Preparation method of high-polymerization metal chelate polymer antibody label carrier material

Abstract

The present invention discloses a method for preparing a metal chelate polymer antibody label carrier material with a high degree of polymerization. The method uses an amphiphilic macromolecular reversible addition-scission chain transfer reagent combined with an emulsion polymerization method to prepare a polymer backbone with a high degree of polymerization, and then functionalizes the end groups and side groups and grafts chelate groups through a chemical modification strategy. The present invention innovatively prepares a polymer backbone with a high degree of polymerization, a narrow molecular weight distribution, and high-end base fidelity through a RAFT emulsion polymerization method. As a label carrier, it can introduce more metal chelate units, which can not only load a larger number of metal atoms and improve the detection signal intensity of the labeled antibody, but also ensure that the polymer carrier can graft some new chelating agents with lower efficiency and load more types of metal isotopes, providing a metal detection signal with high sensitivity and a large number of available metal isotope channels in mass spectrometry flow detection, which is of great significance for achieving higher precision and higher throughput single-cell protein analysis and detection.

Description

Technical Field

[0001] The present invention relates to the field of biomedical detection technology, and in particular to a high-polymerization metal chelate polymer antibody label carrier material for labeling antibodies based on single-cell protein detection technology of mass spectrometry flow cytometry and a preparation method thereof. Background Art

[0002] In recent years, time-of-flight mass cytometry (CyTOF), which has high-dimensional detection capabilities and can simultaneously detect protein parameters in more than 50 channels for millions of single cells, has become the most powerful technology for large-scale cell analysis and biomarker discovery. CyTOF uses rare heavy metal isotopes that are not usually present in biological samples to label antibodies and specifically bind to proteins on cells. The time of flight (TOF) of each metal isotope is then detected by a mass spectrometer to "read" these isotopes. The TOF of each atom is determined by its mass-to-charge ratio, and the signal intensity is proportional to the number of atoms. Therefore, the signal intensity of the corresponding TOF can be used to determine the composition and content of metal atoms on each cell, and then to associate the types and quantities of proteins in individual cells. Currently, high-throughput quantitative analysis of genes and proteins at the single-cell level using CyTOF has become an important technical means for hematology, oncology, and immunology research and the precise diagnosis and treatment of related major diseases.

[0003] However, the current mass spectrometry technology still has problems such as low sensitivity and a small number of multi-dimensional detection channels. On the one hand, because the number of metal atoms loaded by the existing commercial single polymer antibody label carrier is small (less than 100), and the detection sensitivity of CyTOF is positively correlated with the number of metals, it means that the existing metal labels can only have a good detection effect on cells expressing proteins with high abundance, and there are still sensitivity limitations for the labeling detection of proteins expressed in low abundance. On the other hand, the insertion efficiency of effective chelators of most metal elements on polymers is low, resulting in the current commercial polymer antibody labels being able to only load specific metal elements through chelators with high insertion rates, including lanthanides, In, Y, Bi, etc., a total of more than 50 metal isotopes, which limits the number of CyTOF detection channels to more than 50. Most metal isotope channels have not yet been applied. Therefore, CyTOF is difficult to cover more target proteins in the face of certain complex detection systems, limiting the application and development of multi-channel high-dimensional labeling detection of single-cell proteins.

[0004] However, improving the signal sensitivity of existing polymer antibody labels and expanding the available metal isotope channels are difficult problems to solve. One of the main reasons is that the existing polymer metal antibody label carrier synthesis process relies on RAFT solution polymerization to synthesize polymer carriers with a narrow molecular weight distribution. In order to ensure a high retention rate of the functional chain ends of the polymer products, the product polymerization degree is usually low, which limits the number of metal chelating units and makes it difficult to achieve a higher metal loading. For new metal elements, due to the low insertion rate of their effective chelating units on the polymer, the loading of new metals on the polymer is even lower, and even the normal labeling sensitivity of high-expression protein systems cannot be achieved. Therefore, it is difficult to further break through the current number of metal isotope channels. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for preparing a high-polymerization metal chelate polymer antibody label carrier material for mass spectrometry flow cytometry technology. Compared with existing commercial metal antibody labels, the metal loading capacity is greatly increased, the detection sensitivity is significantly improved, and the number of metal isotope detection channels is greatly broadened.

[0006] To achieve the above-mentioned purpose, the technical solution adopted by the present invention is to provide a method for preparing a metal chelate polymer antibody tag carrier material with a high degree of polymerization:

[0007] (1) Synthetic polymer backbone:

[0008] 20 to 80 parts by weight of water, 0.01 to 1.5 parts by weight of an amphiphilic macromolecular reversible addition chain-scission chain transfer reagent, and 1 to 100 parts by weight of a functional monomer are added to a reactor, stirred and mixed, nitrogen is passed through until the air is completely replaced, and the temperature is raised to 40 to 80° C., and then 0.002 to 0.04 parts by weight of a water-soluble initiator is added to initiate polymerization for 10 to 60 minutes. An aqueous solution containing 0.01 to 2 parts by weight of an alkali is added, and after polymerization for 0.5 to 8 hours, the resulting emulsion is demulsified, washed, and dried to obtain a polymer backbone, i.e., an intermediate product A.

