CN112147335A

CN112147335A - Labeled ligand composition based on click chemistry, kit and system

Info

Publication number: CN112147335A
Application number: CN201910577780.9A
Authority: CN
Inventors: 邝俊韬; 许城秀
Original assignee: Shenzhen Dymind Biotechnology Co Ltd
Current assignee: Shenzhen Dymind Biotechnology Co Ltd
Priority date: 2019-06-28
Filing date: 2019-06-28
Publication date: 2020-12-29

Abstract

The application relates to the technical field of chemistry and biomedicine, and particularly discloses a click chemistry-based labeled ligand composition, a kit and a system, wherein the click chemistry-based labeled ligand composition comprises: a ligand containing one of an azide group or an azide-reactive group; a signal protein containing the other of an azide group or an azide-reactive group; wherein the signal protein is linked to the ligand by a click chemistry reaction between the azide group and the azide-reactive group to form the labeled ligand. Through the mode, the problems of unstable marking, low marking efficiency, large difference between marking batches and high marking cost generally existing when two proteins are marked by adopting a different-double-function cross-linking agent in the prior art can be solved.

Description

Labeled ligand composition based on click chemistry, kit and system

Technical Field

The application relates to the technical field of chemistry and biomedicine, in particular to a labeled ligand composition based on click chemistry, a kit and a system.

Background

The exploration and discovery of high-sensitivity analysis and detection methods for important biomolecules such as proteins, nucleic acids, polypeptides and the like in organisms and in the life process are hot spots and difficulties in the research of the biomedical field. Biomarker technology is an indispensable research tool in this field. General biomarker methods can be classified into radioactive labeling, chromogenic labeling, enzyme labeling, fluorescent labeling, and the like. Biomarker technology is a challenging task that requires that such chemical reactions be performed efficiently and specifically under physiological conditions without side reactions with various active substances present in biological systems.

Disclosure of Invention

The technical problem mainly solved by the application is to provide a labeled ligand composition, a kit and a system based on click chemistry, and solve the problems of unstable labeling, low labeling efficiency, large labeling batch difference and high labeling cost generally existing when a heterobifunctional cross-linking agent is adopted to label two proteins in the prior art.

In order to solve the technical problem, the application adopts a technical scheme that: there is provided a click chemistry-based tagged ligand composition comprising: a ligand containing one of an azide group or an azide-reactive group; a signal protein containing the other of an azide group or an azide-reactive group; wherein the signal protein is linked to the ligand by a click chemistry reaction between the azide group and the azide-reactive group to form the labeled ligand.

Wherein the azide-reactive group comprises: alkyne which forms a triazazole ring with an azide group, or cycloalkene which forms a diazacyclo ring with an azide group.

Wherein the signal protein comprises at least one of alkaline phosphatase, horseradish peroxidase, phycoerythrin, phycocyanin, phycoerythrin or allophycocyanin.

Wherein the ligand is at least one of an antibody, an antigen, a hapten-carrier conjugate or avidin.

Wherein the antibody comprises at least one of an angiotensin I antibody, a NT-proBNP antibody, a troponin antibody, an alpha-fetoprotein antibody, a carcinoembryonic antigen antibody, an HIV antigen antibody, a hepatitis B surface antigen antibody, a thyroglobulin antibody, a troponin I or T antibody, or a myoglobin antibody; antigens include: at least one of angiotensin I antigen, NT-proBNP antigen, troponin antigen, alpha-fetoprotein antigen, carcinoembryonic antigen, HIV antigen, hepatitis B surface antigen, thyroglobulin antigen, troponin I or T antigen, or myoglobin antigen; the hapten-carrier conjugate comprises at least one of aldosterone, aldosterone hapten protein conjugate, HA hyaluronic acid, hyaluronic acid hapten protein conjugate, angiotensin I-carrier conjugate, angiotensin II-carrier conjugate or HABP hyaluronic acid binding protein.

In order to solve the technical problem, the application adopts a technical scheme that: there is provided a method for preparing a labeled ligand composition based on click chemistry, the method for preparing the labeled ligand composition as described above, the method comprising: providing a ligand and a signal protein; labeling the ligand with a first click chemistry reagent, wherein the first click chemistry reagent comprises one of an azide group or an azide-reactive group; labeling the signal protein with a second click chemistry reagent, wherein the second click chemistry reagent contains the other of an azide group or an azide-reactive group; and carrying out click chemical reaction on the labeled ligand and the labeled signal protein to connect the ligand with the signal protein to form the labeled ligand composition.

Wherein the first click chemistry reagent and the second click chemistry reagent comprise at least one of a DBCO reagent, a Tetrazine reagent, an Azide reagent, an Alkyne reagent, or a TCO reagent.

In order to solve the technical problem, the application adopts a technical scheme that: providing a kit comprising: a kit body; a first reagent holding position arranged on the kit body and used for holding at least one labeled ligand composition; and the second reagent holding position is used for holding at least one immunoassay composition, and the immunoassay composition is matched with the labeled ligand composition to obtain a detection complex.

Wherein the labeled ligand composition is a labeled antibody composition; the immunoassay composition includes a capture antibody composition and/or a labeled antigen.

