CN106146672B - Acridine marks conjugate and preparation method thereof, chemiluminescence immune detection reagent kit - Google Patents
Acridine marks conjugate and preparation method thereof, chemiluminescence immune detection reagent kit Download PDFInfo
- Publication number
- CN106146672B CN106146672B CN201610522380.4A CN201610522380A CN106146672B CN 106146672 B CN106146672 B CN 106146672B CN 201610522380 A CN201610522380 A CN 201610522380A CN 106146672 B CN106146672 B CN 106146672B
- Authority
- CN
- China
- Prior art keywords
- acridine
- linking agent
- intermediate product
- functional cross
- marked
- 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
- DZBUGLKDJFMEHC-UHFFFAOYSA-N acridine Chemical compound C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 title claims abstract description 198
- 238000001514 detection method Methods 0.000 title claims abstract description 21
- 238000002360 preparation method Methods 0.000 title claims abstract description 21
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 15
- 239000003431 cross linking reagent Substances 0.000 claims abstract description 162
- 125000000641 acridinyl group Chemical group C1(=CC=CC2=NC3=CC=CC=C3C=C12)* 0.000 claims abstract description 79
- 125000001841 imino group Chemical group [H]N=* 0.000 claims abstract description 34
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims abstract description 27
- 239000013067 intermediate product Substances 0.000 claims description 128
- 238000006243 chemical reaction Methods 0.000 claims description 62
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 52
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 claims description 36
- RXNXLAHQOVLMIE-UHFFFAOYSA-N phenyl 10-methylacridin-10-ium-9-carboxylate Chemical group C12=CC=CC=C2[N+](C)=C2C=CC=CC2=C1C(=O)OC1=CC=CC=C1 RXNXLAHQOVLMIE-UHFFFAOYSA-N 0.000 claims description 33
- -1 amino, carboxyl Chemical group 0.000 claims description 31
- 229920001184 polypeptide Polymers 0.000 claims description 25
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 25
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 25
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 20
- 230000004913 activation Effects 0.000 claims description 20
- 108060003552 hemocyanin Proteins 0.000 claims description 17
- 229940042795 hydrazides for tuberculosis treatment Drugs 0.000 claims description 16
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 15
- 238000004132 cross linking Methods 0.000 claims description 13
- 239000003795 chemical substances by application Substances 0.000 claims description 11
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 10
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims description 9
- 239000000427 antigen Substances 0.000 claims description 9
- 102000036639 antigens Human genes 0.000 claims description 9
- 108091007433 antigens Proteins 0.000 claims description 9
- 239000002253 acid Substances 0.000 claims description 5
- 238000013519 translation Methods 0.000 claims description 5
- AORNIVFSXFONEC-UHFFFAOYSA-N acridine-1-carboxamide Chemical compound C1=CC=C2C=C3C(C(=O)N)=CC=CC3=NC2=C1 AORNIVFSXFONEC-UHFFFAOYSA-N 0.000 claims description 4
- BDQRMEBGHYKVLA-UHFFFAOYSA-N acridine-1-sulfonamide Chemical compound C1=CC=C2C=C3C(S(=O)(=O)N)=CC=CC3=NC2=C1 BDQRMEBGHYKVLA-UHFFFAOYSA-N 0.000 claims description 4
- YHUVMHKAHWKQBI-UHFFFAOYSA-N quinoline-2,3-dicarboxylic acid Chemical compound C1=CC=C2N=C(C(O)=O)C(C(=O)O)=CC2=C1 YHUVMHKAHWKQBI-UHFFFAOYSA-N 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 2
- 238000006467 substitution reaction Methods 0.000 claims 1
- 125000003396 thiol group Chemical class [H]S* 0.000 claims 1
- 238000003018 immunoassay Methods 0.000 abstract description 19
- 230000000694 effects Effects 0.000 abstract description 13
- 239000003550 marker Substances 0.000 abstract description 12
- 230000035945 sensitivity Effects 0.000 abstract description 12
- 239000000872 buffer Substances 0.000 description 34
- DZBUGLKDJFMEHC-UHFFFAOYSA-O acridine;hydron Chemical compound C1=CC=CC2=CC3=CC=CC=C3[NH+]=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-O 0.000 description 33
- 239000000243 solution Substances 0.000 description 33
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 21
- 239000007788 liquid Substances 0.000 description 19
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical compound CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 description 17
- 150000001718 carbodiimides Chemical group 0.000 description 17
- 238000011033 desalting Methods 0.000 description 17
- 102000009843 Thyroglobulin Human genes 0.000 description 16
- 108010034949 Thyroglobulin Proteins 0.000 description 16
- 239000006227 byproduct Substances 0.000 description 16
- 230000000052 comparative effect Effects 0.000 description 16
- 229960002175 thyroglobulin Drugs 0.000 description 16
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 15
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 15
- 239000002904 solvent Substances 0.000 description 13
- 102000035118 modified proteins Human genes 0.000 description 12
- 108091005573 modified proteins Proteins 0.000 description 12
- 150000001720 carbohydrates Chemical class 0.000 description 11
- 241000251468 Actinopterygii Species 0.000 description 9
- 102000003886 Glycoproteins Human genes 0.000 description 9
- 108090000288 Glycoproteins Proteins 0.000 description 9
- 238000000034 method Methods 0.000 description 8
- 239000000126 substance Substances 0.000 description 8
- KZNICNPSHKQLFF-UHFFFAOYSA-N dihydromaleimide Natural products O=C1CCC(=O)N1 KZNICNPSHKQLFF-UHFFFAOYSA-N 0.000 description 7
- 238000004020 luminiscence type Methods 0.000 description 7
- 150000001251 acridines Chemical class 0.000 description 6
- 239000007853 buffer solution Substances 0.000 description 6
- 239000000523 sample Substances 0.000 description 6
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 5
- 238000002372 labelling Methods 0.000 description 5
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 5
- 229940126619 mouse monoclonal antibody Drugs 0.000 description 5
- 229960002317 succinimide Drugs 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- PEEHTFAAVSWFBL-UHFFFAOYSA-N Maleimide Chemical compound O=C1NC(=O)C=C1 PEEHTFAAVSWFBL-UHFFFAOYSA-N 0.000 description 4
- 108010048233 Procalcitonin Proteins 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 238000007796 conventional method Methods 0.000 description 4
- 230000036039 immunity Effects 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- CWCXERYKLSEGEZ-KDKHKZEGSA-N procalcitonin Chemical compound C([C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](C(C)C)C(=O)NCC(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)NCC(O)=O)[C@@H](C)O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCSC)NC(=O)[C@H]1NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(N)=O)NC(=O)CNC(=O)[C@@H](N)CSSC1)[C@@H](C)O)[C@@H](C)O)[C@@H](C)O)C1=CC=CC=C1 CWCXERYKLSEGEZ-KDKHKZEGSA-N 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 4
- 230000009870 specific binding Effects 0.000 description 4
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 3
- 239000007987 MES buffer Substances 0.000 description 3
- 125000003368 amide group Chemical group 0.000 description 3
- 239000012491 analyte Substances 0.000 description 3
- 239000011324 bead Substances 0.000 description 3
- 230000007547 defect Effects 0.000 description 3
- 238000004090 dissolution Methods 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- BDNKZNFMNDZQMI-UHFFFAOYSA-N 1,3-diisopropylcarbodiimide Chemical compound CC(C)N=C=NC(C)C BDNKZNFMNDZQMI-UHFFFAOYSA-N 0.000 description 2
- GXYLXFITCXXCQV-UHFFFAOYSA-N 10-methylacridin-10-ium Chemical compound C1=CC=C2[N+](C)=C(C=CC=C3)C3=CC2=C1 GXYLXFITCXXCQV-UHFFFAOYSA-N 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000003139 buffering effect Effects 0.000 description 2
- 235000019253 formic acid Nutrition 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- JQWHASGSAFIOCM-UHFFFAOYSA-M sodium periodate Chemical compound [Na+].[O-]I(=O)(=O)=O JQWHASGSAFIOCM-UHFFFAOYSA-M 0.000 description 2
- 102000006395 Globulins Human genes 0.000 description 1
- 108010044091 Globulins Proteins 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 125000002252 acyl group Chemical group 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 150000001491 aromatic compounds Chemical class 0.000 description 1
- 102000015736 beta 2-Microglobulin Human genes 0.000 description 1
- 108010081355 beta 2-Microglobulin Proteins 0.000 description 1
- 229960004015 calcitonin Drugs 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000005081 chemiluminescent agent Substances 0.000 description 1
- 238000010612 desalination reaction Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 150000002466 imines Chemical class 0.000 description 1
- 229940127121 immunoconjugate Drugs 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 230000009871 nonspecific binding Effects 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- 210000001685 thyroid gland Anatomy 0.000 description 1
- 238000000108 ultra-filtration 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
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/04—Endocrine or metabolic disorders
- G01N2800/046—Thyroid disorders
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/70—Mechanisms involved in disease identification
- G01N2800/7095—Inflammation
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Genetics & Genomics (AREA)
- Analytical Chemistry (AREA)
- Biomedical Technology (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Urology & Nephrology (AREA)
- Gastroenterology & Hepatology (AREA)
- Hematology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Bioinformatics & Computational Biology (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Plasma & Fusion (AREA)
- Food Science & Technology (AREA)
- Peptides Or Proteins (AREA)
- Plural Heterocyclic Compounds (AREA)
Abstract
The invention discloses a kind of acridine label conjugates and preparation method thereof, chemiluminescence immune detection reagent kit.Acridine label conjugate includes sequentially connected acridine substituent, connection carrier, bi-functional cross-linking agent and object to be marked.Acridine substituent reacts to form-CO-NH- structure to connect acridine substituent with carrier is connect with the amino connecting on carrier.Pass through connection carrier and the bi-functional cross-linking agent containing dimaleoyl imino and hydrazide group, chemistry key connection does not occur directly for acridine substituent and object to be marked, it avoids the active site that acridine substituent is treated on marker and forms interference, so that the activity of acridine label conjugate is relatively high, the sensitivity of immunoassay is improved.
