Method for Orienting Enzyme-Labeled Antibody and Its Application

Abstract

The present invention relates to the technical field of immunological detection, and particularly to a method for directionally enzyme-labeling antibodies and its application. In the present invention, cysteine residues are inserted through site-directed mutagenesis at the CH1 position of the heavy chain of the antibody by Thiomab technology, and then through reduction and re-oxidation, the sulfhydryl groups in the cysteine residues are freed, and an SMCC-activated enzyme is added for labeling, thereby obtaining an enzyme directionally labeled antibody. The labeling rate can be as high as over 95%. The prepared directionally labeled antibody can significantly reduce the detection limit of low-concentration samples, and greatly reduce the probability of false positives, and can be widely applied.

Description

Technical Field

[0001] The present invention relates to the technical field of immunological detection, and particularly to a method for directionally enzyme-labeling antibodies and its application. Background Art

[0002] Enzyme immunoassay combines the high efficiency of enzyme-catalyzed reactions and the high specificity of immune reactions, and can quantitatively detect various analytes such as proteins, toxins, microorganisms, etc. It is an immunological detection technology with high sensitivity, strong adaptability, and has been popularized in production and clinical practice. In enzyme-catalyzed chemiluminescence immunoassay detection, antigen or antibody enzyme-labeled substances play an important role in the sensitivity and specificity of immunoassay, and are key reagents in enzyme-catalyzed chemiluminescence immunoassay detection.

[0003] Currently, the traditional methods for preparing enzyme-labeled antibodies mainly include the glutaraldehyde method and the sodium periodate method. Among them, the sodium periodate method is the method with the highest yield for coupling enzymes and glycoproteins. This method uses sodium periodate to oxidize the sugar chain groups on glycoproteins (antibodies, glycosylated enzymes, etc.) into aldehyde groups, which form Schiff bases with antibodies under certain conditions, and then sodium borohydride reduces the Schiff bases to stable carbon-nitrogen single bonds and the excess aldehyde groups in the glycoprotein molecules. Compared with the glutaraldehyde method, the efficiency of the sodium periodate method is increased by at least 3-4 times. Currently, horseradish peroxidase (HRP), alkaline phosphatase, glucose oxidase, etc. are widely used, among which HRP is the most widely used and is widely used in clinical diagnosis and reagents for immunoassay, etc.

[0004] However, due to the presence of multiple cross-linking sites on the surface of antibodies, the sodium periodate labeling method has problems such as many cross-linking sites, non-site-directed coupling, and too large or uneven aggregates, resulting in problems such as abnormal samples (false positives), poor test gradients, and skipped wells in immunological detection of individual special items. Therefore, there is an urgent need to develop a method for enzyme-labeling antibodies with site-directed, small and uniform aggregates, and stable process to overcome the above problems.

[0005] An antibody-drug conjugate (ADC) is produced by conjugating a small molecule drug with biological activity to a monoclonal antibody (mAb) via a linker. Currently, the vast majority of ADCs are composed of antibodies targeting tumor antigens conjugated with small molecule chemical drugs with high cytotoxicity. Utilizing the specific binding property of the antibody to the target antigen, the small molecule drug is targeted and delivered to tumor cells to exert the effect of killing tumors. The site-specific conjugation technology realizes the position of drug conjugation by controlling the position of the conjugable site on the antibody. Among them, the Thiomab technology is a classic site-specific conjugation technology, first reported by Junutula et al. from Genentech. By using genetic engineering technology to insert cysteine residues at specific positions of V110C in the light chain and A114C in the heavy chain of trastuzumab, and then conjugating the sulfhydryl group on cysteine with monomethyl auristatin E (MMAE), a site-specific antibody-drug conjugate was synthesized. However, the existing site-specific conjugation technology is mainly in the field of ADCs, and the main linker molecules are small molecule drugs. It has not yet achieved site-specific conjugation of macromolecular proteins such as horseradish peroxidase, and there has been no report on its use in immunoassay technology. Summary of the Invention

[0006] In view of this, the present invention provides a method for directionally enzyme-labeling antibodies and its application. The enzyme-directionally labeled antibodies of the present invention have a labeling rate of up to more than 95%. The prepared directionally labeled antibodies can significantly reduce the detection limit of low-concentration samples and greatly reduce the probability of false positives.

[0007] To achieve the above-mentioned invention purposes, the present invention provides the following technical solutions:

[0008] The present invention provides a method for preparing enzyme-labeled antibodies, including the following steps:

[0009] Step (1): Insert cysteine into the CH1 of the antibody, or mutate any residue on the CH1 of the antibody into cysteine to obtain a mutant antibody;

[0010] Step (2): React an enzyme with an activator to obtain an activated enzyme;

[0011] Step (3): The mutant antibody obtained in step (1) is reduced, oxidized, and cross-linked with the activated enzyme obtained in step (2) to obtain the enzyme-labeled antibody.

[0012] In some specific embodiments of the present invention, in step (2) of the above preparation method:

[0013] The reaction includes mixing the activator and the enzyme at a molar ratio of 1:10 - 30, and reacting at 20 - 30 °C for 3 - 5 h;

[0014] After the reaction, it further includes the step of dialyzing the activated enzyme in PBS buffer for 5 to 14 hours;

[0015] The activator includes sodium 4-(N-maleimidomethyl)cyclohexane-1-carboxylate sulfosuccinimide ester or succinimide 4-(N-maleimidomethyl)cyclohexane-1-carboxylate-(6-aminohexanoic acid);

[0016] The concentration of the enzyme is 4 to 16 mg / mL; and / or

[0017] The concentration of the activator is 2 to 5 mg / mL; and / or

[0018] The buffer in the reaction includes PBS buffer with a concentration of 0.01 M and a pH of 7.2; and / or

[0019] The pH during the reaction is 7.75 to 8.0.

[0020] In some specific embodiments of the present invention, the reduction in step (3) of the above preparation method includes: mixing a reducing agent with the mutant antibody, reacting at 20 to 30 °C for 3 to 6 hours, and dialyzing for 12 to 14 hours to remove the residual reducing agent;

[0021] The molar ratio of the reducing agent to the mutant antibody is (10 to 80):1; and / or

[0022] The concentration of the reducing agent is 2 to 5 mg / mL; and / or

[0023] The reducing agent includes tris(2-carboxyethyl)phosphine or dithiothreitol; and / or

[0024] The concentration of the mutant antibody is 2 to 5 mg / mL; and / or

[0025] The solvent of the mutant antibody includes PBS buffer with a concentration of 0.01 M and a pH of 7.2; and / or

[0026] The dialysis solution used for dialysis includes PBS buffer with a concentration of 0.01 M and a pH of 7.2.

