Preparation method of horse radish peroxidase labeled antibody

Abstract

The invention provides a preparation method of a horse radish peroxidase labeled antibody. Compared with the prior art, the method has the advantages that firstly, amino on horse radish peroxidase is sealed by the sealing agent, so that coupling reaction between active aldehyde groups after enzyme activation and self amino is greatly reduced, and self-polymerization of horse radish peroxidase is avoided; an o-hydroxyl structure of a sugar ring in an enzyme molecule is oxidized and subjected to ring opening by utilizing the oxidability of sodium periodate to generate an aldehyde group, and the activation reaction is terminated by utilizing an inorganic reducing agent, so that the reaction time is shortened, and the activation efficiency is improved; finally, in the presence of a reducing agent, coupling reaction with amino of the antibody is carried out, so that the cross-linking efficiency of the horse radish peroxidase and the antibody protein is improved, meanwhile, the labeling period is greatly shortened, and the complexity of experimental operation is reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of immunological detection, and particularly relates to a method for preparing horseradish peroxidase-labeled antibody. Background Art

[0002] Immunological detection technology has the advantages of high detection sensitivity, fast speed, low cost, and strong selectivity. It combines the amplification effect of enzyme-catalyzed reactions with the high specificity and selectivity of antigen-antibody affinity reactions, and uses enzyme-labeled antigens or antibodies as key reagents for immunoassays, so it has high sensitivity. The enzymes used to label antigens or antibodies need to have the following characteristics: high activity and sensitivity; easy manifestation of reaction products; and can be commercially produced. Currently, horseradish peroxidase, alkaline phosphatase, glucose oxidase, etc. are widely used, among which horseradish peroxidase is the most widely used and is widely used in clinical diagnosis and immunoassay reagents, etc.

[0003] The methods for preparing enzyme-labeled substances by bioconjugation mainly include carbodiimide method, glutaraldehyde method, sodium periodate method, etc. Each method has its own advantages and disadvantages, and the selection of specific cross-linking methods also needs to be determined according to the available active functional groups (carboxyl group, amino group, aldehyde group, hydroxyl group, mercapto group, etc.) of the target molecule. According to the molecular characteristics of horseradish peroxidase, the sodium periodate method is mostly used for the preparation of enzyme-labeled antigens and antibodies. The basic principle of the traditional sodium periodate method is: using the oxidizing property of sodium periodate to oxidize the adjacent hydroxyl groups in the horseradish peroxidase molecule to aldehyde groups, and then reacting with primary amino groups to form Schiff bases, and reducing the Schiff bases to stable carbon-nitrogen single bonds through a reducing agent.

[0004] However, in the traditional sodium periodate method, after horseradish peroxidase is activated by sodium periodate, it needs to be terminated with ethylene glycol. The reaction is an organic reaction, with a long and insufficient reaction time. Ethylene glycol will react with aldehyde groups to form hemiacetal and acetal structures, which are relatively stable and not easy to form aldehyde group structures again, thus greatly reducing the efficiency of subsequent coupling reactions.

[0005] Moreover, the basic process of labeling antibodies by the traditional sodium periodate method is that the activated enzyme first reacts with the antibody for 24 hours, and then sodium borohydride, a strong reducing agent, is added as a reducing agent for reaction. Sodium borohydride can not only reduce the formed Schiff bases, but also reduce the active aldehyde groups obtained in the previous activation step. In addition, the imine structure formed by the reaction of the activated enzyme and the antibody in this process is very unstable and can hardly remain in the buffer solution for a long time. After 24 hours of reduction, most of the formed imine structures will be hydrolyzed and cannot participate in the next reduction reaction. Summary of the Invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide a method for preparing horseradish peroxidase-labeled antibody, which has a high coupling efficiency.

[0007] The present invention provides a method for preparing horseradish peroxidase-labeled antibody, comprising the following steps:

[0008] S1) React horseradish peroxidase with an amino-blocking agent to obtain amino-blocked horseradish peroxidase;

[0009] S2) React the amino-blocked horseradish peroxidase with sodium periodate for a period of time, and then add an inorganic reducing agent to terminate the reaction to obtain activated horseradish peroxidase;

[0010] S3) Mix the activated horseradish peroxidase, antibody and a reducing agent, and carry out a cross-linking reaction to obtain horseradish peroxidase-labeled antibody.

[0011] Preferably, step S1) is specifically:

[0012] Add an aqueous solution of the amino-blocking agent to an aqueous solution of horseradish peroxidase for reaction to obtain a reaction solution containing amino-blocked horseradish peroxidase.

[0013] Preferably, the amino-blocking agent is selected from Sulfo-NHS-COO-(CH 3 ) m and / or NHS-COO-(CH 3 ) m ; wherein, m is an integer from 1 to 10;

[0014] The molar ratio of the horseradish peroxidase to the amino-blocking agent is 1:6 to 1:20.

[0015] Preferably, the temperature of the reaction is 20°C to 30°C; the reaction time is 1 to 3 h.

[0016] Preferably, in step S2), sodium periodate is added in the form of an aqueous solution of sodium periodate to the solution of amino-blocked horseradish peroxidase for activation reaction. After reacting for a period of time, an inorganic reducing agent is added to terminate the reaction.