[0009] The intermediate product A has the structural formula:

[0010]

[0011] (2) End group functionalization: 1 to 10 parts by weight of the intermediate product A, 1 to 200 parts by weight of a solvent, and 0.05 to 5 parts by weight of a first functionalization reagent are added to a reactor, stirred and reacted at room temperature for 2 to 12 hours. After the reaction is completed, the intermediate product B is obtained by washing with a solvent and then removing the solvent.

[0012] The structural formula of the intermediate product B is:

[0013]

[0014] (3) Side group functionalization: ① Add 1 to 10 parts by weight of intermediate product B, 1 to 200 parts by weight of solvent, and 1 to 20 parts by weight of the second functionalization reagent to the reactor, and stir and react at room temperature for 2 to 12 hours. ② Wash the product and remove the solvent, then add 1 to 200 parts by weight of solvent and 1 to 20 parts by weight of grafting agent to the reactor, and stir and react at room temperature for 2 to 12 hours. ③ Wash the product and remove the solvent, then add 1 to 200 parts by weight of solvent and 1 to 100 parts by weight of chelating agent, stir and mix at room temperature, wash with solvent after the reaction, and then remove the solvent. The final polymer solution is then freeze-dried below -20°C to obtain a metal chelate polymer antibody label carrier.

[0015] The first functionalizing agent is selected from ammonia, ammonium bicarbonate, urea, methylamine, dimethylamine, trimethylamine, ethylamine, ethylenediamine, triethylamine, propylamine, isopropylamine, propylenediamine, tripropylamine, ethanolamine, triethanolamine, aniline, benzylamine, and cyclohexylamine; and the second functionalizing agent is selected from formic acid, acetic acid, trifluoroacetic acid, phenol, 1,3-dimethoxybenzene, hydrochloric acid, sulfuric acid, nitric acid, NaH, and HBr.

[0016] The metal chelate polymer antibody tag carrier structure is:

[0017]

[0018] The general chemical structure formula of the amphiphilic macromolecular reversible addition-fragmentation chain transfer reagent is:

[0019]

[0020] Wherein, the Z group is selected from: phenyl, benzyl, methyl, ethyl, propyl, isopropyl, butyl and its isomers, pentyl and its isomers, ethoxy, methoxy mercaptan, ethyl mercaptan, isopropyl mercaptan, butyl mercaptan, C 12 The R group is selected from the group consisting of 1-methylbenzyl, 1,1-dimethylbenzyl, isopropanoyl, 2-diisobutyryl, 2-isobutyronitrile, cyanopentanoyl, and 3-benzoyl. The lipophilic monomer includes styrene, acrylates, and methacrylates. The hydrophilic monomer is selected from acrylic acid and methacrylic acid. The ratio of n to m is 3 to 7. The molecular weight of the amphiphilic macromolecular reversible addition-fragmentation chain transfer reagent is 1,000 to 10,000.

[0021] Furthermore, the functional monomers of the synthetic polymer main chain are one or more of tert-butyl acrylate and tert-butyl methacrylate in any combination; the degree of polymerization of the functional monomers is greater than 100; and the water-soluble initiator is selected from persulfate and azo.

[0022] The aqueous solution containing 0.01 to 2 parts by weight of an alkali, wherein the alkali is selected from sodium hydroxide, potassium hydroxide, aqueous ammonia, sodium carbonate, potassium carbonate, sodium bicarbonate, and potassium bicarbonate; and water is 0.1 to 1 part by weight.

[0023] Furthermore, the grafting agent has an NH2-R structure, wherein the terminal group of R is selected from amino, N-hydroxysuccinimide, pentafluorophenol ester, carboxyl, aldehyde, thiol, maleimide, vinyl sulfone, azide, alkynyl, hydroxyl, dihydroxyboron, chlorosiloxane, and o-diphenol.

[0024] Furthermore, the chelating agent is selected from:

[0025]

[0026] The names of the above chelating agents are: porphyrin, nitrilotriacetic acid, ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid, 1,4,7-triazacyclononane-1,4,7-triacetic acid, hydrazinonicotinamide, dipyridylamine, dehydrogenated phenylcyclopentene, disalicyclic aldehyde diamine, R1~R 12 is other substituents, determined according to the group connected to the tail of the grafting agent NH2-R, as shown in Table 1:

[0027] Table 1: Reaction combinations of groups connected to grafting agents and chelating agents

[0028]

[0029]

[0030] The solvent is selected from toluene, xylene, acetonitrile, dioxane, tetrahydrofuran, acetone, methyl ethyl ketone, dimethyl sulfoxide, dichloromethane, chloroform, tetrachloroethane, methanol, ethanol, isopropyl alcohol, ether, and water.

