CA15-3 antibody biotin labeled compound, kit and preparation method and application of CA15-3 antibody biotin labeled compound and kit

Abstract

The invention belongs to the field of chemiluminescence immunoassay, and particularly relates to a CA15-3 antibody biotin labeled compound, a kit and a preparation method and application of the CA15-3 antibody biotin labeled compound. An aldehyde group is introduced to the tail end part of sugar chain galactose of the CA15-3 antibody modified by a galactose oxidase method and then reacts with amino biotin, and due to site fixation, on the basis that a large amount of biotin is marked as much as possible, combination of an antigen and the antibody is not affected, so that the stability of a working solution during CA15-3 chemiluminescence immunodiagnosis is improved, and the CA15-3 chemiluminescence immunodiagnosis efficiency is improved. And the signal-to-noise ratio is effectively improved.

Description

Technical Field

[0001] The present application belongs to the field of chemiluminescent immunoassay, and specifically relates to a CA15-3 antibody-biotin labeled complex, a kit, and a preparation method and application thereof. Background Art

[0002] Chemiluminescent immunoassay (CLIA) is a technique that combines highly sensitive chemiluminescent assay technology with highly specific immune reactions, and is used for the detection and analysis of various antigens, haptens, antibodies, hormones, enzymes, fatty acids, vitamins, and drugs. The Biotin-Avidin (SA) System (BAS) is a new type of biological reaction amplification system developed in the late 1970s, which can be combined with almost all successfully studied markers. The strong binding of biotin and avidin with high affinity and the multi-stage amplification effect make BAS immunolabeling and related tracer analysis more sensitive. It has become a new technology widely used in qualitative and quantitative detection of trace antigens and antibodies and localization observation research.

[0003] In recent years, CA15-3 has been used as a biomarker for breast cancer. CA15-3 is located on the tumor cell membrane. When the cell becomes cancerous, glycosyltransferase is activated, resulting in abnormal cell surface glycosylation, excessive expression of cell surface mucin and release into the blood circulation, and increased serum CA15-3 content. CA15-3 is a large molecular glycoprotein, part of its antigenic determinant is sugar and part is polypeptide. In in vitro serum diagnosis, it is generally identified by double antibody sandwich method.

[0004] When traditional in vitro diagnosis is performed, after the CA15-3 antibody-labeled acridine conjugate is prepared into a reagent working solution, when conducting thermal stability experiments, problems such as low signal-to-noise ratio and poor thermal stability will occur. Summary of the invention

[0005] Based on this, one embodiment of the present application provides a CA15-3 antibody-biotin labeled complex.

[0006] On the one hand, the present application provides a CA15-3 antibody-biotin labeled complex, the composition of which includes: a CA15-3 antibody-biotin chemical complex formed by chemically coupling an oxidatively modified CA15-3 antibody with biotin.

[0007] The oxidative modification refers to oxidizing the C-6 position of the galactose molecule in the sugar chain region of the CA15-3 antibody from a hydroxyl group to an aldehyde group; the chemical coupling refers to a condensation reaction between the aldehyde group and the active group of the biotin; the active group includes an amino group.

[0008] On the other hand, the present application provides a CA15-3 chemiluminescent detection kit, which includes the CA15-3 antibody-biotin labeled complex and a magnetic bead coating.

[0009] In one embodiment, the magnetic bead coating is coated with CA15-3 antibody, and the CA15-3 antigen to be detected can specifically bind to the magnetic bead coating and the CA15-3 antibody biotin-labeled complex.

[0010] In one embodiment, the kit further comprises one or more of a standard, a dilution buffer, a protein protectant, and a luminescence excitation solution.

