypeptide chain skeleton in straight chain, and other amino acids and amino acid derivatives are not required to be arranged in special order except that amino acid or amino acid derivative with side chain containing mercapto group is designed at the amino terminal or carboxyl terminal of the polypeptide chain skeleton.
The polypeptide chain skeleton used in this example consisted of 1 cysteine, 10 asparagine, 10 glutamine, 10 lysine, 10 arginine in a linear form. In addition to cysteine designs at the amino-or carboxy-terminus of the polypeptide chain backbone, other amino acids may be randomly arranged. The polypeptide chain skeleton involved in the following experiment is specifically CKNQRKNRQKRNQKRQNKQRNKQNRQKNRQKRNQNKRQNRK. C=cysteine Cys, k=lysine lys, n=asparagine Asn, q=glutamine Gln, r=arginine Arg. Is synthesized by national peptide organisms according to requirements.
Step two, primary amine group on polypeptide chain skeleton side chain modifies diethylenetriamine pentaacetic acid (DTPA)
1) 1g of DTPA and 10mL of water were weighed into a 100mL single-port flask and the pH of the solution was adjusted to 9.0 using 1M sodium hydroxide.
2) 500mg of 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholine hydrochloride (DMTMM) are weighed into 10mL of water and added dropwise to the DTPA solution of step 1) using a pipette and reacted for 1h at room temperature.
3) 500mg of the polypeptide chain backbone of step one was weighed, dissolved in 10mL of water, immediately added to the DTPA solution of step 2), and reacted overnight at room temperature.
4) After ultrafiltration of the product 4 times using a 10kDa ultrafiltration tube, the resulting product was freeze-dried and the reaction product was weighed.
5) And taking 10mg of the obtained product for nuclear magnetic resonance detection, and calculating the modification efficiency of the DTPA on the polypeptide chain skeleton. The higher the modification efficiency, the stronger the detection signal value of the labeled metal antibody.
Step three, thiol-modified Bismaleimide (BMI) on polypeptide chain skeleton side chain
1) 10mg of BMI was weighed and dissolved in 500. Mu.L of dimethyl sulfoxide (DMSO) or Dimethylformamide (DMF) (DMSO in this example) to prepare a 20mg/mL BMI solution.
2) 50mg of the DTPA modified polypeptide chain backbone prepared in step two of this example was weighed and added to 1mL of 20mM Dithiothreitol (DTT) or tris (2-carboxyethyl) phosphine (TCEP) solution (DTT in this example) and reacted at 37℃for 4 hours.
3) The reaction was terminated by adding 4mL of acetate buffer at pH 3.5, and after ultrafiltration concentration using a 10kDa ultrafiltration tube, the reaction product was rinsed 6 times with acetate buffer.
4) After adding 100. Mu.L of phosphate buffer with pH 7.2 to the 10kDa ultrafiltration tube of step 3), 50. Mu.L of 20mg/mL BMI solution was added and reacted overnight at room temperature.
5) After rinsing 9 times with pure water in the 10kDa ultrafiltration tube of step 4), the freeze-dried concentrate recovers the final product of the metal antibody-labeled supported metal conjugate based on the polypeptide chain backbone.
6) To determine the efficiency of BMI modification on the polypeptide chain backbone, 10mg of the final product was taken for nuclear magnetic resonance detection. The higher the modification efficiency, the higher the efficiency of labeling the polypeptide chain backbone onto the antibody (pure antibody).
7) The final product can be stored at-80 deg.C, -20 deg.C and 4 deg.C in a refrigerator, preferably-20 deg.C for a long period of time.
Example 2
Preparation of metal antibody labeling based on polypeptide chain skeleton applied to metal antibody labeling of supported metal conjugate
Step one, reagent consumable preparation
1) Take out in a refrigerator at 4 DEG C4 buffers (L, R, C, W-Buffer) of X8M mu Ltimetal Labeling Kit were left at room temperature for 20min or more until they were returned to room temperature.
2) Taking out in-20deg.C refrigeratorThe polymer of X8 mu Ltimetal Labeling Kit (i.e., commercial high molecular polymer) and the polypeptide chain backbone-based metal conjugate prepared in example 1 were applied to a metal antibody-labeled supported metal conjugate, and centrifuged in a palm centrifuge at room temperature for more than 20min until returning to room temperature.