[0017] Preferably, the mass ratio of the amino-blocked horseradish peroxidase to sodium periodate is 1:1 to 1:5;

[0018] The temperature of the reaction is 20°C to 30°C; the oxidation reaction time is 1 to 3 h.

[0019] Preferably, the inorganic reducing agent is selected from one or more of sodium iodide, potassium iodide, sodium thiosulfate and sodium sulfide;

[0020] The molar ratio of the inorganic reducing agent to sodium periodate is 1:1 to 5:1.

[0021] The temperature of the reaction is 20°C to 30°C; the reduction reaction time is 3 - 10 min.

[0022] Preferably, in the step S3), the mass ratio of the activated horseradish peroxidase to the antibody is 1:1 to 1:5;

[0023] The reducing agent is selected from one or more of sodium cyanoborohydride and sodium triacetoxyborohydride;

[0024] The molar ratio of the antibody to the reducing agent is 1:10 to 1:50.

[0025] Preferably, the temperature of the cross-linking reaction is 20°C to 30°C; the cross-linking reaction time is 6 - 15 h.

[0026] Preferably, after the termination reaction in the step S2), the reaction solution is dialyzed to obtain the activated horseradish peroxidase; the buffer solution used for dialysis is 0.01 mol / L PBS buffer solution; the number of dialysis times is 2 - 6 times;

[0027] After the cross-linking reaction in the step S3), the reaction solution is dialyzed to obtain the horseradish peroxidase-labeled antibody; the buffer solution used for dialysis is 0.01 mol / L PBS buffer solution; the number of dialysis times is 2 - 6 times;

[0028] The pH value of the 0.01 mol / L PBS buffer solution is 7 - 7.5.

[0029] The present invention provides a method for preparing a horseradish peroxidase-labeled antibody, which includes the following steps: S1) reacting horseradish peroxidase with an amino-blocking agent to obtain amino-blocked horseradish peroxidase; S2) reacting the amino-blocked horseradish peroxidase with sodium periodate, then adding an inorganic reducing agent to terminate the reaction, and obtaining the activated horseradish peroxidase after dialysis; S3) mixing the activated horseradish peroxidase, the antibody and the reducing agent to carry out a cross-linking reaction, and obtaining the horseradish peroxidase-labeled antibody after dialysis. Compared with the prior art, the present invention first performs a blocking treatment on the amino groups on the horseradish peroxidase with a blocking agent, greatly reducing the coupling reaction between the active aldehyde groups and the self-amino groups after enzyme activation, and avoiding the self-polymerization of horseradish peroxidase; then uses the oxidizing property of sodium periodate to oxidize and open the adjacent hydroxyl structure of the sugar ring in the enzyme molecule to form aldehyde groups, and uses an inorganic reducing agent to terminate the activation reaction, shortening the reaction time and improving the activation efficiency; finally, in the presence of a reducing agent, it carries out a coupling reaction with the amino groups of the antibody, improving the cross-linking efficiency of horseradish peroxidase and antibody protein, while greatly shortening the labeling period and reducing the complexity of experimental operations. Specific embodiments

[0030] Next, in combination with the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0031] The present invention provides a method for preparing horseradish peroxidase-labeled antibody, which includes the following steps: S1) reacting horseradish peroxidase with an amino-blocking agent to obtain amino-blocked horseradish peroxidase; S2) reacting the amino-blocked horseradish peroxidase with sodium periodate under light-shielded conditions, then adding an inorganic reducing agent to terminate the reaction, and obtaining activated horseradish peroxidase after dialysis; S3) mixing the activated horseradish peroxidase, antibody and a reducing agent, performing a cross-linking reaction, and obtaining horseradish peroxidase-labeled antibody after dialysis.

[0032] Among them, the present invention does not have special restrictions on the sources of all raw materials, and they can be commercially available. In the present invention, HBsAb (hepatitis B surface antibody) is selected as a representative antibody for cross-linking reaction.

[0033] Reacting horseradish peroxidase with an amino-blocking agent to obtain amino-blocked horseradish peroxidase; the amino-blocking agent is preferably Sulfo-NHS-COO-(CH 3 ) m and / or NHS-COO-(CH 3 ) m ; wherein, m is preferably an integer from 1 to 10, more preferably an integer from 1 to 8, still more preferably an integer from 1 to 6, still more preferably an integer from 1 to 4, and most preferably 1 or 2; the smaller the value of m, the smaller the steric hindrance of the amino-blocking agent, and the more beneficial it is to achieve the blocking effect; the molar ratio of the horseradish peroxidase to the amino-blocking agent is preferably 1:6 to 1:20; optionally, the molar ratio of the horseradish peroxidase to the amino-blocking agent is 1:6, 1:8, 1:10, 1:12, 1:14, 1:16, 1:18, 1:20 or the range between any two of the above ratios; the reaction temperature is preferably 20°C to 30°C, more preferably 22°C to 28°C, still more preferably 25°C; the reaction time is preferably 1 to 3 h, more preferably 1.5 to 2.5 h, still more preferably 2 h.