Description
Technical field
The present invention relates to vitro detection fields more particularly to a kind of acridine label conjugate and preparation method thereof, chemistry hair
Light immunity detection reagent.
Background technique
Chemiluminescent labeling immunoassay is also known as chemiluminescence immune assay (CLIA), is directly marked with chemiluminescent agent
Antigen, haptens, antibody or carbohydrate immunoassay method.The chemiluminescent substance for being usually used in label has acridine
It closes object (acridinium ester, AE).It is by starting luminescence reagent (NaOH, H2O2) act on and shine, strong direct hair
Light is completed in one second, is quick flashing.
Acridine compound is used for immunoassay as marker, chemical reaction is simple, quickly, without catalyst, inspection
It surveys small molecule antigens and uses competition law, macromolecular antigen or antibody then use sandwich method, and non-specific binding is few, and background is low,
And the combination of macromolecular will not reduce generated light quantity, to increase sensitivity.Acridine compound can labelled antigen antibody
Conjugate is marked etc. acridine is formed.The quality of acridine label conjugate quality is directly related to the success of Chemiluminescence Immunoassay
Whether, therefore be a very crucial reagent in kit.Acridine label conjugate generally passes through acridine compound and special
Property antibody be formed by connecting through proper method, acridine compound and antibody will directly affect the luminous production of acridine label conjugate
The abilities such as rate, background interference and affinity.
The preparation method of currently used acridine label conjugate is carbodiimide cross-linking method, with the difunctional friendship of carbodiimide
Connection agent is bridge, makes acridine substituent in conjunction with object to be marked.However, in the label conjugate of acridine made from conventional method, due to
Acridine substituent needs to bind directly by carbodiimide with object to be marked, and acridine substituent is often treated on labelled protein
Active site forms interference, and the active site that combination may be selected on object to be marked is few, causes the activity drop of acridine label conjugate
It is low, influence the sensitivity of immunoassay.
Summary of the invention
Based on this, it is necessary to provide a kind of acridine that activity is relatively high label conjugate and preparation method thereof, chemistry hair
Light immunity detection reagent.
A kind of acridine marks conjugate, including sequentially connected acridine substituent, connection carrier, bi-functional cross-linking agent and to
Marker；
The bi-functional cross-linking agent is the crosslinking agent containing dimaleoyl imino and hydrazide group；
The object to be marked is the albumen containing sulfydryl, carbonyl or carboxyl, modified protein, polypeptide, modified polypeptides or carbon water
Compound；
The connection carrier contains amino and carboxyl, and the acridine substituent reacts shape with the amino on the connection carrier
Carboxyl and institute at-CO-NH- structure to connect the acridine substituent and the connection carrier, on the connection carrier
State the hydrazide group on bi-functional cross-linking agent react to be formed-CO-NH-NH- structure to by the connection carrier with it is described difunctional
Crosslinking agent connects, and the dimaleoyl imino on the bi-functional cross-linking agent reacts to be formed with the sulfydryl on the object to be markedStructure is to which the bi-functional cross-linking agent to be connect with the object to be marked；
Alternatively, the connection carrier contains amino and disulfide bond, the ammonia on the acridine substituent and the connection carrier
Base reacts to form-CO-NH- structure to connect the acridine substituent and the connection carrier, on the connection carrier
Disulfide bond reacts to be formed with the dimaleoyl imino on the bi-functional cross-linking agentStructure is thus by the company
It carries body to connect with the bi-functional cross-linking agent, the hydrazide group on the bi-functional cross-linking agent and the carbonyl on the object to be marked
Or carboxyl reacts to form-NH-NH-CO- structure to which the bi-functional cross-linking agent to be connect with the object to be marked.
In one embodiment, the acridine substituent is acridinium ester, acridinic acid, acridinium carboxamide or acridine sulfonamide.
In one embodiment, the connection carrier is the hemocyanin containing amino, carboxyl and disulfide bond.
In one embodiment, the object to be marked is antigen, haptens or antibody.
A kind of preparation method of above-mentioned acridine label conjugate, includes the following steps:
It reacts to obtain intermediate product B with the acridine substituent after activation with carrier is connect, wherein the intermediate product B is
Acridine substituent-connection carrier conjugates；And
Acridine label conjugate will be obtained after the intermediate product B, bi-functional cross-linking agent and object cross-linking reaction to be marked,
Wherein, acridine label conjugate includes sequentially connected acridine substituent, connection carrier, bi-functional cross-linking agent and to be marked
Object, the connection carrier contain amino and carboxyl, the acridine substituent reacted with the amino on the connection carrier to be formed-
CO-NH- structure is to connect the acridine substituent and the connection carrier, the carboxyl on the connection carrier and described pair
Hydrazide group on functional cross-link agent reacts to form-CO-NH-NH- structure thus by the connection carrier and the difunctional crosslinking
Agent connects, and the dimaleoyl imino on the bi-functional cross-linking agent reacts to be formed with the sulfydryl on the object to be markedStructure is to which the bi-functional cross-linking agent to be connect with the object to be marked；Alternatively, the connection carrier contains
There are amino and disulfide bond, the acridine substituent reacts to form-CO-NH- structure thus will with the amino on the connection carrier
The acridine substituent and the connection carrier connect, on the disulfide bond and the bi-functional cross-linking agent on the connection carrier
Dimaleoyl imino reacts to be formedStructure so that the connection carrier be connect with the bi-functional cross-linking agent,
Hydrazide group on the bi-functional cross-linking agent on the object to be marked carbonyl or carboxyl react to be formed-NH-NH-CO- structure from
And the bi-functional cross-linking agent is connect with the object to be marked.
In one embodiment, it will be obtained after the intermediate product B, bi-functional cross-linking agent and object cross-linking reaction to be marked
To acridine label conjugate operation specifically includes the following steps:
It is reacted with intermediate product B with bi-functional cross-linking agent and generates intermediate product D, wherein the intermediate product D takes for acridine
For object-connection carrier-bi-functional cross-linking agent conjugate, the bi-functional cross-linking agent is containing dimaleoyl imino and hydrazide group
Crosslinking agent, the carboxyl on connection carrier in the intermediate product B reacted with the hydrazide group on the bi-functional cross-linking agent to be formed-
CO-NH-NH- structure is to which the intermediate product B to be connect with the bi-functional cross-linking agent；And
By the intermediate product D obtained in conjunction with object to be marked acridine label conjugate, wherein the object to be marked be containing
There are albumen, modified protein, polypeptide, modified polypeptides or the carbohydrate of sulfydryl, carbonyl or carboxyl, in the intermediate product D
Dimaleoyl imino on bi-functional cross-linking agent reacts to be formed with the sulfydryl on the object to be markedStructure from
And the intermediate product D is connect with the object to be marked.
In one embodiment, it will be obtained after the intermediate product B, bi-functional cross-linking agent and object cross-linking reaction to be marked
To acridine label conjugate operation specifically includes the following steps:
Intermediate product B is activated, so that the disulfide bond on the connection carrier in the intermediate product B activates shape
At sulfydryl, intermediate product F is obtained, wherein the intermediate product F is acridine substituent-connection carrier conjugates containing sulfydryl；
Object to be marked is reacted with bi-functional cross-linking agent and generates intermediate product I, wherein the intermediate product I is to be marked
Object-bi-functional cross-linking agent conjugate, the object to be marked be the albumen containing sulfydryl, carbonyl or carboxyl, modified protein, polypeptide,
Modified polypeptides or carbohydrate, the bi-functional cross-linking agent is the crosslinking agent containing dimaleoyl imino and hydrazide group, described
Hydrazide group on bi-functional cross-linking agent on the object to be marked carbonyl or carboxyl react and to form-NH-NH-CO- structure to will
The bi-functional cross-linking agent is connect with the object to be marked；And
The intermediate product I is obtained to acridine label conjugate in conjunction with the intermediate product F, wherein the intermediate production
Dimaleoyl imino reacts to be formed with the sulfydryl on the intermediate product F on bi-functional cross-linking agent in object I
Structure is to which the intermediate product I to be connect with the intermediate product F.
