Methods for orienting antibodies to solid supports, solid supports having antibodies oriented thereto, and kits

Abstract

This application relates to the field of in vitro diagnostic technology, and particularly to a method for directionally conjugating antibodies onto a solid-phase carrier, a solid-phase carrier for antibody conjugation, and a kit, and further to a method for detecting analytes. The method involves a solid-phase carrier surface modified with a coupling group, which can react with an aldehyde group to form a covalent link. The method includes: sequentially connecting galactose and sialic acid to the Fc N-glycan terminus of an antibody to prepare an Fc N-glycan-terminated sialylated antibody; and treating the Fc N-glycan-terminated sialylated antibody with an oxidizing agent to generate an aldehyde group from the sialic acid at the Fc N-glycan terminus, and then directionally conjugating the antibody onto the surface of the solid-phase carrier by reacting with the coupling group to prepare the solid-phase carrier for antibody conjugation. This method combines high conjugation efficiency with low antibody activity loss, and the resulting solid-phase carrier for antibody conjugation significantly enhances signal response and signal-to-noise ratio when used for detection.

Description

Technical Field

[0001] This application relates to the field of in vitro diagnostic technology, and in particular to a method for directional conjugation of antibodies on a solid-phase carrier, a solid-phase carrier for conjugated antibodies and a kit, and further to a method for detecting analytes. Background Technology

[0002] Immunoassay is a detection method based on the specific binding reaction of antigen and antibody. It features high specificity, high sensitivity, and ease of operation, and is widely used in clinical diagnostics, food safety, and environmental monitoring. In immune reactions, bound immune complexes are difficult to separate from free labels. Solid-phase carriers, by immobilizing antibodies / antigens on a solid surface, achieve the physical separation of bound and free states, a crucial step in immunoassay. Taking chemiluminescence immunoassay as an example, it accounts for over 40% of the current in vitro diagnostics market, becoming one of the mainstream technologies. Chemiluminescence immunoassay is a detection method based on immune reactions, relying on the specific antigen-antibody reaction. It typically uses magnetic nanobeads coated with antigens or antibodies as the solid phase, combined with chemiluminescent substances to label antibodies or antigens. The instrument then detects the luminescence signal to quantitatively analyze the target analyte. It boasts advantages such as high sensitivity and a wide detection range, and is widely used in medical diagnostics, biological research, and drug development.

[0003] In the process of conjugating antibodies to solid-phase carriers, the common conjugation scheme targets the reaction site with the ε-amino or N-amino group of the lysine side chain on the protein surface. However, since lysine is randomly distributed across various positions on the antibody, this conjugation method has considerable randomness. When the reaction occurs at the amino group of the antigen-binding region at the Fab end of the antibody, it will affect the antibody's antigen-binding ability. With the development of bioconjugation technology, site-specific conjugation has become possible. Reported site-specific conjugation techniques include cysteine-based site-specific conjugation, insertion of non-natural amino acids, site-directed insertion of short peptide tag sequences, and conjugation of antibody glycoproteins. However, these site-specific conjugation techniques are generally used in antibody drugs and are less commonly used in the preparation of solid-phase carrier coatings.

[0004] Currently, the conventional approach for targeted conjugation involves oxidizing the hydroxyl groups on the Fc-terminal Fc N-glycan to form aldehyde groups, which are then coupled to amino groups on the surface of a solid support. Alternatively, the disulfide bonds in the antibody hinge region can be reduced to expose free thiol groups, which can then be coupled using a maleimide-based crosslinking agent to achieve targeted antibody conjugation.

[0005] In the aforementioned technology, the coupling reaction of oxidized antibody Fc N-glycan mainly involves the oxidation of the cis-vicinal diol in the Fc N-glycan structure to an aldehyde group, followed by further de-amino reactions to ultimately complete the coupling. However, only the terminal galactose and sialic acid in the antibody Fc N-glycan structure contain oxidizable cis-vicinal diol structures, and sialic acid exhibits significantly higher reactivity than galactose.

[0006] Taking IgG molecules as an example, in the IgG molecule, N-acetylglucosamine (GlcNAc) and mannose are linked by an N-glycosidic bond at the Asn-297 position of the CH2 region at the Fc end, forming an antenna-shaped Fc N-glycan backbone. Based on whether galactose is attached to both ends of the antenna, it is classified into G0, G1, and G2, representing no galactose, one galactose, and two galactose at the antenna ends, respectively. Very rarely, it contains sialic acid. It has been reported that in recombinant IgG molecules, the proportion of G0 (galactose-free) glycoforms is typically 60-80%, while G1 (containing one galactose) and G2 (containing two galactoses) are less common, and the proportion containing sialic acid is less than 5%. Therefore, during the oxidation of the antibody molecule's Fc N-glycan, the proportion of galactose and sialic acid that can be effectively oxidized is relatively small, resulting in low coupling efficiency.

[0007] In the scheme of coupling antibody hinge region disulfide bonds, because the antibody needs to be reduced, the process of treating the antibody may also reduce the disulfide bonds in the Fab region, resulting in the final coupling to Fab; in addition, the reduction treatment of the antibody will destroy the integrity of the antibody to a certain extent, which may affect the activity of the antibody. Summary of the Invention

[0008] Therefore, it is necessary to provide a method for directional conjugation of antibodies on a solid-phase carrier, a solid-phase carrier for conjugating antibodies, and a kit.

[0009] In a first aspect, a method for directionally coupling antibodies to a solid-phase support is provided, wherein the surface of the solid-phase support is modified with a coupling group, and the coupling group can react with an aldehyde group to form a covalent link;

[0010] The method includes: sequentially linking galactose and sialic acid to the Fc N-glycan terminus of the antibody to prepare an Fc N-glycan-terminated sialylated antibody; and...

[0011] The antibody with sialylated Fc N-glycan ends is treated with an oxidant to generate aldehyde groups from the sialic acid at the Fc N-glycan ends. The antibody is then directionally coupled to the surface of the solid-phase support by reacting with the coupling group, thus preparing the solid-phase support for the coupled antibody.

[0012] In an optional embodiment, the reaction of attaching galactose to the Fc N-glycan end comprises: contacting the Fc N-glycan of the antibody with β1,4-galactosyltransferase in the presence of a galactosyl donor, the galactosyl donor providing galactose residues for the enzymatic reaction.

[0013] In an optional embodiment, the β1,4-galactosyltransferase includes at least one of wild type and mutant.

[0014] In an optional embodiment, the β1,4-galactosyltransferase includes at least one of B4GALT1, B4GALT2, B4GALT3, B4GALT4, B4GALT5, B4GALT6, and B4GALT7.

[0015] In an optional embodiment, the galactosyl donor includes UPD-galactose.

[0016] In an optional embodiment, the reaction system in which galactose is attached to the Fc N-glycan ends also contains alkaline phosphatase.

[0017] In an optional embodiment, the reaction of linking sialic acid to the Fc N-glycan end comprises: contacting an antibody with galactose linked to the Fc N-glycan end with a sialate transferase in the presence of a sialate donor, wherein the sialate donor provides sialic acid residues for the enzymatic reaction; the sialate transferase is selected from at least one of α2,6-sialate transferase and α2,3-sialate transferase.