[0027] In some specific embodiments of the present invention, in step (3) of the above preparation method:

[0028] The oxidation includes: mixing the mutant antibody after the reduction with an oxidizing agent, reacting at 20 to 30 °C for 3 to 6 hours, and dialyzing to remove the residual oxidizing agent;

[0029] The molar ratio of the oxidizing agent to the mutant antibody is (20 to 80):1; and / or

[0030] The concentration of the oxidant is 2 to 5 mg / mL; and / or

[0031] The oxidant includes dehydroascorbic acid; and / or

[0032] The concentration of the mutant antibody is 2 to 5 mg / mL; and / or

[0033] The dialysis fluid used for dialysis includes a PBS buffer solution with a concentration of 0.01 M and a pH of 7.2; and / or

[0034] The temperature of the crosslinking is 20 to 30 °C; and / or

[0035] The molar ratio of the activated enzyme to the mutant antibody includes (1.5 to 5):1; and / or

[0036] The time of the crosslinking is 3 to 14 h; and / or

[0037] The pH of the crosslinking is 8 to 9; and / or

[0038] After the crosslinking, it further includes the step of subjecting the enzyme-labeled antibody to dialysis for 10 to 14 h.

[0039] In some specific embodiments of the present invention, the antibody in step (1) of the above preparation method includes a cardiac troponin I antibody, a human thyroid stimulating hormone receptor antibody, a thyroxine antibody, a triiodothyronine antibody or a CA724 antibody; and / or

[0040] Any residue on the CH1 of the antibody includes the first amino acid on the CH1 of the antibody; and / or

[0041] The enzyme in step (2) includes horseradish peroxidase.

[0042] In some specific embodiments of the present invention, the antibody in the above preparation method includes a monoclonal antibody;

[0043] The monoclonal antibody includes a murine monoclonal antibody, a rabbit monoclonal antibody, a sheep monoclonal antibody, a human monoclonal antibody or a chimeric monoclonal antibody.

[0044] In some specific embodiments of the present invention, the antibody in step (1) of the above preparation method is an anti-cardiac troponin I murine monoclonal antibody, an anti-human thyroid stimulating hormone receptor human monoclonal antibody or an anti-CA724 murine monoclonal antibody.

[0045] The present invention also provides an enzyme-labeled antibody prepared by the above preparation method.

[0046] In some specific embodiments of the present invention, the above enzyme-labeled antibody includes:

[0047] (I), the light chain of the enzyme-labeled antibody has:

[0048] (1) The amino acid sequence shown in SEQ ID NO.1; or

[0049] (2) An amino acid sequence obtained by substituting, deleting or adding one or more residues to the amino acid sequence shown in (1), and having the same or similar function as that in (1); or

[0050] (3) An amino acid sequence having at least 70% homology with the amino acid sequence shown in (1) or (2);

[0051] The heavy chain of the enzyme-labeled antibody has:

[0052] (4) The amino acid sequence shown in SEQ ID NO.2; or

[0053] (5) An amino acid sequence obtained by substituting, deleting or adding one or more residues to the amino acid sequence shown in (4), and having the same or similar function as that in (4); or

[0054] (6) An amino acid sequence having at least 70% homology with the amino acid sequence shown in (4) or (5);

[0055] Or

[0056] (II), the light chain of the enzyme-labeled antibody has:

[0057] (7) The amino acid sequence shown in SEQ ID NO.3; or

[0058] (8) An amino acid sequence obtained by substituting, deleting or adding one or more residues to the amino acid sequence shown in (7), and having the same or similar function as that in (7); or

[0059] (9) An amino acid sequence having at least 70% homology with the amino acid sequence shown in (7) or (8);

[0060] Its heavy chain has:

[0061] (10) The amino acid sequence shown in SEQ ID NO.4; or

[0062] (11) An amino acid sequence obtained by substituting, deleting or adding one or more residues to the amino acid sequence shown in (10), and having the same or similar function as that in (10); or

[0063] (12) An amino acid sequence having at least 70% homology with the amino acid sequence shown in (10) or (11);

[0064] Or

[0065] (III). The light chain of the enzyme-labeled antibody has:

[0066] (13) The amino acid sequence shown in SEQ ID NO.5; or

[0067] (14) An amino acid sequence obtained by substituting, deleting or adding one or more residues to the amino acid sequence shown in (13), and having the same or similar function as (13); or

[0068] (15) An amino acid sequence having at least 70% homology with the amino acid sequence shown in (13) or (14);

[0069] The heavy chain of the enzyme-labeled antibody has:

[0070] (16) The amino acid sequence shown in SEQ ID NO.6; or

[0071] (17) An amino acid sequence obtained by substituting, deleting or adding one or more residues to the amino acid sequence shown in (16), and having the same or similar function as (16); or

[0072] (18) An amino acid sequence having at least 70% homology with the amino acid sequence shown in (16) or (17);

[0073] Said plurality is from 2 to 50.

[0074] The present invention also provides a reagent, a kit or a device, comprising the above-mentioned enzyme-labeled antibody, and acceptable excipients, adjuvants and / or components.

[0075] Compared with the prior art, the method of the present invention has the following effects:

[0076] (1) The present invention uses the site-directed conjugation technology in the ADC field to site-directly label the antibody, so that the finally prepared enzyme-labeled antibody has a uniform size, one or two HRPs are cross-linked to the antibody, and only a small amount of naked antibody remains, which can significantly reduce the problems of false positives and abnormal samples during subsequent detection.

[0077] (2) Using the site-directed labeled antibody prepared by the present invention for detection, the linear correlation is excellent, and the R 2 reaches more than 0.98, having a broader application market. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art.

