CN116004221B - New application of cyclic peptide, acridine labeled complex, preparation method and detection kit - Google Patents
New application of cyclic peptide, acridine labeled complex, preparation method and detection kit Download PDFInfo
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- CN116004221B CN116004221B CN202211618320.4A CN202211618320A CN116004221B CN 116004221 B CN116004221 B CN 116004221B CN 202211618320 A CN202211618320 A CN 202211618320A CN 116004221 B CN116004221 B CN 116004221B
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- 102000001189 Cyclic Peptides Human genes 0.000 title claims abstract description 65
- 108010069514 Cyclic Peptides Proteins 0.000 title claims abstract description 65
- 238000002360 preparation method Methods 0.000 title claims abstract description 19
- DZBUGLKDJFMEHC-UHFFFAOYSA-N acridine Chemical compound C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 title claims description 131
- 238000001514 detection method Methods 0.000 title abstract description 60
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 34
- 150000001413 amino acids Chemical class 0.000 claims abstract description 31
- 229920001184 polypeptide Polymers 0.000 claims abstract description 30
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 30
- 230000002209 hydrophobic effect Effects 0.000 claims abstract description 15
- 108010016626 Dipeptides Proteins 0.000 claims abstract description 8
- RXNXLAHQOVLMIE-UHFFFAOYSA-N phenyl 10-methylacridin-10-ium-9-carboxylate Chemical class C12=CC=CC=C2[N+](C)=C2C=CC=CC2=C1C(=O)OC1=CC=CC=C1 RXNXLAHQOVLMIE-UHFFFAOYSA-N 0.000 claims description 20
- 238000012360 testing method Methods 0.000 claims description 20
- 238000002372 labelling Methods 0.000 claims description 19
- -1 acridine ester compound Chemical class 0.000 claims description 15
- 239000000427 antigen Substances 0.000 claims description 12
- 102000036639 antigens Human genes 0.000 claims description 12
- 108091007433 antigens Proteins 0.000 claims description 12
- 230000003213 activating effect Effects 0.000 claims description 11
- 238000006243 chemical reaction Methods 0.000 claims description 7
- 230000008878 coupling Effects 0.000 claims description 6
- 238000010168 coupling process Methods 0.000 claims description 6
- 238000005859 coupling reaction Methods 0.000 claims description 6
- 239000012634 fragment Substances 0.000 claims description 6
- AAYSIOJYGRABGS-UHFFFAOYSA-N 3-[9-[4-(2,5-dioxopyrrolidin-1-yl)oxycarbonyl-2,6-dimethylphenoxy]carbonylacridin-10-ium-10-yl]propane-1-sulfonate Chemical compound C=1C(C)=C(OC(=O)C=2C3=CC=CC=C3[N+](CCCS([O-])(=O)=O)=C3C=CC=CC3=2)C(C)=CC=1C(=O)ON1C(=O)CCC1=O AAYSIOJYGRABGS-UHFFFAOYSA-N 0.000 claims description 4
- KQFCNGKUXYNDPF-UHFFFAOYSA-N 3-[9-[[4-(2,5-dioxopyrrolidin-1-yl)oxy-4-oxobutyl]-(4-methylphenyl)sulfonylcarbamoyl]acridin-10-ium-10-yl]propane-1-sulfonate Chemical compound C1=CC(C)=CC=C1S(=O)(=O)N(C(=O)C=1C2=CC=CC=C2[N+](CCCS([O-])(=O)=O)=C2C=CC=CC2=1)CCCC(=O)ON1C(=O)CCC1=O KQFCNGKUXYNDPF-UHFFFAOYSA-N 0.000 claims description 4
- PEEHTFAAVSWFBL-UHFFFAOYSA-N Maleimide Chemical compound O=C1NC(=O)C=C1 PEEHTFAAVSWFBL-UHFFFAOYSA-N 0.000 claims description 3
- 125000000304 alkynyl group Chemical group 0.000 claims description 3
- IVRMZWNICZWHMI-UHFFFAOYSA-N azide group Chemical group [N-]=[N+]=[N-] IVRMZWNICZWHMI-UHFFFAOYSA-N 0.000 claims description 3
- FVZVCSNXTFCBQU-UHFFFAOYSA-N phosphanyl Chemical group [PH2] FVZVCSNXTFCBQU-UHFFFAOYSA-N 0.000 claims description 3
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 2
- 150000003573 thiols Chemical class 0.000 claims 1
- 239000003550 marker Substances 0.000 abstract description 37
- 238000000034 method Methods 0.000 abstract description 15
- 230000035945 sensitivity Effects 0.000 abstract description 5
- 230000008569 process Effects 0.000 abstract description 4
- 230000000052 comparative effect Effects 0.000 description 51
- 239000000243 solution Substances 0.000 description 45
- 238000004020 luminiscence type Methods 0.000 description 17
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 15
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 14
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- 238000003018 immunoassay Methods 0.000 description 9
- 238000000746 purification Methods 0.000 description 9
- 210000002966 serum Anatomy 0.000 description 9
- 238000000108 ultra-filtration Methods 0.000 description 9
- 150000001875 compounds Chemical class 0.000 description 8
- 230000004048 modification Effects 0.000 description 7
- 238000012986 modification Methods 0.000 description 7
- 239000000126 substance Substances 0.000 description 7
- 150000001251 acridines Chemical class 0.000 description 6
- 239000011324 bead Substances 0.000 description 6
- 239000003761 preservation solution Substances 0.000 description 6
- 239000012224 working solution Substances 0.000 description 6
- 239000004471 Glycine Substances 0.000 description 5
- 102000013674 S-100 Human genes 0.000 description 5
- 108700021018 S100 Proteins 0.000 description 5