[0018] In an optional embodiment, the sialic acid donor includes cytidine 5'-monophosphate sialic acid.

[0019] In an optional embodiment, the coupling group includes at least one selected from amino, hydrazide, aminooxy, and hydroxylamine.

[0020] In an optional embodiment, the oxidant includes sodium periodate.

[0021] In an optional embodiment, the working concentration of sodium periodate is 0.5~5 mmol / L, and more preferably 0.5~1.5 mmol / L.

[0022] In an optional embodiment, the method further includes treating the Fc N-glycan-terminated sialylated antibody with an oxidant and then quenching the oxidant to terminate the oxidation reaction.

[0023] In an optional embodiment, the surface of the solid support is modified with amino groups, and the method further includes: the aldehyde group of the Fc N-glycan reacts with the amino group modified on the surface of the solid support to form a Schiff base, and then the reaction product is treated with a reducing agent to form an alkylamine bond between the Fc N-glycan and the solid support under reducing agent conditions.

[0024] In an optional embodiment, the reducing agent includes sodium borohydride.

[0025] In an optional embodiment, the working concentration of the sodium borohydride is 15~30 mmol / L.

[0026] In optional embodiments, the solid support includes: microparticles, nanoparticles, membranes, sheets, microporous plates, columns, polymer chains, and microfluidic chips.

[0027] In an optional embodiment, the solid-phase support is a microparticle, and the mass ratio of the Fc N-glycan-terminated sialylated antibody to the microparticle is (10~40) μg:1 mg.

[0028] In a further optional embodiment, the mass ratio of the Fc N-glycan-terminated sialylated antibody to the microparticle is (15~20) μg:1 mg.

[0029] In a second aspect, a solid-phase carrier for conjugating antibodies is provided, wherein the solid-phase carrier is prepared using the method for directional conjugation of antibodies to a solid-phase carrier as described in the first aspect.

[0030] Thirdly, a detection kit is provided, the detection kit comprising a solid-phase carrier of the conjugated antibody as described in the second aspect.

[0031] In an optional embodiment, the test kit contains reagents for performing at least one of the following detection methods: chemiluminescent immunoassay, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, enzyme-linked immunospot assay, immunochromatographic assay, and latex agglutination assay.

[0032] In an optional embodiment, the detection kit is a kit for performing chemiluminescent immunoassay, and further includes a chemiluminescent reagent-labeled secondary antibody, wherein the solid-phase support is magnetic microparticles.

[0033] In an optional embodiment, the analytes in the test kit include N-terminal pro-brain peptide sodium, high-sensitivity troponin, phosphorylated Tau181 protein, and cancer antigen 15-3.

[0034] Fourthly, a method for detecting an analyte is provided, comprising capturing the analyte in a sample to be tested using a solid-phase carrier of an antibody conjugated as described in the second aspect.

[0035] In an optional implementation, the detection method includes detecting the analyte in the sample to be tested using the detection kit described in the third aspect.

[0036] The method for targeted antibody conjugation on a solid-phase carrier provided in this application uses the Fc N-glycan structure at the Fc end of the antibody as the conjugated portion to the solid-phase carrier. Since the Fc N-glycan structure is far from the antigen-binding region and does not involve the antibody's amino acid sequence, it does not affect antibody activity. Furthermore, by performing a two-step processing on the antibody's Fc N-glycan portion, a sialic acid-rich Fc N-glycan structure is obtained, which is more conducive to subsequent oxidation reactions, greatly improving conjugation efficiency. During the oxidation process, only a very low amount of oxidant is required, without damaging other antibody structures and reducing their impact on antibody activity. Using the solid-phase carrier of the conjugated antibody obtained by this method, and the detection kit containing this solid-phase carrier, significantly improves signal and signal-to-noise ratio. Attached Figure Description

[0037] To more clearly illustrate the technical solutions in the embodiments and examples of this application, and to more completely understand this application and its beneficial effects, the accompanying drawings used in the description of the embodiments or examples will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of this application. Those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0038] Figure 1 Schematic diagram of the Fc N-glycan structure of IgG molecules G0, G1 and G2;

[0039] Figure 2 This is a schematic diagram of the reaction in which galactose is transferred from the substrate UDP-Gal to the Fc N-glycan terminus of β1,4-GalTβ catalyzed by the reaction.

[0040] Figure 3 This is a schematic diagram of the reaction catalyzed by α2,6-SiaT to transfer the substrate CMP-Sia sialic acid to the Fc N-glycan terminus.

[0041] Figure 4 A schematic diagram illustrating the reaction mechanism of antibody oxidation to aldehyde groups by sialylated Fc N-glycans and their reaction with a solid support.

[0042] Figure 5 The protein residue before and after the reaction of the NT-ProBNP antibody coated with magnetic beads without sialylation treatment in Example 1;

[0043] Figure 6 The protein residue in Example 1 is the amount of NT-ProBNP antibody coated with magnetic beads after sialylation treatment before and after the reaction. Detailed Implementation

[0044] The present application will be further described in detail below with reference to the accompanying drawings, embodiments, and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. For example, features described or illustrated as part of one embodiment can be combined in a suitable manner in another embodiment to produce new embodiments. Furthermore, numerous details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0045] Unless otherwise defined, 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 application belongs. The terminology used herein in the specification of this application is for descriptive purposes only and is not intended to be limiting of the application.

[0046] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0047] The terms “and / or,” “or / and,” and “and / or” as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. “Any and all combinations” includes any two related listed items, any more related listed items, or a combination of all related listed items. For example, “A and / or B” includes three parallel options: A, B, and “a combination of A and B.”

[0048] In this application, the terms "multiple", "various", "multiple times", "several", "several", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more or more.

[0049] In this application, "optionally", "optional", and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without".

[0050] In this application, the technical features or solutions described in open-ended language include both closed-ended technical features or solutions consisting of the listed contents and open-ended technical features or solutions that include the listed contents.

[0051] In this application, where the method flow involves multiple steps, unless otherwise explicitly stated herein, there is no strict order restriction on the execution of these steps; they can be executed in any order other than those described. Moreover, any step may include multiple sub-steps or multiple stages, which are not necessarily completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be performed alternately or simultaneously with other steps or parts of the sub-steps or stages of other steps.

[0052] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0053] In this application, the term "solid support" refers to a solid support used in detection technology to bind and immobilize reactants. It fixes reactants to its surface through physical adsorption or chemical coupling, and is not easily detached after repeated washing and long-term storage, without affecting the reactivity of the immobilized substances. This application does not limit the morphology of the solid support. In optional embodiments, the solid support includes, but is not limited to, microparticles, nanoparticles, membranes, sheets, microplates, columns, polymer chains, and microfluidic chips. In optional embodiments, microparticles include, but are not limited to, microspheres, microsheets, and microrods.