3) The single mass number metal ion solution (169 Tm (thulium metal ion with atomic mass number 169) for CD3 metal antibody labeling, 170Er (erbium metal ion with atomic mass number 170), 175Lu (lutetium metal ion with atomic mass number 175) for CD16 metal antibody labeling) and the pure anti-antibodies (CD 3, CD 16) to be labeled were taken out in a refrigerator at 4 ℃.
Step two, metal-supported metal conjugate connection and purification based on polypeptide chain skeleton applied to metal antibody labeling
1) 200ug of polymer was dissolved with 97.5. Mu. L L-Buffer, 200ug of loaded metal conjugate applied to metal antibody labeling based on polypeptide chain backbone was dissolved with 97.5. Mu. L L-Buffer, and blown down until the polymer and loaded metal conjugate applied to metal antibody labeling based on polypeptide chain backbone were completely dissolved, respectively, and 2.5. Mu.L of 100mM 170Er metal solution was added to the polymer. To the supported metal conjugate applied to metal antibody labeling based on the polypeptide chain backbone was added 2.5 μl of 100mm 169tm metal solution. All were incubated in a PCR apparatus at 37℃for 30min.
Polymer-labeled 175Lu-CD16 metal antibody, polypeptide chain skeleton applied to metal antibody-labeled supported metal conjugate-labeled 175Lu-CD16 metal antibody, 170Er and 169Tm in step 1) are both replaced by 175Lu.
2) 200 mu L L-Buffer (300 mu L of 3kDa tube system) is added into two 3kDa ultrafiltration tubes respectively, then incubated metal-polymer and metal-based polypeptide chain skeletons are applied to metal antibody-labeled supported metal conjugate solutions, transferred into the two 3kDa ultrafiltration tubes respectively, and gently blown for 5 times for mixing, centrifuged at 12000 g/room temperature for 25min, and the supernatant is discarded.
3) 300 mu L C-Buffer and 12000 g/normal temperature centrifugation are respectively added into the two 3kDa ultrafiltration tubes of the step 2), and the supernatant is discarded.
Step three, pure anti-reduction
1) An appropriate amount of R-Buffer is added into each of the two 50kDa ultrafiltration tubes, 100ug of CD3 antibody pure anti-protein is added (if the CD16 antibody pure anti-protein is aimed at, the step 1) is added for dissolution, the volume of the mixed solution is 400 mu L,12000 g/normal temperature is centrifuged for 10min, and the supernatant is not discarded.
2) Then 180 mu L R-Buffer is respectively added into the two 50kDa ultrafiltration tubes in the step 1), the mixture is gently blown and mixed uniformly, pure anti-solution OD280 in a sixth measurement tube is measured and recorded, and the step is to observe whether the liquid level of the residual liquid in the 50kDa ultrafiltration tube is higher than a white filter membrane or not before R-Buffer is added, if not, the volume of the residual liquid is considered to be 20 mu L, and if so, 12000 g/normal temperature continues to centrifuge until the liquid level of the residual liquid is lower than the white filter membrane; and centrifuging at normal temperature for 5min at 12000g, and discarding the supernatant.
3) The water bath was brought to 37 ℃ and calibrated with a thermometer.
4) A4 mM TCEP solution was prepared, 100. Mu.L of the 4mM TCEP solution was added to each of the two 50kDa ultrafiltration tubes of step 2), gently blown 5 times, and immediately placed in a 37℃water bath for incubation, and the time was strictly controlled for 30 minutes.
5) After the incubation was completed, the reduction reaction was stopped by adding 300. Mu. L C-Buffer, centrifuging at 12000 g/room temperature for 5min, and discarding the supernatant.
6) 400 mu L C-Buffer and 12000 g/normal temperature are added into the two 50kDa ultrafiltration tubes in the step 5), and the supernatant is discarded.
Step four, pure anti-linking metal-supported metal conjugate based on polypeptide chain skeleton applied to metal antibody labeling
The metal-polymer solution in the 3kDa ultrafiltration tube and the metal-supported metal conjugate solution based on the polypeptide chain skeleton applied to the metal antibody labeling in the 3kDa ultrafiltration tube are respectively transferred to the corresponding 50kDa ultrafiltration tube, are lightly blown for 5 times with the reduced antibody solution, are uniformly mixed, and are put into a water bath at 37 ℃ for incubation for 90min.