In one embodiment, it reacts to obtain the behaviour of intermediate product B with the acridine substituent after activation with carrier is connect
In work, the molar ratio of acridine substituent and the connection carrier after the activation is 1~5000:1.
In one embodiment, it is reacted with intermediate product B with bi-functional cross-linking agent in the operation for generating intermediate product D,
The molar ratio of the bi-functional cross-linking agent and the intermediate product B are 1~10000:1.
A kind of chemiluminescence immune detection reagent kit, including acridine described in any of the above embodiments mark conjugate.
This acridine label conjugate include sequentially connected acridine substituent, connection carrier, bi-functional cross-linking agent and to
Marker.Acridine substituent reacted with the amino connecting on carrier to be formed-CO-NH- structure to by acridine substituent with connect
Carrier connection.Carboxyl on connection carrier reacts to form-CO-NH-NH- structure thus will with the hydrazide group on bi-functional cross-linking agent
Connection carrier is connect with bi-functional cross-linking agent, and the dimaleoyl imino on bi-functional cross-linking agent is reacted with the sulfydryl on object to be marked
It is formedStructure is to which bi-functional cross-linking agent to be connect with object to be marked.Or another way, connect carrier
On disulfide bond react to be formed with the dimaleoyl imino on bi-functional cross-linking agentStructure will be to connect load
Body is connect with bi-functional cross-linking agent, the hydrazide group on bi-functional cross-linking agent on object to be marked carbonyl or carboxyl react to be formed-
NH-NH-CO- structure is to which bi-functional cross-linking agent to be connect with object to be marked.By containing connection carrier and containing maleimide
The bi-functional cross-linking agent of amido and hydrazide group, acridine substituent and object to be marked directly by chemical bonds, do not avoid
Acridine substituent treats the active site on marker and forms interference, so that the activity of acridine label conjugate is relatively high, mentions
The sensitivity of high immunoassay.In addition, dimaleoyl imino and hydrazide group in bi-functional cross-linking agent can with contain sulfydryl, carbonyl
Or the object to be marked specific binding of carboxyl, avoid previous acridine substituent cannot with containing sulfydryl, carbonyl or carboxyl to
The defect that marker combines, expands the range of choice of object to be marked, so that the specificity of acridine label conjugate is high, increases a word used for translation
Pyridine label conjugate uses sensitivity.
Detailed description of the invention
Fig. 1 is the flow chart that the acridine of an embodiment marks the preparation method of conjugate；
Fig. 2 is an embodiment by obtaining a word used for translation after intermediate product B, bi-functional cross-linking agent and object cross-linking reaction to be marked
The preparation flow figure of pyridine label conjugate；
Fig. 3 is another embodiment by obtaining after intermediate product B, bi-functional cross-linking agent and object cross-linking reaction to be marked
The preparation flow figure of acridine label conjugate；
Fig. 4 is series of concentrations thyroglobulin antibody Sample result scatter plot obtained in test case 4.
Specific embodiment
In order to make the foregoing objectives, features and advantages of the present invention clearer and more comprehensible, with reference to the accompanying drawing and specific real
Applying example, specific embodiments of the present invention will be described in detail.Be explained in the following description many details in order to
Fully understand the present invention.But the invention can be embodied in many other ways as described herein, art technology
Personnel can do similar improvement without violating the connotation of the present invention, therefore the present invention is not by following public specific implementation
Limitation.
A kind of acridine marks conjugate, including sequentially connected acridine substituent, connection carrier, bi-functional cross-linking agent and to
Marker.Bi-functional cross-linking agent is the crosslinking agent containing dimaleoyl imino and hydrazide group.Object to be marked is to contain sulfydryl, carbonyl
Or albumen, modified protein, polypeptide, modified polypeptides or the carbohydrate of carboxyl.
In one embodiment, connection carrier contains amino and carboxyl, and acridine substituent is reacted with the amino connecting on carrier
Formation-CO-NH- structure is to connect acridine substituent with carrier is connect.Connect the carboxyl and bi-functional cross-linking agent on carrier
On hydrazide group react to form-CO-NH-NH- structure and connect with bi-functional cross-linking agent so that carrier will be connected, bi-functional cross-linking agent
On dimaleoyl imino react to be formed with the sulfydryl on object to be markedStructure is thus by bi-functional cross-linking agent
It is connect with object to be marked.
In another embodiment, connection carrier contains amino and disulfide bond, acridine substituent and the amino connecting on carrier
Reaction formation-CO-NH- structure is to connect acridine substituent with carrier is connect.Connect carrier on disulfide bond with it is difunctional
Dimaleoyl imino on crosslinking agent reacts to be formedStructure connects to connect carrier and bi-functional cross-linking agent
Connect, the hydrazide group on bi-functional cross-linking agent on object to be marked carbonyl or carboxyl react and to form-NH-NH-CO- structure to will be double
Functional cross-link agent is connect with object to be marked.
Specifically, acridine substituent is acridinium ester, acridinic acid, acridinium carboxamide or acridine sulfonamide.Label acridinium ester, tool
Body can be AE-NHS (10- methylacridinium -9-N- succinimide ester formic acid esters), DMAE-NHS (2 ', 6 '-dimethyl -4 ' -
(N- succinimidyloxycarbonyl) phenyl-acridine -9- formic acid esters), ME-DMAE-NHS (2 ', 6 '-dimethyl-carbonyl phenyl -10-
Methyl -9- acridine formic acid esters -4 '-N- succinimide ester-trifluoromethyl sulfonic acid) etc..
Specifically, connection carrier can be the hemocyanin (KLH) containing amino, carboxyl and disulfide bond.Certainly, other
Carrier simultaneously containing amino, carboxyl and disulfide bond can also be used as the connection carrier in this acridine label conjugate.Connection carries
Body contains these three functional groups of amino, carboxyl and disulfide bond simultaneously, can occur with bi-functional cross-linking agent or object to be marked
A variety of combinations.
Specifically, bi-functional cross-linking agent is the crosslinking agent containing dimaleoyl imino and hydrazide group.It is specifically as follows EMCH
(N- [ε-maleimide acetic acid]-hydrazides ■ trifluoroacetic acid), KMUH (N- [κ-maleimidbundecanoic acid]-hydrazides ■ tri-
Fluoroacetic acid) or MPBH (4- [4-N- maleimide phenyl]-butyric acid hydrazides ■ hydrochloric acid) etc..
Specifically, object to be marked can be inherently to have sulfydryl, the albumen of carbonyl or carboxyl, modified protein, polypeptide, change
Property polypeptide or carbohydrate, or by introduced after modification the albumen of sulfydryl, carbonyl or carboxyl, modified protein, polypeptide,
Modified polypeptides or carbohydrate.
In present embodiment, object to be marked is antigen, haptens or antibody.
Using acridine substituent as acridinium ester, connection carrier be the hemocyanin (KLH) containing amino, carboxyl and disulfide bond,
Bi-functional cross-linking agent be containing dimaleoyl imino for, acridine label conjugate have the following structure formula:
Wherein,Indicate acridine compound,Indicate hemocyanin,It is to be marked
Object.
The acridine label conjugate of another embodiment has the following structure formula:
Wherein,Indicate acridine compound,Indicate hemocyanin,It is to be marked
Object.
This acridine label conjugate include sequentially connected acridine substituent, connection carrier, bi-functional cross-linking agent and to
Marker.Pass through the connection carrier containing amino, carboxyl and disulfide bond and double function containing dimaleoyl imino and hydrazide group
Energy crosslinking agent, acridine substituent can not be bound directly by carbodiimide with object to be marked, avoid acridine substituent and treat
Active site on labelled protein forms interference, so that the activity of acridine label conjugate is relatively high, improves immunoassay
Sensitivity.
In addition, dimaleoyl imino and hydrazide group in bi-functional cross-linking agent can with containing sulfydryl, carbonyl or carboxyl to
Marker specific binding, avoiding previous acridine substituent cannot be in conjunction with the object to be marked containing sulfydryl, carbonyl or carboxyl
Defect, expand the range of choice of object to be marked, so that the specificity of acridine label conjugate is high, increases acridine label and combine
Object uses sensitivity.The corresponding object to be marked of the detected material can be connected accordingly, because wait mark according to the needs of detection
Remember that the range of choice of object is wide, even the object to be marked containing sulfydryl, carbonyl or carboxyl also can by bi-functional cross-linking agent and
Connection carrier forms acridine label conjugate in conjunction with acridine substituent, therefore when detecting, acridine marks conjugate can be with quilt
Detectable substance specific binding, high sensitivity, mark rate are high.
This acridine marks conjugate, acridine substituent reacted with the amino connecting on carrier to be formed-CO-NH- structure from
And by acridine substituent with connect carrier connection, between acridine substituent and object to be marked without directly occur chemical bonding reaction.