[0031] As a second aspect, the present invention provides a metal chelate polymer antibody tag carrier material with a high degree of polymerization prepared by the above preparation method.

[0032] The beneficial effects of the present invention are as follows: the RAFT emulsion polymerization method can break through the limitations of the original polymerization process, synthesize a highly active polymer backbone with a degree of polymerization of 100-1000 and a narrow molecular weight distribution (PDI<1.5), and then prepare a metal chelate polymer antibody tag carrier with a high degree of polymerization through chemical modification. The metal loading capacity of the polymer antibody tag carrier prepared by this method increases exponentially with the increase of the degree of polymerization. Currently, the degree of polymerization of commercial polymer antibody tags is generally 100, so the maximum metal ion loading capacity is only 100, which causes the problem of insufficient signal intensity in the detection of low-expression protein systems; at the same time, for chelating units with low insertion rates, the number of other types of metals that can be loaded by the polymer tag is far less than 100, and sensitive detection cannot be achieved, so that the currently available detection channels are limited to more than 50 metal isotopes such as lanthanides. After the present invention prepares a polymer tag carrier with a degree of polymerization of 100-1000 through the RAFT emulsion polymerization method, the metal chelating units are also greatly increased, and the metal loading capacity of a single polymer tag can be increased by an order of magnitude. On the one hand, compared with the commercial tag signal, the signal intensity and sensitivity are significantly enhanced, which provides convenience for the detection of low-expression protein systems; on the other hand, new chelating agents with lower grafting efficiency can be introduced to ensure that the number of these chelating units is still sufficient, and the number of new types of metal isotopes loaded can achieve a more sensitive signal intensity, thereby greatly increasing the types of metal loadings available for polymer tags and greatly broadening the number of CyTOF channels for high-dimensional detection of single-cell proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Schematic diagram of porphyrin structure chelating metal;

[0034] Figure 2 Schematic diagram of nitrilotriacetic acid structure chelating metal;

[0035] Figure 3 Schematic diagram of ethylenediaminetetraacetic acid structure chelated metal;

[0036] Figure 4 Schematic diagram of metal chelation using diethylenetriaminepentaacetic acid structure;

[0037] Figure 5 Schematic diagram of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid structure chelated metal;

[0038] Figure 6 Schematic diagram of 1,4,7-triazacyclononane-1,4,7-triacetic acid structure chelated metal

[0039] Figure 7 Schematic diagram of hydrazinonicotinamide structure chelating metal

[0040] Figure 8Schematic diagram of the metal chelate structure of dipyridylamine;

[0041] Figure 9 Schematic diagram of the metal chelate structure of dehexahydrotriphenylcyclopentene;

[0042] Figure 10 Schematic diagram of the metal chelate structure of disalicylicaldehyde diamine;

[0043] Figure 11 After marking and staining 100% positive cells for Examples 1-3 and Comparative Example 1, 171 CyTOF signal intensity bar graph detected by Yb channel;

[0044] Figure 12 After marking and staining 100% positive cells for Examples 4 and 5 and Comparative Examples 2 and 3, 120 Sn and 186 CyTOF signal intensity histogram detected by W channel;

[0045] Figure 13 The results of CyTOF labeling of a Sn-loaded polymer antibody tag on a 50%:50% mixed Yin and Yang cells, where (a) is the commercial polymer antibody tag from Comparative Example 2, and (b) is the antibody tag with a degree of polymerization of 1000 from Example 4.

[0046] Figure 14 These are the CyTOF labeling detection results of W-loaded polymer antibody tags on 50%:50% yin and yang mixed cells, where (a) is the commercial polymer antibody tag of Comparative Example 3, and (b) is the antibody tag with a polymerization degree of 1000 in Example 5. DETAILED DESCRIPTION

[0047] The metal chelate polymer antibody tag provided by the present invention is synthesized by the following steps. The first step is to polymerize functional monomers using the RAFT emulsion polymerization method to obtain a polymer backbone. The second step is to convert the RAFT groups at the ends of the backbone into functional thiol end groups through chemical modification. The third step is to chemically modify the groups on the side of the backbone and introduce chelating units at the side positions using a grafting agent to obtain an antibody tag carrier. The final step is to load the metal on the tag carrier, and then use the active end groups to bond with groups on the antibody through a click reaction, ultimately obtaining a metal-containing tag connected to the antibody.

[0048] The present invention will be further described below with reference to specific embodiments and accompanying drawings:

[0049] Example 1

[0050] Synthesis of the main chain: 40g of water, 0.8g of amphiphilic macromolecular reversible addition chain scission transfer reagent C 12 H 25SS=CS-PS3-b-PAA 20 -CH3COOH, 5g of tert-butyl methacrylate was added to the reactor, stirred and mixed evenly, and nitrogen was passed through for 30 minutes (until the air was completely replaced). The temperature was raised to 70°C, and an aqueous solution containing 20mg of potassium persulfate was added to initiate polymerization. After the reaction lasted for 10 minutes, an aqueous solution containing 0.15g of sodium hydroxide was added and the polymerization was continued for 2 hours. After the polymerization was completed, hydrochloric acid was added until the emulsion was completely broken, and the product was neutralized, washed, filtered, and vacuum dried to obtain the polymer backbone product.