Step five, purifying and recycling metal antibody
1) After the incubation was completed, 300. Mu. L W-Buffer was added to each of the two 50kDa ultrafiltration tubes of step four, and the mixture was centrifuged at 12000 g/room temperature for 5min, and the supernatant was discarded.
2) 400 mu L W-Buffer and 12000 g/normal temperature are added into the two 50kDa ultrafiltration tubes of the step 1) respectively, and the supernatant is discarded. And repeating step 2) 2 times.
3) 1 new collecting pipe is taken respectively corresponding to the two 50kDa ultrafiltration pipes in the step 2), marking is carried out, 50 mu L W-Buffer is added into the 50kDa ultrafiltration pipe along the upper ends of the filter membranes at the two sides, then the two filters are inversely buckled in the corresponding new collecting pipe, and the centrifugation is carried out for 2min at 1000 g/normal temperature.
4) And (3) respectively taking 1.5mL of EP pipes corresponding to the step (3), marking, transferring the metal antibody solution in the collecting pipes to the corresponding 1.5mL of EP pipes, adding 50 mu L W-Buffer into the 50kDa ultrafiltration pipe along the upper ends of the filter membranes at the two sides, sucking the 50kDa ultrafiltration pipe bottom solution, flushing along the filter membranes at the two sides again, then inversely buckling in the corresponding collecting pipes, and inversely centrifuging at 1000 g/normal temperature for 2min.
5) The metal antibody solution in the collection tube was again transferred to the corresponding 1.5mL EP tube, the metal antibody volume in the 1.5mL EP tube at this time was measured, OD280 was measured as step six, and recovery was calculated and recorded.
6) According to the quality and volume of the recovered metal antibody, the metal antibody is diluted to a specified concentration (usually 0.2 or 0.4mg/mL, in this example 0.4 mg/mL) by Antibody stabilizer, and the metal antibody is placed in a refrigerator at 4 ℃ for storage at a specified position for subsequent experiments.
Step six, measuring OD280 of the antibody (pure antibody) or the metal antibody
1) The nano100 spectrophotometer was turned on and software Spectrophotometer V2.0, protein a280 was selected.
2) Using buffer solution of the solution to be tested to moisten the sample adding area, wiping the sample adding area, adding 2 mu L of buffer solution of the solution to be tested, selecting 280nm wavelength, and wiping the sample adding area; if the spectrum curve obtained by the blanc is uneven or the OD280 value exceeds 0.005, the blanc operation is repeated.
3) If the solution to be measured is not blown and mixed uniformly in advance, vortex oscillation is carried out for 2s. 2. Mu.L of the solution to be measured was added, the OD value was recorded, and the sample addition area was wiped dry.
4) With ddH 2 And O, cleaning the sample adding area, wiping and repeating for 3 times.
5) The antibody or metal antibody concentration was calculated as formula c=od 280/k (mg/ml), k being 1.4; antibody or metal antibody mass was calculated from the antibody or metal antibody solution volumes and recorded.
Example 3
Detection application of metal antibody labeled by metal conjugate based on application of polypeptide chain skeleton to metal antibody labeling, and detection application of metal antibody labeled by metal conjugate based on application of polypeptide chain skeleton to commercial high-molecular polymerPo of X8M mu Ltimetal Labeling Kitlymer) labeled metal antibodies were compared
The present embodiment is described by taking the labeled CD3 and CD16 metal antibodies as examples (the invention is not limited thereto).
Step one, preparing sample cells to be detected
1) Preparation of single cell suspensions of human Peripheral Blood Mononuclear Cells (PBMCs), cell counts, determination of whether cell concentration and viability meet the experimental requirements (cell count between 1 and 3X 10) 6 The activity rate is more than 90 percent).
2) 1.5 x10 sample cells were taken 6 The PBMCs cells were gently hammered at the tip to resuspend the sample cells, centrifuged at 400g/5min at room temperature and the supernatant discarded.
3) Adding precooled phosphate buffer solution containing bovine serum albumin, centrifuging at 4 ℃ for 400g/5min, and discarding the supernatant; wherein, the phosphate buffer solution containing bovine serum albumin is specifically prepared from 0.5-2mg of bovine serum albumin and 100mL of phosphate buffer solution (GNM 20012).
Step two, determining the death and the activity of the cells
4) 100 mu L of 0.25M of a staining and alive reagent prepared in advance is added into each sample cell, and the sample cells are fully and uniformly mixed and stained on ice for 5min.