[0011] On the other hand, the present application provides a method for preparing a CA15-3 antibody-biotin labeled complex, comprising:

[0012] The CA15-3 antibody and galactose oxidase are mixed in a buffer solution for a first reaction, and after the reaction, a galactose oxidase inactivator is added for a second reaction, and the mixture is centrifuged to obtain a supernatant to prepare a CA15-3 antibody modified intermediate;

[0013] The CA15-3 antibody modified intermediate is reacted with biotin for a third time, and then a termination labeling reaction is performed to obtain a CA15-3 antibody biotin-labeled complex.

[0014] In one embodiment, the step of purification is further included after the termination reaction.

[0015] In one embodiment, the purification process comprises using a desalting column or an AKTA purifier.

[0016] In one embodiment, the CA15-3 antibody is a human glycoprotein monoclonal antibody with a purity of ≥95% and a concentration of 2.4 mg / mL to 2.5 mg / mL.

[0017] In one embodiment, the concentration of the galactose oxidase is 6.5 mg / mL to 7.2 mg / mL.

[0018] In one embodiment, the structural formula of biotin is NH2-(PEG)n-Biotin, and the concentration is 8 mg / mL to 12 mg / mL.

[0019] In one embodiment, the volume ratio of the CA15-3 antibody to the galactose oxidase is (4-7):1.

[0020] In one embodiment, the buffer is 48 mM to 52 mM phosphate buffer with a pH of 6.8 to 7.2.

[0021] In one embodiment, the galactose oxidase inactivator comprises one or more of ethylenediaminetetraacetic acid, cyanide and azide.

[0022] In one embodiment, the conditions of the first reaction include: temperature of 24°C to 26°C, and time of 85 min to 95 min.

[0023] In one embodiment, the conditions of the second reaction include: temperature of 24° C. to 26° C., and time of 25 min to 35 min.

[0024] In one embodiment, the centrifugal treatment conditions include: a rotation speed of 4500g to 5500g and a time of 5min to 15min.

[0025] In one embodiment, the third reaction conditions include: temperature of 24°C to 26°C, and time of 120 min to 125 min.

[0026] In one embodiment, the termination reaction conditions include: temperature of 24°C to 26°C and time of 55min to 65min.

[0027] On the other hand, the present application provides a CA15-3 chemiluminescent detection method, comprising:

[0028] The sample to be tested, the CA15-3 antibody-biotin-labeled complex, the magnetic bead coating, and the SA-labeled acridinium ester are mixed in a buffer solution, incubated, and the mixed solution after incubation is detected.

[0029] In one embodiment, the sample to be tested and the magnetic bead coating are added first, and then the CA15-3 antibody-biotin-labeled complex and the SA-labeled acridinium ester are added.

[0030] In one embodiment, the incubation conditions include: a temperature of 36° C. to 38° C. and a time of 4 min to 6 min.

[0031] In one embodiment, the detection conditions include: using a chemiluminescence immunoassay tester for detection, and the number of detection times is 2 to 3 times.

[0032] The details of one or more embodiments of the present application are set forth in the description which follows, and other features, objects, and advantages of the present application will be apparent from the description and its claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0034] Figure 1 Schematic diagram of CA15-3 antibody after oxidation modification;

[0035] Figure 2 Schematic diagram of the reaction of oxidatively modified CA15-3 antibody with biotin to form a CA15-3 antibody-biotin labeled complex;

[0036] Figure 3 Schematic diagram of the CA15-3 antibody-biotin labeled complex reaction during the detection process. DETAILED DESCRIPTION

[0037] Below in conjunction with embodiment and example, the application is further described in detail.It should be understood that these embodiments and examples are only used to illustrate the application and are not used to limit the scope of the application, and the purpose of providing these embodiments and examples is to make the understanding of the disclosure of the application more thorough and comprehensive.It should also be understood that the application can be implemented in many different forms, is not limited to the embodiment and example described herein, and those skilled in the art can make various changes or modifications without violating the connotation of the application, and the equivalent form obtained also falls within the protection scope of the application.In addition, in the description below, a large number of specific details are given in order to provide a more comprehensive understanding of the application, and it should be understood that the application can be implemented without one or more of these details.