[0051] End-group modification: Weigh 1 g of the above polymer solid into a reactor, add 5 mL of acetone, and stir to dissolve completely. Then, add 0.6 g of benzylamine and stir at room temperature for 12 hours. After the reaction is complete, remove the solvent by rotary evaporation to obtain the end-group functionalized product.

[0052] Pendant Group Modification: Add 20 mL of dichloromethane and 10 mL of acetic acid to the terminal functionalized product, mix, and stir for 12 hours. Filter the solution, wash the polymer with an appropriate amount of dichloromethane, and then remove the solvent by rotary evaporation. Dissolve the polymer in 10 mL of deionized water, then add 5 g of mercaptoethylamine. Stir and react at room temperature for 12 hours. Wash with water and remove water by ultrafiltration. Add 20 mL of water and 30 g of 1,4,7-triazacyclononane-1,4,7-triacetic acid-2-maleimide. Stir and react at room temperature for 1 hour. After the reaction, wash the product with water and remove water by ultrafiltration. Finally, freeze-dry the polymer solution below -20°C and store below -20°C.

[0053] To prepare the metal-antibody tag, 1 mg of the lyophilized polymer powder prepared by the above method was added to 100 μL of a 40 mM YbCl solution (dissolved in PBS buffer, pH 7.0) and allowed to react at 37°C for 1 hour. After the reaction, the mixture was transferred to an ultrafiltration tube, washed, centrifuged, and the supernatant removed. 0.1 mg of the antibody was then added, mixed thoroughly, and allowed to react at 37°C for 0.5 hour. After the reaction, the mixture was transferred to an ultrafiltration tube, centrifuged, and the supernatant removed to obtain the antibody-linked metal tag.

[0054] Example 2

[0055] Synthesis of the main chain: 40g of water, 0.16g of amphiphilic macromolecular reversible addition chain scission transfer reagent C 12 H 25 SS=CS-PS5-b-PAA 20-CH3COOH and 5g of tert-butyl acrylate were added to the reactor, stirred and mixed, and nitrogen was purged for 30 minutes. The temperature was raised to 60°C, and an aqueous solution containing 4mg of azobiscyanovaleric acid was added to initiate polymerization. After 20 minutes of reaction, an aqueous solution containing 0.045g of sodium hydroxide was added, and polymerization was continued for 4 hours. After the polymerization was completed, hydrochloric acid was added until the emulsion was completely broken. The product was neutralized, washed, filtered, and vacuum dried to obtain the polymer backbone product.

[0056] End Group Modification: Weigh 1 g of the above polymer solid into a reactor, add 5 mL of tetrahydrofuran, and stir to dissolve completely. Then, add 0.3 g of propylamine and stir at room temperature for 12 hours. After the reaction is complete, remove the solvent by rotary evaporation to obtain the end-group functionalized product.

[0057] Side group modification: Add 20 mL of chloroform and 10 mL of formic acid to the above-mentioned end-functionalized product, mix and stir to react for 12 hours, then filter out the solution, add an appropriate amount of chloroform to wash the polymer product, and then evaporate to remove the solvent. Then add 10 mL of water to dissolve the polymer, then add 4 g of propylene diamine, stir and react at room temperature for 12 hours, then wash with water, and then ultrafilter to remove water. Then add 20 mL of water and then add 50 g of diethylenetriamine pentaacetic acid, stir and react at room temperature for 1 hour. After the reaction is completed, add water to wash the product, then ultrafilter to remove water, and finally freeze-dry the polymer aqueous solution below -20 degrees, and then seal it at a low temperature below -20 degrees.

[0058] To prepare the metal-antibody tag, 5 mg of the lyophilized polymer powder prepared by the above method was added to 100 μL of a 200 mM YbCl solution (dissolved in PBS buffer, pH 7.0) and allowed to react at 37°C for 1 hour. After the reaction, the mixture was transferred to an ultrafiltration tube, washed, centrifuged, and the supernatant removed. 0.1 mg of the antibody was then added, mixed thoroughly, and allowed to react at 37°C for 0.5 hour. After the reaction, the mixture was transferred to an ultrafiltration tube, centrifuged, and the supernatant removed to obtain the antibody-linked metal tag.