[0054] In this application, the term "signaling substance" refers to a substance capable of providing a detectable signal, which can be directly observed by the naked eye or detected by conventional instruments acceptable in the art. The signaling substance can directly provide a signal, such as color (e.g., colloidal gold, colored microspheres), fluorescence (fluorescent molecules), magnetism, radiation, or luminescence; it can also indirectly provide a signal through a subsequent reaction involving the signaling substance, such as a signal generated by an electrochemiluminescence reaction or a signal generated by an enzyme-catalyzed chemiluminescence reaction. Chemiluminescent reagents can emit light directly or through an enzyme-catalyzed luminescence reaction or an electrochemical reaction. Examples of chemiluminescent reagents include, but are not limited to, at least one of acridine esters and their derivatives, luminol and its derivatives, luciferin, ruthenium bipyridine and its derivatives, dioxane and its derivatives, rofenine and its derivatives, and peroxate and its derivatives. Chemiluminescent reagents may also optionally include, but are not limited to, catalytic enzymes used in enzyme-catalyzed luminescence reactions, including, but not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, and glucose oxidase. Chemiluminescent reagents may also optionally include, but are not limited to, co-reactants in electrochemiluminescence reactions, such as, but not limited to, tripropylamine.

[0055] In this application, the term "working concentration" refers to the concentration of a reagent, solution, or substance at the start of a reaction in a reaction system.

[0056] In one aspect, some embodiments provide a method for directionally coupling an antibody to a solid-phase carrier, wherein the surface of the solid-phase carrier is modified with a coupling group that can react with an aldehyde group to form a covalent link, thereby directionally coupling the antibody to the surface of the solid-phase carrier.

[0057] The method for directionally conjugating antibodies to a solid-phase carrier includes: sequentially linking galactose and sialic acid to the Fc N-glycan terminus of an antibody to prepare an Fc N-glycan-terminated sialylated antibody; and treating the Fc N-glycan-terminated sialylated antibody with an oxidizing agent to generate an aldehyde group from the sialic acid at the Fc N-glycan terminus, and then directionally conjugating the antibody to the surface of a solid-phase carrier by reacting with the conjugating group to prepare a solid-phase carrier for antibody conjugation. Optionally, the antibody directionally conjugated to the solid-phase carrier includes an IgG type antibody.

[0058] The principle of this solid-phase carrier-directed antibody conjugation method is as follows:

[0059] Fc N-glycans are N-linked oligofragments attached to conserved N-glycosylation sites on the Fc fragment (crystallizable fragment) of antibodies. They consist of a pentasaccharide core (GlcNAc2Man3) composed of two molecules of N-acetylglucosamine (GlcNAc) and three molecules of mannose (Man) (see [link to Fc N-glycan]). Figure 1Based on this, a complex dual-antenna structure is formed, with differential modifications at the ends. Depending on whether galactose is attached to the two ends of the dual antennas, they are classified into G0 (no galactose at the antenna ends), G1 (one galactose at one antenna end), and G2 (galactose at both antenna ends). Very few contain sialic acid. The structural diagrams of the Fc N-glycans in IgG molecules G0, G1, and G2 are shown below. Figure 1 As shown.

[0060] The oxidation of antibody Fc N-glycans primarily involves the oxidation of the cis-vicinal diol structure within the Fc N-glycan to form aldehyde groups. These aldehyde groups can then react with coupling groups modified on the solid support, generating covalent bonds and enabling antibody coupling to the solid support surface. In antibody Fc N-glycans, the sugars containing the classic cis-vicinal diol structure are mainly galactose and sialic acid at the Fc N-glycan terminals. In particular, the cis-vicinal diol on the glycerol side chain of sialic acid (C7-C9) is extremely sensitive to oxidation by oxidants (such as periodate) and is the most commonly used reaction site in Fc N-glycan analysis and labeling. However, due to the limitations of the antibody Fc N-glycan structure, galactose and sialic acid are present in relatively small amounts in actual antibody molecules, especially sialic acid. Therefore, the coupling efficiency is low when oxidizing antibody Fc N-glycans for coupling.

[0061] Furthermore, by sequentially linking galactose and sialic acid to the ends of the Fc N-glycan, the Fc N-glycan ends are ultimately sialic acid, providing more reaction sites for subsequent oxidation and coupling steps. It is understood that antibodies typically used for targeted conjugation with antibodies include multiple glycoforms such as G0, G1, and G2. This method involves linking galactose to the two antenna ends of a G0 type to create a G2 type, and then further linking sialic acid to both galactose antenna ends; and linking galactose to one antenna end of a G1 type to create a G2 type, and then further linking sialic acid to both galactose antenna ends. The antibody remodeled from Fc N-glycan can oxidize the cis-vicinal diol in the sialic acid structure to an aldehyde group under mild conditions with lower oxidant concentrations for use in subsequent reaction steps.

[0062] In an optional embodiment, the reaction of linking galactose to the Fc N-glycan terminus includes: contacting the antibody's Fc N-glycan with β1,4-galactosyltransferase (β1,4-GalTβ) in the presence of a galactosyl donor, which provides galactose residues for the enzymatic reaction. By using β1,4-GalTβ and a substrate galactosyl donor to galactosylate the antibody's Fc N-glycan, the antibody molecule, catalyzed by β1,4-GalTβ, uses N-acetylglucosamine at the Fc N-glycan terminus as an acceptor to transfer galactose from the galactosyl donor to the Fc N-glycan terminus. Different glycoforms (G0, G1, and G2) in the IgG molecule will be converted into the G2 glycoform containing two galactoses. In this document, β1,4-galactosyltransferase (β1,4-GalTβ) includes wild-types and mutants of its family. Exemplary β1,4-GalTβ includes, but is not limited to, B4GALT1, B4GALT2, B4GALT3, B4GALT4, B4GALT5, B4GALT6, B4GALT7, or mutants thereof, such as the B4GALT1 mutant β4GalT1-Y289L. In some alternative embodiments, the β1,4-galactosyltransferase is selected from B4GALT1.

[0063] In an optional embodiment, the galactosyl donor includes UPD-galactose (UDP-Gal), and the reaction principle is illustrated in the schematic diagram below. Figure 2 As shown.

[0064] In an optional embodiment, the reaction of attaching sialic acid to the end of Fc N-glycan includes: contacting an antibody with galactose attached to the end of the Fc N-glycan with a sialate transferase in the presence of a sialic acid donor, wherein the sialic acid donor provides sialic acid residues for the enzymatic reaction. The sialate transferase and the substrate sialic acid donor sialylate the galactosylated Fc N-glycan. Under the catalysis of the sialate transferase, the galactosylated Fc N-glycan uses the galactose at the end of the Fc N-glycan as an acceptor to transfer sialic acid from the sialic acid donor to the galactose, resulting in an Fc N-glycan with sialic acid-rich ends. The sialate transferase is selected from at least one of α2,6-sialate transferase (α2,6-SiaT) and α2,3-sialate transferase (α2,3-SiaT), preferably α2,6-sialate transferase.

[0065] In an optional embodiment, the sialic acid donor includes cytidine 5'-monophosphate sialic acid (CMP-Sia), and the reaction principle is illustrated in the schematic diagram below. Figure 3 As shown.

[0066] In an optional embodiment, the oxidant includes sodium periodate.

[0067] In optional embodiments, the working concentration of sodium periodate is 0.5~5 mmol / L, for example, but not limited to 0.5 mmol / L, 1 mmol / L, 1.5 mmol / L, 2 mmol / L, 2.5 mmol / L, 3 mmol / L, 3.5 mmol / L, 4 mmol / L, 4.5 mmol / L or 5 mmol / L, further optionally 0.5~1.5 mmol / L, and further optionally 1 mmol / L.