[0079] Figure 1Schematic diagram showing the directional labeling method of the present invention, taking HRP enzyme as an example;

[0080] Figure 2 HPLC analysis chart of the enzyme-labeled antibody obtained by the directional labeling in Example 1 and the sodium periodate method in Comparative Example 1;

[0081] Figure 3 Electrophoresis chart of the enzyme-labeled antibody obtained by the directional labeling in Example 1; among them, the naked antibody is the cTnI antibody, and Enzyme-label 1 and Enzyme-label 2 respectively represent two repeated experiments in Example 1;

[0082] Figure 4 Electrophoresis chart of the enzyme-labeled antibody obtained by the sodium periodate method in Comparative Example 1; among them, 01 and 02 respectively represent two repeated experiments in Comparative Example 1;

[0083] Figure 5 Linear fitting data of the enzyme-labeled antibodies obtained in Example 1 and Comparative Example 1 during detection; among them, (a) is the linear fitting data of Example 1; (b) is the linear fitting data of Comparative Example 1;

[0084] Figure 6 HPLC analysis chart of the enzyme-labeled antibody obtained by the directional labeling in Example 2 and the sodium periodate method in Comparative Example 2;

[0085] Figure 7 Electrophoresis chart of the enzyme-labeled antibody obtained by the directional labeling in Example 2, where the four lanes from left to right respectively represent: naked antibody; the ratio of HRP to antibody is 1:1.5 and the termination time is 0 h; the ratio of HRP to antibody is 1:2 and the termination time is 1 h; the ratio of HRP to antibody is 1:2 and the termination time is 3 h;

[0086] Figure 8 Linear fitting data of the enzyme-labeled antibodies obtained in Example 2 and Comparative Example 2 during detection; among them, (a) is the linear fitting data of Example 2; (b) is the linear fitting data of Comparative Example 2;

[0087] Figure 9 Electrophoresis chart of the enzyme-labeled antibody obtained by the directional labeling in Experiment 1 of Example 3, Experiment 2 of Example 3, and Comparative Example 5; among them, 01 is Experiment 1 of Example 3; 02 is Experiment 2 of Example 3; 03 is Comparative Example 5;

[0088] Figure 10 Electrophoresis chart of the enzyme-labeled antibody obtained by the directional labeling in Experiments I-V of Example 5;

[0089] Figure 11 Electrophoresis chart of the enzyme-labeled antibody obtained in Comparative Example 3; among them, the antibody is the naked antibody; the 03 is the enzyme-labeled antibody. Detailed implementation method

[0090] The present invention discloses a method for directed enzyme-labeling of antibodies and its applications. Those skilled in the art can draw on the content of this article and appropriately modify the process parameters to achieve the same. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are all considered to be included in the present invention. The method and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously make changes, or appropriate alterations and combinations, to the methods and applications described herein without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

[0091] The present invention provides a method for directed labeling of antibodies and its applications. In the present invention, through the Thiomab technology, cysteine residues are inserted at the CH1 position of the heavy chain of the antibody through directed mutagenesis, and then through reduction and re-oxidation, the sulfhydryl groups in the cysteine residues are freed, and an SMCC-activated enzyme is added for labeling, thus obtaining an enzyme-directed labeled antibody ( Figure 1 ), and the labeling rate can be as high as over 95%. The prepared directed labeled antibody can significantly reduce the detection limit of low-concentration samples, and greatly reduce the probability of false positives, and can be widely applied.

[0092] Specifically, the present invention first provides a method for directed enzyme-labeling of antibodies, and the method includes:

[0093] (1) First, mutate the antibody so that cysteine residues are inserted at the CH1 position of the heavy chain of the antibody;

[0094] (2) Take a reducing agent solution and add it to the mutated antibody solution, react at 20 - 30 °C for 3 - 6 h, and then dialyze to remove the unreacted reducing agent;

[0095] (3) Add an oxidizing agent at 2 - 5 mg / mL to the reduced antibody obtained in step (2) to oxidize the inter-chain disulfide bonds and restore the antibody to its original state, and then dialyze to remove the unreacted oxidizing agent;

[0096] (4) Add SMCC-activated HRP to the antibody obtained in step (3), carry out a cross-linking reaction at 20 - 30 °C for 3 - 14 h, and then dialyze for 10 - 14 h, changing the solution 2 - 3 times during the period, thus obtaining an enzyme-labeled antibody. Among them, the activated HRP is obtained by activating HRP with an activator for 3 - 5 h.

[0097] In an embodiment of the present invention, in step (1), the antibody is an immunoglobulin produced by plasma cells differentiated from B lymphocytes under the stimulation of an antigen by the immune system of an organism, and can specifically bind to the corresponding antigen; preferably, the antibody is a monoclonal antibody; the antibody mutation of the present invention is located on CH1, and therefore, it can be applied to the directed enzyme-labeling process of various antibodies.

[0098] In one embodiment of the present invention, the antibody is derived from murine monoclonal antibodies, rabbit monoclonal antibodies, sheep monoclonal antibodies, human monoclonal antibodies, and chimeric monoclonal antibodies.

[0099] In one embodiment of the present invention, more preferably, the antibody includes but is not limited to any one of CA 724 antibody, thyroxine (T4) antibody, human thyroid stimulating hormone receptor antibody (TSHR), triiodothyronine (T3) monoclonal antibody, and cardiac troponin I (cTnI) antibody; the antibody is preferably an antibody self-produced by Antu Biological.

[0100] In one embodiment of the present invention, in step (1), the mutation method is gene-directed mutagenesis technology.

[0101] In one embodiment of the present invention, in step (2), the solvent of the mutant antibody solution is preferably PBS solution, with a pH of 7.2 and a concentration of 0.01 M.

[0102] In one embodiment of the present invention, in step (2), the reducing agent includes tris(2-carboxyethyl)phosphine (TCEP) or dithiothreitol (DTT), preferably TCEP.

[0103] In one embodiment of the present invention, in step (2), the oxidizing agent includes dehydroascorbic acid (DHAA).

[0104] In one embodiment of the present invention, in step (2), the molar ratio of the reducing agent to the antibody is 10 - 40:1.

[0105] In one embodiment of the present invention, in step (2), the concentration of the reducing agent solution is 2 - 5 mg / mL, and the antibody concentration is 2 - 5 mg / mL.

[0106] In one embodiment of the present invention, in step (3), the molar ratio of the oxidizing agent DHAA to the antibody is 20 - 80:1.

[0107] In one embodiment of the present invention, in steps (2) and (3), the dialysis solution used for dialysis is PBS buffer with a concentration of 0.01 M and a pH of 7.2.

[0108] In one embodiment of the present invention, in step (4), the activation method of HRP is as follows: Mix according to a molar ratio of activator to HRP of 1:20, activate at 25 °C for 3 to 5 hours, and then optionally dialyze in PBS buffer for 5 to 14 hours or choose not to dialyze to obtain activated HRP. Among them, the activator is sodium 4-(N-maleimidomethyl)cyclohexane-1-carboxylate sulfosuccinimide (Sulfo-SMCC) or succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate-(6-aminohexanoic acid) (LC-SMCC).