And traditional method binds directly acridine substituent with object to be marked generally using carbodiimide bi-functional cross-linking agent as bridge, this
Sample mark rate is lower, and activity is bad, and labelling groups are selectively few, cannot be directly combined with the object to be combined containing carbonyl, sulfydryl.
Compared with traditional acridine marks conjugate, acridine label conjugate labeling effciency of the invention is higher, is used directly for chemistry
The detection and quantitative analysis of luminescence immunoassay, and the acridine mark for the preparation that the methods of can solve traditional carbodiimide cross-linking method
Note combines the disadvantages of raw material inactivation, labeling effciency are low, labelling groups selectively lack.
The preparation method of above-mentioned acridine label conjugate as shown in Figure 1, includes the following steps S110~S120.
S110, it reacts to obtain intermediate product B with the acridine substituent after activation with carrier is connect.
Wherein, intermediate product B be acridine substituent-connection carrier conjugates, connection carrier contain amino and carboxyl or
Connection carrier contains amino and disulfide bond, and further, connection carrier can be the connection containing amino, carboxyl and disulfide bond
Carrier, acridine substituent reacted with the amino connecting on carrier to be formed-CO-NH- structure to by acridine substituent with connect load
Body connection.
Specifically, acridine substituent is acridinium ester, acridinic acid, acridinium carboxamide or acridine sulfonamide.It is specifically as follows AE-
((N- succinyl is sub- for 2 ', 6 '-dimethyl -4 '-by NHS (10- methylacridinium -9-N- succinimide ester formic acid esters), DMAE-NHS
Amine oxygen carbonyl) phenyl-acridine -9- formic acid esters), ME-DMAE-NHS (2 ', 6 '-dimethyl-carbonyl phenyl -10- methyl -9- acridines
- the N- of formic acid esters -4 ' succinimide ester-trifluoromethyl sulfonic acid) etc..
Specifically, connection carrier can be the hemocyanin (KLH) containing amino, carboxyl and disulfide bond.Certainly, other
Carrier simultaneously containing amino, carboxyl and disulfide bond can also be used as the connection carrier in this acridine label conjugate.
It reacts to obtain in the operation of intermediate product B with the acridine substituent after activation with carrier is connect, the acridine after activation
Substituent is 1~5000:1 with the molar ratio for connecting carrier.
Further, the acridine substituent after activation is 50~400:1 with the molar ratio for connecting carrier.
Acridine substituent obtains after can activating acridine compound by HOSu NHS, succinimide or carboxylic acid etc.
It arrives.
Acridine substituent is hydroxysuccinimide-activated acridine compounds, connection carrier can be containing amino, carboxylic
For the hemocyanin (KLH) of base and disulfide bond, react to obtain intermediate product B with the acridine substituent after activation with carrier is connect
Reaction equation it is as follows:
Wherein,Indicate acridine compound,Indicate hemocyanin.
The specific reaction process of above-mentioned reaction equation are as follows: KLH is dissolved in PBS buffer solution (pH 7.4), DMSO is used in addition
The acridine ester solution of solvent dissolution, in 20 DEG C~30 DEG C reaction 1h.With the desalting column (Thermo of 5mL 7KD molecular cut off
Fish company) using 150mM PBS (pH 7.4) buffer as liquid buffer is changed, it crosses column 3 times, removes free acridinium ester and anti-
By-product is answered, intermediate product B (acridine substituent-connection carrier conjugates) is obtained, intermediate product B is specially a word used for translation in the present embodiment
Pyridine ester-KLH.
S120, acridine label combination will be obtained after intermediate product B, bi-functional cross-linking agent and object cross-linking reaction to be marked
Object.
Wherein, acridine label conjugate include sequentially connected acridine substituent, connection carrier, bi-functional cross-linking agent and to
Marker, connection carrier contain amino and carboxyl, and acridine substituent reacts to form-CO-NH- structure with the amino connecting on carrier
To which acridine substituent is reacted shape with the hydrazide group on bi-functional cross-linking agent with carrier connection, the carboxyl connected on carrier is connect
It is connect to which carrier will be connected with the bi-functional cross-linking agent at-CO-NH-NH- structure, the maleimide on bi-functional cross-linking agent
Amido reacts to be formed with the sulfydryl on object to be markedStructure is to connect bi-functional cross-linking agent and object to be marked
It connects.Alternatively, connection carrier contains amino and disulfide bond, acridine substituent reacts to form-CO-NH- with the amino connecting on carrier
Structure connects the Malaysia acyl on the disulfide bond and bi-functional cross-linking agent on carrier to connect acridine substituent with carrier is connect
Imido grpup reacts to be formedStructure connect to connect carrier with bi-functional cross-linking agent, on bi-functional cross-linking agent
Hydrazide group on object to be marked carbonyl or carboxyl react to be formed-NH-NH-CO- structure to by bi-functional cross-linking agent with wait mark
Remember object connection.
The intermediate product B obtained by step contains functional groups amino, carboxyl and the disulfide bond on connection carrier
Deng.Intermediate product B can be also possible to first in conjunction with bi-functional cross-linking agent again by bi-functional cross-linking agent in conjunction with object to be marked
First by bi-functional cross-linking agent in conjunction with object to be marked, bi-functional cross-linking agent-object to be marked is formed, is then tied again with intermediate product B
It closes.
In one embodiment, intermediate product B reacted by carboxyl with the hydrazide group on bi-functional cross-linking agent to be formed-
CO-NH-NH- structure connect to connect carrier with bi-functional cross-linking agent, then passes through the maleimide on bi-functional cross-linking agent
Amido reacts to be formed with the sulfydryl on object to be markedStructure is to connect bi-functional cross-linking agent and object to be marked
It connects to obtain acridine label conjugate.Specifically as shown in Fig. 2, S120 includes the following steps S120A and S120B.
S120A, generation intermediate product D is reacted with bi-functional cross-linking agent with intermediate product B.
Wherein, intermediate product D is acridine substituent-connection carrier-bi-functional cross-linking agent conjugate, and bi-functional cross-linking agent is
Crosslinking agent containing dimaleoyl imino and hydrazide group, the carboxyl and bi-functional cross-linking agent on connection carrier in intermediate product B
On hydrazide group react to form-CO-NH-NH- structure so that intermediate product B to be connect with bi-functional cross-linking agent.
Specifically, bi-functional cross-linking agent is the crosslinking agent containing dimaleoyl imino and hydrazide group.It is specifically as follows EMCH
(N- [ε-maleimide acetic acid]-hydrazides ■ trifluoroacetic acid), KMUH (N- [κ-maleimidbundecanoic acid]-hydrazides ■ tri-
Fluoroacetic acid) or MPBH (4- [4-N- maleimide phenyl]-butyric acid hydrazides ■ hydrochloric acid) etc..
It is reacted in the operation for generating intermediate product D with intermediate product B with bi-functional cross-linking agent, bi-functional cross-linking agent and centre
The molar ratio of product B is 1~10000:1.
Further, the molar ratio of bi-functional cross-linking agent and intermediate product B are 5~5000:1.
By taking intermediate product B is EMCH for acridinium ester-KLH, bi-functional cross-linking agent as an example, intermediate product B and difunctional crosslinking
The reaction equation that agent reacts to obtain intermediate product D is as follows:
Wherein,Indicate acridine compound,Indicate hemocyanin.
In present embodiment, intermediate product B and bi-functional cross-linking agent (EMCH) are anti-in carbodiimide solvent (EDC) solution
Answer, carbodiimide solvent can selected from dicyclohexylcarbodiimide or 1- (3- dimethylamino-propyl) -3- ethyl carbodiimide and
N, N- diisopropylcarbodiimide.In present embodiment, EDC selects dicyclohexylcarbodiimide or 1- (3- dimethylamino third
Base) -3- ethyl carbodiimide.Promote intermediate product B to react with bi-functional cross-linking agent (EMCH) by carbodiimide solvent, improves
Product rate.
Specifically, bi-functional cross-linking agent and the molten molar ratio of carbodiimide can be 10:1~1:10.Further
Bi-functional cross-linking agent and the molten molar ratio of carbodiimide are 5:1~1:5.
The specific reaction process of above-mentioned reaction equation are as follows: EDC and EMCH are added in intermediate product B (acridinium ester-KLH).
Wherein, EDC is 10:1~1:10 with EMCH moles.After 20 DEG C~25 DEG C reaction 10min~60min.With 5mL 7KD retention point
The desalting column (Thermo fish company) of son amount is crossed column 3 times using 150mM PBS (pH 7.4) buffer as liquid buffer is changed,
Free EDC and EMCH and by-product are removed, intermediate product D (acridine substituent-connection carrier-bi-functional cross-linking agent knot is obtained
Close object), in present embodiment, intermediate product D is specially acridinium ester-KLH-EMCH.
S120B, intermediate product D is obtained to acridine label conjugate in conjunction with object to be marked.
Wherein, object to be marked is the albumen containing sulfydryl, carbonyl or carboxyl, modified protein, polypeptide, modified polypeptides or carbon water
Compound, the dimaleoyl imino on bi-functional cross-linking agent in intermediate product D react to be formed with the sulfydryl on object to be markedStructure is to which intermediate product D to be connect with object to be marked.
