Method for preparing biotinylated antibodies

Abstract

The present application relates to the field of in vitro diagnosis, in particular to a preparation method of biotinylated antibody. The present application discloses a high-efficiency biotinylated antibody preparation method, which comprises: aminoating the antibody; reacting the antibody with a biotin solution; removing free biotin, adding a protective agent, and obtaining a biotinylated antibody finished product. Through amino modification of the antibody, the present application changes the physicochemical properties of the antibody, increases the reaction sites of the antibody and biotin, changes the isoelectric point of the antibody, and increases the hydrophilicity. In addition, through optimization of the reaction system, the biotinylation efficiency is further improved. The method of the present application is simple to operate, has wide application, and the obtained biotinylated antibody has strong signal amplification capability.

Description

Technical Field

[0001] This invention relates to the field of in vitro diagnostics, and particularly to a method for preparing biotinylated antibodies. Background Technology

[0002] The strong binding affinity and multi-level amplification effect between biotin and avidin make BAS immunolabeling and related tracer analyses more sensitive. It has been widely used in various techniques such as qualitative and quantitative detection of trace antigens and antibodies, and localization observation studies.

[0003] Biotin, also known as vitamin H or vitamin B7, is a water-soluble vitamin that participates in the biochemical reactions of important substances such as fats, sugars, and proteins in the human body. Biotin is widely found in liver, kidneys, yeast, and milk. Currently, through various modification methods, biotin has a wide variety of derivatives, and biotin-labeled protein technology is becoming increasingly mature. The structure of biotin derivatives basically consists of a biotin bicyclic structure, a valeric acid side chain, a spacer arm, and a reactive group. The labeling efficiency of proteins is usually improved by selecting the hydrophilicity / hydrophobicity and length of the spacer arm in the biotin derivative, and optimizing reaction conditions. However, the improvement is often limited. With the widespread application of the biotin-avidin system, the demand for biotinylated antibodies is also increasing. For some biotinylated materials used in large quantities or at high cost, an effective method needs to be found to address this problem. Summary of the Invention

[0004] In view of this, the present invention provides a method for preparing biotinylated antibodies, which is simple to operate, cost-effective, and has a wide range of applications; the obtained biotinylated antibodies have good stability and strong signal amplification ability.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a method for preparing biotinylated antibodies, comprising the following steps:

[0007] Step (1): The antibody is replaced in acidic buffer and mixed with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and aminoPEG amino to undergo the first reaction and obtain the aminated antibody.

[0008] Step (2): The aminated antibody described in step (1) is replaced in a slightly alkaline buffer solution and mixed with biotin to undergo a second reaction to obtain a semi-finished product;

[0009] Step (3): Replace the semi-finished product described in step (2) in the preservation buffer to remove free biotin, and then mix it with the protectant to obtain biotinylated antibody.

[0010] In some specific embodiments of the present invention, the displacement in the above preparation method includes achieving the displacement by dialysis or gel filtration chromatography.

[0011] In some specific embodiments of the present invention, in step (1) of the above preparation method:

[0012] The antibody includes mouse monoclonal antibodies or polyclonal antibodies of any species; and / or

[0013] The acidic buffer solution includes MES buffer solution; and / or

[0014] The pH of the acidic buffer solution is 4.5–6.5; and / or

[0015] The molar ratio of the antibody, the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and the aminoPEG amino group is 1:(20-30):(15-20); and / or

[0016] The amino group of the PEG has a molecular weight of 200–1000; and / or

[0017] The temperature of the first reaction is 2–8 °C; and / or

[0018] The first reaction takes 12 to 16 hours.

[0019] In some specific embodiments of the present invention, in step (1) of the above preparation method:

[0020] The antibody is a mouse monoclonal antibody;

[0021] The concentration of the mouse monoclonal antibody is not less than 1 mg / mL; and / or

[0022] In some specific embodiments of the present invention, in step (1) of the above preparation method:

[0023] The temperature of the first reaction is 4°C; and / or

[0024] The first reaction took 16 hours.