[0109] In one embodiment of the present invention, during the activation process of HRP, the concentration of HRP is 4 to 16 mg / mL, and the concentration of the activator is 2 to 5 mg / mL.

[0110] In one embodiment of the present invention, during the HRP activation process, the buffer is PBS buffer with a concentration of 0.01 M and a pH of 7.2.

[0111] In one embodiment of the present invention, during the HRP activation process, the pH can be adjusted to 7.75 to 8.0 during the activation process.

[0112] In one embodiment of the present invention, in step (4), the pH during the cross-linking reaction can be adjusted to 8 to 9.

[0113] In one embodiment of the present invention, in step (4), after the reaction is completed, glutathione can be used for termination, but it has little impact on the final cross-linking result.

[0114] In one embodiment of the present invention, the pore size of the dialysis bag used for dialysis is 3KD.

[0115] By controlling the insertion site of cysteine residues, the present invention can control the labeling site of HRP, and the labeled antibody is a small oligomer, relatively homogeneous. The antibody cross-links one or two HRPs, with less remaining naked antibody and high labeling efficiency.

[0116] The present invention also provides the application of the above method in the preparation of a kit for in vitro detection.

[0117] The sequences involved in the present invention are as follows:

[0118] cTnI antibody (mouse anti):

[0119] Light chain: SEQ ID NO.1:

[0120] 1 DIVLTQAAFS NPVTLGTSAS ISCRSTKSLL HSNGITFLYW YLQRPGQSPQ LLISQMSTLA

[0121] 61 SGVPDRFSSS GSGTDFTLRI SRVEAEDVGV YYCAQNLELP YTFGGGTKLE IKRADAAPTV

[0122] 121 S

[0123] Heavy chain: SEQ ID NO.2:

[0124] 1 EVQLVESGGD LVKPGGSLKL SCAASGFTFS SFAMSWVRQT PERKLEWVAT VGTGGFYTFY

[0125] 61 PDNVEGRFTV SRDNAKNTLY LQMSSLRSED TAIYYCVRRE EAFAYWGQGT LVTVSAAKTT

[0126] 121 PPSVYPLAPG SAAQTNSMVT LGCLVKGYFP EPVTVTWNSG SLSSGVHTFP AVLQSDLYTL

[0127] 181 SSSVTVPSST WPSETVTCNV AHPASSTKVD KKIVPRDCTS KP

[0128] TSHR antibody (M22) / Human:

[0129] Light chain: SEQ ID NO.3:

[0130] 1 LTVLTQPPSV SGAPRQRVTI SCSGNSSNIG NNAVNWYQQL PGKAPKLLIY YDDQLPSGVS

[0131] 61 DRFSGSRSGT SASLAIRGLQ SEDEADYYCT SWDDSLDSQL FGGGTRLTVL GQPKAAPSVT

[0132] 121 LFPPSSEELQ ANKATLVCLI SDFYPGAVTV AWKADSSPVK AGVETTTPSK QSNNKYAASS

[0133] 181 YLSLTPEQWK SHKSYSCQVT HEGSTVEKTV APTECS

[0134] Heavy chain: SEQ ID NO.4:

[0135] 1 QVQLVQSGAE VKKPGESLKI SCRGSGYRFT SYWINWVRQL PGKGLEWMGR IDPTDSYTNY

[0136] 61 SPSFKGHVTV SADKSINTAY LQWSSLKASD TGMYYCARLE PGYSSTWSVN WGQGTLVTVS

[0137] 121 SASTKGPSVF PLAPSSKSTS GGTAALGCLV KDYFPEPVTV SWNSGALTSG VHTFPAVLQS

[0138] 181 SGLYSLSSVV TVPSSSLGTQ TYICNVNHKP SNTKVDKKVE PKSCDKTS

[0139] CA724 antibody (CC49):

[0140] Light chain: SEQ ID NO.5:

[0141] DIVMSQSPSSLPVSVGEKVTLSCKSSQSLLYSGNQKNYLAWYQQKPGQSPKLLIYWASARESGVPDRFTGSGSGTDFTLSISSVKTEDLAVYYCQQYYSYPLTFGAGTKLVLK

[0142] Heavy chain: SEQ ID NO.6:

[0143] QVQLQQSDAELVKPGASVKISCKASGYTFTDHAIHWVKQNPEQGLEWIGYFSPGNDDFKYNERFKGKATLTADKSSSTAYVQLNSLTSEDSAVYFCTRSLNMAYWGQGTSVTVSS

[0144] The HPLC assay method involved in the present invention: column type Bio SEC5, mobile phase is 0.01M PBS.

[0145] The method for antibody mutation involved in the present invention: gene engineering directed mutation technology.

[0146] The HRP enzyme involved in the present invention is purchased from Roche, product number 10121606103.

[0147] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in the present invention are all ordinary commercially available products and can be purchased from the market.

[0148] The present invention will be further described below in conjunction with embodiments:

[0149] Example 1: Directed labeled antibody

[0150] (1) First, the first amino acid of the heavy chain CH1 of the cTnI antibody (mouse anti, the amino acid sequence of the light chain variable region is shown in SEQ ID NO 1, and the heavy chain variable region is shown in SEQ ID NO 2) is mutated to cysteine by the directed mutagenesis technology of genetic engineering;

[0151] (2) Using tris(2-carboxyethyl)phosphine (TCEP) as a reducing agent, a TCEP solution with a concentration of 4 mg / mL is added to the mutant antibody solution with a concentration of 5 mg / mL obtained in step (1). Among them, the molar ratio of the reducing agent to the antibody is 40:1, and the reaction is carried out at 25 °C for 5 h. Then, it is dialyzed in PBS buffer (0.01 M, pH 7.2) for 12 h (changing the solution twice during the period) to remove the unreacted reducing agent;

[0152] (3) 4 mg / mL of DHAA is added to the reduced antibody obtained in step (2). Among them, the molar ratio of the oxidant DHAA to the antibody is 80:1, and the reaction is carried out at 25 °C for 5 h;

[0153] (4) The activator Sulfo-SMCC is dissolved in 0.01 M PBS buffer to make a solution with a concentration of 5 mg / mL, and then the two are mixed according to the molar ratio of the activator Sulfo-SMCC to HRP (concentration of 4 mg / mL) of 1:20, and activated at 25 °C for 5 h. Then, it is dialyzed in PBS buffer (0.01 M, pH 7.2) for 12 h (changing the solution twice during the period) to obtain the activated HRP;

[0154] (5) The activated HRP is added to the antibody obtained in step (3). Among them, the molar ratio of HRP to the antibody is 5:1, and the cross-linking reaction is carried out at 25 °C for 14 h. Then, it is dialyzed in PBS buffer (0.067 M, pH 6.8) for 12 h (changing the solution twice during the period) to obtain the directed enzyme-labeled antibody.