- 108010063628 acarboxyprothrombin Proteins 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 238000010791 quenching Methods 0.000 description 5
- 230000000171 quenching effect Effects 0.000 description 5
- 238000010668 complexation reaction Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 230000002452 interceptive effect Effects 0.000 description 4
- 125000000641 acridinyl group Polymers C1(=CC=CC2=NC3=CC=CC=C3C=C12)* 0.000 description 3
- 238000004140 cleaning Methods 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
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- 239000004615 ingredient Substances 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 239000003755 preservative agent Substances 0.000 description 3
- 230000002335 preservative effect Effects 0.000 description 3
- 238000002331 protein detection Methods 0.000 description 3
- 239000011265 semifinished product Substances 0.000 description 3
- ATGUDZODTABURZ-UHFFFAOYSA-N thiolan-2-ylideneazanium;chloride Chemical compound Cl.N=C1CCCS1 ATGUDZODTABURZ-UHFFFAOYSA-N 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- OIGKWPIMJCPGGD-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 3-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethoxy]propanoate Chemical compound [N-]=[N+]=NCCOCCOCCOCCOCCC(=O)ON1C(=O)CCC1=O OIGKWPIMJCPGGD-UHFFFAOYSA-N 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 229910000365 copper sulfate Inorganic materials 0.000 description 2
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 2
- 150000002148 esters Chemical class 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 238000011056 performance test Methods 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- QVVDVENEPNODSI-BTNSXGMBSA-N (2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-amino-5-(diaminomethylideneamino)pentanoyl]amino]-5-(diaminomethylideneamino)pentanoyl]amino]-5-(diaminomethylideneamino)pentanoyl]amino]-5-(diaminomethylideneamino)pentanoyl]amino]-5-(diaminomethylidene Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O QVVDVENEPNODSI-BTNSXGMBSA-N 0.000 description 1
- PSZNHSNIGMJYOZ-WDSKDSINSA-N Asp-Arg Chemical compound OC(=O)C[C@H](N)C(=O)N[C@H](C(O)=O)CCCN=C(N)N PSZNHSNIGMJYOZ-WDSKDSINSA-N 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- NKLRYVLERDYDBI-FXQIFTODSA-N Glu-Glu-Asp Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O NKLRYVLERDYDBI-FXQIFTODSA-N 0.000 description 1
- SWQALSGKVLYKDT-ZKWXMUAHSA-N Gly-Ile-Ala Chemical compound NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(O)=O SWQALSGKVLYKDT-ZKWXMUAHSA-N 0.000 description 1
- SWQALSGKVLYKDT-UHFFFAOYSA-N Gly-Ile-Ala Natural products NCC(=O)NC(C(C)CC)C(=O)NC(C)C(O)=O SWQALSGKVLYKDT-UHFFFAOYSA-N 0.000 description 1
- NHHZWPNMYQUNEH-ACRUOGEOSA-N Phe-Tyr-His Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)N[C@@H](CC3=CN=CN3)C(=O)O)N NHHZWPNMYQUNEH-ACRUOGEOSA-N 0.000 description 1
- IMMPMHKLUUZKAZ-WMZOPIPTSA-N Trp-Phe Chemical compound C([C@H](NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)N)C(O)=O)C1=CC=CC=C1 IMMPMHKLUUZKAZ-WMZOPIPTSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 230000009435 amidation Effects 0.000 description 1
- 238000007112 amidation reaction Methods 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000002981 blocking agent Substances 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000000536 complexating effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 239000013024 dilution buffer Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 239000004816 latex Substances 0.000 description 1
- 229920000126 latex Polymers 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 238000006011 modification reaction Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000003335 steric effect Effects 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000004879 turbidimetry Methods 0.000 description 1
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Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Abstract
The invention provides an application of cyclic peptide in preparing chemiluminescent marker, wherein the cyclic peptide has the structure of PG- (AA 1) -ring (Lys- (AA 2) - (AA 3)) - (AA 4); wherein AA3 is selected from Asp or Glu; AA1 is an amino acid, dipeptide or polypeptide comprising from 1 to 4 hydrophilic amino acids; AA2 is a polypeptide comprising 3 to 10 hydrophobic amino acids; AA4 is an amino acid, dipeptide or polypeptide comprising from 1 to 5 hydrophobic amino acids; PG is a reactive group containing one or more reactive sites. The cyclic peptide can be applied to the preparation of chemiluminescent markers, can reduce the background value in the detection process, finally reduces the occurrence of false positive results, and can also ensure that the detection results have higher sensitivity and specificity.
Description
Technical Field
The invention relates to the field of immunoassay, in particular to a novel application of cyclic peptide, an acridine labeling complex, a preparation method and a detection kit.