Specifically, object to be marked can be inherently to have sulfydryl, the albumen of carbonyl or carboxyl, modified protein, polypeptide, change
Property polypeptide or carbohydrate, or by introduced after modification the albumen of sulfydryl, carbonyl or carboxyl, modified protein, polypeptide,
Modified polypeptides or carbohydrate.
In present embodiment, object to be marked is antigen, haptens or antibody.
Intermediate product D is obtained in conjunction with object to be marked acridine label conjugate operation in, intermediate product D with it is to be marked
The molar ratio of object is 0.01~100:1.Further, the molar ratio of intermediate product D and object to be marked is 0.25~5:1.
Using intermediate product D as acridinium ester-KLH-EMCH, object to be marked is centre production for the object to be marked containing sulfydryl
The reaction equation that object D obtains acridine label conjugate in conjunction with object to be marked is as follows:
Wherein,Indicate acridine compound,Indicate hemocyanin,It is to be marked
Object.
The specific reaction process of above-mentioned reaction equation are as follows: by intermediate product D (acridinium ester-KLH-EMCH) addition purify containing
In the object solution to be marked of sulfydryl (- SH), 1h~3h is reacted, obtains acridine label conjugate.
In another embodiment, intermediate product B passes through the dimaleoyl imino on disulfide bond and bi-functional cross-linking agent
Reaction is formedStructure connect to connect carrier with bi-functional cross-linking agent.Hydrazides on bi-functional cross-linking agent
Base on object to be marked carbonyl or carboxyl react and to form-NH-NH-CO- structure to connect bi-functional cross-linking agent and object to be marked
It connects.Specifically as shown in figure 3, in the present embodiment, S120 includes the following steps S120a, S120b and S120c.
S120a, intermediate product B is activated, so that the disulfide bond activation on the connection carrier in intermediate product B
Sulfydryl is formed, intermediate product F is obtained.
Wherein, intermediate product F is acridine substituent-connection carrier conjugates containing sulfydryl.
Specifically, can activate to be formed to the disulfide bond on the connection carrier in intermediate product B by the solution containing sulfydryl
Sulfydryl, to obtain intermediate product F.Solution containing sulfydryl can be DTT (dithiothreitol (DTT)) etc..
By taking intermediate product B is DTT solution for acridinium ester-KLH, activating treatment agent as an example, intermediate product B is carried out at activation
The reaction equation that reason obtains intermediate product F is as follows:
Wherein,Indicate acridine compound,Indicate hemocyanin.
The specific reaction process of above-mentioned reaction equation are as follows: by intermediate product B (acridinium ester-KLH) after purification, final concentration is added
For the DTT of 1mM~10mM, mix, 20 DEG C~30 DEG C reaction 10min~60min.With the desalting column of 5mL7KD molecular cut off
(Thermofish company) crosses column 3 times using 150mM PBS (pH 7.4) buffer as liquid buffer is changed, removes free DTT
And byproduct of reaction, obtain intermediate product F.In present embodiment, intermediate product F is specially acridinium ester-KLH-SH.
S120b, object to be marked is reacted to generation intermediate product I with bi-functional cross-linking agent.
Wherein, intermediate product I be object to be marked-bi-functional cross-linking agent conjugate, object to be marked be containing sulfydryl, carbonyl or
Albumen, modified protein, polypeptide, modified polypeptides or the carbohydrate of carboxyl, bi-functional cross-linking agent are to contain dimaleoyl imino
And the crosslinking agent of hydrazide group, hydrazide group on bi-functional cross-linking agent on object to be marked carbonyl or carboxyl react and to form-NH-
NH-CO- structure is to which bi-functional cross-linking agent to be connect with object to be marked.
Specifically, object to be marked can be inherently to have sulfydryl, the albumen of carbonyl or carboxyl, modified protein, polypeptide, change
Property polypeptide or carbohydrate, or by introduced after modification the albumen of sulfydryl, carbonyl or carboxyl, modified protein, polypeptide,
Modified polypeptides or carbohydrate.
In present embodiment, object to be marked is antigen, haptens or antibody.
Specifically, bi-functional cross-linking agent is the crosslinking agent containing dimaleoyl imino and hydrazide group.It is specifically as follows EMCH
(N- [ε-maleimide acetic acid]-hydrazides ■ trifluoroacetic acid), KMUH (N- [κ-maleimidbundecanoic acid]-hydrazides ■ tri-
Fluoroacetic acid) or MPBH (4- [4-N- maleimide phenyl]-butyric acid hydrazides ■ hydrochloric acid) etc..
Object to be marked is reacted with bi-functional cross-linking agent in the operation for generating intermediate product I, bi-functional cross-linking agent with wait mark
The molar ratio for remembering object is 1~10000:1.Further, the molar ratio of bi-functional cross-linking agent and object to be marked is 50~1000:
1
By taking object to be marked is EMCH for object to be marked, bi-functional cross-linking agent containing carbonyl or carboxyl as an example, object to be marked
The reaction equation that generation intermediate product I is reacted with bi-functional cross-linking agent is as follows:
Wherein,For object to be marked ,-R is-H ,-OH or aromatic compound.
In present embodiment, object to be marked and bi-functional cross-linking agent (EMCH) are anti-in carbodiimide solvent (EDC) solution
Answer, carbodiimide solvent can selected from dicyclohexylcarbodiimide or 1- (3- dimethylamino-propyl) -3- ethyl carbodiimide and
N, N- diisopropylcarbodiimide.In present embodiment, EDC selects dicyclohexylcarbodiimide or 1- (3- dimethylamino third
Base) -3- ethyl carbodiimide.Promote object to be marked to react with bi-functional cross-linking agent (EMCH) by carbodiimide solvent, improves
Product rate.
Specifically, bi-functional cross-linking agent and the molten molar ratio of carbodiimide can be 10:1~1:10.Further
Bi-functional cross-linking agent and the molten molar ratio of carbodiimide are 5:1~1:5.
The specific reaction process of above-mentioned reaction equation are as follows: EDC and EMCH are added in object solution to be marked.Wherein, EDC with
EMCH moles is 10:1~1:10.After 20 DEG C~25 DEG C reaction 10min~60min.With the desalination of 5mL 7KD molecular cut off
Column (Thermo fish company) is crossed column 3 times, is removed free using 150mM PBS (pH 7.4) buffer as liquid buffer is changed
EDC and EMCH and by-product obtain intermediate product I.In present embodiment, intermediate product I is specially object-EMCH to be marked.
S120c, intermediate product I is obtained to acridine label conjugate in conjunction with intermediate product F.
Wherein, dimaleoyl imino with the sulfydryl on intermediate product F reacts shape on the bi-functional cross-linking agent in intermediate product I
AtStructure is to which intermediate product I to be connect with the intermediate product F.
Specifically, intermediate product I is obtained in conjunction with intermediate product F in the operation of acridine label conjugate, intermediate product I
Molar ratio with intermediate product F is 0.01~100:1.Further, the molar ratio of intermediate product I and intermediate product F is 0.2
~20:1.
By taking intermediate product I is object-EMCH to be marked, intermediate product F is acridinium ester-KLH-SH as an example, intermediate product I is in
Between product F combine obtain acridine label conjugate reaction equation it is as follows:
Wherein,Indicate acridine compound,Indicate hemocyanin,It is to be marked
Object.
The specific reaction process of above-mentioned reaction equation are as follows: object-EMCH to be marked is added in acridinium ester-KLH-SH solution, instead
1h~5h is answered, acridine label conjugate is obtained.In present embodiment, it is specially acridinium ester-KLH-EMCH- that acridine, which marks conjugate,
Object to be marked.
The preparation method of this acridine label conjugate, simple process by connection carrier and contain maleimide
The bi-functional cross-linking agent of base and hydrazide group, acridine substituent can not be bound directly by carbodiimide with object to be marked, be avoided
Acridine substituent treats active site on labelled protein and forms interference, so that the activity of acridine label conjugate is relatively
Height improves the sensitivity of immunoassay.Dimaleoyl imino and hydrazide group in bi-functional cross-linking agent can with contain sulfydryl, carbonyl
Or the object to be marked specific binding of carboxyl, avoid previous acridine substituent cannot with containing sulfydryl, carbonyl or carboxyl to
The defect that marker combines, expands the range of choice of object to be marked, so that the specificity of acridine label conjugate is high, increases a word used for translation
Pyridine label conjugate uses sensitivity.It is corresponding to be marked that the detected material can be connected accordingly according to the needs of detection
Object, because the range of choice of object to be marked is wide, even the object to be marked containing sulfydryl, carbonyl or carboxyl also can be by difunctional
Crosslinking agent and connection carrier form acridine label conjugate in conjunction with acridine substituent, therefore when detecting, acridine label knot
Closing object can specifically bind with detected material, improve high sensitivity and mark rate.
The invention also discloses a kind of chemiluminescence immune detection reagent kit, which includes
Above-mentioned acridine marks conjugate.