In order to solve the technical problem, the application adopts a technical scheme that: an immunoassay system is provided, the immunoassay system comprising: the kit is the kit; and a sample analyzer which performs detection of the analyte by using the immunoassay composition and the labeled ligand composition in the kit and outputs a detection result.

In order to solve the technical problem, the application adopts a technical scheme that: providing a use of a labeled ligand composition as described above in an immunoassay; the labeled ligand composition is used for myocardial detection, liver fibrosis detection, hypertension detection, thyroid gland detection, cardiovascular detection, gonad detection, renal function detection, bone metabolism detection, glycometabolism detection, infectious disease detection, autoimmune disease detection, prenatal screening, drug detection, hepatic fibrosis detection, EB virus detection, inflammation monitoring or tumor detection.

The beneficial effect of this application is: in contrast to the state of the art, the present application provides a click chemistry-based tagged ligand composition comprising: a ligand and a signal protein, wherein the ligand contains one of an azide group or an azide reaction group; the signal protein contains the other of an azide group or an azide-reactive group; the signal protein is connected with the ligand through click chemical reaction between the azide groups and the azide reaction groups to obtain the ligand composition containing the labeling groups, and the labeling groups have the advantages of high stability, high reaction speed and high yield, and the number of the labeling groups can be adjusted and controlled. The method also realizes dual labeling of the ligand and the signal protein, can be used for subsequent immunofluorescence analysis, and solves the problems of unstable labeling, low labeling efficiency, large labeling batch difference and high labeling cost generally existing when a heterobifunctional cross-linking agent is used for labeling two proteins in the prior art.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts. Wherein:

FIG. 1 is a schematic flow chart diagram illustrating one embodiment of a method for preparing a tagged ligand composition based on click chemistry according to the present application;

FIG. 2 is a schematic structural diagram of one embodiment of a kit of the present application;

FIG. 3 is a graph showing the change of detection signal with reaction time;

FIG. 4 shows performance results of different groups of tests;

FIG. 5 is a bar graph of different sets of different lot detection signals;

FIGS. 6a-6d are graphs showing the ratio of retention of fluorescence signal as a function of placement conditions and time.

Detailed Description

The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

During the research and development process, the researchers in the application find that glutaraldehyde (homobifunctional cross-linking agent) and heterobifunctional cross-linking agent are generally used for marking in the prior art. Because two identical aldehyde group-targeted groups of glutaraldehyde are amino groups, single protein self-coupling is easily caused in the labeling process of two proteins, the self-coupling is not easy to control, and the labeling lot difference is easily caused. Most of the conventional heterobifunctional cross-linking agents involve the reaction of sulfydryl, the sulfydryl is unstable, proteins containing the sulfydryl are easy to self-link, and the coupling efficiency of the proteins with another protein is reduced.

In view of the foregoing, the present application provides a click chemistry-based labeled ligand composition comprising: a ligand and a signal protein, wherein the ligand contains one of an azide group or an azide reaction group; the signal protein contains the other of an azide group or an azide-reactive group; wherein the signal protein is linked to the ligand by a click chemistry reaction between the azide group and the azide-reactive group to form the labeled ligand.

In particular, click chemistry is through small cellsThe chemical synthesis of the color molecules can be completed quickly and reliably. The method belongs to one of orthogonal reactions, completes modularized framework connection through high-efficiency and high-selectivity chemical reactions, directly clicks modules to construct a novel combined chemical method of various novel compounds, and has the following characteristics: (1) the method accords with green chemistry, has easily obtained raw materials, high yield, good stereoselectivity, easy purification and environment-friendly by-products; (2) fast reaction, high throughput, modular synthesis, e.g., copper-free click chemistry-tetrazine and cyclooctene derivatives with reaction rate constants of about 210-^-1s^-1(PBS, 37 ℃ C.); (3) the reaction condition is mild, is not sensitive to water, temperature and oxygen, and can be carried out under physiological conditions; (4) the reaction module of the cell bioreactor hardly reacts with biomolecules and does not interfere normal physiological functions of cells.

The present embodiment may employ the following three types of click chemistry reactions:

1. copper-catalyzed bioorthogonal reaction (CuAAC), which is the most widely used bioorthogonal reaction, is also representative of click chemistry. The azide and the terminal alkyne keep stable under most chemical conditions, but can be efficiently and specifically converted into the 1, 3-substituted triazole under the catalysis of monovalent copper.

2. The cyclic addition reaction (SPAAC) induced by ring tension is also called copper-free click chemistry, i.e. under the condition of no copper, the reaction is fast, efficient and selective according to the ring expansion tension of an eight-membered ring containing alkynyl functional groups and an azidation reagent.

3. The reaction system is further characterized in that the tetrazine quenches the fluorescence of the dye if modified on the dye, and the tetrazine reacts with cyclic olefin (such as trans-cyclooctene) through IEDDA reaction, the structure of the tetrazine is destroyed, and the fluorescent property of the dye can be recovered, thereby playing the role of fluorescence enhancement.

