Preparation method of labeled antibody

Abstract

The invention discloses a preparation method of a labeled antibody. The preparation method comprises the following steps: mixing an antibody to be labeled with 1, 6-hexanediol to prepare a premixed solution; and reacting the premixed liquid with terpyridyl ruthenium to prepare the labeled antibody. The preparation method can give consideration to marking efficiency and test signals after the working solution is prepared, and has a wide application prospect.

Description

Technical Field

[0001] The present application relates to the field of immunoassay technology, and in particular to a method for preparing a labeled antibody. Background Art

[0002] Electrochemiluminescence, also known as electrochemiluminescence (ECL), refers to the process in which a substance forms an excited state through an electron transfer reaction on the electrode surface, releases energy and emits light. ECL includes two processes: electrochemical reaction and chemiluminescence reaction. Therefore, ECL types are mainly divided into two types, namely annihilation ECL and co-reactant ECL. The former is produced by a single luminophore, and the latter involves an electrochemical reaction between the luminophore and a suitable co-reactant.

[0003] From the above, we can see that ECL has the common advantages of chemiluminescence and electrochemical analysis technology, such as high sensitivity, wide dynamic range, simple operation and good stability. Compared with chemiluminescence, ECL has better spatiotemporal controllability for light emission. Compared with photoluminescence (PL) analysis, ECL does not require an excitation light source, effectively avoiding the interference of stray light and increasing the sensitivity of the analysis. Therefore, it has become an effective analytical method both in basic research and in practical applications.

[0004] At present, the chemiluminescent agent for electrochemiluminescence immunoassay is mainly ruthenium terpyridine [Ru(bpy)3] 2+ , the antibody is labeled with chemiluminescent agent terpyridine ruthenium, and through antigen-antibody reaction and magnetic particle separation technology, the antigen or antibody to be tested is quantitatively or qualitatively determined according to the intensity of light emitted by terpyridine ruthenium on the electrode. The immunoassay products of the famous Roche Diagnostics Company are products of this methodology. At present, terpyridine ruthenium labeled antibodies are mainly coupled with carboxyl terpyridine ruthenium and the amino groups exposed on the antibody surface.

[0005] For the development of in vitro diagnostic reagents, screening out good raw materials is almost equivalent to 90% success, so it is very important to screen out raw materials with good correlation. However, in many cases, the raw materials with good correlation screened out cannot meet the sensitivity requirements. At this time, we generally try to improve the labeling efficiency of the antibody to improve the sensitivity. In most cases, after the antibody is labeled with terpyridine ruthenium under conventional labeling conditions, the reagent working solution prepared for the detection sample will have a high signal, which can meet the labeling requirements of most antibodies. If a higher labeling efficiency is required, for most antibodies, adjusting the salt concentration, surfactant concentration, pH, and the ratio of labeled antibody to terpyridine ruthenium of the labeling medium can basically meet the requirements of improving sensitivity. However, for some antibodies, the reagent sensitivity requirements cannot be met after adjusting the above conditions. For example, for indicators with extremely high sensitivity requirements such as procalcitonin (PCT), cardiac troponin I (CTNI), human chorionic gonadotropin and β subunit (HCG-β), improving labeling efficiency is crucial to solving sensitivity issues. For CA-type tumor indicators, antibodies with good correlation are screened, but the labeling efficiency usually cannot meet the requirements, resulting in a very low signal-to-noise ratio and the inability to prepare diagnostic reagents that meet the requirements.

[0006] Therefore, there is an urgent need in the art for an efficient labeling method that combines both labeling efficiency and detection signal. Summary of the invention

[0007] Based on this, it is necessary to provide at least a method for preparing a labeled antibody.

[0008] In the first aspect of the present application, a method for preparing a labeled antibody is provided, comprising:

[0009] Mixing the antibody to be labeled with 1,6-hexanediol to prepare a premix solution; and,

[0010] The premixed solution is mixed with terpyridine ruthenium to react and prepare a labeled antibody.

[0011] In some embodiments, the concentration of 1,6-hexanediol in the premix is ​​0.1 mg / mL to 0.5 g / mL.

[0012] In some embodiments, the concentration of the terpyridine ruthenium in the initial reaction system is 0.1 mg / mL.