[0034] In a specific embodiment provided by the present invention, step S1) is specifically as follows: An aqueous solution of an amino blocking agent is added to an aqueous solution of horseradish peroxidase for reaction to obtain a reaction solution containing amino-blocked horseradish peroxidase; in the present invention, the aqueous solution of the amino blocking agent is preferably added dropwise; shaking is preferably performed during the addition process; the reaction solution containing amino-blocked horseradish peroxidase obtained after the reaction can be directly used for the next reaction.

[0035] React the amino-blocked horseradish peroxidase with sodium periodate under light-shielded conditions; the mass ratio of the amino-blocked horseradish peroxidase to sodium periodate is preferably 1:1 to 1:5; optionally, the mass ratio of the amino-blocked horseradish peroxidase to sodium periodate is 1:1, 1:2, 1:3, 1:4, 1:5 or the range between any two of the above ratios; the reaction temperature is preferably 20°C to 30°C, more preferably 22°C to 28°C, and still more preferably 25°C; the reaction time is preferably 1 to 3 h, more preferably 1.5 to 2.5 h, and still more preferably 2 h.

[0036] Then, an inorganic reducing agent is added to terminate the reaction to obtain activated horseradish peroxidase; adding the inorganic reducing agent can remove the unreacted sodium periodate; the inorganic reducing agent is preferably one or more of iodides, sodium thiosulfate and sulfides, more preferably one or more of sodium iodide, potassium iodide, sodium thiosulfate and sodium sulfide; the molar ratio of the inorganic reducing agent to sodium periodate is preferably 1:1 to 5:1; optionally, the molar ratio of the inorganic reducing agent to sodium periodate is 1:1, 2:1, 3:1, 4:1, 5:1 or the range between any two of the above ratios; the temperature for terminating the reaction is preferably 20°C to 30°C, more preferably 22°C to 28°C, and still more preferably 25°C; the time for terminating the reaction is preferably 3 to 10 min, more preferably 3 to 8 min, and still more preferably 5 min.

[0037] After terminating the reaction, it is preferable to dialyze the reaction solution to obtain activated horseradish peroxidase; small molecule substances in the system are removed by dialysis; the buffer solution used for dialysis is 0.01 mol / L PBS buffer solution; the pH value of the buffer solution is preferably 7 to 7.5, more preferably 7.2; the dialysis temperature is preferably 20°C to 30°C, more preferably 22°C to 28°C, and still more preferably 25°C; the number of dialysis times is preferably 2 to 6 times, more preferably 3 to 5 times, and still more preferably 4 times; the time for each dialysis is preferably 1 to 3 h, more preferably 1 to 2 h; in the present invention, it is preferable that the time for starting dialysis is less than the time for subsequent dialysis, that is, the dialysis time for the first 1 to 2 times is less than the dialysis time for the last 1 to 2 times.

[0038] Mix the activated horseradish peroxidase, antibody and reducing agent, and carry out a cross-linking reaction to obtain horseradish peroxidase-labeled antibody; the mass ratio of the activated horseradish peroxidase to the antibody is preferably 1:1 to 1:5; optionally, the mass ratio of the activated horseradish peroxidase to the antibody is 1:1, 1:2, 1:3, 1:4, 1:5 or the range between any two of the above ratios; the reducing agent is preferably one or more of sodium cyanoborohydride and sodium triacetoxyborohydride; the molar ratio of the antibody to the reducing agent is preferably 1:10 to 1:50; optionally, the molar ratio of the antibody to the strong reducing agent is 1:10, 1:20, 1:30, 1:40, 1:50 or the range between any two of the above ratios; the temperature of the cross-linking reaction is preferably 20°C to 30°C, more preferably 22°C to 28°C, and still more preferably 25°C; the time of the cross-linking reaction is preferably 6 to 15, more preferably 8 to 12 h.

[0039] In a specific embodiment provided by the present invention, the antibody is preferably dialyzed first and then mixed with the activated horseradish peroxidase and reducing agent; the buffer used for the dialysis treatment is preferably 0.01 mol / L PBS buffer; the temperature of the dialysis is preferably 20°C to 30°C, more preferably 22°C to 28°C, and still more preferably 25°C; the number of dialysis times is preferably 2 to 6 times, more preferably 3 to 5 times, and still more preferably 4 times; the time of each dialysis is preferably 1 to 3 h, more preferably 1 to 2 h; in the present invention, it is preferred that the time for starting dialysis is less than the time for subsequent dialysis, that is, the dialysis time for the first 1 to 2 times is less than the dialysis time for the last 1 to 2 times.

[0040] In a specific embodiment provided by the present invention, after the cross-linking reaction, the reaction solution is dialyzed to obtain horseradish peroxidase-labeled antibody; the buffer used for the dialysis is 0.01 mol / L PBS buffer; the pH value of the buffer is preferably 7 to 7.5, more preferably 7.2; the temperature of the dialysis is preferably 20°C to 30°C, more preferably 22°C to 28°C, and still more preferably 25°C; the number of dialysis times is preferably 2 to 6 times, more preferably 3 to 5 times, and still more preferably 4 times.