[0155] Example 2: Directed labeled antibody

[0156] (1) First, the first amino acid of the heavy chain CH1 of the Fab segment of the TSHR antibody (human anti, the amino acid sequence of the light chain variable region is shown in SEQ ID NO 3, and the heavy chain variable region is shown in SEQ ID NO 4) is mutated to cysteine by the directed mutagenesis technology of genetic engineering;

[0157] (2) Using tris(2-carboxyethyl)phosphine (TCEP) as a reducing agent, a TCEP solution with a concentration of 5 mg / mL was added to the mutant antibody solution with a concentration of 4.5 mg / mL obtained in step (1). Among them, the molar ratio of the reducing agent to the antibody was 40:1, and the reaction was carried out at 25 °C for 5 h. Then, it was dialyzed in PBS buffer (0.01 M, pH 7.2) for 12 h (changing the solution twice during the period) to remove the unreacted reducing agent;

[0158] (3) 4 mg / mL of DHAA was added to the reduced antibody obtained in step (2). Among them, the molar ratio of the oxidant DHAA to the antibody was 80:1, and the reaction was carried out at 25 °C for 4 h;

[0159] (4) The activator Sulfo-SMCC was dissolved in 0.01 M PBS buffer to make a solution with a concentration of 5 mg / mL. Then, according to the molar ratio of the activator Sulfo-SMCC to HRP (concentration of 4 mg / mL) of 1:20, the two were mixed and activated at 25 °C for 5 h. Then, it was dialyzed in PBS buffer (0.01 M, pH 7.2) for 12 h (changing the solution twice during the period) to obtain the activated HRP;

[0160] (5) The activated HRP was added to the antibody obtained in step (3). Among them, the molar ratio of HRP to the antibody was 1.5:1, and the cross-linking reaction was carried out at 25 °C for 14 h. Then, it was dialyzed in PBS buffer (0.067 M, pH 6.8) for 12 h (changing the solution twice during the period) to obtain the directed enzyme-labeled antibody.

[0161] Example 3: Directed Labeling of Antibody

[0162] Experiment 1:

[0163] (1) First, the first amino acid of the heavy chain CH1 of the cTnI antibody (the same as in Example 1) was mutated to cysteine by the site-directed mutagenesis technology of genetic engineering;

[0164] (2) Using tris(2-carboxyethyl)phosphine (TCEP) as a reducing agent, a TCEP solution with a concentration of 5 mg / mL was added to the mutant antibody solution with a concentration of 2 mg / mL obtained in step (1). Among them, the molar ratio of the reducing agent to the antibody was 40:1, and the reaction was carried out at 25 °C for 5 h. Then, it was dialyzed in PBS buffer (0.01 M, pH 7.2) for 14 h (changing the solution three times during the period) to remove the unreacted reducing agent;

[0165] (3) 4 mg / mL of DHAA was added to the reduced antibody obtained in step (2). Among them, the molar ratio of the oxidant DHAA to the antibody was 80:1, and the reaction was carried out at 25 °C for 4 h;

[0166] (4) Dissolve the activator Sulfo - SMCC in 0.01M PBS buffer to make a solution with a concentration of 5mg / mL. Then mix it with HRP (concentration 4mg / mL) according to a molar ratio of 1:20 for the activator Sulfo - SMCC and HRP, and activate at 25°C for 5h. After that, dialyze in PBS buffer (0.01M, pH 7.2) for 10h (changing the solution twice during the period) to obtain the activated HRP;

[0167] (5) Add the activated HRP to the antibody obtained in step (3), where the molar ratio of HRP to the antibody is 5:1. React at 25°C for 14h, and then dialyze in PBS buffer (0.067M, pH 6.8) for 14h (changing the solution three times during the period) to obtain the directed enzyme - labeled antibody.

[0168] Experiment 2:

[0169] In step (2) of Experiment 1 above, the molar ratio of the reducing agent TCEP to the antibody is 80:1, and the remaining operation steps and parameters are the same as those in Experiment 1 of Example 3 to obtain the directed enzyme - labeled antibody.

[0170] Example 4: Directed labeled antibody

[0171] (1) The same as step (1) in Example 2;

[0172] (2) Use tris(2 - carboxyethyl)phosphine (TCEP) as the reducing agent. Add a 5mg / mL TCEP solution to the 5mg / mL mutant antibody solution obtained in step (1), where the molar ratio of the reducing agent to the antibody is 40:1. React at 25°C for 5h, and then dialyze in PBS buffer (0.01M, pH 7.2) for 14h (changing the solution three times during the period) to remove the unreacted reducing agent;

[0173] (3) Add 4mg / mL DHAA to the reduced antibody obtained in step (2), where the molar ratio of the oxidizing agent DHAA to the antibody is 80:1, and react at 25°C for 5h;

[0174] (4) Dissolve the activator Sulfo - SMCC in 0.01M PBS buffer to make a solution with a concentration of 5mg / mL. Then mix it with HRP (concentration 4mg / mL) according to a molar ratio of 1:30 for the activator Sulfo - SMCC and HRP, and activate at 25°C for 5h. After that, dialyze in PBS buffer (0.01M, pH 7.2) for 14h (changing the solution three times during the period) to obtain the activated HRP;

[0175] (5) Add the activated HRP to the antibody obtained in step (3), where the molar ratio of HRP to the antibody is 2:1, and carry out a cross-linking reaction at 25 °C for 14 h. Then dialyze it in a PBS buffer solution (0.067 M, pH 6.8) for 12 h (change the solution twice during the period), and the directed enzyme-labeled antibody can be obtained.