Background
Currently widely used immunological detection techniques include enzyme-linked immunosorbent assay, latex turbidimetry, time-resolved fluoroimmunoassay, chemiluminescent immunoassay, and the like. Among them, chemiluminescent immunoassay has been developed more and more recently due to its advantages of high sensitivity, wide linear range, rapid and simple detection, stable results, etc.
There are a variety of chemiluminescent systems, most notably HRP-luminol luminescent systems, acridinium ester luminescent systems and electrochemiluminescent systems. The acridinium ester is often used as a marker for immunoassay, the marking process is simple and quick, no catalyst is needed, the stability is good, and the reagent period is long. Meanwhile, the acridinium ester emits light in a flash manner, the light is concentrated and strong, and the sensitivity and the precision of the detection result are high.
However, in chemiluminescent immunoassay, there is a case where a negative sample is detected with a high luminescence value and is determined as a positive sample. The main reason for causing false positive is that the interfering substance exists in the sample, and the interfering substance can be combined with an antibody or an antigen in a detection system, so that a luminescent signal is increased to generate false positive. Common interferents are mainly immune-interfering substances such as Heterotrophic Antibodies (HA), rheumatoid Factors (RF), and human anti-animal antibodies (HAAA).
The traditional method is to add a blocking agent to block the binding of an interfering substance and an antibody or antigen in a detection system, so as to achieve the aim of eliminating false positive. However, in some cases, even if these immune blockers are added, the blocking effect is not obvious, mainly because the interfering substances can generate nonspecific interference by factors such as chargeability, hydrophobicity, polarity, metal complexation, and the like, in addition to immune interference. The nonspecific interference is affected by factors such as chargeability, hydrophobicity, polarity, complexing functional groups and the like of magnetic beads, antibodies or antigens in the detection system, so that even if the detection item is the same, the probability of occurrence of false positive is different due to the fact that different magnetic beads, antibodies or antigens are selected.
Also polypeptides (Lys) for conventional methods m —(PEG) n R.ident.m.ltoreq.12, n.ltoreq.4.ltoreq.12, the luminescence signal is increased and the detection sensitivity is improved, but the hydrophilicity of the modified antibody or antigen is enhanced, so that the nonspecific adsorption is very serious. If the interference in the sample is strong in hydrophilicity, false positive phenomenon is easy to occur.
Disclosure of Invention
Based on the above, the invention provides a new application of the cyclic peptide, in particular to an application of the cyclic peptide in preparing a chemiluminescent marker.
The invention is realized by the following technical scheme.
Use of a cyclic peptide having the structure PG- (AA 1) -ring (Lys- (AA 2) - (AA 3)) - (AA 4) for the preparation of a chemiluminescent label;
wherein AA3 is selected from Asp or Glu;
AA1 is an amino acid, dipeptide or polypeptide comprising from 1 to 4 hydrophilic amino acids;
AA2 is a polypeptide comprising 3 to 10 hydrophobic amino acids;
AA4 is an amino acid, dipeptide or polypeptide comprising from 1 to 5 hydrophobic amino acids;
PG is a reactive group containing one or more reactive sites.
In one embodiment, in the AA1, the hydrophilic amino acid is independently selected from Asp, glu, lys or Arg for each occurrence.
In one embodiment, in the AA2, the hydrophobic amino acid is independently selected from Gly, ala, pro, leu, ile, val, phe, trp, his or Tyr for each occurrence.
In one embodiment, in the AA4, the hydrophobic amino acid is independently selected from Gly, ala, pro, leu, ile, val, phe, trp, his or Tyr for each occurrence.
In one embodiment, the reactive sites are independently selected from amino, sulfhydryl, maleimide, alkynyl, phosphino, or azide groups.
In one embodiment, the chemiluminescent label is an acridine-labeled complex.
The invention also provides an acridine-labeled complex, which comprises a body, an activated fragment connected to the body, the cyclic peptide connected to the activated fragment, and an acridine ester compound labeled on the body or simultaneously labeled on the body and the cyclic peptide.
In one embodiment, the body is an antigen, hapten or antibody.
In one embodiment, the acridine ester compound is selected from NSP-DMAE-NHS, NSP-SA-NHS or NSP-DMAE-HEG-NHS.
The invention also provides a preparation method of the acridine marker complex, which comprises the following steps:
activating the body to produce an activated fragment-body complex;
coupling the activated fragment-bulk complex with the cyclic peptide to prepare a cyclic peptide-activated fragment-bulk complex;
labeling acridinium ester compounds on the cyclopeptide-activated fragment-bulk complex; or (b)
Labeling an acridine ester compound on the body to prepare a body-acridine ester compound complex;
activating the bulk-acridine ester compound complex to prepare an activated fragment-bulk-acridine ester compound complex;
and (3) coupling the activated fragment-bulk-acridine ester compound complex with the cyclic peptide.
The invention also provides a detection kit comprising the acridine-labeled complex as described above.