[0038] 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.

[0039] the term

[0040] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0041] The terms "and / or", "or / and", and "and / or" used in this article include any one of two or more related listed items, and also include any and all combinations of related listed items, and the arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0042] In the present application, "plurality", "multiple", "multiple times", "multiples", etc., unless otherwise specified, refer to a number greater than 2 or equal to 2. For example, "one or more" means one or greater than or equal to two.

[0043] As used herein, "combination thereof", "any combination thereof", "any combination thereof" etc. include all suitable combinations of any two or more of the listed items.

[0044] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc., shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0045] In the present application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present application.

[0046] In this application, "optionally", "optional", and "optional" mean optional or dispensable, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0047] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0048] In the present application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0049] The temperature parameters in this application, unless otherwise specified, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0050] In the present application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0051] All documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the invention purpose and / or technical solution of this application, the cited documents involved in this application are cited with all contents and all purposes. When the cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the cited documents are involved in this application, the examples and preferred methods of the cited relevant technical features can also be incorporated into this application as references, but are limited to the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description of this application.

[0052] The term "galactose oxidase" is a copper free radical oxidase that catalyzes the aerobic oxidation of primary alcohols to aldehydes. This enzyme is widely used in biosensors, food, medical and chemical industries. Galactose oxidase has the characteristics of high catalytic efficiency and green environmental protection, but due to the defects of natural galactose oxidase such as poor stability, low catalytic efficiency and high recycling cost, its application in food safety and biomedicine is limited. In order to improve the performance of the enzyme or obtain a recombinant enzyme with new functions, galactose oxidase can be purposefully modified through genetic engineering and protein engineering.

[0053] On the one hand, the present application provides a CA15-3 antibody-biotin labeled complex, the composition of which includes: a CA15-3 antibody-biotin chemical complex formed by chemically coupling an oxidatively modified CA15-3 antibody with biotin.

[0054] Oxidative modification refers to the oxidation of the C-6 position of the galactose molecule in the sugar chain region of the CA15-3 antibody from a hydroxyl group to an aldehyde group; chemical coupling refers to the condensation reaction of the aldehyde group with the active group of biotin; the active group includes an amino group.

[0055] Optionally, the structural formula of aminobiotin is NH2-(PEG)n-Biotin.

[0056] On the other hand, the present application provides a CA15-3 chemiluminescent detection kit, which includes a CA15-3 antibody-biotin labeled complex and a magnetic bead coating.

[0057] In a specific example, the magnetic bead coating is coated with CA15-3 antibody, and the CA15-3 antigen to be detected can specifically bind to the magnetic bead coating and CA15-3 antibody biotin-labeled complex.

[0058] In a specific example, the kit further includes one or more of a standard, a dilution buffer, a protein protectant, and a luminescence excitation solution.

[0059] On the other hand, the present application provides a method for preparing a CA15-3 antibody-biotin labeled complex, comprising:

[0060] The CA15-3 antibody and galactose oxidase are mixed in a buffer for a first reaction, a galactose oxidase inactivator is added for a second reaction, the mixture is centrifuged, and the supernatant is collected to prepare a CA15-3 antibody modified intermediate.

[0061] The CA15-3 antibody modified intermediate is reacted with biotin for a third time, and then the labeling reaction is terminated to obtain a CA15-3 antibody biotin-labeled complex.

[0062] In a specific example, the step of purifying the CA15-3 antibody modified intermediate after the termination reaction with biotin is also included;

[0063] Optionally, the purification treatment comprises treatment with a desalting column or treatment with an AKTA purifier.

[0064] Desalting column treatment is a commonly used technique in biochemical experiments, mainly used to remove small molecular weight impurities such as salt ions, detergents, small molecule dyes, buffers, etc. in samples such as proteins, nucleic acids, peptides, and polysaccharides. This technology uses the principle of size exclusion chromatography (SEC) and the separation and purification method of different molecular sizes to separate large molecular samples from small molecular impurities.