[0041] The present invention first uses a blocking agent to block the amino groups on horseradish peroxidase, greatly reducing the coupling reaction between the active aldehyde groups and the self-amino groups after enzyme activation, and avoiding the self-polymerization of horseradish peroxidase; then uses the oxidizing property of sodium periodate to oxidize and open the adjacent hydroxyl structure of the sugar ring in the enzyme molecule to generate aldehyde groups, and uses an inorganic reducing agent to terminate the activation reaction, shortening the reaction time and improving the activation efficiency; finally, in the presence of a reducing agent, it carries out a coupling reaction with the amino group of the antibody, improving the cross-linking efficiency of horseradish peroxidase and antibody protein, while greatly shortening the labeling period and reducing the complexity of experimental operations.

[0042] To further illustrate the present invention, a method for preparing horseradish peroxidase-labeled antibody provided by the present invention will be described in detail below in conjunction with embodiments.

[0043] All reagents used in the following examples are commercially available. HBsAb (hepatitis B surface antibody) was selected as the representative antibody for cross-linking reaction.

[0044] Example 1

[0045] Step 1: Blocking and terminating the oxidation of horseradish peroxidase

[0046] 1.1 Prepare a 10 mg / mL solution of horseradish peroxidase with deionized water. Dissolve Sulfo-NHS-COO-CH 3 in water to form a 10 mg / mL solution. According to the molar ratio of horseradish peroxidase:Sulfo-NHS-COO-CH 3 = 1:6, slowly add the Sulfo-NHS-COO-CH 3 solution to the horseradish peroxidase solution while dripping and shaking. After the addition is complete, control the temperature at 25 °C and react for 2 h.

[0047] 1.2 Weigh the required amount of sodium periodate according to the mass ratio of amino-blocked horseradish peroxidase to sodium periodate of 1:1. Prepare a 10 mg / mL solution of sodium periodate, and slowly add the sodium periodate solution to the reaction solution obtained in 1.1. React at 25 °C for 2 h to obtain the reaction solution after horseradish peroxidase activation.

[0048] 1.3 Weigh the amount of sodium iodide according to the molar ratio of sodium iodide to sodium periodate of 1:1. Prepare a 10 mg / mL solution of sodium iodide as the activation reaction termination solution. Slowly add the reaction termination solution to the reaction solution after horseradish peroxidase activation while dripping and shaking. React instantaneously at 25 °C for 5 min, and the solution turns brownish-yellow. After the termination reaction is completed, dialyze 4 times with 0.01 M PBS buffer at 25 °C. The first 2 times are dialyzed for 1 h each time, and the last 2 times are dialyzed for 2 h each time. After dialysis, an activated horseradish peroxidase solution is obtained.

[0049] Step 2: Preparation of antibody to be labeled

[0050] Use a pipette to aspirate HBsAb into a dialysis bag, and dialyze 4 times with 0.01 M PBS buffer at 25 °C. The first 2 times are dialyzed for 1 h each time, and the last two times are dialyzed for 2 h each time.

[0051] Step 3: Cross-linking and reduction reaction

[0052] The method for preparing enzyme-labeled antibody is as follows: According to the ratio of enzyme: antibody = 1:1 (mass ratio), appropriate amounts of antibody solution and enzyme solution are aspirated, and the antibody solution is slowly added to the enzyme solution. After thorough mixing, an appropriate volume of sodium triacetoxyborohydride solution is added according to the ratio of enzyme: sodium triacetoxyborohydride = 1:10 (molar ratio). After shaking and mixing, the reaction mixture is allowed to stand overnight at 25°C.

[0053] Step 4: Post-treatment and storage

[0054] After the reaction is completed, the reaction solution is transferred to a dialysis bag, sealed and dialyzed in 0.01M PBS buffer (pH = 7.2) at room temperature for 4 times, and the solution is changed 3 times during dialysis. After dialysis, a solution of horseradish peroxidase-labeled HBsAb is obtained.

[0055] Example 2

[0056] Step 1: Blocking and terminating the oxidation of horseradish peroxidase

[0057] 1.1 Prepare a 10mg / mL solution of horseradish peroxidase with deionized water, and dissolve Sulfo-NHS-COO-CH 3 in water to make a 10mg / mL solution. According to the ratio of horseradish peroxidase: Sulfo-NHS-COO-CH 3 = 1:20 (molar ratio), slowly add the Sulfo-NHS-COO-CH 3 solution dropwise to the horseradish peroxidase solution while shaking. After the addition is complete, control the temperature at 25°C and react for 2h.

[0058] 1.2 Weigh the required amount of sodium periodate according to the mass ratio of amino-blocked horseradish peroxidase to sodium periodate of 1:5, prepare a 10mg / mL solution of sodium periodate, and slowly add the sodium periodate solution to the reaction solution obtained in 1.1. React at 25°C for 2h to obtain the reaction solution after activation of horseradish peroxidase.

[0059] 1.3 Weigh the amount of sodium iodide according to the molar ratio of sodium iodide to sodium periodate of 5:1, prepare a 10mg / mL solution of sodium iodide as the activation reaction termination solution, slowly add the reaction termination solution to the reaction solution after activation of horseradish peroxidase while shaking, and react instantaneously at 25°C for 5min. The solution turns brown-yellow. After the termination reaction is completed, dialyze 4 times at 25°C with 0.01M PBS buffer. The first 2 times of dialysis are 1h each time, and the last 2 times of dialysis are 2h each time. After dialysis, an activated horseradish peroxidase solution is obtained.