5) 1mL of pre-chilled phosphate buffer containing bovine serum albumin was added, the mixture was centrifuged at 4℃for 400g/5min, the supernatant was discarded, and the procedure was repeated 2 times.
Step three, sealing
6) To each sample cell 50 μl of blocking solution was added to resuspend the cell pellet and transferred to a clean 1.5mL EP tube, labeled, and incubated on ice for 20min. The sealing liquid is specifically prepared from 0.5-2mL human immunoglobulin solution (2 mg/mL), 0.5-2mL mouse immunoglobulin solution (2 mg/mL), 0.5-2mL rat immunoglobulin solution (2 mg/mL), 0.5-2mL hamster immunoglobulin solution (2 mg/mL), 0.5-2mg bovine serum albumin and 100mL phosphate buffer solution (GNM 20012).
Step four, extracellular antibody staining
7) To the pre-chilled phosphate buffer containing bovine serum albumin, 0.2ug of commercial high molecular polymer-labeled 170Er-CD3 metal antibody and 0.2ug of metal conjugate-labeled 169Tm-CD3 metal antibody based on a polypeptide chain skeleton applied to the metal label were simultaneously added, and the volume was fixed to 50. Mu.L, namely, the extracellular staining antibody mixture. mu.L of extracellular antibody staining solution was added to one portion of the closed sample cells, mixed well, and stained on ice for 30min.
For CD16 metal antibody labeling, the step 7) is to add 0.2ug of commercial high polymer labeled 175Lu-CD16 metal antibody into precooled phosphate buffer solution containing bovine serum albumin, fix the volume to 50 mu L, then add into a part of closed sample cells, fully and uniformly mix, and dye on ice for 30min;0.2ug of 175Lu-CD16 metal antibody based on polypeptide chain skeleton applied to metal-labeled supported metal conjugate is added into pre-cooled phosphate buffer solution containing bovine serum albumin, the volume is fixed to 50 mu L, and then added into another closed sample cell, fully and uniformly mixed, and the mixture is dyed on ice for 30min.
8) 1mL of pre-chilled phosphate buffer containing bovine serum albumin was added, the mixture was centrifuged at 4℃for 400g/5min, the supernatant was discarded, and the procedure was repeated 2 times.
Step five, cell fixation
9) Using Fix and perm (Standard BioTools) and DNA intercalators 191/193Ir (Standard BioTools), a fixative was prepared at a volume ratio of 2000:1 and other equivalent ratios, 200. Mu.L of fixative was added to one sample cell of step four and left to Fix overnight at 4 ℃.
Step six, rinsing and on-machine detection
10 Taking out the sample cells from the step five from the condition of 4 ℃, centrifuging at 4 ℃ for 800g/5min, and discarding the supernatant.
11 After 1mL of water was added to each EP tube to resuspend the cells, the cell suspension was aspirated with a pipette and transferred through the filter head filter membrane of the flow tube to the corresponding flow tube. After washing the EP tube wall several times by adding 1mL of water again, the washing liquid is sucked by a pipette and filtered by the same flow tube filter head filter membrane to the same flow tube.
12 Centrifugation at 4℃for 800g/5min, and discarding the supernatant.
13 After 1mL of water was added to each of the branched tubes to resuspend the cells, 10. Mu.L of each was taken for cell counting.
14 According to the cell count result, after washing the cell sample with water again for several times, adding a proper amount of balance magnetic beads, detecting on a Helios mass spectrum cell analyzer (Standard Biotools), and processing and analyzing the data.
The commercial high molecular polymer is compared by nuclear magnetic detectionX8M μ Ltimetal Labeling Kit) and the presently disclosed supported metal conjugates based on a polypeptide chain backbone for metal antibody labeling (conjugates of a polypeptide chain backbone), it was observed that the presently disclosed supported metal conjugates based on a polypeptide chain backbone for metal antibody labeling have significant structural feature differences from commercial high molecular polymers, and it was demonstrated that the presently disclosed supported metal conjugates based on a polypeptide chain backbone for metal antibody labeling were obtained from a polypeptide chain backbone via the presently disclosed chemical synthetic pathway (fig. 1), and that the resulting supported metal conjugates based on a polypeptide chain backbone for metal antibody labeling still maintained the structural features of a polypeptide chain backbone (see red arrows in fig. 1), and were not present in the structure of commercial high molecular polymers.