[0059]

[0060] Example 3

[0061] Synthesis of the main chain: 40g of water, 0.08g of amphiphilic macromolecular reversible addition chain scission transfer reagent C 12 H 25 SS=CS-PS6-b-PAA 17-CH3COOH, 5g of tert-butyl methacrylate were added to the reactor, stirred and mixed, and nitrogen was purged for 30 minutes. The temperature was raised to 50°C, and an aqueous solution containing 2mg of azobisisobutylimidazoline hydrochloride was added to initiate polymerization. After 30 minutes of reaction, an aqueous solution containing 0.03g of sodium hydroxide was added, and polymerization was continued for 6 hours. After completion of polymerization, hydrochloric acid was added until the emulsion was completely broken, and the product was neutralized, washed, filtered, and vacuum dried to obtain the polymer backbone product.

[0062] End-group modification: Weigh 1 g of the above polymer solid into a reactor, add 5 mL of isopropanol, and stir to dissolve completely. Then, add 0.5 g of aniline and stir at room temperature for 12 hours. After the reaction is complete, remove the solvent by rotary evaporation to obtain the end-group functionalized product.

[0063] Pendant Group Modification: Add 20 mL of tetrahydrofuran and 25 g of phenol to the terminal functionalized product, mix, and stir for 12 hours. Filter the solution, wash the polymer with 20 mL of tetrahydrofuran, and remove the solvent by rotary evaporation. Dissolve the polymer in 10 mL of water, then add 8 g of alkynylethylamine. Stir and react at room temperature for 12 hours. Wash with water and remove water by ultrafiltration. Add 20 mL of water and 80 g of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-2-azide. Stir and react at room temperature for 1 hour. After the reaction, wash the product with water and remove water by ultrafiltration. Finally, freeze-dry the polymer solution below -20°C and store below -20°C.

[0064] To prepare the metal-antibody tag, 10 mg of the lyophilized polymer powder prepared by the above method was added to 100 μL of a 400 mM YbCl solution (dissolved in PBS buffer, pH 7.0) and allowed to react at 37°C for 1 hour. After the reaction, the mixture was transferred to an ultrafiltration tube, washed, centrifuged, and the supernatant removed. 0.1 mg of the antibody was then added, mixed thoroughly, and allowed to react at 37°C for 0.5 hour. After the reaction, the mixture was transferred to an ultrafiltration tube, centrifuged, and the supernatant removed to obtain the antibody-linked metal tag.

[0065]

[0066] Example 4

[0067] Synthesis of the main chain: 40g of water, 0.08g of amphiphilic macromolecular reversible addition chain scission transfer reagent C 12 H 25 SS=CS-PS6-b-PAA 17-CH3COOH, 5g of tert-butyl acrylate were added to the reactor, stirred and mixed, and nitrogen was purged for 30 minutes. The temperature was raised to 50°C, and an aqueous solution containing 2mg of azobisisobutylimidazoline hydrochloride was added to initiate polymerization. After 30 minutes of reaction, an aqueous solution containing 0.03g of sodium hydroxide was added, and polymerization was continued for 6 hours. After completion of polymerization, hydrochloric acid was added until the emulsion was completely broken, and the product was neutralized, washed, filtered, and vacuum dried to obtain the polymer backbone product.

[0068] End-group modification: Weigh 1 g of the above polymer solid into a reactor, add 5 mL of methanol, and stir to dissolve completely. Then, add 1 g of cyclohexylamine and stir at room temperature for 12 hours. After the reaction is complete, remove the solvent by rotary evaporation to obtain the end-group functionalized product.

[0069] Side group modification: Add 20 mL of ether and 10 mL of trifluoroacetic acid to the above-mentioned end-functionalized product, mix and stir to react for 12 hours, then filter out the solution, add 20 mL of ether to wash the polymer product, and then evaporate to remove the solvent. Then add 10 mL of water to dissolve the polymer, then add 6 g of ethylenediamine, stir and react at room temperature for 12 hours, then wash with water, and then ultrafilter to remove water. Finally, add 20 mL of water to dissolve, then add 120 g of tetracarboxyphenylporphyrin, and stir and react at room temperature for 1 hour. After the reaction is completed, add water to wash the product, then ultrafilter to remove water, and finally freeze-dry the polymer aqueous solution below -20 degrees Celsius and store it at a low temperature below -20 degrees Celsius.

[0070] To prepare the metal-antibody tag, 10 mg of the lyophilized polymer powder prepared by the above method was added to 100 μL of a 400 mM SnCl₂ solution (dissolved in a dilute hydrochloric acid solution at pH 2.0) and reacted at 37°C for 1 hour. After the reaction, the mixture was transferred to an ultrafiltration tube, washed multiple times with pH 7.0 PBS buffer, centrifuged, and the supernatant removed. 0.1 mg of the antibody was then added, mixed thoroughly, and reacted at 37°C for 0.5 hour. After the reaction, the mixture was transferred to an ultrafiltration tube, centrifuged, and the supernatant removed to obtain the antibody-linked metal tag.