[0068] In an optional embodiment, the method further includes treating the Fc N-glycan-terminated sialylated antibody with an oxidant and then quenching the oxidant to terminate the oxidation reaction.

[0069] In an optional embodiment, the oxidant is sodium periodate, and sodium periodate is quenched with ethylene glycol.

[0070] In an optional embodiment, the reaction system in which galactose is linked to the N-glycan of Fc also contains alkaline phosphatase. Alkaline phosphatase is used to hydrolyze the byproduct UDP produced in the reaction, thereby relieving UDP's inhibition of β1,4-GalT production.

[0071] In optional embodiments, the coupling groups modified on the surface of the solid support include, but are not limited to, at least one of amino (-NH2), hydrazide (-NHNH2), aminooxy (-ONH2), and hydroxylamine (-NHOH).

[0072] In an optional embodiment, the surface of the solid-phase support is modified with amino groups. The method further includes reacting the aldehyde groups of Fc N-glycan with the amino groups modified on the solid-phase support surface to form a Schiff base, followed by treatment of the reaction product with a reducing agent. Under reducing agent conditions, stable alkylamine bonds are formed between the Fc N-glycan and the solid-phase support, achieving the directional coupling of the Fc N-glycan portion of the antibody to the solid-phase support. The reaction principle is as follows: Figure 4 As shown.

[0073] In an optional embodiment, the reducing agent includes sodium borohydride.

[0074] In an optional embodiment, the working concentration of sodium borohydride is 15 to 30 mmol / L, for example, but not limited to 15 mmol / L, 20 mmol / L, 25 mmol / L or 30 mmol / L, and more preferably 20 mmol / L.

[0075] In an optional embodiment, the solid-phase carrier is a microparticle, and the mass ratio of the Fc N-glycan-terminated sialylated antibody to the microparticle is (10~40) μg:1 mg, for example, but not limited to, 5 μg:1 mg, 10 μg:1 mg, 15 μg:1 mg, 20 μg:1 mg, 25 μg:1 mg, 30 μg:1 mg, 35 μg:1 mg or 40 μg:1 mg, preferably (15~20) μg:1 mg.

[0076] In an optional embodiment, the method for directionally conjugating antibodies to a solid-phase carrier includes the following steps:

[0077] (a) The Fc N-glycan of the antibody is contacted with β1,4-GalTβ in the presence of UDP-Gal, so that galactose residues are attached to the end of the Fc N-glycan;

[0078] (b) The Fc N-glycan of the antibody treated with the enzyme in step (a) is contacted with α2,6-SiaT in the presence of CMP-Sia, so that sialic acid is attached to the galactose added to the end of the Fc N-glycan in step (a), so that the end of the Fc N-glycan is sialic acid.

[0079] (c) The Fc N-glycan of the antibody treated in step (b) is oxidized with sodium periodate to oxidize the cis-vicinal diol structure of sialic acid to form an aldehyde group;

[0080] (d) The antibody obtained in step (c) is reacted with a solid-phase support with surface-modified amino groups to form a Schiff base, and then a stable alkylamine bond is formed under reducing conditions to achieve directional coupling of the Fc N-glycan portion of the antibody to the solid-phase support.

[0081] Secondly, some embodiments provide a solid-phase carrier for conjugated antibodies, which is prepared using the solid-phase carrier-directed antibody conjugation method of the first aspect. The surface of the solid-phase carrier is directionally conjugated with Fc N-glycans at the Fc end of the antibody, which can fully expose the antigen-binding region of the antibody and significantly improve the signal and signal-to-noise ratio.

[0082] Thirdly, a detection kit is provided that comprises a solid-phase carrier of the conjugated antibody of the second aspect.

[0083] In an optional embodiment, the test kit further comprises reagents for performing at least one of the following detection methods: chemiluminescent immunoassay, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, enzyme-linked immunospot assay, immunochromatographic assay, and latex agglutination assay.

[0084] In an optional implementation, the test kit may also contain a signaling agent.

[0085] In an optional implementation, the detection kit is used to perform chemiluminescent immunoassay and also includes chemiluminescent reagents.

[0086] In an optional embodiment, the test kit further comprises a signal-labeled secondary antibody that binds to different antigenic epitopes of the same antigen with an antibody directionally conjugated on a solid-phase support.

[0087] In an optional embodiment, the test kit further includes reagents for implementing immunoassay technology. Exemplary reagents for implementing immunoassay technology include, but are not limited to, at least one of buffer, washing solution, chromogenic substrate, stop solution, blocking solution, diluent, standard and quality control.

[0088] In an optional embodiment, the detection kit is a kit for performing chemiluminescent immunoassay and further includes a chemiluminescent reagent-labeled secondary antibody, wherein the solid-phase support is magnetic microparticles.

[0089] In an optional embodiment, the analytes in the test kit include N-terminal pro-brain peptide sodium, high-sensitivity troponin, phosphorylated Tau181 protein, and cancer antigen 15-3.

[0090] Fourthly, an immunoassay method for an analyte is also provided, the method comprising capturing the analyte in a test sample using a solid-phase carrier of an antibody conjugated to the test sample, as described in the second aspect.

[0091] In optional embodiments, the immunoassay method may also integrate conventional immunoassay procedures in the art, which typically cover key technical aspects such as sample preparation, immune reaction, signal detection, and result interpretation. Exemplary operational steps include, but are not limited to: in the sample preparation stage, pretreatment methods for the sample to be tested (such as centrifugation, filtration, lysis, or purification), sample dilution strategies to optimize the linear range of detection, and dissociation or denaturation steps required for bound analytes; in the immunoreaction stage, in addition to the aforementioned solid-phase capture, multi-step incubation reactions, sequential addition of immunoassay reagents at each stage, and washing and separation steps using a suitable buffer system between reaction steps to remove non-specific bindings; in the signal detection stage, specific binding reactions with signal substances, such as chemiluminescent reagents, enzyme markers, fluorescent probes, or radioisotopes, and one or more steps to generate detectable signals from the signal substances through substrate color development, light excitation, chemiluminescence reactions, or radiometric counting; furthermore, the method may include quality control procedures (such as standard curve establishment, internal control, and quality control product verification) and steps for qualitative, semi-quantitative, or quantitative analysis based on signal intensity, thereby constructing a complete immunoassay technology solution.

[0092] In an optional implementation, the detection method includes using a third-party detection kit to detect the analyte in the sample to be tested.

[0093] In an optional implementation, the detection method is not for diagnostic or therapeutic purposes.

[0094] The following are some examples.

[0095] The embodiments of this application will be described in detail below with reference to some examples. It should be understood that these embodiments are only for illustrating this application and are not intended to limit the scope of this application. For experimental methods in the following embodiments where conditions are not specified, please refer to the guidelines given in this application first, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0096] In the following examples, the measurement parameters of the raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0097] Example 1

[0098] This embodiment provides an NT-ProBNP (amino-terminal pro-brain peptide sodium) detection kit:

[0099] 1. Key raw material information:

[0100] a. Antibody: NT-ProBNP antibody (manufacturer: Boyue, catalog number: NT-ProBNP101).