Preferably, this chemical luminescence reagent kit further includes centrifugation at least one of desalting column and centrifugal ultrafiltration pipe.
This chemical luminescence reagent kit can connect the quilt in acridine label conjugate according to the needs of detection accordingly
The corresponding object to be marked of detectable substance, because the range of choice of object to be marked is wide, even containing sulfydryl, carbonyl or carboxyl wait mark
Note object also can by bi-functional cross-linking agent and connection carrier be formed with acridine substituent in conjunction with acridine label conjugate, therefore
When detection, acridine label conjugate can be specifically bound with detected material, improve high sensitivity and mark rate.
The following are specific embodiments.
In embodiment, unless specifically indicated, otherwise used reagent is purchased from Sigma-aldrich company, pure to analyze.
The acridinium ester occurred in embodiment is the acridinium ester of n-hydroxysuccinimide activation.KLH indicate containing amino,
The hemocyanin of carboxyl and disulfide bond.EDC indicates dicyclohexylcarbodiimide or 1- (3- dimethylamino-propyl) -3- ethyl carbon two
Imines.DTT indicates dithiothreitol (DTT).
Embodiment 1
KLH is dissolved in 1mL150mM PBS buffer solution (pH 7.4), final concentration of 20nmol/L.10 μ L dissolution is added
In the 10mmol/L acridinium ester of DMSO solvent.In 25 DEG C of reaction 1h, with the desalting column (Thermo of 5mL 7KD molecular cut off
Fish company) using 150mM PBS (pH 7.4) buffer as liquid buffer is changed, it crosses column 3 times, removes free acridinium ester and anti-
By-product is answered, acridinium ester-KLH solution is obtained.
1mg thyroglobulin (producer: biospacific, article No.: J19400,1.5nmol) is dissolved in 1mL 50mM
In MES buffer (pH 4.5), EDC (final concentration of 5mmol/L) and EMCH (final concentration of 10mmol/L), 25 DEG C of conditions are added
After lower reaction 30 minutes, with the desalting column (Thermo fish company) of 5mL 7KD molecular cut off with 150mM PBS (pH
7.4) buffer is crossed column 3 times as liquid buffer is changed, removes free EDC and EMCH and by-product, the first shape after being activated
Gland globulin-EMCH.
By acridinium ester-KLH solution after purification, the DTT processing of final concentration of 5mM is added, mixes, 25 DEG C of reaction 0.5h,
Use the desalting column (Thermofish company) of 5mL 7KD molecular cut off using 150mM PBS (pH 7.4) buffer as changing liquid
Buffer is crossed column 3 times, removes free DTT and byproduct of reaction, obtain acridinium ester-KLH-SH.
Thyroglobulin-EMCH after activation is added in purified acridinium ester-KLH-SH solution, 2h is reacted, obtains
Conjugate (acridinium ester-KLH-EMCH- thyroglobulin) solution is marked to acridine.
Embodiment 2
KLH is dissolved in 1mL150mM PBS buffer solution (pH 7.4), final concentration of 20nmol/L, 10 μ L dissolution is added
In the 10mmol/L acridinium ester of DMSO solvent.In 25 DEG C of reaction 1h, with the desalting column (Thermo of 5mL 7KD molecular cut off
Fish company) using 50mM MES (pH 4.5) buffer as liquid buffer is changed, it crosses column 3 times, removes free acridinium ester and anti-
By-product is answered, acridinium ester-KLH solution is obtained.
EDC (final concentration of 5mmol/L) and EMCH (final concentration of 10mmol/L) are added in acridinium ester-KLH, 25 DEG C
After reaction 30 minutes, with the desalting column (Thermo fish company) of 5mL 7KD molecular cut off with 150mM PBS (pH 7.4)
Buffer is crossed column 3 times as liquid buffer is changed, removes free EDC and EMCH and by-product, the acridinium ester-after being activated
KLH-EMCH；
By the mouse monoclonal antibody antibody of the anti-Procalcitonin of 1mg (producer: Hytest, article No.: 4PC47-6F10,
It 6.67nmol) is dissolved in 150mM PBS (pH 7.4) buffer soln, is handled with the DTT of final concentration 5mM, 25 DEG C of reaction 0.5h,
Use the desalting column (Thermofish company) of 5mL 7KD molecular cut off using 150mM PBS (pH 7.4) buffer as changing liquid
Buffer is crossed column 3 times, removes free DTT and byproduct of reaction, obtain anti-Procalcitonin antibody (the anti-calcitonin of sulfhydrylation
Original antibody-SH).
Acridinium ester-KLH-EMCH after activation is added in purified anti-Procalcitonin antibody-SH solution, 2h is reacted,
Obtain acridine label conjugate (the anti-Procalcitonin mouse monoclonal antibody of acridinium ester-KLH-EMCH-) solution.
Embodiment 3
KLH is dissolved in 1mL150mM PBS buffer solution (pH 7.4), final concentration 20nmol/L, 10 μ L are added and are dissolved in
The 10mmol/L acridinium ester of DMSO solvent, in 25 DEG C of reaction 1h, with desalting column (the Thermo fish of 5mL 7KD molecular cut off
Company) using 150mM PBS (pH 7.4) buffer as liquid buffer is changed, it crosses column 3 times, it is secondary to remove free acridinium ester and reaction
Product obtains acridinium ester-KLH solution.
The beta2 Glycoprotein (producer: abgree, article No.: G7018,16.7nmol) of 1mg is dissolved in 1mL50mM MES buffering
It in liquid (pH 4.5), is added sodium metaperiodate (final concentration of 10mmol/L), after 25 DEG C are reacted 30 minutes, adds the ethylene glycol of 20ul
Quenching reaction 30min, with the desalting column (Thermo fish company) of 5mL 7KD molecular cut off with 150mM PBS (pH 7.4)
Buffer is crossed column 3 times as liquid buffer is changed, and is removed free byproduct of reaction, the EMCH of final concentration of 10mmol/L is added, instead
1h is answered, the lysine reaction 30min of final concentration of 20mmol/L is added, with the desalting column (Thermo of 5mL 7KD molecular cut off
Fish company) using 150mM PBS (pH 7.4) buffer as liquid buffer is changed, it crosses column 3 times, removes free byproduct of reaction,
Beta2 Glycoprotein-EMCH after being activated.
By acridinium ester-KLH solution after purification, the DTT processing of final concentration 5mM is added, mixes, 25 DEG C of reaction 0.5h, uses
The desalting column (Thermofish company) of 5mL 7KD molecular cut off is slow using 150mM PBS (pH 7.4) buffer as liquid is changed
Fliud flushing is crossed column 3 times, removes free DTT and byproduct of reaction, obtain acridinium ester-KLH-SH solution.
Beta2 Glycoprotein-EMCH after activation is added in purified acridinium ester-KLH-SH solution, 2h is reacted, is tied
Close object acridinium ester-KLH-EMCH- glycoprotein solution.
Comparative example 1
This embodiment be activation after acridinium ester under equal conditions with conventional method label thyroglobulin scheme,
1mg thyroglobulin (producer: biospacific, article No.: J19400,1.5nmol) is dissolved in 1mL150mM PBS buffering
In liquid (pH 7.4), the 10mmol/L acridinium ester that 10 μ L are dissolved in DMSO solvent is added, in 25 DEG C of reaction 1h.It is cut with 5mL 7KD
It stays the desalting column (Thermo fish company) of molecular weight using 150mM PBS (pH 7.4) buffer as liquid buffer is changed, crosses column
3 times, free acridinium ester and byproduct of reaction are removed, obtains acridinium ester-thyroglobulin solution.
Comparative example 2
This embodiment is that the acridinium ester after activation under equal conditions marks the mouse of anti-Procalcitonin single with conventional method
The scheme of clonal antibody, by the mouse monoclonal antibody of the anti-Procalcitonin of 1mg (producer: Hytest, article No.: 4PC47-6F10,
It 6.67nmol) is dissolved in 1mL150mM PBS buffer solution (pH 7.4), the 10mmol/L that 10ul is dissolved in DMSO solvent is added
Acridinium ester, in 25 DEG C of reaction 1h, with the desalting column (Thermo fish company) of 5mL 7KD molecular cut off with 150mM PBS
(pH 7.4) buffer is crossed column 3 times as liquid buffer is changed, removes free acridinium ester and byproduct of reaction, obtain acridinium ester-
The mouse monoclonal antibody solution of anti-Procalcitonin.
Comparative example 3
This embodiment be activation after acridinium ester under equal conditions with conventional method label β2-microglobulin scheme, will
1mg beta2 Glycoprotein (producer: abgree, article No.: G7018,16.7nmol) is dissolved in 1mL150mM PBS buffer solution (pH 7.4)
In, the 10mmol/L acridinium ester that 10ul is dissolved in DMSO solvent is added, in 25 DEG C of reaction 1h.With 5mL 7KD molecular cut off
Desalting column (Thermo fish company) is crossed column 3 times using 150mM PBS (pH 7.4) buffer as liquid buffer is changed, and removes trip
From acridinium ester and byproduct of reaction, obtain acridinium ester-beta2 Glycoprotein solution.