Compared with the prior art, the application of the cyclic peptide in preparing the chemiluminescent marker has the following beneficial effects:
the inventor finds that carboxyl in Asp or Glu and amino of Lys react to form a ring, so that the prepared chemiluminescent label has a shorter peptide chain, the aim of reducing the shielding effect of polypeptide steric effect on an antibody or antigen active site can be fulfilled, and the activity of the antibody or antigen is not influenced. Meanwhile, the hydrophobic amino acid introduced into the ring and the other end of Asp or Glu can ensure that the hydrophobicity of the modified antibody or antigen is enhanced, thereby reducing the nonspecific adsorption in a chemiluminescent immunoassay system. In addition, the other end of Lys is introduced with hydrophilic amino acid, which can increase the solubility of cyclic peptide in water solution, so that the subsequent modification reaction can be carried out in water.
Furthermore, the cyclic peptide can be applied to the preparation of chemiluminescent markers, can reduce the background value in the detection process, finally reduces the occurrence of false positive results, and can also ensure that the detection results have higher sensitivity and specificity.
Detailed Description
In order that the invention may be readily understood, a more particular description of the invention will be rendered by reference to specific embodiments that are illustrated in the appended drawings. Preferred embodiments of the present invention are shown in the examples. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Furthermore, the terms "first," "second," and the like, are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include at least one such feature. In the description of the invention, the meaning of "plurality" means at least two, for example, two, three, etc., unless specifically defined otherwise. In the description of the present invention, the meaning of "several" means at least one, such as one, two, etc., unless specifically defined otherwise.
The words "preferably," "more preferably," and the like in the present invention refer to embodiments of the invention that may provide certain benefits in some instances. However, other embodiments may be preferred under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, nor is it intended to exclude other embodiments from the scope of the invention.
When a range of values is disclosed herein, the range is considered to be continuous and includes both the minimum and maximum values for the range, as well as each value between such minimum and maximum values. Further, when a range refers to an integer, each integer between the minimum and maximum values of the range is included. Further, when multiple range description features or characteristics are provided, the ranges may be combined. In other words, unless otherwise indicated, all ranges disclosed herein are to be understood to include any and all subranges subsumed therein.
All percentages, fractions and ratios are calculated on the total mass of the composition of the invention, unless otherwise indicated. All of the mass of the ingredients listed, unless otherwise indicated, are given to the active substance content and therefore they do not include solvents or by-products that may be included in commercially available materials. The term "mass percent" herein may be represented by the symbol "%". All molecular weights herein are weight average molecular weights expressed in daltons, unless indicated otherwise. All formulations and tests herein take place in an environment of 25 ℃, unless otherwise indicated. The terms "comprising," "including," "containing," "having," or other variations thereof herein are intended to cover a non-closed inclusion, without distinguishing between them. The term "comprising" means that other steps and ingredients may be added that do not affect the end result. The compositions and methods/processes of the present invention comprise, consist of, and consist essentially of the essential elements and limitations described herein, as well as additional or optional ingredients, components, steps, or limitations of any of the embodiments described herein. The terms "efficacy," "performance," "effect," "efficacy" are not differentiated herein.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
The invention provides an application of cyclic peptide in preparing chemiluminescent marker, the structure of cyclic peptide is PG- (AA 1) -ring (Lys- (AA 2) - (AA 3)) - (AA 4);
wherein AA3 is selected from Asp or Glu;
AA1 is an amino acid, dipeptide or polypeptide comprising from 1 to 4 hydrophilic amino acids;
AA2 is a polypeptide comprising 3 to 10 hydrophobic amino acids;
AA4 is an amino acid, dipeptide or polypeptide comprising from 1 to 5 hydrophobic amino acids;
PG is a reactive group containing one or more reactive sites.
In a specific example, the carboxyl group in AA3 and the amino group of the Lys side chain undergo amidation to form a ring.
In a specific example, in AA1, each occurrence of a hydrophilic amino acid is independently selected from Asp, glu, lys or Arg.
In a specific example, AA1 is selected from any one of the following peptide chains:
Glu-Glu-Asp, glu-Lys-Arg-Lys or Asp-Arg.
In a specific example, in AA2, each occurrence of a hydrophobic amino acid is independently selected from Gly, ala, pro, leu, ile, val, phe, trp, his or Tyr.
In a specific example, AA2 is selected from any one of the following peptide chains:
Ala-Phe-Tyr-Trp-Leu-Pro-Trp-Phe、
Val-Gly-Ala-Pro-Leu-Val or
Trp-His-Tyr-His-Phe。
In a specific example, in AA4, each occurrence of a hydrophobic amino acid is independently selected from Gly, ala, pro, leu, ile, val, phe, trp, his or Tyr.
In a specific example, AA4 is selected from any one of the following peptide chains:
Gly-Ile-Ala, phe-Tyr-His or Trp-Phe.
In a specific example, the reactive sites are independently selected from amino, sulfhydryl, maleimide, alkynyl, phosphino, or azide groups.
In a specific example, PG is selected from Cys or any one of the following groups:
in one specific example, the chemiluminescent label is an acridine-labeled complex.
In a specific example, the cyclic peptide is selected from any one of the following polypeptides:
Cys-Glu-Glu-Asp-cyclo (Lys-Ala-Phe-Tyr-Trp-Leu-Pro-Trp-Phe-Asp) -Gly-Ile-Ala,
6-Maleimidohexanoic acid-Glu-Lys-Arg-Lys-cyclo (Lys-Val-Gly-Ala-Pro-Leu-Val-Glu) -Phe-Tyr-His or
Alkyne-PEG 6-Asp-Arg-cyclo (Lys-Trp-His-Tyr-His-Phe-Glu) -Trp-Phe.