[0176] Example 5: Directed labeling antibody

[0177] Experiment I:

[0178] First, mutate the first amino acid of the heavy chain CH1 of the CA724 antibody (mouse anti, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO 5, and the heavy chain variable region is as shown in SEQ ID NO 6) to cysteine by the directed mutagenesis technique of genetic engineering;

[0179] (2) Use tris(2-carboxyethyl)phosphine (TCEP) as a reducing agent, and add a TCEP solution with a concentration of 2 mg / mL to the mutant antibody solution with a concentration of 4 mg / mL obtained in step (1). Among them, the molar ratio of the reducing agent to the antibody is 40:1, react at 25 °C for 3 h, and then dialyze it in a PBS buffer solution (0.01 M, pH 7.2) for 12 h (change the solution twice during the period) to remove the unreacted reducing agent;

[0180] (3) Add 2 mg / mL of DHAA to the reduced antibody obtained in step (2), where the molar ratio of the oxidizing agent DHAA to the antibody is 80:1, and react at 25 °C for 5 h;

[0181] (4) Dissolve the activator LC-SMCC in a 0.01 M PBS buffer solution to make a solution with a concentration of 2 mg / mL, and then mix the activator LC-SMCC and HRP (concentration of 4 mg / mL) according to the molar ratio of 1:20, activate at 25 °C for 3 h, and then dialyze it in a PBS buffer solution (0.01 M, pH 7.2) for 12 h (change the solution twice during the period) to obtain the activated HRP;

[0182] (5) Add the activated HRP to the antibody obtained in step (3), where the molar ratio of HRP to the antibody is 5:1, carry out a cross-linking reaction at 25 °C for 3 h, and then dialyze it in a PBS buffer solution (0.067 M, pH 6.8) for 12 h (change the solution twice during the period), and the directed enzyme-labeled antibody can be obtained.

[0183] Experiment II: Adjust the pH of the cross-linking reaction in step (5) to 8.6, and the rest is the same as Experiment I.

[0184] Experiment III: Adjust the pH of the activation reaction in step (4) to 7.75, and the rest is the same as Experiment I.

[0185] Experiment IV: Adjust the pH of the activation reaction in step (4) to 8.0, and the rest is the same as Experiment I.

[0186] Experiment V: Replace the activator in step (4) with LC-SMCC, and the rest is the same as Experiment I.

[0187] Comparative Example 1: Labeling antibody by sodium periodate method

[0188] (1) Prepare a solution of HRP with a concentration of 10 mg / mL, add sodium periodate solution according to the amount of 25 μL of 0.1 M sodium periodate solution required per milligram of HRP, react in the dark at 4 °C for 1 h, then add ethylene glycol to terminate the reaction, react in the dark at 4 °C for 0.5 h, and dialyze in 1 mM acetate buffer at pH 4.4 for 12 h, changing the solution three times during this period; at the same time, dialyze the antibody cTnI to be labeled in 0.01 mM carbonate buffer at pH 9.6 for 12 h, changing the solution three times during this period;

[0189] (2) Then mix according to the molar ratio of HRP to the antibody to be labeled (the same as in Example 1) of 5:1, and adjust the pH of the mixed solution by adding 180 μL of 0.2 M carbonic acid solution with pH 9.6 per mL of HRP, react in the dark at 4 °C for 24 h;

[0190] (3) Terminate the reaction by adding 30 μL of 0.106 M sodium borohydride solution per mg of HRP, react in the dark at 4 °C for 2 h, and then dialyze in PBS buffer at pH 6.8 and 0.067 M at 4 °C for 16 h, changing the solution 3 times during this period.

[0191] Comparative Example 2: Labeling antibody by sodium periodate method

[0192] (1) Prepare a solution of HRP with a concentration of 10 mg / mL, add sodium periodate solution according to the amount of 25 μL of 0.1 M sodium periodate solution required per milligram of HRP, react in the dark at 4 °C for 1 h, then add ethylene glycol to terminate the reaction, react in the dark at 4 °C for 0.5 h, and dialyze in 1 mM acetate buffer at pH 4.4 for 12 h, changing the solution three times during this period; at the same time, dialyze the antibody TSHR-Fab to be labeled in 0.01 mM carbonate buffer at pH 9.6 for 12 h, changing the solution three times during this period;

[0193] (2) Then mix according to the molar ratio of HRP to the antibody to be labeled (the same as in Example 2) of 5:1, and adjust the pH of the mixed solution by adding 180 μL of 0.2 M carbonic acid solution with pH 9.6 per mL of HRP, react in the dark at 4 °C for 24 h;

[0194] (3) Terminate the reaction by adding 30 μL of 0.106 M sodium borohydride solution per mg of HRP, react in the dark at 4 °C for 2 h, and then dialyze in PBS buffer at pH 6.8 and 0.067 M at 4 °C for 16 h, changing the solution three times during the period, and the enzyme-labeled antibody can be obtained.

[0195] Comparative Example 3

[0196] Same as Example 1, except that the activator in step (4) is replaced with long-arm SMCC(PEG)8.

[0197] Comparative Example 4

[0198] Same as Example 1, except that the oxidant in step (3) is replaced with copper sulfate.

[0199] Comparative Example 5

[0200] Same as Experiment 1 of Example 3, except that in step (1), the first amino acid of the heavy chain CH2 of the cTnI antibody (the same as in Example 1) is mutated to cysteine by site-directed mutagenesis technology of genetic engineering, and the remaining operation steps and parameters are the same as those in Experiment 1 of Example 3 above, and the enzyme-labeled antibody is obtained.

[0201] Effect Example 1: Comparison between Example 1 and Comparative Example 1

[0202] The prepared directed enzyme-labeled antibody and non-directed enzyme-labeled antibody (sodium periodate method) in Example 1 and Comparative Example 1 were detected by HPLC (high performance liquid chromatography), and the results are shown in Figure 2 It can be seen that the sizes of the enzyme-labeled antibodies finally prepared in Example 1 are uniform, and one or two HRPs are cross-linked to the antibody, while the sizes of the enzyme-labeled antibodies prepared in Comparative Example 1 are not uniform, the aggregates are too large, and the number of cross-linked HRPs on the antibody is uncertain.

[0203] Figure 3 and Figure 4 are the electrophoresis diagrams of the enzyme-labeled antibodies and naked antibodies of Example 1 and Comparative Example 1 respectively. It can be seen from the electrophoresis diagrams that the number of remaining naked antibodies in the labeling method of Example 1 is small, and the enzyme-labeled antibodies are mainly one antibody cross-linked with one or two HRPs, and the sizes of the enzyme-labeled antibodies are uniform, which is consistent with the HPLC analysis results. In the enzyme-labeled antibodies obtained by the traditional method of Comparative Example 1, the number of HRPs cross-linked to one antibody is uncertain, the sizes of the aggregates are different, and it is a mixture of various cross-linking states. The electrophoresis results are the same as Figure 2 the results.