[0025] In some specific embodiments of the present invention, in step (2) of the above preparation method:

[0026] The alkaline buffer solution includes an amino-free buffer solution; and / or

[0027] The amino-free buffer solution includes borate buffer and / or carbonate buffer; and / or

[0028] The biotin includes water-soluble succinimide-modified biotin; and / or

[0029] The water-soluble succinimide-modified biotin includes sulfo-NHS-LC-LC-Biotin and / or sulfo-NHS-Biotin; and / or

[0030] The molar ratio of the aminolated antibody to the biotin is 1:(40-80); and / or

[0031] The temperature of the second reaction is 20–27°C; and / or

[0032] The second reaction takes 12 to 16 hours.

[0033] In some specific embodiments of the present invention, in step (2) of the above preparation method:

[0034] The biotin is sulfo-NHS-Biotin; and / or

[0035] The molar ratio of the aminolated antibody to the biotin is 1:(40-60); and / or

[0036] In some specific embodiments of the present invention, in step (2) of the above preparation method:

[0037] The temperature of the second reaction is 25°C; and / or

[0038] The second reaction took 16 hours.

[0039] In some specific embodiments of the present invention, in step (3) of the above preparation method:

[0040] The preservation buffer includes a neutral buffer; and / or

[0041] The neutral buffer solution includes a phosphate buffer solution with a pH of 7.0–7.4; and / or

[0042] The protective agent includes trehalose and / or sucrose at a concentration of 80–120 g / L.

[0043] In some specific embodiments of the present invention, in step (3) of the above preparation method:

[0044] The neutral buffer solution is a phosphate buffer with a pH of 7.2; and / or

[0045] The protective agent comprises sucrose at a concentration of 100 g / L.

[0046] The method of the present invention has the following effects:

[0047] This invention is simple to operate, and can be achieved through a two-step cross-linking reaction; it saves costs, as the amount of antibody and biotin can be reduced while achieving the same labeling effect by modifying the antibody; it has a wide range of applications, not only for monoclonal antibodies but also for polyclonal antibodies; the obtained biotinylated antibody has good stability and strong signal amplification capability. Attached Figure Description

[0048] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0049] Figure 1 The bar chart shows the efficacy verification of the antibody prepared by the method of the present invention. Detailed Implementation

[0050] This invention discloses a method for preparing biotinylated antibodies. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0051] The purpose of this invention is to provide an efficient method for preparing biotinylated antibodies. This method is simple to operate, cost-effective, and has a wide range of applications. The biotinylated antibodies obtained have good stability and strong signal amplification capabilities.

[0052] The principle is as follows: The antibody surface has a large number of carboxyl groups exposed. The carboxyl groups on the antibody surface are modified to amino groups by PEGylation. Combined with the amino groups already present on the antibody surface, this greatly increases the probability of reaction with biotin modified with succinimide. In addition, the large amount of PEGylation makes the antibody's isoelectric point higher and its hydrophilicity stronger. By selecting appropriate reaction conditions, the reaction efficiency can be maximized. The increased reaction efficiency allows more biotin to be attached to the antibody, increasing the probability of binding with avidinase, which indirectly affects the amplification of the enzyme signal. The reaction efficiency also affects the signal amplification, ultimately achieving better reaction results with less raw material.

[0053] Specifically, the carboxyl groups on the surface of the antibody are first modified into PEG amino groups using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and aminoPEG amino groups. Combined with the amino groups on the surface of the antibody itself, this greatly increases the total number of exposed amino groups on the antibody surface. At the same time, it changes the isoelectric point and hydrophilicity of the antibody. By adjusting the reaction system, the probability of NHS-modified biotin reacting with the antibody is greatly increased, thereby improving the cross-linking efficiency.

[0054] The method for preparing highly efficient biotinylated antibodies according to the present invention specifically includes the following steps:

[0055] The first step is to replace the antibody storage buffer with an acidic buffer. After the replacement is completed, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and aminoPEG amino are added to the antibody solution in proportion and mixed well. The mixture is reacted at 4°C for 12-16 hours to aminate the antibody.