[0101] b. Enzyme reagents and substrates: B4GALT1 (manufacturer: Aladdin, catalog number: G293642) and substrate UDP-Gal sodium salt (manufacturer: Aladdin, catalog number: U303352); α2,6-SiaT (manufacturer: Aladdin, catalog number: S1439869) and substrate CMP-Sia (sodium salt) (manufacturer: Aladdin, catalog number: C1443710).

[0102] c. Alkaline phosphatase (manufacturer: Aladdin, product number: P755419).

[0103] d. Magnetic beads: Surface-modified amino magnetic beads (manufacturer: Merck, part number: EM2-100 / 40).

[0104] e. Enzyme reaction buffer: 50 mmol / L HEPES, pH 7.5, 20 mmol / L MnCl2.

[0105] f. Ethylene glycol (Manufacturer: Sigma, Product No.: 102466-500ML)

[0106] 2. Fc N-glycan galactosylation:

[0107] a. Take 1 mg of NT-ProBNP antibody, add it to enzyme reaction buffer to a final volume of 425 μL, and mix well.

[0108] b. Transfer to ice, add 25 μL of 0.1 M UDP-Gal sodium salt, and gently blow to mix.

[0109] c. Finally, add 25 μL of 20 U / mL β1,4-GalT enzyme and 25 μL of 200 U / mL alkaline phosphatase, and gently mix.

[0110] d. Immediately transfer to a 37°C constant temperature roller mixer and react for 4 hours.

[0111] e. After the reaction is complete, add 5 μL of 0.5M EDTA and purify it using an ultrafiltration tube (50KD, 2 mL).

[0112] 3. Fc N-glycan sialylation:

[0113] a. Transfer the galactosylated NT-ProBNP antibody to ice, add enzyme reaction buffer to 465 μL, add 10 μL of 50 mmol / L substrate CMP-Sia, and gently mix.

[0114] b. Immediately add 25 μL of 10 U / mL α2,6-SiaT enzyme and gently mix.

[0115] c. Immediately transfer to a 37°C constant temperature roller mixer and react for 4 hours.

[0116] d. After the reaction is complete, add 5 μL of 0.5M EDTA and purify it using an ultrafiltration tube (50KD, 2 mL).

[0117] 4. Antibody-conjugated magnetic beads remodeled from Fc N-glycans:

[0118] a. Take 0.1 mg of the antibody prepared in step 3 in three portions, dilute with MES buffer at pH 5.0 to 1.0 mg / mL, add sodium periodate to make the final concentrations 0.5 mmol / L, 1 mmol / L and 5 mmol / L respectively, and react at 25°C in the dark for 0.5 h.

[0119] b. After the reaction is complete, add 5 μL of ethylene glycol (10%) and continue the reaction for 0.5 h.

[0120] c. Take three 5mg magnetic beads, wash the magnetic beads three times with coupling buffer (pH 9.5 borate buffer), and then resuspend the magnetic beads with 0.5mL coupling buffer.

[0121] d. After the reaction in step b is complete, add it to the magnetic beads in step c, mix well, and place in a constant temperature oven at 2-8℃ for 2 hours of roller reaction.

[0122] e. After the reaction is complete, add reducing agent (sodium borohydride) to a final concentration of 20 mmol / L and continue the reaction for 1 hour.

[0123] f. After the reaction is complete, wash twice with washing buffer (150 mmol / L phosphate containing 1% Tween 20).

[0124] g. After washing, the coated magnetic beads were blocked with blocking buffer (150 mmol / L phosphate) containing BSA for 2 hours to obtain the magnetic bead coating.

[0125] 5. Preparation of control group magnetic bead coating: Following the method in step 4, the unsalicylated NT-ProBNP antibody was treated with sodium periodate at a final concentration of 1.0 mmol / L, and then coated onto magnetic beads to obtain the control group magnetic bead coating.

[0126] 6. Reagent kit:

[0127] This kit contains the magnetic bead coating prepared in step 4 above, combined with an acridil ester-labeled specific antibody complex. The kit utilizes a double-antibody sandwich detection principle: the antibody coated on the coating captures the analyte in the test sample, and the acridil ester-labeled specific antibody, as the secondary antibody, binds to the antigen captured by the antibody coated on the magnetic beads, forming an acridil ester-labeled specific antibody-analyte-antibody-magnetic bead complex. The luminescence intensity of the acridil ester is used for qualitative, semi-quantitative, or quantitative analysis of the test sample. The difference between the magnetic beads in the control kit and those in this kit is that the control kit contains magnetic beads coated with untreated antibodies prepared using the method in step 5 above.

[0128] The ista500 fully automated chemiluminescence analyzer from Shenzhen Zhuorun Biotechnology Co., Ltd. was used for fully automated detection. Each sample was tested three times, and the average value was taken. The signal-to-noise ratio was used as the ratio of the previous gradient luminescence value to the current concentration. The test data are as follows:

[0129] Table 1

[0130]

[0131] The test results in the table above show that the signal level varies under different oxidant concentrations. The best signal level is achieved when the concentration of sodium periodate is 1.0 mmol / L. In addition, compared with the untreated antibody, the signal is significantly improved, the signal-to-noise ratio is improved, the linear range of the test is wider, and the test is more accurate.

[0132] Meanwhile, during the antibody coating process with magnetic beads, to understand the antibody coating efficiency, the amount of residual protein after the antibody-magnetic bead reaction was detected by HPLC (manufacturer: Shimadzu, model: LC-2050C3D). Combined with the amount of protein added before the reaction, the antibody coupling efficiency was calculated. The HPLC results of the residual protein before and after the reaction in the experimental group and the control group treated with 1.0 mmol / L sodium periodate oxidation are shown below. Figure 5 and Figure 6 And as shown in Table 2:

[0133] Table 2

[0134]

[0135] The above liquid phase results show that when the NT-ProBNP antibody is enzymatically remodeled to reshape the Fc N-glycan and sialylate the Fc N-glycan, the coupling efficiency of the antibody is significantly improved in the process of coupling amino magnetic beads by oxidizing the Fc N-glycan.

[0136] Example 2

[0137] This embodiment provides a kit for detecting high-sensitivity troponin (hs-cTnI), and the specific implementation steps are as follows:

[0138] 1. Key raw material information:

[0139] a. Antibody: hs-cTnI antibody (manufacturer: Heavy Chain, catalog number: HA101-11H).

[0140] b. Enzyme reagents and substrates: B4GALT1 (manufacturer: Aladdin, catalog number: G293642) and substrate UDP-Gal sodium salt (manufacturer: Aladdin, catalog number: U303352); α2,6-SiaT (manufacturer: Aladdin, catalog number: S1439869) and substrate CMP-Sia (sodium salt) (manufacturer: Aladdin, catalog number: C1443710).

[0141] c. Alkaline phosphatase (manufacturer: Aladdin, product number: P755419).

[0142] d. Magnetic beads: Carboxyl magnetic beads (manufacturer: Dynabeads, item number: 35401).

[0143] e. Modifier: Polyethylene glycol diamine (manufacturer: Aladdin, product number: P432394).

[0144] f. Enzyme reaction buffer: 50 mmol / L HEPES, pH 7.5, 20 mmol / L MnCl2.