[0060] Step 2: Preparation of antibody to be labeled

[0061] Use a pipette to aspirate HBsAb into a dialysis bag, and dialyze it 4 times with 0.01M PBS buffer at 25°C. The first 2 times are dialyzed for 1 hour each time, and the last 2 times are dialyzed for 2 hours each time.

[0062] Step 3: Crosslinking reduction reaction

[0063] The method for preparing enzyme-labeled antibody is as follows: According to enzyme: antibody = 1:5 (mass ratio), aspirate appropriate amounts of antibody solution and enzyme solution, and slowly add the antibody solution to the enzyme solution. After fully mixing, then add an appropriate volume of triacetoxyborohydride solution according to enzyme: triacetoxyborohydride = 1:50 (molar ratio). After shaking and mixing, let it stand and react overnight at 25°C.

[0064] Step 4: Post-treatment and storage

[0065] After the reaction is completed, transfer the reaction solution to a dialysis bag, seal it and dialyze it in 0.01M PBS buffer (pH = 7.2) at room temperature 4 times, and change the solution 3 times during dialysis. After dialysis, a solution of horseradish peroxidase-labeled HBsAb is obtained.

[0066] Example 3

[0067] Step 1: Blocking oxidation termination of horseradish peroxidase

[0068] 1.1 Prepare a 10mg / mL solution of horseradish peroxidase with deionized water, and dissolve Sulfo-NHS-COO-CH 3 in water to form a 10mg / mL solution. According to horseradish peroxidase: Sulfo-NHS-COO-CH 3 = 1:10 (molar ratio), slowly drip the Sulfo-NHS-COO-CH 3 solution into the horseradish peroxidase solution while dripping and shaking. After dripping, control the temperature at 25°C and react for 2 hours.

[0069] 1.2 Weigh the required amount of sodium periodate according to the mass ratio of amino-blocked horseradish peroxidase to sodium periodate of 1:2, prepare a 10mg / mL solution of sodium periodate, and slowly drip the sodium periodate solution into the reaction solution obtained in 1.1, and react at 25°C for 2 hours to obtain the reaction solution after activation of horseradish peroxidase.

[0070] 1.3 Weigh the amount of sodium iodide according to the molar ratio of sodium iodide to sodium periodate of 2:1, and make a 10 mg / mL solution of sodium iodide as the activation reaction termination solution. Slowly add the reaction termination solution dropwise to the reaction solution after the activation of horseradish peroxidase, and shake while adding. React instantaneously for 5 min at 25 °C. The solution turns brownish-yellow. After the reaction ends, dialyze 4 times with 0.01 M PBS buffer at 25 °C. The first 2 times are dialyzed for 1 h each time, and the last 2 times are dialyzed for 2 h each time. After dialysis, an activated horseradish peroxidase solution is obtained.

[0071] Step 2: Preparation of antibody to be labeled

[0072] Use a pipette to aspirate HBsAb into a dialysis bag, and dialyze 4 times with 0.01 M PBS buffer at 25 °C. The first 2 times are dialyzed for 1 h each time, and the last two times are dialyzed for 2 h each time.

[0073] Step 3: Cross-linking reduction reaction

[0074] The method for preparing enzyme-labeled antibody is as follows: According to enzyme:antibody = 1:2 (mass ratio), aspirate appropriate volumes of antibody solution and enzyme solution, and slowly add the antibody solution to the enzyme solution. After thorough mixing, then add an appropriate volume of sodium triacetoxyborohydride solution according to enzyme:sodium triacetoxyborohydride = 1:20 (molar ratio). After shaking and mixing evenly, let it stand and react overnight at 25 °C.

[0075] Step 4: Post-treatment and storage

[0076] After the reaction ends, transfer the reaction solution to a dialysis bag, seal it and dialyze in 0.01 M PBS buffer (pH = 7.2) at room temperature 4 times, and change the solution 3 times during dialysis. After dialysis, a solution of horseradish peroxidase-labeled HBsAb is obtained.

[0077] Example 4

[0078] Step 1: Blocking, oxidation and termination of horseradish peroxidase

[0079] 1.1 Prepare a 10 mg / mL solution of horseradish peroxidase with deionized water, and dissolve Sulfo-NHS-COO-CH 3 in water to make a 10 mg / mL solution. According to horseradish peroxidase:Sulfo-NHS-COO-CH 3 = 1:10 (molar ratio), slowly add the Sulfo-NHS-COO-CH 3 solution dropwise to the horseradish peroxidase solution, and shake while adding. After adding, control the temperature at 25 °C and react for 2 h.

[0080] 1.2 Weigh the required amount of sodium periodate according to the mass ratio of amino-blocked horseradish peroxidase to sodium periodate of 1:2. Prepare a 10 mg / mL solution of sodium periodate, and slowly add the sodium periodate solution dropwise to the reaction solution obtained in 1.1. React at 25 °C for 2 h to obtain the reaction solution after the activation of horseradish peroxidase.