[0056] The second step is to place the modified antibody solution in a slightly alkaline buffer solution. After the displacement is completed, add the dissolved biotin solution to the antibody solution in proportion and mix well. React at 25°C for 12-16 hours.

[0057] The third step involves placing the product in a biotinylated antibody preservation buffer to remove the free biotin from the reaction product, adding a protective agent, and obtaining the finished biotinylated antibody.

[0058] In the first step of the above-mentioned biotinylated antibody preparation method, the antibody storage buffer is replaced with an acidic buffer, which is a MES buffer with a pH of 4.5 to 6.5. The antibody is a mouse monoclonal antibody or a polyclonal antibody from other species, with a concentration of not less than 1 mg / mL.

[0059] After the antibody buffer replacement in the first step is completed, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and aminoPEG amino are added to the antibody solution in a certain proportion and mixed well. The molar ratio is antibody: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride: aminoPEG amino = 1:20~30:15~20. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and aminoPEG amino are dissolved in ultrapure water. The aminoPEG amino fraction is selected to be 200~1000.

[0060] In the second step, the modified antibody solution is placed in a slightly alkaline buffer solution, which is an amino-free buffer solution with a pH of 8.0 to 8.2, such as borate buffer or carbonate buffer.

[0061] In the second step, the dissolved biotin solution is added to the antibody solution in a certain proportion. The molar ratio of antibody to biotin is 1:40 to 60. The biotin is a commercially available water-soluble succinimide-modified biotin (sulfo-NHS-LC-LC-Biotin, sulfo-NHS-Biotin, etc.), which is dissolved in ultrapure water.

[0062] In the third step, the reaction is terminated by placing the product in a biotinylated antibody preservation buffer, and the free biotin in the reaction product is removed. A protective agent is then added, which is 10% (w / v, 10g / 100mL) of trehalose or sucrose.

[0063] The third step involves placing the product in a biotinylated antibody preservation buffer solution, removing the free biotin from the reaction product, and adding a protective agent. The buffer solution used is a neutral buffer solution with a pH of 7.2 to 7.4.

[0064] In the first and third steps, buffer replacement can be performed using either dialysis or gel filtration chromatography.

[0065] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in this invention are all commercially available products and can be purchased from the market.

[0066] The present invention will be further illustrated below with reference to the embodiments:

[0067] Example 1: Preparation of biotinylated antibodies

[0068] 1. Antibody modification

[0069] The antibody to be labeled was dialyzed into pH 4.5 MES buffer at a volume ratio of 1:50, with the buffer changed twice during the process, with an interval of no less than 4 hours between the two changes. After dialysis, the antibody was added in a molar ratio of antibody: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride: aminoPEG amino (molecular weight 200) = 1:20:15. After mixing, the mixture was reacted at 4°C for 16 hours.

[0070] 2. Crosslinking reaction

[0071] After the reaction was completed, the antibody was placed in pH 8.0 carbonate buffer and dialyzed at a volume ratio of 1:50, with the buffer changed twice during the process, with an interval of no less than 4 hours between buffer changes. After dialysis, the modified antibody and biotin (sulfo-NHS-Biotin) were added at a molar ratio of 1:40, mixed well, and reacted at 25°C for 16 hours.

[0072] 3. Post-processing

[0073] After the reaction was complete, the biotinylated antibody was placed in pH 7.2 phosphate buffer and dialyzed at a volume ratio of 1:40, with the buffer changed twice, with an interval of no less than 4 hours between changes. After dialysis, the volume was measured, the concentration of the biotinylated antibody was recalculated, 10% sucrose was added and mixed well, and the mixture was stored at -20°C.