In contrast to the state of the art, the present application provides a click chemistry-based tagged ligand composition comprising: a ligand and a signal protein, wherein the ligand contains one of an azide group or an azide reaction group; the signal protein contains the other of an azide group or an azide-reactive group; the signal protein is connected with the ligand through click chemical reaction between the azide groups and the azide reaction groups to obtain the ligand composition containing the labeling groups, and the labeling groups have the advantages of high stability, high reaction speed and high yield, and the number of the labeling groups can be adjusted and controlled. The method also realizes dual labeling of the ligand and the signal protein, can be used for subsequent immunofluorescence analysis, and solves the problems of unstable labeling, low labeling efficiency, large labeling batch difference and high labeling cost generally existing when a heterobifunctional cross-linking agent is used for labeling two proteins in the prior art.

Wherein, the azide-reactive group comprises: alkyne which forms a triazazole ring with an azide group, or cycloalkene which forms a diazacyclo ring with an azide group.

In principle any protein can be used as the signal protein of the present application, preferably the signal protein has the following properties: 1) the surface of the material has carboxyl (-COOH) and amino (-NH)₂) Sulfhydryl (-SH), and other proteins with chemically active functional groups, so as to couple with azide reactive groups or azide groups; 2) proteins with sufficient molecular capacity for conjugation; 3) in order to prevent the generation of non-specific binding, the signal protein should be selected from proteins that are not homologous to the subject; 4) should have sufficient stability and should be inexpensive and readily available. Thus, signal proteins include, but are not limited to: horseradish peroxidase, bovine serum albumin, hemocyanin, chicken egg albumin, bovine IgG, murine IgG, ovine IgG, rabbit IgG, alkaline phosphatase, acid phosphatase, glucose oxidase, beta-galactosidase, or the like.

Preferably, the signal protein includes at least one of alkaline phosphatase, horseradish peroxidase, phycoerythrin, phycocyanin, phycoerythrin or allophycocyanin.

In the present application, a ligand is a molecule that can be specifically recognized by a tag, such as an antibody, an antigen, avidin, or the like.

Preferably, the ligand may be an antibody; the antibody is at least one of a troponin antibody, an angiotensin I antibody, or a myoglobin antibody.

Preferably, the ligand is a hapten-carrier conjugate; the hapten-carrier conjugate is at least one of aldosterone, aldosterone hapten protein conjugate, HA hyaluronic acid, hyaluronic acid hapten protein conjugate, angiotensin I-carrier conjugate, angiotensin II-carrier conjugate or HABP hyaluronic acid binding protein.

The aldosterone and aldosterone hapten protein conjugates can comprise aldosterone-BSA, aldosterone-OVA, aldosterone-KLH, aldosterone-various animal IgG, and the like. The HA hyaluronic acid and hyaluronic acid hapten protein conjugate can include hyaluronic acid-BSA, hyaluronic acid-OVA, hyaluronic acid-KLH, hyaluronic acid IgG and the like. The angiotensin I-carrier conjugate may include angiotensin I-BSA, angiotensin I-OVA, angiotensin I-KLH, angiotensin I-various animal IgG, etc. The angiotensin II-carrier conjugate may include angiotensin II-BSA, angiotensin II-OVA, angiotensin II-KLH, angiotensin II-various animal IgG, and the like.

The application also provides a use of the click chemistry-based labeled ligand composition as described above in an immunoassay; the labeled ligand composition is used for myocardial detection, liver fibrosis detection, hypertension detection, thyroid gland detection, cardiovascular detection, gonad detection, renal function detection, bone metabolism detection, glycometabolism detection, infectious disease detection, autoimmune disease detection, prenatal screening, drug detection, hepatic fibrosis detection, EB virus detection, inflammation monitoring or tumor detection.

In order to solve the technical problem, the application adopts a technical scheme that: there is provided a method for preparing a labeled ligand composition based on click chemistry, for preparing the labeled ligand composition as described above, the method comprising the steps of:

s101: a ligand and a signal protein are provided.

Wherein the ligand may be an antibody; the antibody is at least one of a troponin antibody, an angiotensin I antibody, or a myoglobin antibody.

The ligand is a hapten-carrier conjugate; the hapten-carrier conjugate is at least one of aldosterone, aldosterone hapten protein conjugate, HA hyaluronic acid, hyaluronic acid hapten protein conjugate, angiotensin I-carrier conjugate, angiotensin II-carrier conjugate or HABP hyaluronic acid binding protein. The aldosterone and aldosterone hapten protein conjugates can comprise aldosterone-BSA, aldosterone-OVA, aldosterone-KLH, aldosterone-various animal IgG, and the like. The HA hyaluronic acid and hyaluronic acid hapten protein conjugate can include hyaluronic acid-BSA, hyaluronic acid-OVA, hyaluronic acid-KLH, hyaluronic acid IgG and the like. The angiotensin I-carrier conjugate may include angiotensin I-BSA, angiotensin I-OVA, angiotensin I-KLH, angiotensin I-various animal IgG, etc. The angiotensin II-carrier conjugate may include angiotensin II-BSA, angiotensin II-OVA, angiotensin II-KLH, angiotensin II-various animal IgG, and the like.

Signal proteins include, but are not limited to: horseradish peroxidase, bovine serum albumin, hemocyanin, chicken egg albumin, bovine IgG, murine IgG, ovine IgG, rabbit IgG, alkaline phosphatase, acid phosphatase, glucose oxidase, beta-galactosidase, or the like. Preferably, the signal protein includes at least one of alkaline phosphatase, horseradish peroxidase, phycoerythrin, phycocyanin, phycoerythrin or allophycocyanin.