[0081] 1.3 Weigh the amount of sodium sulfide according to the molar ratio of sodium sulfide to sodium periodate of 2:1. Prepare a 10 mg / mL solution of sodium sulfide as the activation reaction termination solution. Slowly add the reaction termination solution dropwise to the reaction solution after the activation of horseradish peroxidase, shaking while adding. React instantaneously at 25 °C for 5 min, and the solution turns brownish-yellow. After the reaction ends, dialyze 4 times with 0.01 M PBS buffer at 25 °C. The first 2 times of dialysis are 1 h each time, and the last 2 times of dialysis are 2 h each time. After dialysis, an activated horseradish peroxidase solution is obtained.

[0082] Step 2: Preparation of antibody to be labeled

[0083] Use a pipette to aspirate HBsAb into a dialysis bag, and dialyze 4 times with 0.01 M PBS buffer at 25 °C. The first 2 times of dialysis are 1 h each time, and the last two times of dialysis are 2 h each time.

[0084] Step 3: Crosslinking reduction reaction

[0085] The method for preparing enzyme-labeled antibody is as follows: According to enzyme:antibody = 1:2 (mass ratio), aspirate appropriate amounts of antibody solution and enzyme solution, and slowly add the antibody solution to the enzyme solution. After thorough mixing, add an appropriate volume of triacetoxyborohydride solution according to enzyme:sodium cyanoborohydride = 1:20 (molar ratio). After shaking and mixing evenly, let it stand and react overnight at 25 °C.

[0086] Step 4: Post-treatment and storage

[0087] After the reaction ends, transfer the reaction solution to a dialysis bag, seal it and dialyze it in 0.01 M PBS buffer (pH = 7.2) at room temperature for 4 times, and change the solution 3 times during dialysis. After dialysis, a solution of horseradish peroxidase-labeled HBsAb is obtained.

[0088] Example 5

[0089] Step 1: Blocking, oxidation and termination of horseradish peroxidase

[0090] 1.1 Prepare a 10 mg / mL solution of horseradish peroxidase with deionized water, and dissolve Sulfo-NHS-COO-CH 3 in water to form a 10 mg / mL solution. According to horseradish peroxidase:Sulfo-NHS-COO-CH 3= 1:10 (molar ratio), slowly add the Sulfo-NHS-COO-CH solution to the horseradish peroxidase solution while shaking. After the addition is complete, control the temperature at 25 °C and react for 2 h. 3 Slowly add the solution while shaking. After the addition is complete, control the temperature at 25 °C and react for 2 h.

[0091] 1.2 Weigh the required amount of sodium periodate according to the mass ratio of amino-blocked horseradish peroxidase to sodium periodate of 1:2. Prepare a 10 mg / mL solution of sodium periodate, and slowly add the sodium periodate solution to the reaction solution obtained in 1.1. React at 25 °C for 2 h to obtain the reaction solution after horseradish peroxidase activation.

[0092] 1.3 Weigh the amount of sodium thiosulfate according to the molar ratio of sodium thiosulfate to sodium periodate of 2:1. Prepare a 10 mg / mL solution of sodium thiosulfate as the activation reaction termination solution. Slowly add the reaction termination solution to the reaction solution after horseradish peroxidase activation while shaking. React instantaneously at 25 °C for 5 min, and the solution turns brownish-yellow. After the reaction is terminated, dialyze 4 times with 0.01 M PBS buffer at 25 °C. The first 2 times are dialyzed for 1 h each time, and the last 2 times are dialyzed for 2 h each time. After dialysis, an activated horseradish peroxidase solution is obtained.

[0093] Second step: Preparation of antibody to be labeled

[0094] Use a pipette to aspirate HBsAb into a dialysis bag, and dialyze 4 times with 0.01 M PBS buffer at 25 °C. The first 2 times are dialyzed for 1 h each time, and the last two times are dialyzed for 2 h each time.

[0095] Third step: Cross-linking reduction reaction

[0096] The method for preparing enzyme-labeled antibody is as follows: According to enzyme: antibody = 1:2 (mass ratio), aspirate an appropriate amount of antibody solution and enzyme solution, and slowly add the antibody solution to the enzyme solution. After thorough mixing, add an appropriate volume of triacetoxyborohydride solution according to enzyme: sodium cyanoborohydride = 1:20 (molar ratio). After shaking and mixing evenly, let it stand and react overnight at 25 °C.

[0097] Fourth step: Post-treatment and storage

[0098] After the reaction is completed, transfer the reaction solution to a dialysis bag, seal it and dialyze it in 0.01 M PBS buffer (pH = 7.2) at room temperature 4 times, and change the solution 3 times during dialysis. After dialysis, a solution of horseradish peroxidase-labeled HBsAb is obtained.

[0099] Comparative example 1

[0100] First step: Oxidation termination of horseradish peroxidase

[0101] Calculate the amount of horseradish peroxidase required according to the labeled ratio of horseradish peroxidase / antibody (mass ratio) of 1 / 1.5. Weigh the enzyme and prepare a 4 mg / ml enzyme solution with ultrapure water. Prepare a 0.1 M sodium periodate solution (i.e., 21.4 mg / mL) with ultrapure water. Slowly add the sodium periodate solution to the enzyme solution according to 51 μL of 0.1 M sodium periodate solution added to 1 mg of horseradish peroxidase. Gently pipette and mix well, place it in a brown fresh-keeping box, and let it react at 4°C in the dark for 1 h. Terminate the reaction by adding 26 μL of ethylene glycol to 1 mg of horseradish peroxidase. Gently pipette and mix well, and let it react at 4°C in the dark for 0.5 h. Place it in a 1 mM acetate buffer solution at pH = 4.4 and let it dialyze at 4°C in the dark overnight. Dialyze 3 times and change the solution 2 times during this period. Unbind the dialysis bag to obtain the activated horseradish peroxidase solution.