[0204] The prepared enzyme-labeled antibodies in Example 1 and Comparative Example 1 were evaluated. The evaluation method was: magnetic particle chemiluminescence, the reaction mode was competitive method, and the standard value was the data measured by the corresponding Roche kit. The evaluation results are shown in Figure 5, It can be seen that the enzyme-labeled antibody obtained by directional labeling has better linearity at low concentrations than the enzyme-labeled antibody obtained by using traditional sodium periodate labeling in Comparative Example 1, and R 2 is greater than 0.98, indicating that the enzyme-labeled antibody obtained by directional labeling can perform more accurate quantitative determination at low concentrations.

[0205] The enzyme-labeled antibodies prepared in Example 1 and Comparative Example 1 were used for actual detection. The detection method was magnetic particle chemiluminescence, and the reaction mode was a competitive method. The corresponding enzyme-labeled antibody of Roche was used as a control. When the corresponding measured concentration value was less than 2, it indicated that the sample was negative, otherwise it was false positive. The results are shown in Table 1 below (where different sample numbers represent different samples prone to false positive results). It can be seen that the probability of false positive in the detection of Comparative Example 1 is relatively high, while the test results of Example 1 are consistent with Roche and no false positive occurs. It can be seen that the enzyme-labeled antibody prepared by the directional enzyme labeling process of the present invention can significantly reduce problems such as false positive and sample abnormality.

[0206] Table 1 Interference data of the enzyme-labeled antibodies of Example 1, Comparative Example 1 and Roche

[0207] Sample Number Example 1 Comparative Example 1 Roche Type 110 0.77 2.34 ＜0.8 Against interference of enzyme-labeled antibody 111 0.69 2.98 ＜0.8 Against interference of enzyme-labeled antibody 112 0.90 1.89 ＜0.8 Against interference of enzyme-labeled antibody 113 0.67 2.64 ＜0.8 Against interference of enzyme-labeled antibody 114 0.87 1.74 ＜0.8 Against interference of enzyme-labeled antibody 115 0.51 2.29 ＜0.8 Against interference of enzyme-labeled antibody 116 0.75 3.05 ＜0.8 Against interference of enzyme-labeled antibody

[0208] Effect Example 2: Comparison between Example 2 and Comparative Example 2

[0209] The directional enzyme-labeled antibody and non-directional enzyme-labeled antibody prepared in Example 2 and Comparative Example 2 were respectively detected by HPLC (high performance liquid chromatography). The results are shown in Figure 6 It can be seen that the sizes of the enzyme-labeled antibodies finally prepared in Example 2 are uniform, and one antibody crosslinks one HRP, while the sizes of the enzyme-labeled antibodies prepared in Comparative Example 2 are not uniform, the aggregates are too large, and the number of antibody crosslinks is uncertain.

[0210] Figure 7 is the electrophoresis pattern of the enzyme-labeled antibody and naked antibody of Example 2 and Comparative Example 2. Among them, the three experiments are the enzyme-labeled antibodies after changing the experimental parameters in Example 2 (respectively, the ratio of HRP to antibody is 1:1.5 and the termination time is 0 h; the ratio of HRP to antibody is 1:2 and the termination time is 1 h; the ratio of HRP to antibody is 1:2 and the termination time is 3 h). It can be seen from the electrophoresis pattern that the remaining amount of naked antibody in the labeling method of this example is small, and the enzyme-labeled antibody mainly crosslinks one HRP per antibody, and the sizes of the enzyme-labeled antibodies are uniform, which is consistent with the HPLC analysis results.

[0211] The evaluation method is the same as that in Effect Example 1. The evaluation results are shown in Figure 8 , It can be seen that the enzyme-labeled antibody obtained by directional labeling has better linearity at low concentrations than the enzyme-labeled antibody obtained by using traditional sodium periodate labeling in Comparative Example 2, and R 2It is 0.9861, indicating that the enzyme-labeled antibody obtained by the directional labeling is more suitable for the determination of antigens at low concentrations.

[0212] The enzyme-labeled antibodies prepared in Example 2 and Comparative Example 2 were used for actual detection. The method was the same as that in Effect Example 1. Among them, different sample numbers represented different samples prone to false positive results. The results are shown in Table 2. It can be seen that the enzyme-labeled antibody prepared by the directional enzyme labeling process of the present invention can significantly reduce problems such as false positives and sample anomalies.

[0213] Table 2 Interference data of enzyme-labeled antibodies in Example 2, Comparative Example 2 and Roche

[0214] Sample Number Example 2 Comparative Example 2 Roche Type 887 0.97 3.51 1.06 Against interference of enzyme-labeled antibody 1820 0.88 2.40 ＜0.8 Against interference of enzyme-labeled antibody 1924 0.43 2.24 ＜0.8 Against interference of enzyme-labeled antibody 5513 1.05 2.26 ＜0.8 Against interference of enzyme-labeled antibody 206 0.66 1.97 ＜0.8 Against interference of enzyme-labeled antibody 241 0.87 2.45 ＜0.8 Against interference of enzyme-labeled antibody

[0215] Effect Example 3: Verification of the effect of Example 3 and comparison with Comparative Example 5

[0216] By measuring the electrophoresis data of the three enzyme-labeled antibodies in Experiment 1 of Example 3, Experiment 2 of Example 3, and Comparative Example 5 (see Figure 9 ), it can be found that one antibody in the enzyme-labeled antibodies obtained in Experiment 1 and Experiment 2 of Example 3 is connected to one or two HRPs, and the size is uniform, which can well reduce the occurrence of problems such as false positives and sample anomalies. In Comparative Example 5, when the mutation site becomes the first amino acid of the heavy chain CH2, the antibody cannot or rarely cross-links with HRP. The results show that the selection of the mutation site is very important.

[0217] Effect Example 4: Verification of the effect of Example 4

[0218] By measuring the HPLC and electrophoresis of the enzyme-labeled antibody, it can also be found that one antibody in the enzyme-labeled antibody obtained in Example 4 is connected to one HRP, and the size is uniform, which can well reduce the occurrence of problems such as false positives and sample anomalies.

[0219] Effect Example 5: Verification of the effect of Example 5

[0220] The enzyme-labeled antibodies prepared in Experiments I-V of Example 5 were subjected to electrophoresis tests. The results are as Figure 10 shown. It can be seen that the remaining naked antibodies in Experiments I-V are relatively few, indicating a high enzyme-labeling efficiency. Among the prepared enzyme-labeled antibodies, one antibody molecule cross-links 1 or 2 HRPs, and the size is uniform. The antibody molecules prepared in Experiment V are mainly enzyme-labeled antibodies with one HRP connected. The prepared enzyme-labeled antibody can improve the linear correlation of sample detection at low concentrations and can greatly reduce the occurrence rate of false positives and abnormal samples during the detection process.