S102: the ligand is labeled with a first click chemistry.

Wherein the first click chemistry reagent comprises one of an azide group or an azide-reactive group.

The first click chemistry reagent and the second click chemistry reagent include at least one of a DBCO reagent, a Tetrazine reagent, an Azide reagent, an Alkyne reagent, or a TCO reagent.

Specifically, the first click chemistry reagent can be a fluorescent tag containing a modification of a DBCO group, a Tetrazine group, an Azide group, an Alkyne group, or a TCO group.

Wherein, the DBCO group-modified fluorescent marker can be DBCO-C6-NHS Ester, DBCO-Sulfo-NHS Ester, DBCO-PEG4-NHS Ester, DBCO-PEG5-NHS Ester, DBCO-PEG13-NHS Ester and PC DBCO-NHS Ester which react with amino; DBCO-Amine, DBCO-PEG4-Amine, Sulfo DBCO-PEG4-Amine which react with carboxyl; DBCO-Maleimide, DBCO-PEG4-Maleimide reacted with thiol.

The fluorescent marker modified by the Tetrazine group can be Tetrazine-NHS Ester, Methetyl-NHS Ester, Tetrazine-Sulfo-NHS Ester, Tetrazine-PEG5-NHS Ester, Methetyl-PEG 4-NHS Ester, Methetyl-PEG 5-NHS Ester, Methetyl-PEG 4-Sulfo-NHS Ester and PC Methetyl-NHS Ester which react with amino; Methyletrazine-Amine, Tetrazine-Amine, Methyletrazine-Propylamine, Methyletrazine-PEG 4-Amine which reacts with carboxyl; Methyltrazine-PEG 4-Maleimide reacted with a thiol group.

The fluorescent marker modified by the Azide group can be PC Azido-NHS Ester reacted with amino; Azido-Propylamine reacted with carboxyl groups.

The fluorescent marker modified by the Alkyne group can be propagyl-NHS Ester and PC Alkyne-NHS Ester which react with amino; propagyl-Maleimide reacted with a thiol group.

The fluorescent marker modified by the TCO group can be TCO-NHS Ester, TCO-PEG4-NHS Ester and TCO-PEG12-NHS Ester which react with amino; TCO-PEG3-Amine reacted with carboxyl; TCO-PEG3-Maleimide reacted with thiol.

S103: the signal protein is labeled with a second click chemistry reagent.

Wherein the second click chemistry reagent comprises the other of an azide group or an azide-reactive group.

Specifically, the second click chemistry reagent may be a fluorescent label containing a modification of a DBCO group, a Tetrazine group, an Azide group, an Alkyne group, or a TCO group.

S104: and carrying out click chemical reaction on the labeled ligand and the labeled signal protein to connect the ligand with the signal protein to form the labeled ligand composition.

Specifically, when the labeled ligand and the labeled signal protein are subjected to click chemistry reaction, the molar ratio of the labeled ligand to the labeled signal protein is 1:1-1:3, such as 1:1, 1:2, or 1: 3. Buffers used for click chemistry reactions include, but are not limited to: PBS, Tris-HCl, BS, MES, HEPES, MOPS, acetate buffer, glycine buffer, citrate buffer, carbonate buffer, and the like.

The present application provides a kit 300, the kit 300 comprising: the reagent cartridge 300 includes a body, a first reagent holding site 310, and a second reagent holding site 320.

A first reagent holding site 310 is provided on the body of the kit 300 for holding at least one labeled ligand composition as previously described; the second reagent holding portion 320 is disposed on the body of the kit 300, and is used for holding at least one immunoassay composition, and the immunoassay composition is matched with the labeled ligand composition to obtain a detection complex.

The present application provides an immunoassay system comprising: the kit comprises a kit and a sample analyzer, wherein the kit is the kit; the sample analyzer performs detection of the analyte by using the immunodetection composition and the labeled ligand composition in the kit, and outputs a detection result.

Specifically, the immunoassay system comprises: a kit; and a sample analyzer for detecting different analytes by using the immunoassay composition and the detection antibody composition in the kit, and outputting the detection results.

In this embodiment, the detection antibody used in the sample analyzer is connected to the metal nanoparticles and the fluorescent marker, and the plasma resonance generated by the metal nanoparticles enhances the fluorescent signal emitted by the fluorescent marker, thereby effectively improving the detection sensitivity.

The immunofluorescence analyzer includes a flow cytometry analyzer, a microplate reader or a fluorescence microscope and the like which carry out qualitative and/or quantitative detection through fluorescence signals. The specific technical benefits and technical details of the kit have been explained in detail above and are therefore not described in detail herein.

The immunoassay system is used for myocardial detection, liver fibrosis detection, hypertension detection, thyroid gland detection, cardiovascular detection, gonad detection, renal function detection, bone metabolism detection, glycometabolism detection, infectious disease detection, autoimmune disease detection, prenatal screening, drug detection, hepatic fibrosis detection, EB virus detection, inflammation monitoring or tumor detection.

The technical scheme of the present application will be explained in detail by the following examples (taking Alpha Fetoprotein (AFP) (hereinafter referred to as AFP) detection as an example, wherein antibody b-AFP is selected as a ligand and has a molecular weight of 150kD, and RPE is selected as a signal protein and has a molecular weight of 250 kD).