[0102] Step 2: Preparation of the antibody to be labeled

[0103] Take the HBsAb to be labeled and dialyze it at 4°C overnight with 0.01 M CB buffer (carbonate buffer, pH = 9.6). Dialyze 2 times and change the solution 1 time during this period.

[0104] Step 3: Cross-linking reduction reaction (performed on an ice plate)

[0105] According to the labeled ratio of horseradish peroxidase / antibody (mass ratio) of 1 / 1.5, calculate the volume of horseradish peroxidase based on the concentration of the activated horseradish peroxidase and add it to the antibody solution. Adjust the pH value of the reaction by adding 153 μL of 0.2 M CB buffer (pH = 9.6) to 1 mL of horseradish peroxidase. Place the reaction container in a brown fresh-keeping box and let it react at 4°C in the dark for 24 - 26 hours. Calculate the amount of sodium borohydride required, weigh sodium borohydride, and prepare a 4 mg / mL sodium borohydride solution with ultrapure water. Terminate the reaction by adding 0.106 M sodium borohydride solution (4 mg / mL), gently shake the bottle holder to mix well, place it in a brown fresh-keeping box, and let it react at 4°C in the dark for 2 h. Dialyze it at 4°C in the dark with 0.067 M PBS buffer (pH = 6.8) (single dialysis ratio not less than 1:50). Dialyze 5 times and change the solution 4 times during this period. After dialysis, accurately measure the volume of the solution, add an equal volume of glycerol to the enzyme conjugate, mix well, and store it at -20°C in the dark at low temperature.

[0106] The differences between the example and Comparative Example 1 are shown in Table 1.

[0107] Table 1 Process differences between the example and Comparative Example 1

[0108]

[0109] Perform immunochemiluminescence tests on the enzyme conjugates obtained in the example and Comparative Example 1. The specific operation steps are as follows:

[0110] 1. After preparing the test calibration product and placing it correctly, click the start button to perform the calibration program or sample detection program, and the instrument will perform the following operations:

[0111] ⑴ Transfer the sample rack to the sampling position and load the reaction vessel to the reagent adding position.

[0112] ⑵ When performing the calibration program, dispense 75 μL of calibration product, 20 μL of magnetic particle suspension, and 30 μL of sample diluent.

[0113] ⑶ Mix and incubate the reaction solution at a temperature of 37 °C for 15 minutes.

[0114] ⑷ After incubation, wash and separate the reaction solution with the cleaning solution.

[0115] ⑸ Dispense 50 μL of enzyme conjugate.

[0116] ⑹ Mix and incubate the reaction solution at a temperature of 37 °C for 17 minutes.

[0117] ⑺ After incubation, wash and separate the reaction solution with the cleaning solution.

[0118] ⑻ Dispense 50 μL of substrate solution A and 50 μL of substrate solution B.

[0119] ⑼ Mix the reaction solution and detect the luminescence intensity.

[0120] The equipment and reagents used in the experiment are: purified water, reaction vessels, sample containers, sample racks, chemiluminescence detectors (suitable for Autolumo A2000, Autolumo A2000 Plus, Autolumo A2000 Plus B fully automatic chemiluminescence analyzers), 1 set of calibration product (containing HBsAg) (concentrations are approximately 0, 0.05, 0.4, 4, 40, 250 IU / ml), substrate solution for fully automatic immunoassay system (record number: Yu Zheng Medical Equipment Preparation No. 2014002), cleaning solution (record number: Yu Zheng Medical Equipment Preparation No. 20140021), sample diluent (record number: Yu Zheng Medical Equipment Preparation No. 20140022).

[0121] 2. Result calculation:

[0122] The instrument automatically calculates the sample test results based on the calibration curve and the luminescence values obtained from the sample tests. The test results are shown in Table 2.

[0123] Table 2 Signal value data of the enzyme-labeled antibody for the verification of the HBsAg item

[0124]

[0125]

[0126] In summary, the present invention improves and optimizes the reducing agent used in the traditional sodium periodate method, greatly enhancing the activation efficiency of horseradish peroxidase, improving the cross-linking efficiency between horseradish peroxidase and antibody protein, shortening the labeling period, and reducing the complexity of experimental operations. Specifically, its advantages are as follows:

[0127] 1) Enhance the activation efficiency. Compared with the traditional method of labeling antibodies with sodium periodate, after the activation of horseradish peroxidase with sodium periodate in the present invention, the traditional terminator is changed from ethylene glycol to inorganic small molecule sodium iodide. This not only shortens the time for terminating the reaction. The termination reaction with potassium iodide is an inorganic reaction with a fast reaction rate and is almost completed instantaneously, while the termination reaction with ethylene glycol is an organic reaction with a longer and incomplete reaction time. In addition, sodium iodide does not react with the aldehyde group generated after enzyme activation, while ethylene glycol will react with the aldehyde group to form hemiacetal and acetal structures, which are relatively stable and not easily reform into aldehyde group structures, thus greatly reducing the efficiency of subsequent coupling reactions.