[0013] In some embodiments, the antibody to be labeled is a procalcitonin antibody or a carbohydrate antigen 19-9 antibody.

[0014] In some embodiments, the antibody to be labeled is a procalcitonin antibody, and its concentration in the initial reaction system of the reaction is 0.1 mg / mL to 0.2 mg / mL. In some embodiments, the antibody to be labeled is a carbohydrate antigen 19-9 antibody, and its concentration in the initial reaction system of the reaction is 0.25 mg / mL to 5 mg / mL.

[0015] In some embodiments, the reaction conditions are 35° C. to 39° C. in a water bath for 1 h to 4 h.

[0016] In some embodiments, the termination method of the reaction comprises adding a glycine solution to the reaction system.

[0017] In some embodiments, the volume ratio of the glycine solution to the mixed solution obtained after the reaction is 1:17-1:15, and the concentration of the glycine solution is 1M-1.1M.

[0018] In some embodiments, the conditions for terminating the reaction are: standing at room temperature for 5 min to 20 min.

[0019] In some embodiments, the preparation method further comprises the step of purifying the labeled antibody.

[0020] In some embodiments, the means of purification comprises dialysis.

[0021] In some embodiments, dialysis is performed using a 14KD dialysis bag and a 0.05M to 0.1M phosphate buffer at 2°C to 8°C.

[0022] In a second aspect of the present application, there is provided use of 1,6-hexanediol in preparing a product for electrochemiluminescence immunoassay.

[0023] In the third aspect of the present application, a labeled antibody prepared by the preparation method described in the first aspect is provided.

[0024] The method for preparing the labeled antibody in the present application, in some embodiments, significantly improves the labeling efficiency of the ruthenium-labeled antibody, while having almost no effect on the background of the reagent, which is of great help in improving the reagent performance of this indicator; it can take into account both the labeling efficiency and the test signal after being prepared into a working solution, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the implementation methods and examples 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 use in the description of the implementation methods or examples. Obviously, the drawings described below are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without creative work. It should also be noted that the drawings are all drawn in a simplified form and are only used to conveniently and clearly assist in explaining the present application.

[0026] Figure 1 The test signal of sample 1 and the change of 1,6-hexanediol concentration during labeling in one embodiment of the present application.

[0027] Figure 2 The test signal of sample 2 and the change of 1,6-hexanediol concentration during labeling in one embodiment of the present application.

[0028] Figure 3 The test signal of sample 3 and the change of 1,6-hexanediol concentration during labeling in one embodiment of the present application. DETAILED DESCRIPTION

[0029] In order to facilitate the understanding of the present application, the present application will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present application are given in the drawings. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0031] In this application, unless otherwise specified, "one or more" means any one of the listed items or any combination of the listed items. Similarly, "one or more" and the like, when used in other ways to mean "one or more", are also understood in the same way unless otherwise specified.

[0032] The terms "combination thereof", "any combination thereof", "any combination thereof" and the like used in this application include all suitable combinations of any two or more of the listed items.

[0033] In this application, the word "suitable" in "suitable combination", "suitable method", "any suitable method", etc. is 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.

[0034] In this application, "further", "further", "particularly", "for example", "such as", "example", "for example", etc. are used for descriptive purposes, indicating that the previous and subsequent different technical solutions are related in terms of the content covered, but should not be understood as a limitation on the previous technical solution, nor can it be understood as a limitation on the protection scope of this application. In this application, unless otherwise specified, A (such as B) means that B is a non-limiting example of A, and it can be understood that A is not limited to B.

[0035] In this application, "optionally", "optional", and "optional" mean optional, that is, any one of the two parallel solutions of "yes" or "no". If there are multiple "optional" in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent. Unless otherwise specified, the descriptions of "optionally include", "optionally contain", etc. in this application, taking "optionally include" as an example, mean "may include or not include".

[0036] The terms "contain", "include" and "include" used in this application are synonymous, which are inclusive or open-ended and do not exclude additional, uncited members or features. Members or features include materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features include actions, conditions for the occurrence of actions, timing, states, etc.

[0037] In this application, the technical features or technical solutions described in an open language include closed technical features or technical solutions composed of the listed contents, and also include open technical features or technical solutions containing the listed contents.