Comparative example 1

Glutaraldehyde method labeled link b-AFP-RPE and performance detection thereof

1. Glutaraldehyde method for marking and connecting b-AFP-RPE

The experimental procedure was as follows:

a) a sufficient amount of 0.1M PBS buffer containing 2% glutaraldehyde was prepared for use.

b) 1mg (6.7. mu. mol) of b-AFP and 1.7mg (6.8. mu. mol) of RPE were added to 0.1mM PBS buffer containing 2% glutaraldehyde to give a final reaction volume of 1mL, and the reaction was carried out at 37 ℃ for 2 hours with slow shaking.

c) After the reaction, the reaction mixture was purified by an ultrafiltration tube to remove glutaraldehyde, and 1mL of 0.1M PBS solution was added to obtain b-AFP-RPE, which was stored at-20 ℃.

d) The above steps were repeated to prepare three batches of b-AFP-RPE.

2. b-AFP-RPE and performance testing thereof

The experimental procedure was as follows:

b) adding 100 μ L of prepared b-AFP-RPE (diluted 1000 times) for reaction for 5min, magnetic separating and cleaning, and detecting fluorescence signal.

c) The above test was performed on three batches of b-AFP-RPE.

Comparative example 2:

connection of b-AFP-RPE by SPDP + SMCC method and performance detection thereof

1. Labeling and connecting b-AFP-RPE by SPDP + SMCC method

The experimental procedure was as follows:

a) b-AFP-SPDP preparation: 1mg (6.7. mu. mol) of b-AFP antibody was collected, undiluted, added with 20mM SPDP (molar ratio of b-AFP: SPDP: 1:20), mixed well, and reacted at 37 ℃ for 30min in the absence of light. The product was purified using an ultrafiltration tube and the Ab concentration was adjusted to 5-10mg/mL using 1M MOPS (pH 8.0). Since the reaction of SPDP and SMCC requires a free thiol group, the product obtained above needs to be treated with DTT. A sufficient amount of 10mg/mL DTT solution was prepared from 1M MOPS (pH 8.0), 10. mu.L of 10mg/mL DTT was added to 100. mu.L of the previously obtained product, the mixture was pipetted and mixed well, and the mixture was treated with light at room temperature for 30 min. After the reaction, the mixture was purified by an ultrafiltration tube, and finally 0.1M PBS (pH 7.3) was added to adjust the concentration of b-AFP-SPDP to 10mg/mL for use.

b) RPE-SMCC preparation: 1.7mg (6.8. mu. mol) of the signal protein RPE was added to 100mM PBS (pH 7.3) and 20mM SMCC (the final reaction concentration of RPE was 10mg/mL, and the molar ratio of RPE: SMCC was 1:10), mixed well, protected from light, and left at 37 ℃ for 30 min. After the reaction was completed, purification was performed using an ultrafiltration tube, and the RPE-SMCC concentration was adjusted to 10mg/mL by adding 0.1M PBS (pH 7.3) to the final product.

c) b-AFP-RPE preparation: and (c) adding the liquid b-AFP-SPDP collected in the step (a) into the RPE-SMCC obtained in the step (b), uniformly mixing, centrifuging, and reacting at 37 ℃ overnight. The label after the reaction was finished was stored at-20 ℃.

d) The above steps were repeated to prepare three batches of b-AFP-RPE.

2. b-AFP-RPE and performance testing thereof

The experimental procedure was as follows:

a) taking a magnetic ball coated with the antibody a-AFP and AFP samples of 0, 10, 100, 1000 and 2000ng/mL for reaction for 5min, and carrying out magnetic separation and cleaning.

b) Adding 100 μ L of prepared b-AFP-RPE (diluted 5000 times) for reaction for 5min, magnetic separating and cleaning, and detecting fluorescence signal.

c) The above test was performed on three batches of b-AFP-RPE.

Example 1:

connection of b-AFP-RPE by CuAAC method and performance detection thereof

1. Labeling and connecting b-AFP-RPE by CuAAC method

The experimental procedure was as follows:

a) b-AFP-Azide preparation: taking 1mg (6.7 mu mol) of b-AFP antibody, adding 20mM Azide-NHS (the molar ratio of b-AFP: Azide-NHS is 1:20) (or other coupling methods can be adopted to mark Azide on the antibody), mixing uniformly, and reacting for 30min at 37 ℃ in a dark place. After the reaction, the mixture was purified by an ultrafiltration tube, and finally 0.1M PBS (pH 7.3) was added to adjust the concentration of b-AFP-Azide to 10mg/mL for use.

b) Preparation of RPE-Alkyne: taking 1.7mg (6.8 mu mol) of signal protein RPE, adding 20mM Alkyne-NHS (the molar ratio of RPE to Alkyne-NHS is 1:20) (or adopting other coupling modes to mark Alkyne on an antibody), mixing uniformly, and reacting for 30min at 37 ℃ in a dark place. After the reaction, the mixture was purified by an ultrafiltration tube, and finally 0.1M PBS (pH 7.3) was added to adjust the concentration of RPE-Alkyne to 10mg/mL for further use.

c) b-AFP-RPE preparation: and (c) adding the liquid b-AFP-Azide collected in the step (a) into the RPE-Alkyne obtained in the step (b), uniformly mixing, adding a copper catalyst, uniformly mixing, and reacting at 37 ℃ for 45 min. And (4) storing the marker after the reaction is finished at-20 ℃ for later use.

d) The above steps were repeated to prepare three batches of b-AFP-RPE.