CD3 is a cell surface protein expressed at high levels on the surface of T cells, and is capable of specifically identifying T lymphocytes; CD16 is a cell surface protein expressed on natural killer cells, mononuclear/macrophages and granulocytes, the expression level is lower than that of CD3 (taking CD3 and CD16 as examples, taking CD3 as a high-expression high-abundance detection protein test representative and CD16 as a low-abundance and low-expression detection protein test representative for consideration of research and development cost, test sample availability and easy testing, and evaluating the performance index of the invention). The polypeptide chain skeleton-based metal antibody-labeled supported metal conjugate and commercial high molecular polymer are used for respectively carrying out the labeling and performance comparison of the CD3 and CD16 metal antibodies by adopting the same metal antibody labeling technical flow (example 2) and the detection flow (example 3). It was observed that in detecting the high-expression protein CD3, the dye signal specificity and the signal intensity on the peripheral blood sample are both equivalent to those of the commercial high-molecular polymer-labeled metal antibody based on the application of the polypeptide chain skeleton to the metal antibody-labeled metal-loaded conjugate (fig. 2, fig. 3). In the detection of the cell surface protein of CD16 protein having a relatively low expression level, the effect of detection of the cell surface protein is not weaker than that of commercial reagents, and the influence on the conclusion of the detection data due to the variation in sensitivity of the metal detection signal is avoided by labeling the same mass number of metal isotopes (fig. 4).
Claims (11)
1. The metal conjugate loaded on the metal antibody label based on the polypeptide chain skeleton is characterized by comprising a polypeptide chain skeleton, wherein the polypeptide chain skeleton comprises a side chain group for modifying a metal ion group capable of being combined, a side chain group for modifying an antibody bridging structure, and a connecting structure which can be arranged among the polypeptide chain skeleton, the polypeptide chain skeleton and the metal ion group capable of being combined and between the polypeptide chain skeleton and the antibody bridging structure and avoids steric hindrance from affecting the labeling effect.
2. The supported metal conjugate for metal antibody labeling based on a polypeptide chain backbone according to claim 1, wherein the polypeptide chain backbone comprises an amino acid or/and an amino acid derivative having a side chain comprising a group for modifying a bindable metal ion group, an amino acid or/and an amino acid derivative having a side chain comprising a group for modifying an antibody bridging structure, and an amino acid or/and an amino acid derivative, if any, for avoiding steric hindrance affecting the labeling effect.
3. The supported metal conjugate for metal antibody labeling based on a polypeptide chain backbone according to claim 2, wherein the polypeptide chain backbone comprises at least 1 amino acid or/and amino acid derivative having a side chain comprising a group for modifying a bindable metal ion group, 1 amino acid or amino acid derivative having a side chain comprising a group for modifying an antibody bridging structure, and possibly an amino acid or/and amino acid derivative avoiding steric hindrance affecting the labeling effect.
4. The supported metal conjugate for metal antibody labeling based on a polypeptide chain backbone of claim 3, wherein the polypeptide chain backbone comprises at least 1 amino acid or/and amino acid derivative having a primary amine group in the side chain, 1 amino acid or amino acid derivative having a sulfhydryl group in the side chain, and possibly an amino acid or/and amino acid derivative that avoids steric hindrance from affecting the labeling effect;
amino acid with a side chain containing primary amine groups or/and amino acid derivative with a side chain modified by primary amine groups can be combined with metal ion groups to provide a binding site of a polypeptide chain skeleton and metal ions, and amino acid with a side chain containing sulfhydryl groups or sulfhydryl groups on the side chain of the amino acid derivative modify an antibody bridging structure to provide a binding site of the polypeptide chain skeleton and an antibody to be marked, and amino acid or/and amino acid derivative with steric hindrance effect is/are prevented from extending the chain length of the polypeptide chain skeleton to avoid steric hindrance effect.
5. The supported metal conjugate for metal antibody labeling based on a polypeptide chain backbone according to claim 4, wherein the amino acid with a side chain containing a primary amine group is one or more of asparagine, glutamine, lysine, arginine, the amino acid with a side chain containing a thiol group is cysteine, and the amino acid avoiding steric hindrance effects is alanine or glycine or other amino acid with a side chain containing neither a thiol group nor a primary amine group.