[0038] In the present application, exemplary descriptions such as "in some implementation modes (or examples)" and "in one implementation mode (or example)" may include but are not limited to the following meanings: these solutions may be combined with other solutions in a suitable manner to form new technical solutions.

[0039] In the present application, in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc., the terms "first", "second", "third", "fourth", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0040] In the present application, when it comes to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the optional numerical values ​​in the numerical interval is considered to be continuous, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical interval, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to an integer in the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints, which is equivalent to directly listing each integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical range disclosed herein should be understood to include any and all sub-ranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. "Numerical interval" allows broadly including numerical interval types such as percentage intervals, ratio intervals, and ratio intervals.

[0041] In this application, if there are multiple steps involved in the method flow, unless there is a clear different description in this document, there is no strict order restriction for the execution of these steps, and they can be executed in other orders than described. Moreover, any step can include multiple sub-steps or multiple stages, and these sub-steps or stages are not necessarily executed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be executed in turn, alternating or simultaneously with other steps or parts of sub-steps or stages of other steps.

[0042] The term "room temperature" in this application refers to 25°C to 35°C, for example, 30±5°C.

[0043] In a first aspect of the present application, a method for preparing a labeled antibody is provided, the method comprising sequentially adding 1,6-hexanediol and a terpyridine ruthenium mother solution to a solution of the antibody to be tested.

[0044] Unless otherwise specified, the term "labeling" in this application refers to covalently linking a marker (such as a radioisotope, fluorescein or enzyme) to another compound (such as an antibody) for the purpose of identification and detection, forming a multi-component complex through a specific reaction between the labeled compound and the object to be detected. After separation from the unbound marker, the object to be detected can be identified and detected by a simpler method.

[0045] In some embodiments, the preparation method comprises:

[0046] Mixing the antibody to be labeled with 1,6-hexanediol to prepare a premix solution; and,

[0047] The premixed solution is mixed with terpyridine ruthenium to react and prepare a labeled antibody.

[0048] In some embodiments, the concentration of 1,6-hexanediol in the premix is ​​0.1 mg / mL to 0.5 g / mL.

[0049] Without wishing to be limited by any theory, the inventors have found that the higher the concentration of 1,6-hexanediol added, the better. It may be that the addition of 1,6-hexanediol has a certain effect on the structure of the antibody, resulting in the inactivation of the antibody aggregates during the labeling process. Even if the labeling efficiency is greatly improved, the activity of the labeled antibody is significantly reduced, resulting in a still low test signal after being prepared into a working solution. It can be seen that it is necessary to select a suitable concentration to take into account both the labeling efficiency and the test signal after being prepared into a working solution. Taking into account these two effects is generally the focus and difficulty of labeling methods in this field.

[0050] In some embodiments, the concentration of 1,6-hexanediol in the premix is ​​0.1 mg / mL, 0.15 mg / mL, 0.2 mg / mL, 0.25 mg / mL, 0.3 mg / mL, 0.35 mg / mL, 0.4 mg / mL, 0.45 mg / mL, 0.5 mg / mL, or a range or value between any two values.

[0051] In some embodiments, the concentration of the terpyridine ruthenium in the initial reaction system is 0.05 mg / mL to 0.15 mg / mL.

[0052] In this application, unless otherwise specified, the term "initial reaction system of the reaction" refers to the reaction system after the premix and terpyridine ruthenium are mixed but before the reaction starts or at the beginning of the reaction. Without wishing to be bound by any theory, it is believed that at this time, the premix and terpyridine ruthenium have not yet been consumed by the reaction.

[0053] In some embodiments, the concentration of the terpyridine ruthenium in the initial reaction system of the reaction is 0.05 mg / mL, 0.06 mg / mL, 0.07 mg / mL, 0.08 mg / mL, 0.09 mg / mL, 0.1 mg / mL, 0.11 mg / mL, 0.12 mg / mL, 0.13 mg / mL, 0.14 mg / mL, 0.15 mg / mL, or a range or value between any two values. In some embodiments, the concentration of the terpyridine ruthenium in the initial reaction system of the reaction is 0.1 mg / mL.

[0054] In some embodiments, the antibody to be labeled is procalcitonin antibody (PCT) or carbohydrate antigen 19-9 antibody.