The invention also provides an acridine labeling compound, which comprises a body, an activation fragment connected to the body, the cyclic peptide connected to the activation fragment, and an acridine ester compound marked on the body or marked on the body and the cyclic peptide at the same time.
In a specific example, the entity is an antigen, hapten or antibody.
In a specific example, the acridine ester compound is selected from NSP-DMAE-NHS, NSP-SA-NHS or NSP-DMAE-HEG-NHS.
The invention also provides a preparation method of the acridine marker compound, which comprises the following steps:
activating the body to prepare an activated fragment-body complex;
coupling the activated fragment-body complex with a cyclic peptide to prepare the cyclic peptide-activated fragment-body complex;
labeling acridinium ester compounds on the cyclopeptide-activated fragment-bulk complex; or (b)
Labeling an acridine ester compound on a body to prepare a body-acridine ester compound complex;
activating the bulk-acridine ester compound complex to prepare an activated fragment-bulk-acridine ester compound complex;
and (3) coupling the activated fragment-bulk-acridine ester compound complex with a cyclic peptide.
The invention also provides a detection kit comprising the acridine-labeled complex.
In a specific example, the detection kit further comprises coated capture antibodies, and magnetic beads of antigen; wherein, the coated capture antibody can form a sandwich structure of the coated capture antibody-substance to be detected-acridine labeled complex with the substance to be detected and the acridine labeled complex in an immunological combination mode.
In a specific example, the test kit further comprises one or more of a calibrator, a dilution buffer, a sample diluent, a protein protectant, and a luminescent substrate.
The novel application of the cyclic peptide, the acridine labeling complex, the preparation method and the detection kit of the invention are described in further detail below by referring to specific examples. The raw materials used in the following examples are all commercially available products unless otherwise specified.
The cyclic peptides provided in the examples were all purchased from the company Shanghai, inc. of Biotechnology.
Example 1
The embodiment provides an acridine labeling compound containing cyclic peptide and a preparation method thereof, and the acridine labeling compound comprises the following specific steps:
1. cyclic peptides
The cyclic peptides provided in this example are (seq_1 to seq_2):
Cys-Glu-Glu-Asp-cyclo (Lys-Ala-Phe-Tyr-Trp-Leu-Pro-Trp-Phe-Asp) -Gly-Ile-Ala; wherein Cys is a reactive group;
the structural formula is as follows:
2. acridine labeling complex and preparation method thereof
In this embodiment, the S100 protein detection item is taken as a body target, and the specific steps are as follows:
1. detection of antibody-labeled acridine
(1) 1mg of S100 protein detection antibody is taken, PBS solution (50 mM, pH 7.4) is added, and the concentration is 1mg/mL;
(2) Acridine ester NSP-DMAE-NHS powder is dissolved in DMSO to prepare 5mg/mL acridine ester solution;
(3) Adding 30uL of acridinium ester solution into the S100 protein detection antibody solution, and reacting for 3 hours at 25 ℃;
(4) Adding 100uL of glycine PBS solution with the concentration of 10g/L, and performing quenching reaction;
(5) Purification was performed using a 10kDa ultrafiltration tube to remove the free acridinium ester.
2. Activated detection antibody acridine marker
(1) The detection antibody acridine marker is added into PBS solution (50 mM, pH 7.4) with the concentration of 1mg/mL;
(2) SM (PEG) is added 4 20uL of DMSO solution (10 mg/mL), at 25℃for 2h;
(3) Adding 100uL of glycine PBS solution with the concentration of 10g/L, and performing quenching reaction;
(4) Purification was performed using a 10kDa ultrafiltration tube to remove the free acridinium ester.
3. Cyclic peptide modified detection antibody acridine marker
(1) The activated detection antibody acridine marker is added into PBS solution (50 mM, pH 7.4) with the concentration of 10mg/mL;
(2) Dissolving the cyclic peptide in 15% DMF solution with concentration of 2mg/mL;
(3) 100uL of cyclopeptide solution is taken and added to the activated detection antibody acridine marker, and the reaction is carried out for 6 hours at 25 ℃.
(4) Purification was performed using a 10kDa ultrafiltration tube to remove unreacted cyclic peptide.
(5) The cyclopeptide-modified acridine label is stored after dilution with a preservative solution.