Test case 1
Conjugate obtained in embodiment 1 and comparative example 1 is respectively used to the change of thyroglobulin antibody (Anti-Tg)
Learn luminescence immunoassay detection.Into the thyroglobulin antibody sample of 500IU/mL, 40 μ g thyroglobulin packets are added
The magnetic bead of quilt, then it is separately added into 4nmoL acridinium ester label thyroglobulin prepared by embodiment 1 and comparative example 1, incubation reaction
After clean, 100 μ L HNO are successively added3-H2O2The sodium hydroxide solution of solution and 100 μ L, with iFlash3000 type chemiluminescence
Immunity analysis instrument measures luminous value, parallel to carry out 3 parts of measurements, is averaged.It the results are shown in Table 1.The hair of immunoassay acquired results
Light value is bigger, illustrates that the activity of acridinium ester label is better.
Table 1: analyte detection Anti-Tg comparing result is combined obtained in embodiment 1 and comparative example 1
| Acridinium ester label | Average irradiance (RLU) |
| Embodiment 1 | 2015672 |
| Comparative example 1 | 431557 |
As seen from the results in Table 1, the activity for the acridine label conjugate being prepared using embodiment 1 is significantly better than comparative example
The conjugate of 1 preparation.
Test case 2
Conjugate obtained in embodiment 2 and comparative example 2 is respectively used to the chemiluminescence immunoassay point of Procalcitonin (PCT)
Analysis detection.Into the Procalcitonin sample of 30ng/ml, the coated magnetic of mouse monoclonal antibody of the 40 anti-Procalcitonins of μ g is added
Pearl, then it is separately added into 10nmol acridinium ester label thyroglobulin prepared by embodiment 2 and comparative example 2, it is clear after incubation reaction
It washes, 100 μ L HNO is successively added3-H2O2The sodium hydroxide solution of solution and 100 μ L, with iFlash3000 type chemiluminescence immunoassay
Analyzer measures luminous value, parallel to carry out 3 parts of measurements, is averaged, the results are shown in Table 2.The luminous value of immunoassay acquired results
It is bigger, illustrate that the activity of acridinium ester label is better.
Table 2: analyte detection PCT comparing result is combined obtained in embodiment 2 and comparative example 2
| Acridinium ester label | Average irradiance (RLU) |
| Embodiment 2 | 6358457 |
| Comparative example 2 | 865473 |
As seen from the results in Table 2, the activity for the acridine label conjugate being prepared using embodiment 2 is significantly better than comparative example
The conjugate of 2 preparations.
Test case 3
Conjugate obtained in embodiment 3 and comparative example 3 is respectively used to the chemiluminescence immunoassay of anti-beta 2 glycoprotein I antibody
Analysis detection.Into the anti-beta 2 glycoprotein I antibody sample of 50AU/mL, the 40 coated magnetic beads of μ g beta2 Glycoprotein are added, then add respectively
Enter 1nmol acridinium ester label beta2 Glycoprotein prepared by embodiment 3 and comparative example 3, is cleaned after incubation reaction, 100 μ L are successively added
HNO3-H2O2The sodium hydroxide solution of solution and 100 μ L measures luminous value with iFlash3000 type chemical illumination immunity analysis instrument,
It is parallel to carry out 3 parts of measurements, it is averaged and the results are shown in Table 3.The luminous value of immunoassay acquired results is bigger, illustrates acridinium ester label
The activity of object is better.
Table 3: the anti-beta 2 glycoprotein I antibody comparing result of analyte detection is combined obtained in embodiment 3 and comparative example 3
As shown in Table 3, the activity for the acridine label conjugate being prepared using embodiment 3 is significantly better than comparison
Conjugate prepared by example 3.
Test case 4
Acridinium ester-KLH-EMCH- thyroglobulin solution obtained in embodiment 1 is used for the thyroid gland of series of concentrations
The chemiluminescence immune assay of globulin antibody (Anti-Tg) detects.Add respectively into the Anti-Tg sample solution of series of concentrations
Enter the coated magnetic bead reagent of 40 μ g thyroglobulins and 4nmoL acridinium ester-KLH-EMCH- thyroglobulin reagent, is incubated for
It is cleaned after reaction, 100 μ L HNO is successively added3-H2O2The sodium hydroxide solution of solution and 100 μ L, with iFlash3000 type chemistry
Luminescence immunoassay instrument measures luminous value, the results are shown in Table 4.Using thyroglobulin antibody concentration as X-axis, it is with relative light unit
Y-axis is mapped by 4 data of table, obtains Fig. 4.
Table 4: the immune detection result of series of concentrations thyroglobulin antibody sample
By table 4 and Fig. 4 result it is found that embodiment 1 prepares acridinium ester-KLH-EMCH- can be applied to of thyroglobulin
It learns luminescence immunoassay and works well.Acridinium ester-KLH-EMCH- marker preparation method prepared by embodiment 1 has good
Applicability.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
Limitations on the scope of the patent of the present invention therefore cannot be interpreted as.It should be pointed out that for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to guarantor of the invention
Protect range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Claims (8)
1. a kind of acridine marks conjugate, which is characterized in that by sequentially connected acridine substituent, connection carrier, difunctional friendship
Join agent and object to be marked composition；
The acridine substituent is acridinium ester, acridinic acid, acridinium carboxamide or acridine sulfonamide；
The bi-functional cross-linking agent is N- [ε-maleimide acetic acid]-hydrazides trifluoroacetic acid, N- [κ-maleimide 11
Alkanoic acid]-hydrazides trifluoroacetic acid or 4- [4-N- maleimide phenyl]-butyric acid hydrazides hydrochloric acid；
The object to be marked is the albumen containing sulfydryl, carbonyl or carboxyl or the polypeptide containing sulfydryl, carbonyl or carboxyl；
The connection carrier is the hemocyanin containing amino, carboxyl and disulfide bond, and the acridine substituent and the connection carry
Amino on body reacts to form-CO-NH- structure to the acridine substituent and the connection carrier be connected, the connection
Carboxyl on carrier reacts to form-CO-NH-NH- structure thus by the connection with the hydrazide group on the bi-functional cross-linking agent
Carrier is connect with the bi-functional cross-linking agent, on the dimaleoyl imino on the bi-functional cross-linking agent and the object to be marked
Sulfydryl reacts to be formedStructure is to which the bi-functional cross-linking agent to be connect with the object to be marked；
Alternatively, the connection carrier is the hemocyanin containing amino, carboxyl and disulfide bond, the acridine substituent and the company
Carry the amino on body react to be formed-CO-NH- structure to by the acridine substituent and the connection carrier connection, it is described
Disulfide bond on connection carrier reacts to be formed with the dimaleoyl imino on the bi-functional cross-linking agentStructure from
And the connection carrier is connect with the bi-functional cross-linking agent, the hydrazide group on the bi-functional cross-linking agent with it is described to be marked
Carbonyl or carboxyl on object react to form-NH-NH-CO- structure to which the bi-functional cross-linking agent to be connect with the object to be marked.
2. acridine according to claim 1 marks conjugate, which is characterized in that the object to be marked is antigen, haptens
Or antibody.
3. a kind of preparation method of acridine label conjugate according to any one of claims 1 to 2, which is characterized in that including
Following steps:
It reacts to obtain intermediate product B with the acridine substituent after activation with carrier is connect, wherein the intermediate product B is acridine
Substituent-connection carrier conjugates；And
Acridine label conjugate will be obtained after the intermediate product B, bi-functional cross-linking agent and object cross-linking reaction to be marked,
In, acridine label conjugate includes sequentially connected acridine substituent, connection carrier, bi-functional cross-linking agent and to be marked
Object, the bi-functional cross-linking agent are N- [ε-maleimide acetic acid]-hydrazides trifluoroacetic acid, N- [κ-maleimide 11
Alkanoic acid]-hydrazides trifluoroacetic acid or 4- [4-N- maleimide phenyl]-butyric acid hydrazides hydrochloric acid, the connection carrier be containing
Have a hemocyanin of amino, carboxyl and disulfide bond, the acridine substituent reacted with the amino on the connection carrier to be formed-
CO-NH- structure is to connect the acridine substituent and the connection carrier, the carboxyl on the connection carrier and described pair
Hydrazide group on functional cross-link agent reacts to form-CO-NH-NH- structure thus by the connection carrier and the difunctional crosslinking
Agent connects, and the dimaleoyl imino on the bi-functional cross-linking agent reacts to be formed with the sulfydryl on the object to be markedStructure is to which the bi-functional cross-linking agent to be connect with the object to be marked；Alternatively, the connection carrier is
Hemocyanin containing amino, carboxyl and disulfide bond, the acridine substituent with it is described connection carrier on amino react to be formed-
CO-NH- structure to which the acridine substituent and the connection carrier be connected, the disulfide bond connected on carrier with it is described
Dimaleoyl imino on bi-functional cross-linking agent reacts to be formedStructure is thus by the connection carrier and described pair
Functional cross-link agent connection, hydrazide group on the bi-functional cross-linking agent on the object to be marked carbonyl or carboxyl react shape
At-NH-NH-CO- structure to which the bi-functional cross-linking agent to be connect with the object to be marked.