[0055] In some embodiments, the antibody to be labeled is a procalcitonin antibody, and its concentration in the initial reaction system is 0.1 mg / mL to 0.2 mg / mL.

[0056] In some embodiments, the antibody to be labeled is a carbohydrate antigen 19-9 antibody, and its concentration in the initial reaction system of the reaction is 0.25 mg / mL to 5 mg / mL. In some embodiments, its concentration in the initial reaction system of the reaction is 0.25 mg / mL, 0.3 mg / mL, 0.5 mg / mL, 0.8 mg / mL, 1 mg / mL, 1.5 mg / mL, mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 3.5 mg / mL, 4 mg / mL, 4.5 mg / mL, 5 mg / mL, or a range or value between any two values. In some embodiments, the concentration of the carbohydrate antigen 19-9 antibody in the initial reaction system of the reaction is 2 mg / mL.

[0057] In some embodiments, the concentration ratio of the terpyridine ruthenium to the antibody to be labeled is 1:30 to 1:15.

[0058] In some embodiments, the reaction conditions are 35°C to 39°C water bath for 1 h to 4 h. In some embodiments, the temperature in the reaction conditions is 35°C, 36°C, 37°C, 38°C, 39°C, or a range or value between any two values. Without wishing to be bound by any theory, it is believed that too high a temperature may affect the activity of the antibody, and too low a temperature may affect the labeling efficiency.

[0059] In some embodiments, the reaction is carried out in a 37° C. water bath for 1 h to 4 h.

[0060] In the present application, the method for terminating the reaction can be conventional in the art. For example, the labeling of the antibody with terpyridine ruthenium can be terminated by glycine.

[0061] In some embodiments, the termination method of the reaction comprises adding a glycine solution to the reaction system.

[0062] In some embodiments, the volume ratio of the glycine solution to the mixed solution obtained after the reaction is 1:17 to 1:15, and the concentration of the glycine solution is 1M to 1.1M. In some embodiments, the volume ratio of the glycine solution to the mixed solution obtained after the reaction is 1:17. In some embodiments, the volume ratio of the glycine solution to the mixed solution obtained after the reaction is 1:16. In some embodiments, the volume ratio of the glycine solution to the mixed solution obtained after the reaction is 1:15. In some embodiments, the concentration of the glycine solution is 1M. In some embodiments, the concentration of the glycine solution is 1.01M, 1.02M, 1.03M, 1.04M, 1.05M, 1.06M, 1.07M, 1.08M, 1.09M, 1.1M, or a range or value between any two values.

[0063] In some embodiments, the volume ratio of the glycine solution to the mixed solution obtained after the reaction is 1:17, and the concentration of the glycine solution is 1M.

[0064] In some embodiments, the conditions for terminating the reaction are: standing at room temperature for 5 min to 20 min. In some embodiments, the conditions for terminating the reaction are: standing at room temperature for 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min, 20 min, or a range or value between any two values.

[0065] In some embodiments, the preparation method further comprises the step of purifying the labeled antibody.

[0066] In some embodiments, the means of purification comprises dialysis.

[0067] In some embodiments, dialysis is performed using a 14KD dialysis bag and a 0.05M-0.1M phosphate buffer at 2°C to 8°C. In some embodiments, a 0.05M phosphate buffer is used. In some embodiments, a 0.06M phosphate buffer is used. In some embodiments, a 0.07M phosphate buffer is used. In some embodiments, a 0.08M phosphate buffer is used. In some embodiments, a 0.09M phosphate buffer is used. In some embodiments, a 0.1M phosphate buffer is used.

[0068] In some embodiments, the dialysis temperature is 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, or a range or value between any two values.

[0069] The following is an example of a 255 μL (250 μL premix + 5 μL terpyridine ruthenium mother solution) initial reaction system to exemplify the preparation method of the labeled antibody in the present application. In the following embodiments, the concentration of the antibody mother solution (storage medium is, for example, 0.1 M PBS) is greater than 2 mg / mL, so it also includes the operation of antibody dilution.

[0070] 1. Calculate the volume of antibody stock solution according to the concentration of antibody stock solution to ensure that the concentration after dilution is the antibody reaction concentration of 2 mg / mL, and add the corresponding volume of antibody into a 2 mL centrifuge tube.