Example 2
The embodiment provides an acridine labeling compound containing cyclic peptide and a preparation method thereof, and the acridine labeling compound comprises the following specific steps:
1. cyclic peptides
The cyclic peptide provided in this example is (seq_3): 6-maleimidocaprooic acid-Glu-Lys-Arg-Lys-cyclo (Lys-Val-Gly-Ala-Pro-Leu-Val-Glu) -Phe-Tyr-His; wherein 6-maleimidoca acid is a reactive group;
the structural formula is as follows:
the cyclic peptide derivatives provided in this example are: 6-Maleimidohexanoic acid-Glu-Lys-Arg-Lys-cyclo (Lys-Val-Gly-Ala-Pro-Leu-Val-Glu) -Phe-Tyr-His-NH 2 。
2. Acridine labeling complex and preparation method thereof
In this embodiment, PIVKA-II detection items are taken as body targets, and the specific steps are as follows:
1. activated detection antibodies
(1) 1mg PIVKA-II detection antibody is taken, PBS solution (100 mM, pH 8.0) is added, and the concentration is 2mg/mL;
(2) Traut's Reagent is dissolved in water to prepare a solution of 10mg/mL;
(3) Adding 10uL of Traut's Reagent solution into the PIVKA-II detection antibody solution, and reacting for 1.5 hours at 25 ℃;
(4) Purification was performed using a 30kDa ultrafiltration tube to remove free Traut's Reagent.
2. Cyclic peptide modified detection antibody
(1) The activated detection antibody is added into PBS solution (100 mM, pH 7.2) with the concentration of 5mg/mL;
(2) Dissolving the cyclic peptide derivative in 20% DMF solution with the concentration of 2mg/mL;
(3) Adding 100uL of cyclopeptide solution into the activated detection antibody solution, and reacting for 2 hours at 25 ℃;
(4) Purification was performed using a 10kDa ultrafiltration tube to remove the free acridinium ester.
3. Cyclic peptide modified detection antibody labeled acridine
(1) The cyclopeptide modified detection antibody was added to PBS (50 mM, pH 7.4) at a concentration of 1mg/mL;
(2) Acridine ester NSP-SA-NHS powder is dissolved in DMSO to prepare 5mg/mL acridine ester solution;
(3) Adding 30uL of acridinium ester solution into the cyclopeptide modified detection antibody solution, and reacting for 2 hours at 25 ℃;
(4) Adding 100uL of glycine PBS solution with the concentration of 10g/L, and performing quenching reaction;
(5) Purification was performed using a 10kDa ultrafiltration tube to remove free acridinium ester;
(6) The cyclopeptide-modified acridine label is stored after dilution with a preservative solution.
Example 3
The embodiment provides an acridine labeling compound containing cyclic peptide and a preparation method thereof, and the acridine labeling compound comprises the following specific steps:
1. cyclic peptides
The cyclic peptide provided in this example is (seq_4):
Alkyne-PEG 6-Asp-Arg-cyclo (Lys-Trp-His-Tyr-His-Phe-Glu) -Trp-Phe; wherein Alkyne-PEG6 is a reactive group;
the structural formula is as follows:
2. acridine labeling complex and preparation method thereof
In this embodiment, the FDP detection item is taken as an ontology target, and the specific steps are as follows:
1. detection of antibody-labeled acridine
(1) FDP detection antibody is taken, PBS solution (50 mM, pH 7.4) is added, and the concentration is 1mg/mL;
(2) Acridine ester NSP-DMAE-HEG-NHS powder is dissolved in DMSO to prepare 5mg/mL acridine ester solution;
(3) Adding 20uL of acridinium ester solution into the FDP detection antibody solution, and reacting for 3 hours at 25 ℃;
(4) Adding 100uL of glycine PBS solution with the concentration of 10g/L, and performing quenching reaction;
(5) Purification was performed using a 10kDa ultrafiltration tube to remove the free acridinium ester.
2. Activated detection antibody acridine marker
(1) The detection antibody acridine marker is added into PBS solution (50 mM, pH 7.4) with the concentration of 1mg/mL;
(2) NHS-PEG4-Azide DMSO solution (10 mg/mL) was added to the mixture and the mixture was reacted at 25℃for 2 hours;
(3) Adding 100uL of glycine PBS solution with the concentration of 10g/L, and performing quenching reaction;
(4) Purification was performed using a 10kDa ultrafiltration tube to remove free NHS-PEG4-Azide.
3. Cyclic peptide modified detection antibody acridine marker
(1) The activated detection antibody acridine marker is added into PBS solution (150 mM, pH 7.4) with the concentration of 10mg/mL;
(2) Dissolving the cyclic peptide in 10% DMF solution with the concentration of 4mg/mL;
(3) Adding 500uL of cyclopeptide solution into the activated detection antibody acridine marker, adding copper sulfate solution to make the final concentration of copper sulfate be 0.5mM, and reacting for 1h at 37 ℃;
(4) Purification was performed using a 10kDa ultrafiltration tube;
(5) The cyclopeptide-modified acridine label is stored after dilution with a preservative solution.
Comparative example 1
The procedure for preparing the acridine marker of the detection antibody provided in this comparative example was almost the same as in example 1, except that the step of activating the acridine marker of the detection antibody and the step of modifying the polypeptide were omitted in comparative example 1, and the final concentration of the marker in the preservation solution was the same as in example 1.
That is, comparative example 1 provides a detection antibody with no polypeptide modification in the acridine label, relative to example 1.
Comparative example 2
The procedure for the preparation of the acridine marker of the detection antibody provided in this comparative example was almost identical to that of example 1, except that the cyclic peptide sequence used in comparative example 2 was Cys-Glu-Glu-Asp-ring (Lys-Lys-Lys-Lys-Lys-Lys-Lys-Asp) -Gly-Ile-Ala, and the final concentration of the marker in the preservation solution was the same as in example 1.