[0065] AKTA Purifier is an efficient and flexible chromatography system, which is widely used in the purification of biomolecules such as proteins, peptides and nucleic acids. It supports a variety of chromatography techniques, such as affinity chromatography, ion exchange chromatography, size exclusion chromatography (SEC), hydrophobic chromatography and reverse phase chromatography.

[0066] Optionally, the CA15-3 antibody is a human glycoprotein monoclonal antibody with a purity of ≥95% and a concentration of 2.4 mg / mL to 2.5 mg / mL. For example, the concentration is 2.41 mg / mL, 2.42 mg / mL, 2.43 mg / mL, 2.44 mg / mL, 2.45 mg / mL, 2.46 mg / mL, 2.47 mg / mL, 2.48 mg / mL, 2.49 mg / mL, 2.50 mg / mL. Optionally, the concentration is 2.486 mg / mL;

[0067] Further optionally, the concentration of galactose oxidase is 6.5 mg / mL to 7.2 mg / mL; in a specific embodiment, the concentration of galactose oxidase is 6.5 mg / mL, and in another specific embodiment, the concentration of lactose oxidase is 7.2 mg / mL. It is understood that in other embodiments, the concentration of galactose oxidase can also be 6.6 mg / mL, 6.7 mg / mL, 6.8 mg / mL, 6.9 mg / mL, 7.0 mg / mL, 7.1 mg / mL and other values.

[0068] Optionally, the concentration of biotin is 8 mg / mL to 12 mg / mL. In a specific embodiment, the concentration of galactose oxidase is 8 mg / mL, and in another specific embodiment, the concentration of lactose oxidase is 12 mg / mL. It is understood that in other embodiments, the concentration of galactose oxidase can also be 9 mg / mL, 9.5 mg / mL, 10 mg / mL, 10.5 mg / mL, 11 mg / mL, 11.5 mg / mL and other values.

[0069] In a specific example, the volume ratio of CA15-3 antibody to galactose oxidase is (4-7): 1. In a specific embodiment, the volume ratio of CA15-3 antibody to galactose oxidase is 4: 1, and in another specific embodiment, the volume ratio of CA15-3 antibody to galactose oxidase is 7: 1. It is understood that in other embodiments, the volume ratio of CA15-3 antibody to galactose oxidase is 4: 1, 5: 1, 6: 1 and other values.

[0070] In a specific example, the buffer is a 48 mM to 52 mM phosphate buffer with a pH of 6.8 to 7.2, such as a buffer concentration of 48 mM, 49 mM, 50 mM, 51 mM or 52 mM, and a pH of 6.8, 6.9, 7.0, 7.1 or 7.2.

[0071] Optionally, the galactose oxidase inactivator includes, but is not limited to, one or more of ethylenediaminetetraacetic acid, cyanide, and azide.

[0072] In a specific example, the conditions of the first reaction include: a temperature of 24°C to 26°C, and a time of 85 min to 95 min. In a specific example, the temperature is 24°C, 25°C, 26°C, and the time is 85 min, 86 min, 87 min, 88 min, 89 min, 90 min, 91 min, 92 min, 93 min, 94 min, 95 min.

[0073] In a specific example, the conditions of the second reaction include: a temperature of 24°C to 26°C, and a time of 25 min to 35 min. In a specific example, the temperature is 24°C, 25°C, 26°C, and the time is 25 min, 26 min, 27 min, 28 min, 29 min, 30 min, 31 min, 32 min, 33 min, 34 min, 35 min.

[0074] Optionally, the centrifugal treatment conditions include: a rotation speed of 4500g to 5500g, and a time of 5min to 15min. In a specific example, the rotation speed is 4500g, 4600g, 4700g, 4800g, 4900g, 5000g, 5100g, 5200g, 5300g, 5400g or 5500g, and the time is 5min, 6min, 7min, 8min, 9min, 10min, 11min, 12min, 13min, 14min or 15min.