[0074] Example 2: Preparation of biotinylated antibodies

[0075] 1. Antibody modification

[0076] The antibody to be labeled was dialyzed into pH 6.5 MES buffer at a volume ratio of 1:50, with the buffer changed twice during the process, with an interval of no less than 4 hours between the two changes. After dialysis, the antibody was added in a molar ratio of antibody: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride: aminoPEG amino (molecular weight 1000) = 1:30:20. After mixing, the mixture was reacted at 4°C for 16 hours.

[0077] 2. Crosslinking reaction

[0078] After the reaction was completed, the antibody was placed in pH 8.0 carbonate buffer and dialyzed at a volume ratio of 1:40, with the buffer changed twice during the process, with an interval of no less than 4 hours between buffer changes. After dialysis, the modified antibody and biotin (sulfo-NHS-Biotin) were added at a molar ratio of 1:60, mixed well, and reacted at 25°C for 16 hours.

[0079] 3. Post-processing

[0080] After the reaction was complete, the biotinylated antibody was placed in pH 7.2 phosphate buffer and dialyzed at a volume ratio of 1:40, with the buffer changed twice, with an interval of no less than 4 hours between changes. After dialysis, the volume was measured, the concentration of the biotinylated antibody was recalculated, 10% sucrose was added and mixed well, and the mixture was stored at -20°C.

[0081] Comparative Example 1: Preparation of Biotinylated Antibodies

[0082] 1. Antibody modification

[0083] The antibody to be labeled was dialyzed into pH 4.5 MES buffer at a volume ratio of 1:50, with the buffer changed twice during the process, with an interval of no less than 4 hours between the two changes. After dialysis, the antibody was added in a molar ratio of antibody: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride: aminoPEG amino (molecular weight 200) = 1:20:50. After mixing, the mixture was reacted at 4°C for 16 hours.

[0084] 2. Crosslinking reaction

[0085] After the reaction was completed, the antibody was placed in pH 8.0 carbonate buffer and dialyzed at a volume ratio of 1:40, with the buffer changed twice during the process, with an interval of no less than 4 hours between buffer changes. After dialysis, the modified antibody and biotin (sulfo-NHS-Biotin) were added at a molar ratio of 1:40, mixed well, and reacted at 25°C for 16 hours.

[0086] 3. Post-processing

[0087] After the reaction was complete, the biotinylated antibody was placed in pH 7.2 phosphate buffer and dialyzed at a volume ratio of 1:40, with the buffer changed twice, with an interval of no less than 4 hours between changes. After dialysis, the volume was measured, the concentration of the biotinylated antibody was recalculated, 10% sucrose was added and mixed well, and the mixture was stored at -20°C.

[0088] Comparative Example 2: Preparation of Biotinylated Antibodies

[0089] 1. Antibody modification

[0090] The antibody to be labeled was dialyzed into pH 4.5 MES buffer at a volume ratio of 1:50. The buffer was changed twice during the dialyzing process, with an interval of no less than 4 hours between the two changes. After the dialyzing was completed, the antibody was added in a molar ratio of antibody: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride: aminoPEG amino (molecular weight 200) = 1:40:15. The mixture was then reacted at 4°C for 16 hours.

[0091] 2. Crosslinking reaction

[0092] After the reaction was completed, the antibody was placed in pH 8.0 carbonate buffer and dialyzed at a volume ratio of 1:40, with the buffer changed twice during the process, with an interval of no less than 4 hours between buffer changes. After dialysis, the modified antibody and biotin (sulfo-NHS-Biotin) were added at a molar ratio of 1:40, mixed well, and reacted at 25°C for 16 hours.

[0093] 3. Post-processing

[0094] After the reaction was complete, the biotinylated antibody was placed in pH 7.2 phosphate buffer and dialyzed at a volume ratio of 1:40, with the buffer changed twice, with an interval of no less than 4 hours between changes. After dialysis, the volume was measured, the concentration of the biotinylated antibody was recalculated, 10% sucrose was added and mixed well, and the mixture was stored at -20°C.