That is, the sequence of AA2 of the cyclic peptide provided in comparative example 2 was changed to:
Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys。
comparative example 3
The procedure for preparing the acridine marker of the detection antibody provided in this comparative example was almost the same as in example 2, except that the step of activating the detection antibody and the step of modifying the polypeptide were omitted in comparative example 3, and the final concentration of the marker in the preservation solution was the same as in example 2.
That is, comparative example 3 provides a detection antibody with no polypeptide modification in the acridine label, relative to example 2.
Comparative example 4
The procedure for the preparation of the acridine marker of the detection antibody provided in this comparative example was almost identical to that of example 2, except that the cyclic peptide sequence used in comparative example 4 was 6-maleimidocaprooic acid-Glu-Lys-Arg-Lys-cyclo (Lys-Arg-Arg-Arg-Arg-Arg-Arg-Glu) -Phe-Tyr-His, and the final concentration of the marker in the preservation solution was the same as in example 2.
That is, relative to example 2, the sequence of AA2 of the cyclic peptide provided in comparative example 4 was changed to:
Arg-Arg-Arg-Arg-Arg-Arg。
comparative example 5
The procedure for preparing the acridine marker of the detection antibody provided in this comparative example was almost the same as in example 3, except that the step of activating the acridine marker of the detection antibody and the step of modifying the polypeptide were omitted in comparative example 5, and the final concentration of the marker in the preservation solution was the same as in example 3.
That is, the detection antibody provided in comparative example 5 has no polypeptide modification in the acridine label, relative to example 3.
Comparative example 6
The procedure for the preparation of the acridine marker of the detection antibody provided in this comparative example was almost identical to that of example 3, except that the cyclic peptide sequence used in comparative example 6 was Alkyne-PEG 6-Asp-Arg-cyclo (Lys-Glu-Arg-Asp-Lys-Asp-Glu) -Trp-Phe, and the final concentration of the marker in the preservation solution was the same as in example 3.
That is, the sequence of AA2 of the cyclic peptide provided in comparative example 6 was changed to:
Glu-Arg-Asp-Lys-Asp。
effect verification test
Performance test 1
The acridinium ester markers obtained in example 1, comparative example 1 and comparative example 2 were applied to chemiluminescent immunoassay detection of S100 protein respectively, and a semi-finished product of the S100 protein chemiluminescent kit was tested by using an iStar500 fully automated chemiluminescent determinator (Zuo Biotechnology Co., shenzhen Co., ltd.). The method comprises the following specific steps: taking different samples of 50uL, respectively adding 50uL of magnetic bead working solution coated with capture antibody and 50uL of working solution prepared in example 1, comparative example 1 and comparative example 2, reacting at 37 ℃ for 10min, magnetically separating and cleaning for 3 times, respectively adding 100uL of HNO into each component 3 -H 2 O 2 The luminescence values were measured with an iStar500 full-automatic chemiluminescence analyzer using 100uL NaOH solution and 3 measurements in parallel, taking the average. The sample contains calibrator, negative sample and positive sampleAnd a sample of false positive. The false positive samples are 5 samples with very high luminous values screened from 2000 negative serum, and are confirmed to be negative by the Rogowski kit test. The test results are shown in Table 1.
TABLE 1
As can be seen from Table 1, the use of the polypeptide modified acridinium ester markers of example 1 reduced the luminescence value of negative serum, while the signal of medium and high value calibrator and positive samples did not change much with comparative example 2 without polypeptide modification. The luminescence of negative serum measured using the polypeptide-modified acridine marker of comparative example 2 was higher than that measured in comparative example 1, which was not modified. For the 5-example pseudo-positive samples, the test luminescence values of comparative example 1 and comparative example 2 are high, positive results are shown, and the test is carried out by using the polypeptide modified antibody acridine marker of example 1 instead, the luminescence values are low, and the test results are consistent with Rogowski.
Performance detection II
The acridinium ester markers obtained in example 2, comparative example 3 and comparative example 4 were applied to chemiluminescent immunoassay detection of PIVKA-II, respectively, and the semi-finished products of the PIVKA-II chemiluminescent kit were tested by using an iStar500 fully automated chemiluminescent determinator (Zuo Biotechnology Co., shenzhen Co., ltd.). The method comprises the following specific steps: taking different samples of 10uL, respectively adding 50uL of magnetic bead working solution coated with capture antibody, reacting at 37 ℃ for 5min, adding 50uL of working solution prepared in example 2, comparative example 3 and comparative example 4, reacting at 37 ℃ for 5min, magnetically separating and cleaning for 3 times, respectively adding 100uLHNO into each component 3 -H 2 O 2 The luminescence values were measured with an iStar500 full-automatic chemiluminescence analyzer using 100uL NaOH solution and 3 measurements in parallel, taking the average. The sample contains calibrator, negative sample positiveSex specimens and pseudo-positive specimens. The false positive samples are 5 samples with very high luminous values screened from 2000 negative serum, and are confirmed to be negative through the test of the yaban kit. The test results are shown in Table 2.