[0075] In a specific example: the specific conditions for the reaction of the modified CA15-3 antibody with biotin include:

[0076] 2 μL of 10 mg / mL aminobiotin was added to the CA15-3 antibody modified intermediate solution, and the reaction was carried out at 25° C. for 2 h. Subsequently, L-lysine hydrochloride buffer was added and the reaction was carried out at 25° C. for 1 h to terminate the labeling reaction.

[0077] On the other hand, the present application also provides a CA15-3 chemiluminescent immunoassay method, comprising:

[0078] The sample to be tested is mixed with the CA15-3 antibody biotin-labeled complex, the magnetic bead coating, and the SA-labeled acridinium ester, incubated, and the luminescence value is detected.

[0079] In a specific example, the sample to be tested and the magnetic bead coating are added first, and then the CA15-3 antibody biotin-labeled complex and the SA-labeled acridinium ester are added.

[0080] Among them, SA-labeled acridinium ester (NSP-SA-NHS) is a highly efficient chemiluminescent marker with stability, activity and sensitivity, which make it surpass radioisotopes in some aspects. It can react with proteins containing primary amino groups. Under alkaline conditions, NHS is replaced as a leaving group to form a stable amide bond with the protein. After the reaction is completed, the excess acridinium salt can be removed by a desalting column.

[0081] Optionally, the incubation conditions include: a temperature of 36° C. to 38° C., and a time of 4 min to 6 min. For example, the temperature is 36° C., 37° C. or 38° C., and the time is 4 min, 5 min or 6 min.

[0082] Further optionally, the conditions for detecting the luminescence value include: using an iFlash 3000C chemiluminescence immunoassay instrument and measuring 2 to 3 times.

[0083] The present application provides a CA15-3 antibody-biotin labeled complex, the composition of which includes: a sandwich structure formed by chemical coupling of an oxidatively modified CA15-3 antibody and biotin. Among them, oxidative modification refers to the oxidation of the C-6 position on the galactose molecule in the sugar chain region of the CA15-3 antibody from a hydroxyl group to an aldehyde group, and chemical coupling refers to the condensation reaction of the aldehyde group with the active group of the biotin; the active group includes an amino group. The present application introduces an aldehyde group into the terminal part of the sugar chain galactose of the CA15-3 antibody through galactose oxidase modification, and then reacts with amino-biotin. Because the site is fixed, while labeling as much biotin as possible, it does not affect the binding of the antigen and the antibody, thereby improving the stability of the working fluid during chemiluminescent immunoassay of CA15-3, and effectively improving the signal-to-noise ratio.

[0084] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0085] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0086] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0087] Example 1

[0088] This embodiment provides a method for preparing a CA15-3 antibody-biotin labeled complex, which specifically comprises the following steps:

[0089] (1) Preparation of CA15-3 antibody modified intermediates

[0090] Weigh 6.8 mg of galactose oxidase (purchased from Sigma-Aldrich) and dissolve it in 1 mL of phosphate buffer at pH 7.0. After it is fully dissolved, take 15 μL and add it to 100 μg of CA15-3 antibody (purchased from Cell Signaling Technology) solution with a purity of 98% and a concentration of 2.486 mg / mL. Carry out the first reaction at 25°C for 90 min.

[0091] After the reaction is completed, 50 μL of 1 mg / mL EDTA solution is added to inhibit the unreacted oxidase activity, and after mixing evenly, a second reaction is carried out at 25°C for 30 minutes. Subsequently, the mixture is centrifuged at 5000 g for 10 minutes to remove the excess inactivated enzyme, and the supernatant is taken to obtain the CA15-3 antibody modified intermediate.