[0221] Effect Example 6: Verification of the effect of Comparative Example 3

[0222] Through the same test as in Example 1, it was found that the proportion of the remaining naked antibody was very high (see the electrophoresis diagramFigure 11 )。It shows that not any activator can effectively achieve the directional labeling process and obtain a high enzyme labeling efficiency. The activator of the present invention is preferably Sulfo-SMCC and / or LC-SMCC.

[0223] Effect Example 7: Verification of the Effect of Comparative Example 4

[0224] The results show that the addition of copper sulfate will cause precipitation of the antibody, resulting in the failure of the experiment.

[0225] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing an enzyme-labeled antibody, characterized in that, It includes the following steps: Step (1): Insert cysteine into the CH1 of the antibody, or mutate any residue on the CH1 of the antibody into cysteine to obtain a mutant antibody; Step (2): React an enzyme with an activator to obtain an activated enzyme; Step (3): The mutant antibody described in step (1) is reduced, oxidized, and cross-linked with the activated enzyme described in step (2) to obtain the enzyme-labeled antibody; In step (2), the reaction includes mixing the activator and the enzyme at a molar ratio of 1:10 - 30, and reacting at 20 - 30 °C for 3 - 5 h; In step (3), the molar ratio of the activated enzyme to the mutant antibody is (1.5 - 5):1; The activator includes sodium 4-(N-maleimidomethyl)cyclohexane-1-carboxylate sulfosuccinimide ester or succinimide 4-(N-maleimidomethyl)cyclohexane-1-carboxyl-(6-aminohexanoic acid); The oxidant includes dehydroascorbic acid.

2. The preparation method according to claim 1, characterized in that, In step (2): After the reaction, it further includes the step of dialyzing the activated enzyme in PBS buffer for 5 - 14 h; The concentration of the enzyme is 4 - 16 mg / mL; and / or The concentration of the activator is 2 - 5 mg / mL; and / or The buffer in the reaction includes PBS buffer with a concentration of 0.01M and a pH of 7.2; and / or The pH during the reaction is 7.75 - 8.

0.

3. The preparation method according to claim 1 or 2, characterized in that, The reduction described in step (3) includes: mixing a reducing agent with the mutant antibody, reacting at 20 - 30 °C for 3 - 6 h, and dialyzing for 12 - 14 h to remove the residual reducing agent; The molar ratio of the reducing agent to the mutant antibody is (10 - 80):1; and / or The concentration of the reducing agent is 2 - 5 mg / mL; and / or The reducing agent includes tris(2-carboxyethyl)phosphine or dithiothreitol; and / or The concentration of the mutant antibody is 2 - 5 mg / mL; and / or The solvent of the mutant antibody includes PBS buffer with a concentration of 0.01M and a pH of 7.2; and / or The dialysis solution used for dialysis includes PBS buffer with a concentration of 0.01M and a pH of 7.

2.

4. The preparation method according to claim 3, characterized in that, In step (3): The oxidation includes: mixing the mutant antibody after the reduction with an oxidant, reacting at 20 - 30 °C for 3 - 6 h, and dialyzing to remove the residual oxidant; The molar ratio of the oxidant to the mutant antibody is (20 - 80):1; and / or The concentration of the oxidant is 2 - 5 mg / mL; and / or The concentration of the mutant antibody is 2 - 5 mg / mL; and / or The dialysis solution used for dialysis includes PBS buffer with a concentration of 0.01M and a pH of 7.2; and / or The temperature of the cross-linking is 20 - 30 °C; and / or The time of the cross-linking is 3 - 14 h; and / or The pH of the cross-linking is 8 - 9; and / or After the cross-linking, it further includes the step of dialyzing the enzyme-labeled antibody for 10 - 14 h.

5. The preparation method according to claim 4, characterized in that, The antibody described in step (1) includes cardiac troponin I antibody, human thyroid stimulating hormone receptor antibody, thyroxine antibody, triiodothyronine antibody, or CA724 antibody; and / or Any residue on the CH1 of the antibody includes the first amino acid on the CH1 of the antibody; and / or The enzyme described in step (2) includes horseradish peroxidase.

6. The preparation method according to claim 5, wherein The antibody includes a monoclonal antibody; The monoclonal antibody includes a murine monoclonal antibody, a rabbit monoclonal antibody, a sheep monoclonal antibody, a human monoclonal antibody or a chimeric monoclonal antibody.

7. The preparation method according to claim 6, characterized in that, The antibody described in step (1) is a murine monoclonal antibody against cardiac troponin I, a human monoclonal antibody against human thyroid stimulating hormone receptor or a murine monoclonal antibody against CA724.

8. An enzyme-labeled antibody prepared by the preparation method according to any one of claims 1 to 7.

9. The enzyme-labeled antibody according to claim 8, wherein Comprising: (I), The light chain of the enzyme-labeled antibody has: (1), The amino acid sequence shown in SEQ ID NO.1; or (2), An amino acid sequence obtained by substituting, deleting or adding one or more residues to the amino acid sequence shown in (1), and having the same or similar function as (1); The heavy chain of the enzyme-labeled antibody has: (3), The amino acid sequence shown in SEQ ID NO.2; or (4), An amino acid sequence obtained by substituting, deleting or adding one or more residues to the amino acid sequence shown in (3), and having the same or similar function as (3); Or (II), The light chain of the enzyme-labeled antibody has: (5), The amino acid sequence shown in SEQ ID NO.3; or (6), An amino acid sequence obtained by substituting, deleting or adding one or more residues to the amino acid sequence shown in (5), and having the same or similar function as (5); Its heavy chain has: (10), The amino acid sequence shown in SEQ ID NO.4; or (11), An amino acid sequence obtained by substituting, deleting or adding one or more residues to the amino acid sequence shown in (10), and having the same or similar function as (10); Or (III), The light chain of the enzyme-labeled antibody has: (13), The amino acid sequence shown in SEQ ID NO.5; or (14), An amino acid sequence obtained by substituting, deleting or adding one or more residues to the amino acid sequence shown in (13), and having the same or similar function as (13); The heavy chain of the enzyme-labeled antibody has: (16), The amino acid sequence shown in SEQ ID NO.6; or (17), An amino acid sequence obtained by substituting, deleting or adding one or more residues to the amino acid sequence shown in (16), and having the same or similar function as (16); The plurality is from 2 to 50.

10. A reagent, kit or device, characterized in that, Comprising the enzyme-labeled antibody according to claim 8 or 9, and acceptable excipients, adjuvants and / or components.

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