TABLE 2
As can be seen from Table 2, the use of the polypeptide modified acridinium ester markers of example 2 reduced the luminescence value of negative serum, while the signal of medium and high value calibrator and positive samples and comparative example 3 without polypeptide modification increased slightly. The luminescence of negative serum measured with the polypeptide-modified acridine marker of comparative example 4 was higher than that measured with comparative example 3, which was not modified. For the 5-example pseudo-positive samples, the test luminescence values of comparative example 3 and comparative example 4 are high, positive results are shown, and the test is carried out by using the polypeptide modified antibody acridine marker of example 2 instead, the luminescence values are low, and the test results are consistent with those of the yapei samples.
Performance test three
The acridinium ester markers obtained in example 3, comparative example 5 and comparative example 6 were applied to chemiluminescent immunoassay detection of FDP, respectively, and were tested using an iStar500 fully automated chemiluminescent assay (Zuo Biotechnology Co., shenzhen Co., ltd.) using an FDP chemiluminescent kit semi-finished product. The method comprises the following specific steps: taking different samples of 10uL, respectively adding 50uL of magnetic bead working solution coated with capture antibody and 50uL of working solution prepared in example 3, comparative example 5 and comparative example 6, reacting at 37 ℃ for 10min, magnetically separating and cleaning for 3 times, respectively adding 100uL of HNO into each component 3 -H 2 O 2 The luminescence values were measured with an iStar500 full-automatic chemiluminescence analyzer using 100uL NaOH solution and 3 measurements in parallel, taking the average. The samples include calibrator, negative sample positive sample and false positive sample. The false positive samples are 4 samples with very high luminous values screened from 1000 negative serum, and are confirmed to be negative by the test of the Hissen-Meikang kit. The test results are shown in Table 3.
TABLE 3 Table 3
As can be seen from Table 3, the use of the polypeptide modified acridinium ester markers of example 3 reduced the luminescence value of negative serum, while the signal of medium and high value calibrator and positive samples and comparative example 5 without polypeptide modification were slightly reduced. The luminescence of negative serum measured with the polypeptide-modified acridine marker of comparative example 6 was higher than that measured with comparative example 5, which was not modified. For the 4-example pseudo-positive samples, the test luminescence values of comparative example 5 and comparative example 6 are high, positive results are shown, and the test is carried out by using the polypeptide modified antibody acridine marker of example 3 instead, the luminescence values are low, and the test results are consistent with the test results of the Hissezian.
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples merely represent a few embodiments of the present invention, which facilitate a specific and detailed understanding of the technical solutions of the present invention, but are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. It should be understood that, based on the technical solutions provided by the present invention, those skilled in the art may obtain technical solutions through logical analysis, reasoning or limited experiments, which are all within the scope of protection of the appended claims. The scope of the patent of the invention should therefore be determined with reference to the appended claims, which are to be construed as in accordance with the doctrines of claim interpretation.
Claims (7)
1. An acridine-labeled complex, comprising a body, an activating fragment linked to the body, a cyclic peptide linked to the activating fragment, and an acridine ester compound labeled on the body or on both the body and the cyclic peptide;
the preparation method of the acridine labeling complex comprises the following steps:
activating the body to produce an activated fragment-body complex;
coupling the activated fragment-bulk complex with the cyclic peptide to prepare a cyclic peptide-activated fragment-bulk complex;
labeling acridinium ester compounds on the cyclopeptide-activated fragment-bulk complex; or (b)
Labeling an acridine ester compound on the body to prepare a body-acridine ester compound complex;
activating the bulk-acridine ester compound complex to prepare an activated fragment-bulk-acridine ester compound complex;
coupling the activated fragment-bulk-acridine ester compound complex with the cyclic peptide;
wherein,
the cyclic peptide has the structure of PG- (AA 1) -ring (Lys- (AA 2) - (AA 3)) - (AA 4),
AA3 is selected from Asp or Glu,
AA1 is an amino acid, dipeptide or polypeptide comprising from 1 to 4 hydrophilic amino acids,
AA2 is a polypeptide comprising 3 to 10 hydrophobic amino acids,
AA4 is an amino acid, dipeptide or polypeptide comprising from 1 to 5 hydrophobic amino acids,
PG is a reactive group containing one or more reactive sites;
the body is an antigen, hapten or antibody.
2. The acridine-labeled complex according to claim 1, wherein in AA1, the hydrophilic amino acid is independently selected from Asp, glu, lys or Arg for each occurrence.
3. The acridine-labeled complex according to claim 1, wherein the hydrophobic amino acid in AA2 is independently selected from Gly, ala, pro, leu, ile, val, phe, trp, his or Tyr for each occurrence.
4. The acridine-labeled complex according to claim 1, wherein the hydrophobic amino acid in AA4 is independently selected from Gly, ala, pro, leu, ile, val, phe, trp, his or Tyr for each occurrence.
5. The acridine-labeled complex according to claim 1, wherein the reaction sites are independently selected from amino, thiol, maleimide, alkynyl, phosphino, or azide groups.
6. The acridine-labeled complex according to claim 1, wherein the acridine ester compound is selected from NSP-DMAE-NHS, NSP-SA-NHS or NSP-DMAE-HEG-NHS.
7. A test kit comprising the acridine-labeled complex according to any one of claims 1 to 6.
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