[0092] (2) Preparation of CA15-3 antibody-biotin labeled complex

[0093] Add 2 μL of 10 mg / mL aminobiotin to the CA15-3 antibody modified intermediate solution, react at 25°C for 2 h, then add L-lysine hydrochloride buffer and react at 25°C for 1 h to terminate the labeling reaction. Finally, use a 2 mL 7KD molecular weight cutoff desalting column (Thermo fish) with 150 mM PBS (pH 7.4) buffer as the column pressure buffer, pass the column twice to remove the unreacted biotin, and prepare the CA15-3 antibody biotin-labeled complex.

[0094] Example 2

[0095] This embodiment provides a method for preparing a CA15-3 antibody-biotin labeled complex, which specifically comprises the following steps:

[0096] (1) Preparation of CA15-3 antibody modified intermediates

[0097] Weigh 6.5 mg of galactose oxidase and dissolve it in 1 mL of phosphate buffer at pH 6.8. After it is fully dissolved, take 15 μL and add it to 100 μg of CA15-3 antibody solution with a purity of 98%. Perform the first reaction at 25°C for 90 minutes.

[0098] After the reaction is completed, 50 μL of 1 mg / mL EDTA solution is added to inhibit the unreacted oxidase activity, and the mixture is mixed and reacted at 25° C. for 30 min. The mixture is then centrifuged at 5000 g for 10 min to remove excess inactivated enzymes, and the supernatant is taken to obtain the CA15-3 antibody modified intermediate.

[0099] (2) Preparation of CA15-3 antibody-biotin labeled complex

[0100] Add 2 μL of 8 mg / mL aminobiotin to the CA15-3 antibody modified intermediate solution, react at 25°C for 2 h, then add L-lysine hydrochloride buffer and react at 25°C for 1 h to terminate the labeling reaction. Finally, use a 2 mL 7KD molecular weight cutoff desalting column (Thermo fish) with 150 mM PBS (pH 7.4) buffer as the column pressure buffer, pass the column twice to remove the unreacted biotin, and prepare the CA15-3 antibody biotin-labeled complex.

[0101] Example 3

[0102] This embodiment provides a method for preparing a CA15-3 antibody-biotin labeled complex, which specifically comprises the following steps:

[0103] (1) Preparation of CA15-3 antibody modified intermediates

[0104] Weigh 7.2 mg of galactose oxidase and dissolve it in 1 mL of phosphate buffer at pH 7.2. After it is fully dissolved, take 15 μL and add it to 100 μg of CA15-3 antibody solution with a purity of 98%. Perform the first reaction at 25°C for 90 minutes.

[0105] After the reaction is completed, 50 μL of 1 mg / mL EDTA solution is added to inhibit the unreacted oxidase activity, and after mixing evenly, a second reaction is carried out at 25°C for 30 minutes. Subsequently, the mixture is centrifuged at 5000 g for 10 minutes to remove the excess inactivated enzyme, and the supernatant is taken to obtain the CA15-3 antibody modified intermediate.

[0106] (2) Preparation of CA15-3 antibody-biotin labeled complex

[0107] Add 2 μL of 12 mg / mL aminobiotin to the CA15-3 antibody modified intermediate solution, react at 25°C for 2 h, then add L-lysine hydrochloride buffer and react at 25°C for 1 h to terminate the labeling reaction. Finally, use a 2 mL 7KD molecular weight cutoff desalting column (Thermo fish) with 150 mM PBS (pH 7.4) buffer as the column pressure buffer, pass the column three times to remove the unreacted biotin, and prepare the CA15-3 antibody biotin-labeled complex.

[0108] Comparative Example 1

[0109] This comparative example provides a method for preparing a CA15-3 antibody marker that is labeled with biotin but not chemically coupled in a conventional manner, which specifically comprises the following steps:

[0110] Take 100 μg of unmodified CA15-3 antibody solution and add it to a phosphate buffer solution with a pH of 7.4. The reaction concentration is 0.5 mg / mL. After mixing evenly, add 2 μL of 10 mg / mL aminobiotin and react at 25°C for 2 hours. Then add L-lysine hydrochloride buffer and react at 25°C for 1 hour to terminate the labeling reaction.