4. the preparation method of acridine according to claim 3 label conjugate, which is characterized in that by the intermediate product B,
Obtained after bi-functional cross-linking agent and object cross-linking reaction to be marked acridine label conjugate operation specifically includes the following steps:
It is reacted with intermediate product B with bi-functional cross-linking agent and generates intermediate product D, wherein the intermediate product D is acridine substitution
Object-connection carrier-bi-functional cross-linking agent conjugate, carboxyl on connection carrier in the intermediate product B with it is described difunctional
Hydrazide group on crosslinking agent reacts to form-CO-NH-NH- structure to connect the intermediate product B and the bi-functional cross-linking agent
It connects；And
The intermediate product D is obtained into acridine label conjugate in conjunction with object to be marked, wherein double in the intermediate product D
Dimaleoyl imino on functional cross-link agent reacts to be formed with the sulfydryl on the object to be markedStructure to
The intermediate product D is connect with the object to be marked.
5. the preparation method of acridine according to claim 3 label conjugate, which is characterized in that by the intermediate product B,
Obtained after bi-functional cross-linking agent and object cross-linking reaction to be marked acridine label conjugate operation specifically includes the following steps:
Intermediate product B is activated, so that the disulfide bond on the connection carrier in the intermediate product B activates to form mercapto
Base obtains intermediate product F, wherein the intermediate product F is acridine substituent-connection carrier conjugates containing sulfydryl；
Object to be marked is reacted with bi-functional cross-linking agent and generates intermediate product I, wherein the intermediate product I is that object-to be marked is bis-
Functional cross-link agent conjugate, hydrazide group on the bi-functional cross-linking agent on the object to be marked carbonyl or carboxyl react shape
At-NH-NH-CO- structure to which the bi-functional cross-linking agent to be connect with the object to be marked；And
The intermediate product I is obtained to acridine label conjugate in conjunction with the intermediate product F, wherein in the intermediate product I
Bi-functional cross-linking agent on dimaleoyl imino react to be formed with the sulfydryl on the intermediate product FStructure from
And the intermediate product I is connect with the intermediate product F.
6. the preparation method of acridine label conjugate according to claim 3, which is characterized in that taken with the acridine after activation
It reacts to obtain in the operation of intermediate product B with carrier is connect for object, the acridine substituent after the activation and the connection carrier
Molar ratio be 1~5000:1.
7. the preparation method of acridine according to claim 4 label conjugate, which is characterized in that with intermediate product B and double
Functional cross-link agent reaction generates in the operation of intermediate product D, and the molar ratio of the bi-functional cross-linking agent and the intermediate product B is
1~10000:1.
8. a kind of chemiluminescence immune detection reagent kit, which is characterized in that including a word used for translation according to any one of claims 1 to 2
Pyridine marks conjugate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610522380.4A CN106146672B (en) | 2016-07-05 | 2016-07-05 | Acridine marks conjugate and preparation method thereof, chemiluminescence immune detection reagent kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610522380.4A CN106146672B (en) | 2016-07-05 | 2016-07-05 | Acridine marks conjugate and preparation method thereof, chemiluminescence immune detection reagent kit |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106146672A CN106146672A (en) | 2016-11-23 |
| CN106146672B true CN106146672B (en) | 2019-11-29 |
Family
ID=58061282
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610522380.4A Active CN106146672B (en) | 2016-07-05 | 2016-07-05 | Acridine marks conjugate and preparation method thereof, chemiluminescence immune detection reagent kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106146672B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018006269A1 (en) * | 2016-07-05 | 2018-01-11 | 深圳市亚辉龙生物科技股份有限公司 | Acridine-marker conjugate and preparation method thereof, and chemiluminescence immunoassay kit |
| CN109852669A (en) * | 2019-01-04 | 2019-06-07 | 南京航思生物科技有限公司 | A kind of method of acridinium ester label nucleic acid |
| CN113049805B (en) * | 2021-03-22 | 2022-09-23 | 深圳市亚辉龙生物科技股份有限公司 | Antibody compound, preparation method thereof and detection kit |
| CN116004221B (en) * | 2022-12-15 | 2023-12-19 | 湖南卓润生物科技有限公司 | New application of cyclic peptide, acridine labeled complex, preparation method and detection kit |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999056130A1 (en) * | 1998-04-29 | 1999-11-04 | Queen Mary & Westfield College | Method for assaying a plurality of analytes |
| CN102269762A (en) * | 2010-06-04 | 2011-12-07 | 深圳迈瑞生物医疗电子股份有限公司 | Preparation method of conjugate and relative kit |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090068635A1 (en) * | 2007-09-06 | 2009-03-12 | Muerhoff Anthony S | Indirectly labelled assay conjugates and methods of preparing and using same |
-
2016
- 2016-07-05 CN CN201610522380.4A patent/CN106146672B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999056130A1 (en) * | 1998-04-29 | 1999-11-04 | Queen Mary & Westfield College | Method for assaying a plurality of analytes |
| CN102269762A (en) * | 2010-06-04 | 2011-12-07 | 深圳迈瑞生物医疗电子股份有限公司 | Preparation method of conjugate and relative kit |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106146672A (en) | 2016-11-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106146672B (en) | Acridine marks conjugate and preparation method thereof, chemiluminescence immune detection reagent kit | |
| CN108700584B (en) | Marker complex, preparation method thereof, kit, application and detection system | |
| CN106053443A (en) | Acridine marker conjugate and preparation method thereof and chemiluminescent kit | |
| CN110146692A (en) | One kind being based on acridinium ester chemiluminescent, Streptavidin MagneSphere-biotin iodine system and detection kit | |
| CA2661995C (en) | Antigenic protein conjugates and process for preparing same | |
| WO2007140696A1 (en) | Elisa kit for detecting sudan red and method thereof | |
| CN102269762B (en) | Preparation method of conjugate and relative kit | |
| CN101949943B (en) | Thyrotropic hormone quantitative detection kit | |
| WO2018006269A1 (en) | Acridine-marker conjugate and preparation method thereof, and chemiluminescence immunoassay kit | |
| CN109682967A (en) | Application of the PAMAM in the reagent that preparation is used for immune detection | |
| CN109239348B (en) | Antibody for gastrin-releasing peptide precursor detection kit and kit | |
| CN101149375A (en) | An immunoassay method and kit to leucomalachite green and malachite green | |
| CN108535486A (en) | A kind of chloramphenicol immunofluorescence assay method based on europium label | |
| CN106124777A (en) | Acridine labelling conjugate and preparation method thereof, chemical luminescence reagent kit | |
| WO2018006268A1 (en) | Acridine-marker conjugate and preparation method thereof, and chemiluminescence kit | |
| WO2023124154A1 (en) | Magnetic bead coating, preparation method therefor, and test kit | |
| CN109283347A (en) | The method that nano biological sensor based on immunomagnetic beads and fluorescence quantum quickly detects Enrofloxacin in broiler chicken | |
| WO2018006264A1 (en) | Acridine marker conjugate and preparation method therefor and chemiluminescent kit | |
| CN109180507B (en) | Ketoprofen hapten, artificial antigen and antibody, and preparation method and application thereof | |
| CN112592473B (en) | Synthesis method and application of branched polyethyleneimine-enrofloxacin | |
| CN112326971A (en) | Novel method and kit for NMDAR antibody quantitative detection | |
| CN109633159A (en) | A kind of method for detecting content of melamine and its dedicated colloid gold test paper | |
| CN113307863B (en) | Preparation method and application of polyaspartic acid and salt antibody thereof | |
| CN108333358A (en) | A kind of screening technique of immunoaffinity purification eluent | |
| CN109134292B (en) | Atenolol hapten, artificial antigen and antibody, and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB02 | Change of applicant information | ||
| CB02 | Change of applicant information |
Address after: 518000 Yahuilong Biotechnology Plant, Baolong Street, Longgang District, Shenzhen City, Guangdong Province Applicant after: SHENZHEN YHLO BIOTECH Co.,Ltd. Address before: 518000, Shenzhen, Guangdong, Nanshan District Nanshan Road, Lai Shan Industrial Zone, 5, 1-4 Applicant before: SHENZHEN YHLO BIOTECH Co.,Ltd. |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| EE01 | Entry into force of recordation of patent licensing contract | ||
| EE01 | Entry into force of recordation of patent licensing contract |
Application publication date: 20161123 Assignee: Hunan Yahuilong Biotechnology Co.,Ltd. Assignor: SHENZHEN YHLO BIOTECH Co.,Ltd. Contract record no.: X2023980047609 Denomination of invention: Acridine labeled conjugate and its preparation method, chemiluminescence immunoassay kit Granted publication date: 20191129 License type: Common License Record date: 20231120 |