2. b-AFP-RPE and performance testing thereof

The experimental procedure was as follows:

c) The above test was performed on three batches of b-AFP-RPE.

Example 2:

SPAAC method for connecting b-AFP-RPE and performance detection thereof

1. Labeling of the ligation b-AFP-RPE by the SPAAC method

The experimental procedure was as follows:

b) RPE-DBCO preparation: taking 1.7mg (6.8 mu mol) of signal protein RPE, adding 20mM DBCO-NHS (the molar ratio of RPE to DBCO-NHS is 1:20) (other coupling modes can be adopted to label DBCO on the antibody), mixing uniformly, and reacting for 30min at 37 ℃ in a dark place. After the reaction, the reaction mixture was purified by an ultrafiltration tube, and finally 0.1M PBS (pH 7.3) was added to adjust the concentration of RPE-DBCO to 10mg/mL for use.

c) b-AFP-RPE preparation: and (c) adding the liquid b-AFP-Azide collected in the step (a) into the RPE-DBCO obtained in the step (b), uniformly mixing, and reacting for 30min at 37 ℃. And (4) storing the marker after the reaction is finished at-20 ℃ for later use.

d) The above steps were repeated to prepare three batches of b-AFP-RPE.

2. b-AFP-RPE and performance testing thereof

The experimental procedure was as follows:

c) The above test was performed on three batches of b-AFP-RPE.

Example 3:

IEDDA method for connecting b-AFP-RPE and performance detection thereof

1. IEDDA method for marking the junction b-AFP-RPE

The experimental procedure was as follows:

a) b-AFP-Tetrazine preparation: taking 1mg (6.7 mu mol) of b-AFP antibody, diluting, adding 20mM Tetrazine-NHS (the molar ratio of b-AFP: Tetrazine-NHS is 1:20) (or using other coupling methods to label Tetrazine on the antibody), mixing, and reacting at 37 ℃ for 30min in dark. After the reaction, the mixture was purified by an ultrafiltration tube, and finally 0.1M PBS (pH 7.3) was added to adjust the concentration of b-AFP-Tetrazine to 10mg/mL for use.

b) Preparation of RPE-TCO or RPE-DBCO: taking 1.7mg (6.8 mu mol) of signal protein RPE, adding 20mM TCO-NHS or DBCO-NHS (the molar ratio of RPE to TCO-NHS or DBCO-NHS is 1:20) (or adopting other coupling modes to mark TCO or DBCO on the antibody), mixing uniformly, and reacting for 30min at 37 ℃ in a dark place. After the reaction, the mixture was purified by an ultrafiltration tube, and finally 0.1M PBS (pH 7.3) was added to adjust the concentration of RPR-TCO or RPE-DBCO to 10mg/mL for use.

c) b-AFP-RPE preparation: and (c) adding the liquid b-AFP-Tetrazine collected in the step (a) into the RPR-TCO or RPE-DBCO obtained in the step (b), uniformly mixing, and reacting for 15min at 37 ℃. And (4) storing the marker after the reaction is finished at-20 ℃ for later use.

d) The above steps were repeated to prepare three batches of b-AFP-RPE.

2. b-AFP-RPE and performance testing thereof

The experimental procedure was as follows:

c) The above test was performed on three batches of b-AFP-RPE.

Example 4

Comparative experiments to test 1000ng/mL AFP samples for comparative examples 1-2 and examples 1-3 above

Specifically, a 1000ng/mL AFP sample is tested by using the b-AFP-RPE labeled ligand composition prepared in comparative examples 1-2 and examples 1-3 to obtain fluorescence signals at various time points, the experimental result data are shown in Table 1, the connection time/min (minutes) of b-AFP and RPE is used as an abscissa, the 1000ng/mL fluorescence signal is used as an ordinate, the data in Table 1 are processed, and a graph showing the change of the test signals in FIG. 3 along with the reaction time is obtained by plotting.

TABLE 1 Table of relationship between detection signal and reaction time

The time required for the b-AFP-RPE ligation reaction of comparative examples 1-2, examples 1-3 to reach the end of the reaction is shown in Table 2.

TABLE 2 time required to reach the end of the reaction for the different groups

The time required for the reaction to link b-AFP and RPE is shown in Table 2, and can be seen from Table 2:

the reaction times for examples 1-3 are all significantly shorter than for comparative examples 1-2, with example 3 requiring the shortest reaction time being the more preferred embodiment.

Example 5:

immunofluorescence assay experiments of comparative examples 1-2 and examples 1-3

Specifically, AFP samples with different concentrations were tested using the b-AFP-RPE labeled ligand composition obtained in comparative examples 1-2 and examples 1-3 to obtain the strength values of the test signals, and the data of the test results are shown in Table 3, wherein the AFP sample concentration (ng/mL) is plotted on the abscissa and the strength value of the test signal is plotted on the ordinate, and the data in Table 3 are plotted to show the performance results of different groups in FIG. 4.