[0111] A 2 mL 7KD molecular weight cutoff desalting column (Thermo fish) was used with 150 mM PBS (pH 7.4) buffer as the column pressure buffer. The column was passed twice to remove the unreacted biotin and prepare a biotin-labeled CA15-3 antibody marker that was not chemically coupled.

[0112] Verification results:

[0113] 1. Chemiluminescence result verification:

[0114] The CA15-3 antibody biotin-labeled complex prepared in Example 1 and the CA15-3 antibody marker without chemical coupling treatment prepared in Comparative Example 1 were used for chemiluminescent immunoassay test of CA15-3, which specifically includes the following steps:

[0115] CA15-3 calibrators Cal 1, Cal 2 and Cal 3, quality control products QC 1 and QC 2, and 10 samples of random concentrations (referred to as samples 1 to 10) were tested. The CA15-3 antibody biotin-labeled complex prepared in Example 1 was recorded as marker B, and the CA15-3 antibody marker without chemical coupling treatment prepared in Comparative Example 1 was recorded as marker C.

[0116] First, add 50 μL of magnetic bead-coated CA15-3 antibody working solution and 50 μL of the sample to be tested, incubate at 37°C for 5 min, separate the magnetic beads, and then wash once, then add the working solution composed of marker B in Example 1 and marker C in Comparative Example 1 and SA-labeled acridinium ester, incubate at 37°C for 5 min, then perform magnetic separation again, and then wash once. Finally, use iFlash3000C chemiluminescence immunoassay to measure its luminescence value, measure 2 times on average, take the average value for analysis, and the results are shown in Table 1:

[0117] Table 1: Comparison of chemiluminescence values ​​of markers in Example 1 and Comparative Example 1

[0118]

[0119] From the results in Table 1, it can be seen that the test signal-to-noise ratio of the marker B in Example 1 which was enzymatically modified and then labeled with aminobiotin was improved to about 1.5 times compared with the marker C in Comparative Example 1 which was not subjected to any modification treatment. The background of the two markers was similar and did not increase significantly.

[0120] 2. Stability analysis and verification:

[0121] The marker B obtained in Example 1 and the marker C obtained in Comparative Example 1 were used for the accelerated stability analysis of the CA15-3 chemiluminescent kit. Equal amounts of B and C were prepared into working solutions and placed at 4°C and 37°C for 7 days to test their stability. The test results are shown in Table 2:

[0122] Table 2: Comparison of the stability of the markers of Example 1 and Comparative Example 1 at room temperature and low temperature

[0123]

[0124]

[0125] The results are shown in Table 2. The signal value of the marker B in Example 1, which was modified by the enzyme method and then labeled with aminobiotin, decreased by about 10% after being placed at 37°C for 7 days, while the signal value of the marker C in Comparative Example 1, which was not treated, decreased by about 30%. Therefore, it can be shown that the CA15-3 antibody-biotin labeled complex and the preparation method thereof of the present application can improve the stability of the reagent working solution.

[0126] The above-described embodiments only express several implementation methods of the present application, which is convenient for understanding the technical solution of the present application in detail, but it cannot be understood as a limitation on the scope of protection of the patent application. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present application, several deformations and improvements can be made, which all belong to the protection scope of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, the technicians in this field can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the protection scope of the present application. It should also be understood that the technical solutions obtained by the technicians in this field through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the protection scope of the claims attached to the present application. Therefore, the protection scope of the patent of the present application shall be based on the content of the attached claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. A CA15-3 antibody-biotin labeled complex, comprising: A CA15-3 antibody-biotin chemical complex formed by chemically coupling an oxidatively modified CA15-3 antibody with biotin; The oxidative modification refers to oxidizing the C-6 position of the galactose molecule in the sugar chain region of the CA15-3 antibody from a hydroxyl group to an aldehyde group; the chemical coupling refers to a condensation reaction between the aldehyde group and the active group of the biotin; the active group includes an amino group.