[0128] 2) Reduce the self-polymerization state of horseradish peroxidase. Compared with the traditional method of labeling antibodies with sodium periodate, before enzyme activation in the present invention, the amino group in the enzyme structure is acylated and blocked with an active ester, which greatly reduces the coupling reaction between the active aldehyde group and its own amino group after enzyme activation, reducing the self-polymerization of horseradish peroxidase and the formation of large aggregates.

[0129] 3) Shorten the antibody labeling period. Compared with the traditional method of labeling antibodies with sodium periodate, the present invention uses sodium triacetoxyborohydride with mild reducibility and adds it at the beginning of the coupling reaction. In the traditional method of labeling antibodies with sodium periodate, the activated enzyme reacts with the antibody for 24 hours first, and then sodium borohydride with strong reducibility is added as a reducing agent for reaction. Sodium borohydride can not only reduce the generated Schiff base, but also reduce the active aldehyde group obtained in the previous activation step. In addition, the imine structure generated by the reaction between the activated enzyme and the antibody in this process is very unstable and can hardly remain in the buffer for a long time. Reducing it after 24 hours causes most of the generated imine structures to be hydrolyzed and unable to participate in the next reduction reaction. The reducing agent selected in the present invention not only shortens the experimental period but also reduces the problems of unstable decomposition of imine and reduction of aldehyde groups, improving the cross-linking efficiency.

[0130] 4) Convenient experimental operation: All reactions are carried out at a temperature of 25 °C, and only one kind of dialysis solution is used. Compared with the traditional process, it saves the cumbersome operation steps of temperature control and the "damage" to proteins caused by changing different buffers.

[0131] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing a horseradish peroxidase-labeled antibody, characterized in that: The following steps are involved: S1) reacting horseradish peroxidase with an amino blocking agent to obtain amino-blocked horseradish peroxidase; S2) reacting the amino-blocked horseradish peroxidase with sodium periodate for a period of time, and then adding an inorganic reducing agent to terminate the reaction to obtain activated horseradish peroxidase; S3) Mixing the activated horseradish peroxidase, the antibody and the reducing agent to perform a cross-linking reaction to obtain the horseradish peroxidase labeled antibody.

2. The preparation method according to claim 1, characterized in that: The step S1) is specifically as follows: The aqueous solution of the amino-blocking agent is added to the aqueous solution of horseradish peroxidase to react, thereby obtaining a reaction solution containing amino-blocked horseradish peroxidase.

3. The preparation method according to claim 2, characterized in that: The amino blocking agent is selected from Sulfo-NHS-COO-(CH3) m and / or NHS-COO-(CH3) m ; Wherein, m is an integer from 1 to 10; The molar ratio of the horseradish peroxidase to the amino blocking agent is 1:6 to 1:

20.

4. The preparation method according to claim 2, characterized in that: The reaction temperature is 20°C to 30°C; the reaction time is 1 to 3 hours.

5. The preparation method according to claim 1, characterized in that: In the step S2), sodium periodate is added to the amino-blocked horseradish peroxidase solution in the form of sodium periodate aqueous solution to perform activation reaction. After a period of reaction, an inorganic reducing agent is added to terminate the reaction.

6. The preparation method according to claim 5, characterized in that: The mass ratio of the amino-blocked horseradish peroxidase to sodium periodate is 1:1 to 1:5; The reaction temperature is 20°C to 30°C; the oxidation reaction time is 1 to 3 hours.

7. The preparation method according to claim 5, characterized in that: The inorganic reducing agent is selected from one or more of sodium iodide, potassium iodide, sodium thiosulfate and sodium sulfide; The molar ratio of the inorganic reducing agent to sodium periodate is 1:1 to 5:

1. The reaction temperature is 20°C to 30°C; the reduction reaction time is 3 to 10 minutes.

8. The preparation method according to claim 1, characterized in that: In the step S3), the mass ratio of activated horseradish peroxidase to antibody is 1:1 to 1:5; The reducing agent is selected from one or more of sodium cyanoborohydride and sodium triacetoxyborohydride; The molar ratio of the antibody to the reducing agent is 1:10 to 1:

50.

9. The preparation method according to claim 1, characterized in that: The temperature of the cross-linking reaction is 20° C. to 30° C.; the time of the cross-linking reaction is 6 to 15 hours.

10. The preparation method according to claim 1, characterized in that: After the reaction is terminated in step S2), the reaction solution is dialyzed to obtain activated horseradish peroxidase; the buffer used for the dialysis is 0.01 mol / L PBS buffer; the number of dialysis is 2 to 6 times; After the cross-linking reaction in step S3), the reaction solution is dialyzed to obtain a horseradish peroxidase labeled antibody; the buffer used for the dialysis is a 0.01 mol / L PBS buffer; the number of dialysis is 2 to 6 times; The pH value of the 0.01 mol / L PBS buffer solution is 7-7.5.

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