[0095] Comparative Example 3: Preparation of Biotinylated Antibodies

[0096] 1. Antibody modification

[0097] The antibody to be labeled was dialyzed into pH 4.5 MES buffer at a volume ratio of 1:50, with the buffer changed twice during the process, with an interval of no less than 4 hours between the two changes. After dialysis, the antibody was added in a molar ratio of antibody: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride: aminoPEG amino (molecular weight 200) = 1:20:15. After mixing, the mixture was reacted at 4°C for 16 hours.

[0098] 2. Crosslinking reaction

[0099] After the reaction was completed, the antibody was placed in pH 6.0 phosphate buffer and dialyzed at a volume ratio of 1:40, with the buffer changed twice during the process, with an interval of no less than 4 hours between buffer changes. After dialysis, the modified antibody and biotin (sulfo-NHS-Biotin) were added at a molar ratio of 1:40, mixed well, and reacted at 25°C for 16 hours.

[0100] 3. Post-processing

[0101] After the reaction was complete, the biotinylated antibody was placed in pH 7.2 phosphate buffer and dialyzed at a volume ratio of 1:40, with the buffer changed twice, with an interval of no less than 4 hours between changes. After dialysis, the volume was measured, the concentration of the biotinylated antibody was recalculated, 10% sucrose was added and mixed well, and the mixture was stored at -20°C.

[0102] Comparative Example 4: Preparation of Biotinylated Antibodies

[0103] 1. Antibody modification

[0104] The antibody to be labeled was dialyzed into pH 4.5 MES buffer at a volume ratio of 1:50, with the buffer changed twice during the process, with an interval of no less than 4 hours between the two changes. After dialysis, the antibody was added in a molar ratio of antibody: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride: aminoPEG amino (molecular weight 200) = 1:20:15. After mixing, the mixture was reacted at 4°C for 16 hours.

[0105] 2. Crosslinking reaction

[0106] After the reaction was completed, the antibody was placed in pH 9.0 carbonate buffer and dialyzed at a volume ratio of 1:40, with the buffer changed twice during the process, with an interval of no less than 4 hours between buffer changes. After dialysis, the modified antibody and biotin (sulfo-NHS-Biotin) were added at a molar ratio of 1:40, mixed well, and reacted at 25°C for 16 hours.

[0107] 3. Post-processing

[0108] After the reaction was complete, the biotinylated antibody was placed in pH 7.2 phosphate buffer and dialyzed at a volume ratio of 1:40, with the buffer changed twice, with an interval of no less than 4 hours between changes. After dialysis, the volume was measured, the concentration of the biotinylated antibody was recalculated, 10% sucrose was added and mixed well, and the mixture was stored at -20°C.

[0109] Comparative Example 5: Preparation of Biotinylated Antibodies

[0110] 1. Antibody modification

[0111] The antibody to be labeled was dialyzed into pH 7.0 phosphate buffer at a volume ratio of 1:50, with the buffer changed twice during the process, with an interval of no less than 4 hours between the two changes. After dialysis, the antibody was added in a molar ratio of antibody: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride: aminoPEG amino (molecular weight 1000) = 1:30:20. After mixing, the mixture was reacted at 4°C for 16 hours.

[0112] 2. Crosslinking reaction

[0113] After the reaction was completed, the antibody was placed in pH 8.0 carbonate buffer and dialyzed at a volume ratio of 1:40, with the buffer changed twice during the process, with an interval of no less than 4 hours between buffer changes. After dialysis, the modified antibody and biotin (sulfo-NHS-Biotin) were added at a molar ratio of 1:60, mixed well, and reacted at 25°C for 16 hours.

[0114] 3. Post-processing

[0115] After the reaction was complete, the biotinylated antibody was placed in pH 7.2 phosphate buffer and dialyzed at a volume ratio of 1:40, with the buffer changed twice, with an interval of no less than 4 hours between changes. After dialysis, the volume was measured, the concentration of the biotinylated antibody was recalculated, 10% sucrose was added and mixed well, and the mixture was stored at -20°C.