[0071] 2. Add 5 μL of 5 mg / mL 1,6-hexanediol (0.01% g / mL) to the diluted antibody solution, and then add 0.1 M PBS buffer to make up to 250 μL.

[0072] 3. Add 5 μL of 5 mg / mL (DMSO dissolved) terpyridine ruthenium stock solution to the above 0.5 mg antibody solution and mix well.

[0073] 4. Place the above solution in a 37°C water bath for 2 hours, then remove the ruthenium-labeled antibody from the water bath, add 15 μL of 1 M glycine solution, mix well, and let stand at room temperature for 10 minutes to terminate the reaction.

[0074] 5. Take out the above-mentioned labeled antibody, put it into a 14KD dialysis bag, and dialyze it with 0.1M PB buffer at 2℃-8℃, 4L / time, for a total of 4 times, with a fluid change interval of at least 4h-6h.

[0075] In a second aspect of the present application, there is provided use of 1,6-hexanediol in preparing a product for electrochemiluminescence immunoassay.

[0076] In the third aspect of the present application, a labeled antibody prepared by the preparation method described in the first aspect is provided.

[0077] In some embodiments, the product may be, for example, a kit.

[0078] Illustratively, the kit comprises different units, which are physically existing separate partitions or separate spaces.

[0079] In some embodiments, one unit in the kit comprises the 1,6-hexanediol. In some embodiments, another unit in the kit comprises a terpyridine ruthenium mother solution. In some embodiments, the concentration of the terpyridine ruthenium mother solution is at least 5 mg / mL. In some embodiments, the solvent of the terpyridine ruthenium mother solution is DMSO.

[0080] Some examples are provided below.

[0081] 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 the conditions are not specified in the following examples are preferably referred to the guidance given in the present application, and can also be based on the experimental manual or normal conditions in the art, or can also be based on the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0082] Example 1

[0083] The carbohydrate antigen 19-9 (CA199) indicator antibody (purchased from Fantai Biotechnology, catalog number: FT-PD7166S) was used to demonstrate the application of 1,6-hexanediol in the labeling of terpyridine ruthenium antibodies. For this antibody, it is difficult to increase the labeling signal using conventional labeling methods. The following adds 1,6-hexanediol during the labeling process to observe its effect on the labeling efficiency.

[0084] The marking method is as follows:

[0085] 6. The concentration of the antibody mother solution (storage medium is 0.1M PBS) is greater than 2mg / mL. Set the total volume of the labeling reaction system to 250 μL. Calculate the volume of the antibody mother solution according to the concentration of the antibody mother solution, and add the corresponding volume of antibody into a 2ml centrifuge tube (ensure that the concentration after dilution is the antibody reaction concentration of 2mg / mL).

[0086] 7. Add 5 μL of 5 mg / mL 1,6-hexanediol (0.01% g / mL) to the diluted antibody solution, and then add 0.1 M PBS buffer to make up to 250 μL.

[0087] 8. Add 5 μL of 5 mg / mL (DMSO dissolved) terpyridine ruthenium stock solution to the above 0.5 mg antibody solution and mix well.

[0088] 9. Place the above solution in a 37°C water bath for 2 hours, then remove the ruthenium-labeled antibody from the water bath, add 15 μL of 1 M glycine solution, mix well, and let stand at room temperature for 10 minutes to terminate the reaction.

[0089] 10. Take out the above-mentioned labeled antibody, put it into a 14KD dialysis bag, and dialyze it with 0.1M PB buffer at 2℃-8℃, 4L / time, for a total of 4 times, with a fluid change interval of at least 4h-6h.

[0090] The ruthenium-labeled antibody after dialysis was sucked out from the dialysis bag and placed in a 2 mL cryotube. The protein concentration was tested with a micro-protein concentration detector. The label paper was affixed and the mother solution antibody information was written. The above solution was stored at -20°C.

[0091] According to the above operation steps, several other concentrations were also tried. See Table 1 below for details.

[0092] Table 1

[0093]

[0094] Based on the above results, it can be seen that adding 0.01%-0.05% (0.1 mg / mL~0.5 g / mL) of 1,6-hexanediol to the labeling buffer can significantly improve the labeling efficiency.