[0145] g. Ethylene glycol (manufacturer: Sigma, item number: 102466-500ML).

[0146] 2. Fc N-glycan galactosylation:

[0147] Take 1 mg of hs-cTnI antibody, and perform Fc N-glycan galactosylation using the same method as step 2 in Example 1.

[0148] 3. Fc N-glycan sialylation:

[0149] The galactosylated hs-cTnI antibody was sialylated according to step 3 of Example 1.

[0150] 4. Amination of magnetic beads:

[0151] a. Take 30 mg of magnetic beads, wash 3 times with coating buffer (15 mmol / L MES, pH 6.0), and resuspend the magnetic beads in 1.2 mL of coating buffer (15 mmol / L MES, pH 6.0).

[0152] b. Add 0.6 mL of activator (0.3 mL each of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide sulfonate sodium salt, dissolved in DMSO to a concentration of 10 mg / mL) and mix and react at 25 °C for 30 min.

[0153] c. After the reaction is complete, perform magnetic separation, wash once with coating buffer (15 mmol / L MES, pH 6.0), resuspend in 3.0 mL of coating buffer (15 mmol / L MES, pH 6.0), add 0.6 mg of polyethylene glycol diamine (water-soluble), and mix and react at 25 °C for 3 h.

[0154] d. After the reaction is complete, wash twice with washing buffer (150 mmol / L phosphate containing 1% Tween 20).

[0155] e. After washing, store in washing buffer (150 mmol / L phosphate containing 1% Tween 20).

[0156] 5. Fc N-glycan-remodeled antibody-coupled amino-modified magnetic beads:

[0157] Take 0.1 mg of hs-cTnI antibody treated with Fc N-glycan sialylation, and couple it to the aminated magnetic beads prepared in step 4 of this example according to step 4 of Example 1 (selecting a sodium periodate concentration of 1.0 mmol / L), and finally obtain the magnetic bead coating.

[0158] 6. Preparation of control group magnetic bead coating: The hs-cTnI antibody without sialylation treatment was coated onto magnetic beads according to the method in step 5 to obtain the control group magnetic bead coating.

[0159] 7. Reagent kit:

[0160] This kit contains the magnetic bead coating prepared in step 5 above, combined with a complex of acridinium ester-labeled specific antibody. The detection principle of this kit is the same as in Example 1. The difference between the magnetic beads in the control kit and the kit provided in this example is that the magnetic bead coating in the control kit is coated with untreated antibody-coated magnetic beads prepared using the method in step 6 above. Fully automated detection was performed using an istar500 fully automated chemiluminescence analyzer from Shenzhen Zhuorun Biotechnology Co., Ltd. Each sample was tested three times, and the average value was taken. The signal-to-noise ratio was used as the ratio of the previous gradient luminescence value to the current concentration. The test data are as follows:

[0161] Table 3

[0162]

[0163] The test results in the table above show that in the process of coupling the oxidized antibody Fc N-glycan to the amino-modified magnetic beads, when the hs-cTnI antibody is enzymatically treated to reshape the Fc N-glycan and sialylate it, the signal is significantly improved compared to the unsialylated antibody, and the low-end sensitivity is improved.

[0164] Example 3

[0165] This embodiment provides a P-Tau181 (phosphorylated Tau181 protein) detection kit, which is prepared according to the following method:

[0166] 1. Key raw material information:

[0167] a. Antibody: P-Tau181 antibody (manufacturer: Medix, catalog number: R13321 SPTN-5).

[0168] b. Enzyme reagents and substrates: B4GALT1 (manufacturer: Aladdin, catalog number: G293642) and substrate UDP-Gal sodium salt (manufacturer: Aladdin, catalog number: U303352); α2,6-SiaT (manufacturer: Aladdin, catalog number: S1439869) and substrate CMP-Sia (sodium salt) (manufacturer: Aladdin, catalog number: C1443710).

[0169] c. Alkaline phosphatase (manufacturer: Aladdin, product number: P755419).

[0170] d. Magnetic beads: Carboxyl magnetic beads (manufacturer: Dynabeads, item number: 35401).

[0171] e. Modifier: Polyethylene glycol diamine (manufacturer: Aladdin, product number: P432394).

[0172] f. Enzyme reaction buffer: 50 mmol / L HEPES, pH 7.5, 20 mmol / L MnCl2.

[0173] g. Ethylene glycol (manufacturer: Sigma, item number: 102466-500ML).

[0174] 2. Fc N-glycan galactosylation:

[0175] Take 1 mg of P-Tau181 antibody and perform Fc N-glycan galactosylation as described in step 2 of Example 1.

[0176] 3. Fc N-glycan sialylation:

[0177] The galactosylated P-Tau181 antibody was added to an enzyme reaction buffer to a volume of 465 μL, and the subsequent steps were the same as step 3 of Example 1 for sialylation.

[0178] 4. Amination of magnetic beads:

[0179] a. Take 100 mg of magnetic beads, wash 3 times with coating buffer (15 mmol / L MES, pH 6.0), and resuspend the magnetic beads in 4 mL of coating buffer (15 mmol / L MES, pH 6.0).

[0180] b. Add 2 mL of activator (1.0 mL each of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide sulfonate sodium salt, dissolved in DMSO to 10 mg / mL) and mix and react at 25 °C for 30 min.

[0181] c. After the reaction is complete, perform magnetic separation, wash once with coating buffer (15 mmol / L MES, pH 6.0), resuspend in 10 mL of coating buffer (15 mmol / L MES, pH 6.0), add 2 mg of polyethylene glycol diamine (water-soluble), and mix and react at 25°C for 3 h.

[0182] d. After the reaction is complete, wash twice with washing buffer (150 mmol / L phosphate containing 1% Tween 20).

[0183] e. After washing, store in washing buffer (150 mmol / L phosphate containing 1% Tween 20).

[0184] 5. Fc N-glycan-remodeled antibody-coupled amino-modified magnetic beads:

[0185] a. Take four portions of Fc N-glycan sialylated antibody, namely 0.05 mg, 0.1 mg, 0.15 mg, and 0.2 mg, respectively, dilute them to 1.0 mg / mL with MES buffer at pH 5.0, add sodium periodate to a final concentration of 1 mmol / L, and react at 25°C in the dark for 0.5 h.

[0186] b. After the reaction is complete, add 5 μL of ethylene glycol (10%) and continue the reaction for 0.5 h.

[0187] c. Take 4 portions of the amino-modified magnetic beads prepared in step 4, each 10 mg, wash the magnetic beads 3 times with coupling buffer (pH 9.5 borate buffer), and then resuspend the magnetic beads in 1 mL of coupling buffer.

[0188] d. After the reaction in step b is complete, add the processed antibody to the magnetic beads in step c, mix well, and place in a constant temperature incubator at 25°C for 2 hours of roller reaction.

[0189] e. After the reaction is complete, add reducing agent (sodium borohydride) to a final concentration of 20 mmol / L and continue the reaction for 1 hour.

[0190] f. After the reaction is complete, wash twice with washing buffer (150 mmol / L phosphate containing 1% Tween 20).

[0191] g. After washing, the coated magnetic beads were blocked with blocking buffer (150 mmol / L phosphate) containing BSA for 2 hours to obtain the magnetic bead coating.