[0116] Comparative Example 6: Preparation of Biotinylated Antibodies

[0117] 1. Antibody modification

[0118] The antibody to be labeled was dialyzed into pH 6.5 MES buffer at a volume ratio of 1:50, with the buffer changed twice during the process, with an interval of no less than 4 hours between the two changes. After dialysis, the antibody was added in a molar ratio of antibody: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride: aminoPEG amino (molecular weight 5000) = 1:30:20. After mixing, the mixture was reacted at 4°C for 16 hours.

[0119] 2. Crosslinking reaction

[0120] After the reaction was completed, the antibody was placed in pH 8.0 carbonate buffer and dialyzed at a volume ratio of 1:60, with the buffer changed twice during the process, with an interval of no less than 4 hours between buffer changes. After dialysis, the modified antibody and biotin (sulfo-NHS-Biotin) were added at a molar ratio of 1:60, mixed well, and reacted at 25°C for 16 hours.

[0121] 3. Post-processing

[0122] After the reaction was complete, the biotinylated antibody was placed in pH 7.2 phosphate buffer and dialyzed at a volume ratio of 1:40, with the buffer changed twice, with an interval of no less than 4 hours between changes. After dialysis, the volume was measured, the concentration of the biotinylated antibody was recalculated, 10% sucrose was added and mixed well, and the mixture was stored at -20°C.

[0123] Comparative Example 7: Preparation of Biotinylated Antibodies

[0124] 1. Antibody modification

[0125] The antibody to be labeled was dialyzed into pH 6.5 MES buffer at a volume ratio of 1:50. The buffer was changed twice during the dialyzing process, with an interval of no less than 4 hours between the two changes. After the dialyzing was completed, the antibody:carbodiimide:ethylenediamine was added at a molar ratio of 1:30:20. After mixing, the mixture was reacted at 4°C for 16 hours.

[0126] 2. Crosslinking reaction

[0127] After the reaction was completed, the antibody was placed in pH 8.0 carbonate buffer and dialyzed at a volume ratio of 1:40, with the buffer changed twice during the process, with an interval of no less than 4 hours between buffer changes. After dialysis, the modified antibody and biotin (sulfo-NHS-Biotin) were added at a molar ratio of 1:60, mixed well, and reacted at 25°C for 16 hours.

[0128] 3. Post-processing

[0129] After the reaction was complete, the biotinylated antibody was placed in pH 7.2 phosphate buffer and dialyzed at a volume ratio of 1:40, with the buffer changed twice, with an interval of no less than 4 hours between changes. After dialysis, the volume was measured, the concentration of the biotinylated antibody was recalculated, 10% sucrose was added and mixed well, and the mixture was stored at -20°C.

[0130] Comparative Example 8: Preparation of Biotinylated Antibodies

[0131] 1. Antibody modification

[0132] The antibody to be labeled was dialyzed into pH 6.5 MES buffer at a volume ratio of 1:50, with the buffer changed twice during the process, with an interval of no less than 4 hours between the two changes. After dialysis, the antibody was added in a molar ratio of antibody: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride: aminoPEG amino (molecular weight 1000) = 1:30:20. After mixing, the mixture was reacted at 4°C for 16 hours.

[0133] 2. Crosslinking reaction

[0134] After the reaction was completed, the antibody was placed in pH 8.0 carbonate buffer and dialyzed at a volume ratio of 1:40, with the buffer changed twice during the process, with an interval of no less than 4 hours between buffer changes. After dialysis, the modified antibody and biotin (sulfo-NHS-Biotin) were added at a molar ratio of 1:80, mixed well, and reacted at 25°C for 16 hours.

[0135] 3. Post-processing

[0136] After the reaction was complete, the biotinylated antibody was placed in pH 7.2 phosphate buffer and dialyzed at a volume ratio of 1:40, with the buffer changed twice, with an interval of no less than 4 hours between changes. After dialysis, the volume was measured, the concentration of the biotinylated antibody was recalculated, 10% sucrose was added and mixed well, and the mixture was stored at -20°C.