[0095] The above antibodies were prepared into CA199 reagent working solution with an antibody concentration of 2 μg / mL. The clinical sample results of CA199 index were tested with biotin-labeled antibody (2 μg / mL) and streptavidin magnetic beads (1 mg / mL) as shown in Table 2 below.

[0096] Table 2

[0097]

[0098] It can be seen from the experimental data that adding 0.025% (0.025 g / 100 mL) of 1,6-hexanediol can significantly improve the test signal, indicating that the labeling efficiency of terpyridine ruthenium can be significantly improved under this method.

[0099] The working solution contains 0.1M phosphate buffer, 0.5% (M / V) BSA, 1% (M / V) sucrose, 0.1% (V / V) surfactant (Triton-100), 1% (M / V) trehalose, and 0.1% (V / V) preservative.

[0100] The results showed that the addition of 1,6-hexanediol can significantly improve the labeling efficiency of ruthenium-labeled CA199 antibody, while having almost no effect on the background of the reagent, which is of great help in improving the reagent performance of this indicator. The optimal concentration range of 1,6-hexanediol is 0.01%-0.05% (0.01 g / 100 mL-0.05 g / 100 mL).

[0101] The conclusion that using 1,6-hexanediol in the labeling buffer can improve the labeling efficiency has a certain degree of generalization. For example, PCT antibody can also significantly improve the labeling efficiency of antibody-labeled terpyridine ruthenium through this method, but it should be noted that the optimal concentration of 1,6-hexanediol for each antibody may be slightly different, and it needs to be screened according to the specific situation. For example, the optimal addition concentration of PCT antibody (purchased from FANTIBODY, catalog No. FAB-B002, Lot No. 2020120902) is 0.01%-0.02% (0.1 mg / mL~0.2 g / mL), and the specific experimental method is the same as CA199. The experimental data are shown in the following table.

[0102] Table 3

[0103]

[0104] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0105] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims, and the description and drawings may be used to interpret the contents of the claims.

Claims

1. A method for preparing a labeled antibody, characterized in that: It includes: Mix the antibody to be labeled with 1,6-hexanediol to prepare a premix solution; as well as, The premixed solution is mixed with terpyridine ruthenium to react and prepare a labeled antibody.

2. The preparation method according to claim 1, characterized in that The concentration of the 1,6-hexanediol in the premix is ​​0.1 mg / mL to 0.5 g / mL.

3. The preparation method according to claim 1 or 2, characterized in that: The concentration of the terpyridine ruthenium in the initial reaction system is 0.05 mg / mL to 0.15 mg / mL.

4. The preparation method according to claim 1 or 2, characterized in that: The antibody to be labeled is procalcitonin antibody or carbohydrate antigen 19-9 antibody.

5. The preparation method according to claim 4, characterized in that: The antibody to be labeled is a procalcitonin antibody, and the concentration of the procalcitonin antibody in the initial reaction system of the reaction is 0.1 mg / mL to 0.2 mg / mL.

6. The preparation method according to claim 1 or 2, characterized in that: The antibody to be labeled is carbohydrate antigen 19-9 antibody, and the concentration of the carbohydrate antigen 19-9 antibody in the initial reaction system of the reaction is 0.25 mg / mL to 5 mg / mL.

7. The preparation method according to claim 1 or 2, characterized in that: The reaction conditions are 35℃~39℃ water bath for 1 h~4 h.

8. The preparation method according to claim 1 or 2, characterized in that: The termination method of the reaction includes adding a glycine solution to the reaction system; Optionally, the volume ratio of the glycine solution to the mixed solution obtained after the reaction is 1:17-1:15, and the concentration of the glycine solution is 1M-1.1M; Optionally, the conditions for terminating the reaction are: standing at room temperature for 5 min to 20 min.

9. The preparation method according to claim 1 or 2, characterized in that: The preparation method also includes the step of purifying the labeled antibody; Optionally, the purification comprises dialysis; Optionally, dialysis is performed using a 14KD dialysis bag and 0.05M-0.1M phosphate buffer at 2°C-8°C.

10. Application of 1,6-hexanediol in the preparation of products for electrochemiluminescent immunoassay.

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