[0192] 6. Preparation of coating material for control group magnetic beads:

[0193] a. Take 4 portions of magnetic beads, each 10 mg, wash 3 times with coating buffer (15 mmol / L MES, pH 6.0), and resuspend the magnetic beads in 0.4 mL of coating buffer (15 mmol / L MES, pH 6.0).

[0194] b. Add 0.2 mL of activator (0.1 mL each of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide sulfonate sodium salt, dissolved in DMSO to 10 mg / mL) and mix and react at 25 °C for 30 min.

[0195] c. After the reaction is complete, magnetic separation is performed, and the mixture is washed once with coating buffer (15 mmol / L MES, pH 6.0). The mixture is then resuspended in 1.0 mL of coating buffer (15 mmol / L MES, pH 6.0), and 0.1 mg, 0.2 mg, 0.3 mg, and 0.4 mg of P-Tau antibody (without sialylation treatment) are added respectively. The mixture is then placed at 25 °C for 3 h to react.

[0196] d. After the reaction is complete, wash twice with washing buffer (150 mmol / L phosphate containing 1% Tween 20).

[0197] e. After washing, the coated magnetic beads are blocked with blocking buffer containing BSA (150 mmol / L phosphate) for 2 hours to obtain the magnetic bead coating.

[0198] 7. Reagent kit:

[0199] This kit contains the magnetic bead coating prepared in step 5 above, combined with a complex of acridinium ester-labeled specific antibody. The detection principle of this kit is the same as in Example 1. The difference between the magnetic beads in the control kit and the kit provided in this example is that the magnetic bead coating in the control kit is coated with untreated antibody-coated magnetic beads prepared using the method in step 6 above. Fully automated detection was performed using an istar500 fully automated chemiluminescence analyzer from Shenzhen Zhuorun Biotechnology Co., Ltd. Each sample was tested three times, and the average value was taken. The signal-to-noise ratio was used as the ratio of the previous gradient luminescence value to the current concentration. The test data are as follows:

[0200] Table 4

[0201]

[0202] The results showed that, compared with the conventional conjugation method that did not involve sialylation, the P-Tau181 antibody, after enzymatic remodeling of Fc N-glycan and sialylation of Fc N-glycan, could achieve the same signal level as the conventional conjugation method with a lower coating amount and had a better signal-to-noise ratio.

[0203] Example 4

[0204] This embodiment provides a CA153 (cancer antigen 15-3) detection kit, which is prepared according to the following method:

[0205] 1. Raw material information:

[0206] a. Antibody: CA153 antibody (manufacturer: Phytotech, catalog number: CA153-REAB-C1-004).

[0207] b. Enzyme reagents and substrates: B4GALT1 (manufacturer: Aladdin, product number: G293642) and substrate UDP-Gal sodium salt (manufacturer: Aladdin, product number: U303352); α2,6-SiaT (manufacturer: Aladdin, product number: S1439869) and substrate CMP-Sia (sodium salt) (manufacturer: Aladdin, product number: C1443710).

[0208] c. Alkaline phosphatase (manufacturer: Aladdin, product number: P755419).

[0209] d. Magnetic beads: Carboxyl magnetic beads (manufacturer: Dynabeads, part number: 35401).

[0210] e. Modifier: Polyethylene glycol diamine (manufacturer: Aladdin, product number: P432394).

[0211] f. Enzyme reaction buffer: 50 mmol / L HEPES, pH 7.5, 20 mmol / L MnCl2.

[0212] g. Ethylene glycol (manufacturer: Sigma, item number: 102466-500ML).

[0213] 2. Fc N-glycan galactosylation:

[0214] Take 1 mg of CA153 antibody and perform Fc N-glycan galactosylation using the same method as step 2 in Example 1.

[0215] 3. Fc N-glycan sialylation:

[0216] The galactosylated CA153 antibody was added to an enzyme reaction buffer to a volume of 465 μL, and the subsequent steps were the same as step 3 of Example 1 for sialylation.

[0217] 4. Amination of magnetic beads:

[0218] 100 mg of polyethylene glycol-modified magnetic beads were prepared, and the experimental steps were carried out according to step 4 of Example 3.

[0219] 5. Fc N-glycan-remodeled antibody-coupled amino-modified magnetic beads:

[0220] a. Take 0.2 mg of the glycan-treated antibody in 4 portions, add sodium periodate to the portions, and react them at 25°C in the dark for 0.5 h to achieve final concentrations of 0.5 mmol / L, 1 mmol / L, 5 mmol / L, and 10 mmol / L respectively.

[0221] b. After the reaction is complete, add 5 μL of ethylene glycol (10% aqueous solution) and continue the reaction for 0.5 h.

[0222] c. Take 4 portions of 10 mg of the above amino-modified magnetic beads, wash the magnetic beads 3 times with coupling buffer (pH 9.5 borate buffer), and then resuspend the magnetic beads with 1 mL of coupling buffer.

[0223] d. After the reaction in step b is complete, add it to the magnetic beads in step c, mix well, and place in a constant temperature oven at 25°C for 2 hours of roller reaction.

[0224] e. After the reaction is complete, add reducing agent (sodium borohydride) to a final concentration of 20 mmol / L and continue the reaction for 1 hour.

[0225] f. After the reaction is complete, wash twice with washing buffer (150 mmol / L phosphate containing 1% Tween 20).

[0226] g. After washing, the coated magnetic beads were blocked with blocking buffer containing BSA (150 mmol / L phosphate) for 2 hours to obtain the magnetic bead coating.

[0227] 6. Preparation of magnetic bead coating material for control group 1:

[0228] Following the method in step 5, the unsalicylated CA153 antibody was coated onto magnetic beads to obtain the control group magnetic bead coating.

[0229] 7. Preparation of the coating material for control group 2 magnetic beads:

[0230] a. Take 5 portions of 0.2 mg untreated antibody, add sodium periodate to the portions, and the final concentrations are 0 mmol / L, 0.5 mmol / L, 1.0 mmol / L, 5.0 mmol / L and 10 mmol / L, respectively. React at 25°C in the dark for 0.5 h.

[0231] b. After the reaction is complete, add 5 μL of ethylene glycol (10% aqueous solution) and continue the reaction for 0.5 h.

[0232] c. Take 5 magnetic beads, 10 mg each, wash 3 times with coating buffer (15 mmol / L MES, pH 6.0), and resuspend the magnetic beads in 0.4 mL of coating buffer (15 mmol / L MES, pH 6.0).

[0233] d. Add 0.2 mL of activator (0.1 mL each of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide sulfonate sodium salt, dissolved in DMSO to 10 mg / mL) and mix and react at 25 °C for 30 min.

[0234] e. After the reaction is complete, perform magnetic separation, wash once with coating buffer (15 mmol / L MES, pH 6.0), resuspend in 1.0 mL of coating buffer (15 mmol / L MES, pH 6.0), add CA153 antibody treated in step b in sequence, and mix and react at 25°C for 3 h.

[0235] f. After the reaction is complete, wash twice with washing buffer (150 mmol / L phosphate containing 1% Tween 20).

[0236] g. After washing, the coated magnetic beads were blocked with blocking buffer (150 mmol / L phosphate) containing BSA for 2 hours to obtain the magnetic bead coating.