6. The metal conjugate for metal antibody labeling based on polypeptide chain skeleton according to claim 4, wherein various amino acids and amino acid derivatives are synthesized into polypeptide chain skeleton in straight chain, and the amino acids and amino acid derivatives are not required to be arranged in special order except that the amino acid or amino acid derivative with side chain containing sulfhydryl group is designed at amino terminal or carboxyl terminal of polypeptide chain skeleton.
7. The supported metal conjugate for metal antibody labeling based on a polypeptide chain backbone of claim 4, wherein the modification of the primary amine groups on the side chains of the amino acid or/and amino acid derivative containing primary amine groups on the side chains can bind metal ion groups including ethylenediamine tetraacetic acid, diethylenetriamine pentaacetic acid, 1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid.
8. The metal-loaded conjugate for metal antibody labeling based on a polypeptide chain backbone according to claim 4, wherein the thiol-modified antibody bridge structure on the side chain of the thiol-containing amino acid or amino acid derivative comprises bismaleimide.
9. The supported metal conjugate for metal antibody labeling based on a polypeptide chain backbone according to claim 4, wherein the primary amine group modification on the side chain of the amino acid or/and amino acid derivative containing a primary amine group is capable of binding to a metal ion group, and the metal ion capable of binding to a metal ion group is an element having one of the following atomic numbers: 3. 4, 11-13, 19-32, 37-42, 44-51, 55-60, 62-83.
10. Use of a supported metal conjugate according to any one of claims 1-9 for metal antibody labelling based on a polypeptide chain backbone for detection of metal antibody labelling based on mass spectrometry detection techniques.
11. The use according to claim 10, wherein the quantitative detection of the relative expression level of the target protein including single cell level or the quantitative detection of the average expression level of the target protein of the total protein extract of the biological sample is based on mass spectrometry detection technique.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311450244.5A CN117466980A (en) | 2023-11-02 | 2023-11-02 | Supported metal conjugate based on polypeptide chain skeleton and applied to metal antibody labeling and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311450244.5A CN117466980A (en) | 2023-11-02 | 2023-11-02 | Supported metal conjugate based on polypeptide chain skeleton and applied to metal antibody labeling and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117466980A true CN117466980A (en) | 2024-01-30 |
Family
ID=89634386
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311450244.5A Pending CN117466980A (en) | 2023-11-02 | 2023-11-02 | Supported metal conjugate based on polypeptide chain skeleton and applied to metal antibody labeling and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117466980A (en) |
Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1990009196A1 (en) * | 1989-02-10 | 1990-08-23 | Celltech Limited | Cross-linked antibodies and processes for their preparation |
| WO1998008548A2 (en) * | 1996-08-28 | 1998-03-05 | Immunomedics, Inc. | Stable radioiodine conjugates and methods for their synthesis |
| US20060111552A1 (en) * | 2002-09-05 | 2006-05-25 | Blower Philip J | Metal binding precursors for the synthesis of peptide-metal conjugates |
| EP1700608A1 (en) * | 2005-03-10 | 2006-09-13 | Schering AG | Chelators for radioactively labeled conjugates comprising a stabilizing sidechain |
| US20150246146A1 (en) * | 2012-10-25 | 2015-09-03 | Life Technologies Corporation | Methods and compositions for enzyme-mediated site-specific radiolabeling of glycoproteins |
| CN109071606A (en) * | 2016-06-13 | 2018-12-21 | 国立大学法人鹿儿岛大学 | Utilize the locus specificity labelled with radioisotope antibody of IgG binding peptide |
| CN110835363A (en) * | 2019-11-14 | 2020-02-25 | 浙江普罗亭健康科技有限公司 | Antibody labeling method based on protein sulfhydryl coupled metal |
| CN111116701A (en) * | 2019-11-14 | 2020-05-08 | 浙江普罗亭健康科技有限公司 | Intermediate for protein metal marking and preparation method and application thereof |
| CN112041337A (en) * | 2018-04-16 | 2020-12-04 | 日本医事物理股份有限公司 | Modified antibody and radiometal-labeled antibody |