[0137] Example 3: Performance Detection of Biotinylated Antibody

[0138] 1. ELISA titer determination: ① Biotinylated mouse monoclonal antibody: solid-phase coated goat anti-mouse secondary antibody, biotinylated antibody added, after reaction, avidinized HRP added, and its reactivity determined based on the signal value; ② Biotinylated polyclonal antibody: taking goat anti-mouse as an example, solid-phase coated mouse IgG, biotinylated secondary antibody added, after reaction, avidinized HRP added, and its reactivity determined based on the signal value.

[0139] 2. Potency determination method: 2 μg / mL 50 μL of solid-phase coating was used for coating. Biotinylated antibodies with different cross-linking conditions were uniformly diluted to 1 μg / mL 50 μL for reaction. After the reaction was completed, 1 / 5K 50 μL of avidinized HRP was added. The potency of the biotinylated antibody was reflected by the magnitude of the luminescence value. The higher the luminescence value, the higher the potency of the biotinylated antibody.

[0140] The control group consisted of unmodified antibodies directly labeled with biotin. Detailed measurement values ​​are shown in Table 1.

[0141] Table 1

[0142]

[0143]

[0144] From the bar chart ( Figure 1 As can be seen from this:

[0145] 1. In Example 1, the titers of Comparative Examples 1, 2, and 8 were basically the same, indicating that the addition of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, aminoPEG-amino, and biotin had little impact on the experimental results, and the reagents were in excess.

[0146] 2. Comparative analysis of Example 1, Comparative Examples 3 and 4 shows that pH has a significant impact on the biotin cross-linking reaction;

[0147] 3. Analysis of Example 2 and Comparative Example 6 shows that the molecular weight of the amino group in aminoPEG affects the potency test results, suggesting the existence of steric hindrance.

[0148] 4. Analysis of Example 2 and Comparative Example 7 shows that when the amino group of amino PEG is replaced by a diamine crosslinking agent, represented by ethylenediamine, without PEG modification, the titer decreases, and the physicochemical properties of the antibody affect the reaction efficiency.

[0149] 5. Analysis of data from examples, controls, and comparative examples shows that the preparation of biotinylated antibodies is affected not only by the physicochemical properties of the antibody itself but also by the cross-linking conditions.

[0150] Conclusion: The biotinylated antibody prepared by the method of the present invention has a high cross-linking efficiency, which is 2 to 3 times higher than that of conventional biotinylated antibody control.

[0151] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing biotinylated antibodies, characterized in that, Includes the following steps: Step (1): The antibody is replaced in acidic buffer and mixed with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and aminoPEG amino to undergo the first reaction to obtain the aminated antibody; Step (2): The aminated antibody described in step (1) is replaced in a slightly alkaline buffer solution and mixed with biotin to undergo a second reaction to obtain a semi-finished product; Step (3): Replace the semi-finished product described in step (2) in the preservation buffer to remove free biotin, and then mix it with the protective agent to obtain biotinylated antibody; In step (1): The antibody is a mouse monoclonal antibody; The concentration of the mouse monoclonal antibody is not less than 1 mg / mL; The acidic buffer solution includes MES buffer solution; The pH of the acidic buffer solution is 4.5~6.5; The molar ratio of the antibody, the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and the aminoPEG amino group is 1:(20~30):(15~20); The amino group of the PEG has a molecular weight of 200-1000. The temperature of the first reaction is 4°C; The first reaction took 16 hours; In step (2): The alkaline buffer solution includes a buffer solution that does not contain amino groups; The non-amino buffer solution includes borate buffer and / or carbonate buffer; The biotin is sulfo-NHS-Biotin; The molar ratio of the amino-modified antibody to the biotin is 1:(40~60); The temperature of the second reaction is 25°C; The second reaction took 16 hours; In step (3): The preservation buffer includes a neutral buffer; The neutral buffer solution is a phosphate buffer solution with a pH of 7.2; The protective agent comprises sucrose at a concentration of 100 g / L; The replacement includes achieving the replacement through dialysis or gel filtration chromatography.

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