[0071]

[0072] Example 5

[0073] Synthesis of the main chain: 40g of water, 0.08g of amphiphilic macromolecular reversible addition chain scission transfer reagent C 12 H 25 SS=CS-PS6-b-PAA 17-CH3COOH, 5g of tert-butyl acrylate were added to the reactor, stirred and mixed, and nitrogen was purged for 30 minutes. The temperature was raised to 50°C, and an aqueous solution containing 2mg of azobisisobutylimidazoline hydrochloride was added to initiate polymerization. After 30 minutes of reaction, an aqueous solution containing 0.03g of sodium hydroxide was added, and polymerization was continued for 6 hours. After completion of polymerization, hydrochloric acid was added until the emulsion was completely broken, and the product was neutralized, washed, filtered, and vacuum dried to obtain the polymer backbone product.

[0074] End-group modification: Weigh 1 g of the above polymer solid into a reactor, add 5 mL of dichloromethane, and stir to dissolve completely. Then, add 0.15 g of triethylamine and stir at room temperature for 12 hours. After the reaction is complete, remove the solvent by rotary evaporation to obtain the end-group functionalized product.

[0075] Side group modification: Add 20 mL of acetone and 10 mL of formic acid to the above-mentioned end-functionalized product, mix and stir to react for 12 hours, then filter out the solution, add 20 mL of acetone to wash the polymer product, and then remove the solvent by rotary evaporation. Then add 10 mL of water to dissolve the polymer, then add 10 g of dihydroxyborylethylamine, stir and react at room temperature for 12 hours, then wash with water, and then ultrafilter to remove water. Then add 20 mL of water to dissolve, then add 100 g of dehydrobenzocyclopentene, stir and react at room temperature for 1 hour. After the reaction is completed, add water to wash the product, then ultrafilter to remove water, and finally freeze-dry the polymer aqueous solution below -20 degrees Celsius and store it at a low temperature below -20 degrees Celsius.

[0076] To prepare the metal-antibody tag, 10 mg of the lyophilized polymer powder prepared by the above method was added to 100 μL of a 400 mM Na₂WO₄.2H₂O aqueous solution and reacted at 37°C for 1 hour. After the reaction, the mixture was transferred to an ultrafiltration tube, washed multiple times with pH 7.0 PBS buffer, centrifuged, and the supernatant removed. 0.1 mg of the antibody was then added, mixed thoroughly, and reacted at 37°C for 0.5 hour. After the reaction, the mixture was transferred to an ultrafiltration tube, centrifuged, and the supernatant removed to obtain the antibody-linked metal tag.

[0077]

[0078] Comparative Example 1

[0079] 1 mg of lyophilized powder of a commercially available polymer antibody tag carrier (fluidium) was added to a reaction tube. 100 μL of a 40 mM YbCl solution (dissolved in PBS buffer, pH 7.0) was then added and the reaction was incubated at 37°C for 1 hour. The tube was transferred to an ultrafiltration tube, washed, centrifuged, and the supernatant removed. 0.1 mg of antibody was then added, mixed thoroughly, and allowed to react at 37°C for 0.5 hour. After the reaction, the tube was centrifuged and the supernatant removed to obtain the antibody-linked metal tag.

[0080] Comparative Example 2

[0081] 1 mg of lyophilized powder of a commercially available polymer antibody tag carrier from Fluidium was added to a reaction tube. 100 μL of a 40 mM SnCl₂ solution (dissolved in dilute hydrochloric acid at pH 2.0) was then added and allowed to react at 37°C for 1 hour. After the reaction, the tube was transferred to an ultrafiltration tube, washed with PBS buffer at pH 7.0, centrifuged, and the supernatant removed. 0.1 mg of antibody was then added, mixed thoroughly, and allowed to react at 37°C for 0.5 hour. After the reaction, the tube was centrifuged and the supernatant removed to obtain the antibody-linked metal tag.

[0082] Comparative Example 3

[0083] 1 mg of lyophilized powder of a commercially available fluidium polymer antibody tag carrier was added to a reaction tube. 100 μL of a 400 mM Na₂WO₄.2H₂O solution was then added and allowed to react at 37°C for 1 hour. After the reaction, the tube was transferred to an ultrafiltration tube, washed with pH 7.0 PBS buffer, centrifuged, and the supernatant removed. 0.1 mg of the antibody was then added, mixed thoroughly, and allowed to react at 37°C for 0.5 hour. After the reaction, the tube was centrifuged and the supernatant removed to obtain the antibody-linked metal tag.

[0084] The degree of polymerization, molecular weight distribution index, and end group retention rate of the polymer backbones of Examples 1-5 are shown in Table 2. The actual number of polymerized units substantially all reached the designed degree of polymerization of 100-1000, while maintaining a narrow molecular weight distribution with a molecular weight distribution index of <1.5 and an end group activity retention rate of >96%.