| CN114555132A (en) * | 2019-10-18 | 2022-05-27 | 日本医事物理股份有限公司 | Method for producing radiometal-labeled antibody |
-
2023
- 2023-11-02 CN CN202311450244.5A patent/CN117466980A/en active Pending
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1990009196A1 (en) * | 1989-02-10 | 1990-08-23 | Celltech Limited | Cross-linked antibodies and processes for their preparation |
| WO1998008548A2 (en) * | 1996-08-28 | 1998-03-05 | Immunomedics, Inc. | Stable radioiodine conjugates and methods for their synthesis |
| US20060111552A1 (en) * | 2002-09-05 | 2006-05-25 | Blower Philip J | Metal binding precursors for the synthesis of peptide-metal conjugates |
| EP1700608A1 (en) * | 2005-03-10 | 2006-09-13 | Schering AG | Chelators for radioactively labeled conjugates comprising a stabilizing sidechain |
| US20150246146A1 (en) * | 2012-10-25 | 2015-09-03 | Life Technologies Corporation | Methods and compositions for enzyme-mediated site-specific radiolabeling of glycoproteins |
| CN109071606A (en) * | 2016-06-13 | 2018-12-21 | 国立大学法人鹿儿岛大学 | Utilize the locus specificity labelled with radioisotope antibody of IgG binding peptide |
| CN112041337A (en) * | 2018-04-16 | 2020-12-04 | 日本医事物理股份有限公司 | Modified antibody and radiometal-labeled antibody |
| US20210170058A1 (en) * | 2018-04-16 | 2021-06-10 | Nihon Medi-Physics Co., Ltd. | Modified antibody and radioactive metal-labelled antibody |
| CN114555132A (en) * | 2019-10-18 | 2022-05-27 | 日本医事物理股份有限公司 | Method for producing radiometal-labeled antibody |
| CN110835363A (en) * | 2019-11-14 | 2020-02-25 | 浙江普罗亭健康科技有限公司 | Antibody labeling method based on protein sulfhydryl coupled metal |
| CN111116701A (en) * | 2019-11-14 | 2020-05-08 | 浙江普罗亭健康科技有限公司 | Intermediate for protein metal marking and preparation method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP5031208B2 (en) | Assay | |
| US6969591B2 (en) | Method for diagnosing nephropathy | |
| CN101945990A (en) | Small molecules and protein analysis devices based on molecular imprinted polymers | |
| CN110346557B (en) | Detection kit | |
| EP1969373A2 (en) | Multiplexed detection of anti-red cell alloantibodies | |
| AU2015239040B2 (en) | Control means for implementing multiplex analysis methods | |
| CN117470819A (en) | Autoimmune myositis disease detection kit and detection method thereof | |
| KR20000048833A (en) | Assay for the detection of antibodies against p53 | |
| CN117466980A (en) | Supported metal conjugate based on polypeptide chain skeleton and applied to metal antibody labeling and application thereof | |
| CN112480244A (en) | Anti-allergic nano antibody composition, antibody determination method and spray | |
| CN115792208A (en) | Protein magnetic bead coating material, preparation method and application thereof, and target antibody detection method | |
| CN113447656B (en) | Kit for detecting anti-filamentous actin cap-forming protein beta-IgG antibody | |
| CN108303541B (en) | Porcine circovirus type 2 antibody detection kit and detection method thereof | |
| CN111724857B (en) | Protein traceability validity and interchangeability evaluation method in immunoassay | |
| EP1340085B1 (en) | Assay for directly detecting a biological cell in a body fluid sample | |
| CN110346556B (en) | Liquid detection reagent and using method thereof | |
| CN110346558B (en) | Use method of detection kit | |
| CN114895023A (en) | Application of reagent for detecting anti-Talin-1-IgG autoantibody in preparation of kit for detecting vascular endothelial injury | |
| JPH02298866A (en) | Oxygen assay kit and method applicable for complete cell | |
| CN116296702B (en) | Dead-living dye capable of customizing detection channel based on mass spectrometry detection technology and preparation method and application thereof | |
| CN108303531B (en) | O-type foot-and-mouth disease antibody detection kit and detection method thereof | |
| EP1406089A1 (en) | A method for selectively staining water soluble protein using reactive dye | |
| CN116041520B (en) | Rabbit monoclonal antibody aiming at Human CD63, and preparation method and application thereof | |
| CN113447648B (en) | Kit for detecting anti-serine/arginine-rich splicing factor 9-IgG antibody | |
| CN117288966B (en) | Kit for combined detection of IGF-1 and IGFBP-3 and lysate and buffer solution used by kit |
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 |