TABLE 3 results of different groups of measurements

As can be seen from table 3 and fig. 2: the detection signals obtained in examples 1 to 3 and comparative example 1 are obviously better than that obtained in comparative example 2, but the white signal strength in comparative example 1 is high, and the performance of comparative example 1, such as the sensitivity of detection, is greatly influenced.

Example 6:

run-to-run experiment for comparative examples 1-2 and examples 1-2

Specifically, three batches of b-AFP-RPE tagged ligand compositions obtained in comparative examples 1-2 and examples 1-2 were used to test AFP samples at different concentrations to obtain test signals, and the experimental data are shown in Table 4. The experimental data in table 4 were plotted in a histogram to obtain histograms of different sets of the detection signals in fig. 5.

TABLE 4 results of signal intensity measurements in different batches with different methods

AFP concentration (ng/mL)	0	10	100	1000	2000
						Comparative example 1 run 1	1530	8734	68794	342279	497821
Comparative example 1 run 2	4595	12101	93191	483419	726735
						Comparative example 1 run 3	801	4467	35397	182239	258910
Comparative example 2 run 1	80	2711	28109	128427	188732
						Comparative example 2 run 2	76	2914	28973	137486	202783
Comparative example 2 run 3	83	2573	27647	121732	181246
						Example 1 run 1	73	4573	44121	278464	407818
Example 1 run 2	80	4324	41848	269723	397242
						Example 1 run 3	77	5016	47874	281467	417686
Example 2 run 1	77	6397	57677	328979	437313
						Example 2 run 2	70	6686	59425	337545	451427
Example 2 run 3	76	5973	56138	306941	413751
						Example 3 run 1	83	10875	78424	408672	598761
Example 3 run 2	74	9246	74846	387415	572812
						Example 3 run 3	78	9532	75657	395765	589876

As can be seen from table 4 and fig. 3: comparative example 1 has a large batch-to-batch difference and a large difference in the measured bottom signal, which is mainly due to randomness of the glutaraldehyde-coupled ligand and the signal protein and the possibility of generating a large amount of by-products, and it is difficult to separate similar products by purification. The batch-to-batch differences for examples 1-2 are significantly better than for comparative examples 1 and 2.

Example 7

Stability test of comparative examples 1 to 2 and examples 1 to 3

Specifically, a part of the b-AFP-RPE labeled ligand composition obtained by different methods is placed at 4 ℃ and 37 ℃ for a certain number of days for detection, fluorescence signals are obtained from 10ng/mL and 1000ng/mL AFP samples, and the stability of the b-AFP-RPE labeled ligand composition obtained by the same preparation method is compared according to the change of the fluorescence signals. The results are shown in FIGS. 6a to 6d, which are graphs showing the retention ratio of fluorescence signal as a function of the standing condition and time. It can be seen from the data that the stability of examples 1-3 is significantly better than that of comparative examples 1-2.

Example 8

Detection sensitivity test for comparative examples 1 to 2 and examples 1 to 3

Specifically, the b-AFP-RPE labeled ligand compositions obtained in comparative examples 1-2 and examples 1-3 were used for the detection of a series of low concentration AFP samples, the experimental data are shown in Table 6, the sensitivity of detection was measured, the experimental results are shown in Table 7, and it can be seen from tables 6-7 that: the sensitivity of examples 1-3 was superior to that of comparative example 1, while the detection sensitivity of example 3 was the best.

TABLE 6 Table of detection signals of b-AFP-RPE labeled ligand compositions prepared by different methods

AFP concentration (ng/mL)	Comparative example 1	Comparative example 2	Example 1	Example 2	Example 3
						0	1530	80	73	77	78
0.0391	1554	79	76	79	87
						0.0781	1437	84	70	69	134
0.1563	1534	90	86	147	267
						0.3125	1498	87	157	278	357
0.625	1527	197	378	467	704
						1.25	1678	389	697	904	1378
2.5	2467	794	1347	1756	2546
						5	4034	1437	2475	3342	4879
10	8820	2754	4573	6397	9532

TABLE 7 sensitivity table for b-AFP-RPE detection prepared by different methods

Group of	Comparative example 1	Comparative example 2	Example 1	Example 2	Example 3
						Sensitivity (ng/mL)	1.25	0.625	0.3125	0.1563	0.0781

In summary, the labeled ligand composition based on click chemistry of the present application has the following advantages:

1) comparative experiments were conducted to examine 1000ng/mL AFP samples for comparative examples 1-2 and examples 1-3 above, and it can be seen that: the reaction times for examples 1-3 are all significantly shorter than for comparative examples 1-2, and the reaction rates for the preparation of labeled ligand compositions for click chemistry are significantly faster;

2) in the immunofluorescence analysis experiments of comparative examples 1-2 and examples 1-3 above, it can be seen that: the detection signal intensity obtained in examples 1-3 and comparative example 1 is obviously superior to that obtained in comparative example 2, but the blank signal intensity in comparative example 1 is high, and the performance influence of comparative example 1 on the sensitivity and the like of detection is large, so that obviously, when the labeled ligand composition prepared by the method is used for immunofluorescence analysis, the detection signal intensity is stronger, and meanwhile, the blank signal intensity is weak, so that the immunofluorescence analysis is facilitated;

3) for the above batch-to-batch difference experiments of comparative examples 1-2 and examples 1-2, it can be seen that: the batch-to-batch differences for examples 1-2 are significantly better than for comparative examples 1 and 2.