[0085] Table 2 Degree of polymerization, molecular weight distribution, and end group retention rate of polymer antibody tag carriers

[0086] Example Design aggregation Actual number of aggregation units Molecular weight distribution index End group retention rate 1 100 102 1.21 99％ 2 500 495 1.31 99％ 3 1000 986 1.35 98％ 4 1000 977 1.42 97％ 5 1000 982 1.43 96％

[0087] The metal ion concentration in the antibody label sample solution was tested by ICP-MS, and the metal loading on a single polymer chain was calculated by the known polymer concentration. The number of chelating agents was characterized by nuclear magnetic resonance, and the results are shown in Table 3. Examples 1-3 show that with the increase in the degree of polymerization, the number of chelating units and the metal loading also doubled, reaching a maximum of nearly 1000. Examples 4 and 5 show that although the insertion rate of chelating agents such as porphyrin and dehydrobenzocyclopentene on the polymer is low, the total number of grafted chelating agents on the high-polymerization polymer antibody label carrier prepared by the present invention can reach more than 100, so that the total amount of new metal elements Sn and W effectively loaded can reach or even exceed the amount of metal in traditional commercial polymer antibody labels. Comparative Examples 1-3 show that the use of commercial polymer antibody labels can only have a high loading efficiency for lanthanide elements such as Yb, while the loading amount for new metal elements such as Sn and W is extremely low.

[0088] Table 3 Metal loading of metal chelating polymer antibody labels

[0089]

[0090]

[0091] Cell staining test experiment:

[0092] (1) Using the eight antibody labeling materials of Examples 1-5 and Comparative Examples 1-3, 100% positive cells (expressing antigen proteins specifically recognized by antibodies) were labeled and stained to characterize the signal intensity.

[0093] (2) Using the antibody labeling materials of Examples 4 and 5 and Comparative Examples 2 and 3, a mixture of 50% negative cells (not expressing the antigen protein specifically recognized by the antibody) and 50% positive cells was labeled and stained to detect the clustering effect.

[0094] The labelled cell staining method is as follows:

[0095] 1. Prepare several groups of normal living cells, about 2×10^6 cells per group, resuspend them in PBS buffer, adjust the volume to 1 mL, add Rh-103, and stain at room temperature for 5 minutes to distinguish between live and dead cells.

[0096] 2. Add 2 mL of 0.5-5 mg / mL bovine serum albumin solution to each group, centrifuge at 500 x g for 5 min, remove the supernatant, add 50 μL of blocking solution (0.5 μL of 0.5-1.5 mg / mL human immunoglobulin solution, 0.5 μL of 0.5-1.5 mg / mL mouse immunoglobulin solution, 0.5 μL of 0.5-1.5 mg / mL rat immunoglobulin solution, 0.5 μL of 0.5-1.5 mg / mL hamster immunoglobulin solution, 48 μL of 0.5-5 mg / mL bovine serum albumin solution), and block on ice for 20 min.

[0097] 3. Add 50 μL of extracellular antibody mixture (polymer antibody label, the antibody concentration is 0.1-1 mg / mL), resuspend the cells, and stain on ice for 30 minutes.

[0098] 4. Add 2 mL of 0.5-5 mg / mL bovine serum albumin solution, centrifuge at 500 x g for 5 min, remove the supernatant, add 1 mL of fix and perm- brane mixture (fluidigm) containing 0.5 v / v‰ single cell indicator 191 / 193Ir, resuspend the cells, and incubate at 4°C overnight.

[0099] 5. Add 2 mL of 0.5-5 mg / mL bovine serum albumin solution, centrifuge at 800 x g for 5 min, remove the supernatant, and repeat twice.

[0100] 6. Add 2 mL of deionized water, centrifuge at 800 x g for 5 min, remove the supernatant, and repeat twice.

[0101] 7. Filter the sample, count the cells, adjust the volume, prepare for mass spectrometry flow cytometry detection.

[0102] The labeling signal intensity of 100% positive cells in Examples 1-3 and Comparative Example 1 is as follows: Figure 11 It can be found that the signal intensity increases exponentially with the degree of polymerization of the polymer tag. The signal intensity of MCP-500 and MCP-1000 is increased by nearly 5 times and 10 times respectively compared with the commercial CMCP tag, which significantly increases the labeling sensitivity.

[0103] The labeling signal intensity of positive cells in Examples 4-5 and Comparative Examples 2-3 is as follows Figure 12 As shown. In Example 4, Sn 120 The signal intensity of the channel is much higher than that of Comparative Example 2, while that of Example 5 is 186The signal intensity of the channel is much higher than that of Comparative Example 3, indicating that the chelating agents of current commercial tags cannot effectively load metals such as Sn and W, resulting in very low metal signal intensity; while the MCP-1000 prepared by the present invention can significantly increase the number of low-insertion rate chelating agents to effectively load sufficient Sn and W, thereby achieving higher detection sensitivity on these metal isotope channels.