2. A CA15-3 chemiluminescent detection kit, characterized in that: The kit comprises the CA15-3 antibody-biotin labeled complex according to claim 1 and a magnetic bead coating.

3. The CA15-3 chemiluminescent detection kit according to claim 2, characterized in that: The magnetic bead coating is coated with CA15-3 antibody, and the CA15-3 antigen to be detected can specifically bind to the magnetic bead coating and the CA15-3 antibody biotin-labeled complex.

4. The CA15-3 chemiluminescent detection kit according to claim 2, characterized in that: The kit also includes one or more of a standard substance, a dilution buffer, a protein protectant and a luminescence excitation solution.

5. A method for preparing a CA15-3 antibody-biotin labeled complex, characterized in that: include: The CA15-3 antibody and galactose oxidase are mixed in a buffer solution for a first reaction, and after the reaction, a galactose oxidase inactivator is added for a second reaction, and the mixture is centrifuged to obtain a supernatant to prepare a CA15-3 antibody modified intermediate; and, The CA15-3 antibody modified intermediate is reacted with biotin for a third time, and then a termination labeling reaction is performed to obtain a CA15-3 antibody biotin-labeled complex.

6. The method for preparing the CA15-3 antibody-biotin labeled complex according to claim 5, characterized in that: After the reaction is terminated, a purification step is also included; Optionally, the purification treatment comprises treatment with a desalting column or treatment with an AKTA purifier.

7. The method for preparing the CA15-3 antibody-biotin labeled complex according to claim 5, characterized in that: The method includes one or more of the following features: (1) The CA15-3 antibody is a human glycoprotein monoclonal antibody with a purity of ≥95% and a concentration of 2.4 mg / mL to 2.5 mg / mL; (2) The concentration of the galactose oxidase is 6.5 mg / mL to 7.2 mg / mL; (3) The structural formula of biotin is NH2-(PEG)n-Biotin, and the concentration is 8 mg / mL to 12 mg / mL; (4) The volume ratio of the CA15-3 antibody to the galactose oxidase is (4-7):1; (5) The buffer is 48 mM to 52 mM phosphate buffer with a pH of 6.8 to 7.2; (6) The galactose oxidase inactivator includes one or more of ethylenediaminetetraacetic acid, cyanide and azide.

8. The method for preparing the CA15-3 antibody-biotin labeled complex according to claim 5, characterized in that: The method includes one or more of the following features: (1) The conditions of the first reaction include: temperature of 24°C to 26°C, time of 85 min to 95 min; (2) The conditions of the second reaction include: temperature of 24°C to 26°C, time of 25 min to 35 min; (3) The conditions of the centrifugal treatment include: a speed of 4500 g to 5500 g and a time of 5 min to 15 min; (4) The third reaction conditions include: temperature of 24°C to 26°C, time of 120 min to 125 min; (5) The termination reaction conditions include: temperature of 24°C to 26°C and time of 55 min to 65 min.

9. A CA15-3 chemiluminescence detection method, characterized in that: include: The sample to be tested is mixed with the CA15-3 antibody biotin-labeled complex of claim 1, the magnetic bead coating of any one of claims 2 to 3, and the SA-labeled acridinium ester in a buffer solution, incubated, and the mixed solution after incubation is detected.

10. The CA15-3 chemiluminescence detection method according to claim 9, characterized in that: The method includes one or more of the following features: (1) first adding the sample to be tested and the magnetic bead coating, and then adding the CA15-3 antibody biotin-labeled complex and SA-labeled acridinium ester; (2) The incubation conditions include: a temperature of 36°C to 38°C and a time of 4 min to 6 min; (3) The detection conditions include: using a chemiluminescence immunoassay tester for detection, and the number of detection times is 2 to 3 times.

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