[0237] 7. Reagent kit:

[0238] This kit contains the magnetic bead coating prepared in step 5 above, combined with a complex of acrid ester-labeled specific antibody. The detection principle of this kit is the same as in Example 1.

[0239] The difference between the magnetic beads in the control kit 1 and the kit provided in this embodiment is that the magnetic beads in the control kit 1 are coated with untreated antibody-coated magnetic beads prepared by the method in step 6 above.

[0240] The difference between the magnetic beads in control kit 2 and the kit provided in this embodiment is that the magnetic beads contained are prepared by the method in step 7 above. The coating of the magnetic beads is an antibody coated with an oxidized material using a conventional coupling scheme. In this scheme, the magnetic beads are not aminated, and the antibody is mainly coupled to the activated magnetic beads through the amino group on the lysine residue.

[0241] The ista500 fully automated chemiluminescence analyzer from Shenzhen Zhuorun Biotechnology Co., Ltd. was used for fully automated detection. Each sample was tested three times, and the average value was taken. The signal-to-noise ratio was used as the ratio of the previous gradient luminescence value to the current concentration. The test data are as follows:

[0242] Table 5

[0243]

[0244] The test results in the table above show that in the process of coupling antibody glycans to amino-modified magnetic beads by oxidizing the antibody, when the CA153 antibody is enzymatically treated to reshape the glycans, it achieves a better signal level at a lower concentration (1 mmol / L NaIO4) compared to the untreated antibody. This indicates that the oxidation efficiency is improved after the FC-terminal glycans of the antibody are sialylated, which is more conducive to the subsequent coupling reaction and results in a higher signal.

[0245] Table 6

[0246]

[0247] In conventional conjugation processes, the signal level is affected to some extent when antibodies are treated with different concentrations of oxidant. Different oxidant concentrations have varying degrees of impact on antibody activity. The test results in the table above show that when the oxidant concentration is in the range of 0-1 mmol / L, the signal level does not change significantly. When the oxidant concentration is increased to 5 mmol / L, the signal level decreases significantly, indicating that at this concentration, it begins to affect antibody activity. When the oxidant concentration is increased to 10 mmol / L, the signal level decreases significantly, indicating that the oxidant at this concentration significantly damages the antibody and affects its activity.

[0248] Based on the data in Table 5 above, it can be seen that after the antibody is treated with enzymes, the glycan ends are rich in sialic acid, which can ensure oxidation efficiency even at a low concentration of oxidant, without destroying the activity of the antibody. Therefore, the oxidant can achieve good signal and signal-to-noise ratio at a low concentration.

[0249] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0250] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A method for directional conjugation of antibodies on a solid-phase support, characterized in that, The surface of the solid support is modified with coupling groups, which can react with aldehyde groups to form covalent bonds. The method includes: sequentially linking galactose and sialic acid to the Fc N-glycan terminus of the antibody to prepare an Fc N-glycan-terminally sialylated antibody; and... The antibody with sialylated Fc N-glycan ends is treated with an oxidant to generate aldehyde groups from the sialic acid at the Fc N-glycan ends. The antibody is then directionally coupled to the surface of the solid-phase support by reacting with the coupling group, thus preparing the solid-phase support for the coupled antibody.

2. The method for directional conjugation of antibodies on a solid-phase support according to claim 1, characterized in that, The reaction of attaching galactose to the Fc N-glycan end includes: contacting the Fc N-glycan of the antibody with β1,4-galactosyltransferase in the presence of a galactosyl donor, which provides galactose residues for the enzymatic reaction; Optionally, the β1,4-galactosyltransferase includes at least one of wild type and mutant; Optionally, the β1,4-galactosyltransferase includes at least one of B4GALT1, B4GALT2, B4GALT3, B4GALT4, B4GALT5, B4GALT6 and B4GALT7; Optionally, the galactosyl donor includes UPD-galactose; Optionally, the reaction system in which galactose is attached to the end of the Fc N-glycan also contains alkaline phosphatase.

3. The method for directional conjugation of antibodies on a solid-phase support according to claim 1, characterized in that, The reaction of linking sialic acid to the Fc N-glycan end comprises: contacting an antibody with galactose linked to the Fc N-glycan end with a sialate transferase in the presence of a sialate donor, wherein the sialate donor provides sialic acid residues for the enzymatic reaction; the sialate transferase is selected from at least one of α2,6-sialate transferase and α2,3-sialate transferase; Optionally, the sialic acid donor includes cytidine 5'-monophosphate sialic acid.

4. The method for directional conjugation of antibodies on a solid-phase support according to claim 1, characterized in that, The coupling group includes at least one of amino, hydrazide, aminooxy, and hydroxylamine. Optionally, the oxidant includes sodium periodate; Optionally, the working concentration of sodium periodate is 0.5~5 mmol / L, and more preferably 0.5~1.5 mmol / L; Optionally, the method further includes treating the Fc N-glycan-terminated sialylated antibody with an oxidant and then quenching the oxidant to terminate the oxidation reaction.

5. The method for directional conjugation of antibodies on a solid-phase support according to claim 4, characterized in that, The solid support surface is modified with amino groups, and the method further includes: the aldehyde group of the Fc N-polysaccharide reacts with the amino group modified on the surface of the solid support to form a Schiff base, and then the reaction product is treated with a reducing agent to form an alkylamine bond between the Fc N-polysaccharide and the solid support under reducing agent conditions; Optionally, the reducing agent includes sodium borohydride; Optionally, the working concentration of the sodium borohydride is 15~30 mmol / L.

6. The method for directional conjugation of antibodies on a solid-phase support according to any one of claims 1 to 5, characterized in that, The solid support includes: microparticles, nanoparticles, membranes, sheets, microporous plates, columns, polymer chains, and microfluidic chips; Optionally, the solid-phase support is a microparticle, and the mass ratio of the Fc N-glycan-terminated sialylated antibody to the microparticle is (10~40) μg:1 mg; Further optionally, the mass ratio of the Fc N-glycan-terminated sialylated antibody to the microparticle is (15~20) μg: 1 mg.

7. A solid-phase support for conjugated antibodies, characterized in that, The antibody was prepared using the method of solid-phase carrier directional conjugation as described in any one of claims 1 to 6.

8. A test kit, characterized in that, Solid-phase carriers including the conjugated antibodies of claim 7.

9. The detection kit according to claim 8, characterized in that, The test kit contains reagents for performing at least one of the following detection methods: chemiluminescent immunoassay, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, enzyme-linked immunospot assay, immunochromatographic assay, and latex agglutination assay. Optionally, the detection kit is a kit for performing chemiluminescent immunoassay, and further includes a secondary antibody labeled with a chemiluminescent reagent, wherein the solid-phase support is magnetic microparticles; Optionally, the analytes in the test kit include N-terminal pro-brain peptide sodium, high-sensitivity troponin, phosphorylated Tau181 protein, and cancer antigen 15-3.

10. A method for detecting an analyte, characterized in that, This includes using a solid-phase carrier containing the conjugated antibody as described in claim 7 to capture the analyte in the sample to be tested; Optionally, the detection method includes detecting the analyte in the sample to be tested using the detection kit of claim 8 or 9.

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