[0104] The staining results of 50%:50% mixed Yin and Yang cells are as follows Figure 13-14 As shown, the horizontal axis 191 Ir represents the staining of DNA, demonstrating the presence of a cell signal; the ordinate represents the corresponding metal isotope signal intensity. It can be seen that in the Sn and W channels, the signal intensities of Comparative Examples 2 and 3 are very low, making it impossible to distinguish between positive and negative cell populations. However, Examples 4 and 5 clearly distinguish between negative and positive populations, with the proportion of the positive population approaching the actual value of 50%, indicating high sensitivity in labeling detection, capable of separating target cells from non-target cells. This demonstrates that the polymer antibody tag prepared by the present invention, by increasing the degree of polymerization and significantly increasing the number of grafted novel chelating agents with low insertion rates, is suitable for sensitive labeling and detection of novel metal elements such as Sn and W.

[0105] The above embodiments are only used to illustrate the present invention, but are not used to limit the scope of implementation of the present invention.

Claims

1. A method for preparing a metal chelate polymer antibody tag carrier material with a high degree of polymerization, characterized in that: The steps include: 20 to 80 parts by weight of water, 0.01 to 1.5 parts by weight of an amphiphilic macromolecular reversible addition chain-scission transfer agent, and 1 to 100 parts by weight of a functional monomer are added to a reactor, stirred and mixed, nitrogen is passed through until the air is completely replaced, the temperature is raised to 40 to 80° C., 0.002 to 0.04 parts by weight of a water-soluble initiator is added, polymerization is carried out for 10 to 60 minutes, an aqueous solution containing 0.01 to 2 parts by weight of an alkali is added, polymerization is carried out for 0.5 to 8 hours, and the resulting emulsion is demulsified, washed, and dried to obtain an intermediate product A; 1 to 10 parts by weight of intermediate product A, 1 to 200 parts by weight of solvent, and 0.05 to 5 parts by weight of first functionalization reagent are stirred and reacted at room temperature for 2 to 12 hours; washed with solvent and the solvent is removed to obtain intermediate product B; 1-10 parts by weight of the intermediate product B, 1-200 parts by weight of a solvent, and 1-20 parts by weight of a second functionalizing agent are added to a reactor, stirred and reacted at room temperature for 2-12 hours, filtered, washed with a solvent, and the solvent removed; 1-200 parts by weight of a solvent and 1-20 parts by weight of a grafting agent are added, stirred and reacted at room temperature for 2-12 hours, washed with a solvent, and the solvent removed; 1-200 parts by weight of a solvent and 1-100 parts by weight of a chelating agent are added, stirred at room temperature until mixed, washed with a solvent, and the solvent removed; and freeze-dried below -20°C to obtain the product. Wherein, the first functionalizing agent is selected from triethylamine, propylamine, aniline, benzylamine, and cyclohexylamine; The second functionalizing agent is selected from formic acid, acetic acid, trifluoroacetic acid, hydrochloric acid, sulfuric acid, nitric acid, and HBr; The general chemical structure formula of the amphiphilic macromolecular reversible addition-fragmentation chain transfer reagent is: wherein the Z group is selected from phenyl, benzyl, methyl, ethyl, propyl, isopropyl, butyl and its isomers, pentyl and its isomers, ethoxy, methoxymercaptan, ethylmercapto, isopropylmercapto, butylmercapto, C 12 The R group is selected from 1-methylbenzyl, 1,1-dimethylbenzyl, isopropyl, 2-diisobutyric, 2-isobutyronitrile, cyanopentanoic acid, and 3-benzoic acid; the lipophilic monomer is selected from styrene, and the hydrophilic monomer is selected from acrylic acid and methacrylic acid, and the ratio of n to m is 3 to 7; The functional monomer is one or more of tert-butyl acrylate and tert-butyl methacrylate in any proportion, and the degree of polymerization of the functional monomer is greater than 100; The grafting agent has an NH2-R structure; The chelating agent is selected from: Among them, R1~R 12 According to the terminal group of the grafting agent NH2-R, the terminal group of the grafting agent NH2-R and R1~R 12 As shown in the following table:

2. The preparation method according to claim 1, wherein The molecular weight of the amphiphilic macromolecular reversible addition-fragmentation chain transfer reagent is 1,000 to 10,000.

3. The preparation method according to claim 1, wherein The water-soluble initiator is selected from persulfate and azo.

4. The preparation method according to claim 1, wherein The aqueous solution containing 0.01 to 2 parts by weight of an alkali, wherein the alkali is selected from sodium hydroxide, potassium hydroxide, aqueous ammonia, sodium carbonate, potassium carbonate, sodium bicarbonate, and potassium bicarbonate; and water is 0.1 to 1 part by weight.

5. The preparation method according to claim 1, wherein The solvent is selected from toluene, xylene, acetonitrile, dioxane, tetrahydrofuran, acetone, methyl ethyl ketone, dimethyl sulfoxide, dichloromethane, chloroform, tetrachloroethane, methanol, ethanol, isopropyl alcohol, ether, and water.

6. A metal chelate polymer antibody tag carrier material with a high degree of polymerization prepared by the preparation method according to any one of claims 1 to 5.

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