4) For the stability tests of comparative examples 1-2 and examples 1-3 above, it can be seen that: the stability of examples 1-3 is significantly better than that of comparative examples 1-2.

5) In the detection sensitivity test of comparative examples 1 to 2 and examples 1 to 3, it can be seen that: the sensitivity of examples 1-3 was superior to that of comparative example 1, while the detection sensitivity of example 3 was the best.

According to the method, through the click chemical reaction between the azide groups and the azide reaction groups, the signal protein is connected with the ligand to obtain the ligand composition containing the marker groups, and the marker groups have the advantages of high stability, high reaction speed and high yield, and the number of the marker groups can be adjusted and controlled. The method also realizes dual labeling of the ligand and the signal protein, can be used for subsequent immunofluorescence analysis, and solves the problems of unstable labeling, low labeling efficiency, large labeling batch difference and high labeling cost generally existing when a heterobifunctional cross-linking agent is used for labeling two proteins in the prior art.

The above description is only for the purpose of illustrating embodiments of the present application and is not intended to limit the scope of the present application, and all modifications of equivalent structures and equivalent processes, which are made by the contents of the specification and the drawings of the present application or are directly or indirectly applied to other related technical fields, are also included in the scope of the present application.

Claims

1. A click chemistry-based tagged ligand composition, comprising:

a ligand containing one of an azide group or an azide-reactive group;

a signal protein containing the other of an azide group or an azide-reactive group;

wherein the signal protein links the ligand to form the labeled ligand by a click chemistry reaction between an azide group and an azide-reactive group.

2. The tagged ligand composition of claim 1,

the azide-reactive group includes: an alkyne which forms a triazazole ring with the azide group, or a cycloalkene which forms a diazacyclo ring with the azide group.

3. The tagged ligand composition of claim 1,

the signal protein comprises at least one of alkaline phosphatase, horseradish peroxidase, phycoerythrin, phycocyanin, phycoerythrin or allophycocyanin.

4. The tagged ligand composition of claim 1,

the ligand is at least one of an antibody, an antigen, a hapten-carrier conjugate or avidin.

5. The tagged ligand composition of claim 1,

the antibody comprises at least one of an angiotensin I antibody, a NT-proBNP antibody, a troponin antibody, an alpha-fetoprotein antibody, a carcinoembryonic antigen antibody, an HIV antigen antibody, a hepatitis B surface antigen antibody, a thyroglobulin antibody, a troponin I or T antibody, or a myoglobin antibody;

the antigen comprises: at least one of angiotensin I antigen, NT-proBNP antigen, troponin antigen, alpha-fetoprotein antigen, carcinoembryonic antigen, HIV antigen, hepatitis B surface antigen, thyroglobulin antigen, troponin I or T antigen, or myoglobin antigen;

the hapten-carrier conjugate comprises at least one of aldosterone, aldosterone hapten protein conjugate, HA hyaluronic acid, hyaluronic acid hapten protein conjugate, angiotensin I-carrier conjugate, angiotensin II-carrier conjugate or HABP hyaluronic acid binding protein.

6. A method for preparing a tagged ligand composition based on click chemistry, for use in preparing a tagged ligand composition according to any one of claims 1-5, the method comprising:

providing a ligand and a signal protein;

labeling the ligand with a first click chemistry, wherein the first click chemistry comprises one of an azide group or an azide-reactive group;

labeling the signal protein with a second click chemistry reagent, wherein the second click chemistry reagent contains the other of an azide group or an azide-reactive group;

and carrying out click chemistry reaction on the labeled ligand and the labeled signal protein, and connecting the signal protein with the ligand to form the labeled ligand composition.

7. The production method according to claim 6,

the first and second click chemistry reagents comprise at least one of a DBCO reagent, a Tetrazine reagent, an Azide reagent, an Alkyne reagent, or a TCO reagent.

8. A kit, comprising:

a kit body;

a first reagent holding site disposed on the kit body for holding at least one labeled ligand composition of any one of claims 1-5;

and the second reagent holding position is arranged on the kit body and is used for holding at least one immunoassay composition, and the immunoassay composition is matched with the labeled ligand composition to obtain a detection complex.

9. The kit according to claim 8,

the labeled ligand composition is a labeled antibody composition;

the immunoassay composition includes a capture antibody composition and/or a labeled antigen.

10. An immunoassay analysis system, the immunoassay analysis system comprising:

a kit according to any one of claims 8 to 9;

a sample analyzer which performs detection of an analyte by using the immunodetection composition and the labeled ligand composition in the kit and outputs a detection result.

11. Use of a labeled ligand composition according to any one of claims 1-5 in an immunoassay;

the labeled ligand composition is used for myocardial detection, liver fibrosis detection, hypertension detection, thyroid gland detection, cardiovascular detection, gonadal detection, renal function detection, bone metabolism detection, glycometabolism detection, infectious disease detection, autoimmune disease detection, prenatal screening, drug detection, hepatic fibrosis detection, EB virus detection, inflammation monitoring or tumor detection.