Alkaline phosphatase antibody label, method of making, detection reagent and kit

Abstract

The application discloses an alkaline phosphatase antibody label and a preparation method, a detection reagent and a kit thereof, relates to the field of protein coupling technology. The alkaline phosphatase antibody label provided by the technical scheme of the application is prepared by activating alkaline phosphatase by a polyethylene glycol compound containing a succinimidyl ester group and SMCC, or is prepared by modifying alkaline phosphatase by a polyethylene glycol molecule with an amino group and then activating by SMCC. Compared with the traditional Traut's-SMCC method, the alkaline phosphatase antibody label has improved hydrophilicity, improved solubility, and certain steric hindrance, so that the possibility of continued reaction of the excess active sites on the alkaline phosphatase can be reduced, the strength of the cross-linking and aggregation of the alkaline phosphatase and the antibody can be reduced without affecting the cross-linking efficiency, so that the stability and yield of the alkaline phosphatase antibody label are improved, and batch differences are reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of protein coupling, in particular to an alkaline phosphatase antibody marker, a preparation method thereof, a detection reagent and a kit. BACKGROUND

[0002] Enzymatic chemiluminescence is a widely used immunodiagnostic technique, in which alkaline phosphatase-labeled antibody and its labeling process are important links, affecting the stability, batch difference, signal-to-noise ratio and other detection performances of the luminescent kit. The commonly used labeling processes for alkaline phosphatase include glutaraldehyde method, carbodiimide method, sodium periodate oxidation method and 2-iminothiolane hydrochloride (Traut's) combined with 4-(N-maleimidomethyl) cyclohexane-1-carboxylate succinimidyl ester (SMCC) method. The first two labeling methods do not have specific cross-linking, which can easily cause self-crosslinking of alkaline phosphatase and / or antibody, thereby reducing the detection efficiency; the oxidation efficiency of natural alkaline phosphatase with a sugar content of less than 5% by the sodium periodate oxidation method is low, and the formed aldehyde group is unstable and has a probability of reacting with its own amino group to form a self-polymer.

[0003] The 2-iminothiolane hydrochloride (Traut's) combined with 4-(N-maleimidomethyl) cyclohexane-1-carboxylate succinimidyl ester (SMCC) method uses the hetero-functional groups of SMCC to connect alkaline phosphatase and antibody, respectively, so the coupling efficiency is relatively high, but the solubility and stability of the alkaline phosphatase-labeled antibody obtained by this method are poor, which ultimately leads to a decrease in the yield of enzyme-labeled antibody and a large batch difference. Currently, some researchers use a coupling agent containing a polyethylene glycol group to replace the SMCC reagent, so that the alkaline phosphatase antibody marker obtained by coupling has the characteristics of high stability, high sensitivity and low non-specific reaction. However, the cost of the hetero-bifunctional coupling agent containing ethylene glycol is high, and the higher the polymerization degree of ethylene glycol, the higher the price. In addition, the use of the hetero-bifunctional coupling agent containing ethylene glycol cannot slow down the reaction of the excess active sites.

[0004] Therefore, it is still of great significance to develop an alkaline phosphatase-labeled antibody with high stability and high sensitivity. SUMMARY

[0005] The main purpose of the present application is to provide an alkaline phosphatase antibody marker, a preparation method thereof, a detection reagent and a kit, which aims to solve the problems of poor stability and poor sensitivity of the existing alkaline phosphatase antibody marker.

[0006] To achieve the above-mentioned purpose, the present application provides an alkaline phosphatase antibody marker, which comprises a first activator, a second activator, an alkaline phosphatase connected to the first activator and the second activator by chemical bonds, and an antibody connected to the first activator by a chemical bond.

[0007] wherein the first activating agent comprises 4-(N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester;

[0008] the second activating agent comprises a polyethylene glycol compound containing a succinimidyl ester group.

[0009] In an embodiment, the polyethylene glycol compound containing a succinimidyl ester group comprises at least one of methoxypolyethylene glycol succinimidyl valerate, methoxypolyethylene glycol succinimidyl carbonate, methoxypolyethylene glycol succinimidyl glutarate, methoxypolyethylene glycol succinimidyl succinate, methoxypolyethylene glycol succinimidyl carboxymethyl ester, methoxypolyethylene glycol succinimidyl butyrate, methoxypolyethylene glycol succinimidyl succinamide and methoxypolyethylene glycol succinimidyl propionate; and / or,

[0010] the mass ratio of the first activating agent to the alkaline phosphatase is 1:(25-30); and / or,

[0011] the mass ratio of the second activating agent to the alkaline phosphatase is 1:(30-40); and / or,

[0012] the mass ratio of the alkaline phosphatase to the antibody is (1-1.4):1.

[0013] The present application provides a preparation method of the alkaline phosphatase antibody marker, the alkaline phosphatase antibody marker comprising a first activating agent, a second activating agent, an alkaline phosphatase connected to the first activating agent and the second activating agent by chemical bonds respectively, and an antibody connected to the first activating agent by a chemical bond; the preparation method of the alkaline phosphatase antibody marker comprising the following steps:

[0014] SA1, mixing the alkaline phosphatase, the first activating agent and the second activating agent, performing activation treatment and desalination purification treatment to obtain the alkaline phosphatase after pretreatment; performing activation treatment and desalination purification treatment on the antibody to obtain the antibody after pretreatment;

[0015] SA2, sequentially performing coupling reaction, blocking treatment and purification treatment on the alkaline phosphatase after pretreatment and the antibody after pretreatment to obtain the alkaline phosphatase antibody marker.

[0016] In an embodiment, in the step SA1, the steps of mixing the alkaline phosphatase, the first activating agent and the second activating agent, performing activation treatment and desalination purification treatment to obtain the alkaline phosphatase after pretreatment; and performing activation treatment and desalination purification treatment on the antibody to obtain the antibody after pretreatment comprise:

[0017] The alkaline phosphatase, the enzyme activation buffer, the first activator and the second activator are mixed, and activation treatment and desalination purification treatment are performed to obtain the alkaline phosphatase after pretreatment; the antibody, the antibody activation buffer and 2-iminothiolane hydrochloride are mixed, and activation treatment and desalination purification treatment are performed to obtain the antibody after pretreatment.

[0018] In an embodiment, in the SA1 step, the enzyme activation buffer comprises a mixed solution of PBS with a molar concentration of 10-100 mM and EDTA-2Na with a molar concentration of 1-5 mM; and / or,

[0019] The pH value of the enzyme activation buffer is 6.5-7.5; and / or,

[0020] The antibody activation buffer comprises a mixed solution of PBS with a molar concentration of 50-100 mM and EDTA-2Na with a molar concentration of 2-5 mM; and / or,

[0021] The pH value of the antibody activation buffer is 7.5-8.0; and / or,

[0022] The mass ratio of the antibody to 2-iminothiolane hydrochloride is (35-45):1.

[0023] In an embodiment, in the SA2 step, the mass ratio of the alkaline phosphatase after pretreatment to the antibody after pretreatment is (1-1.4):1.

[0024] The application provides an alkaline phosphatase antibody marker, which comprises an activator, a modifier, alkaline phosphatase connected to the activator and the modifier by chemical bonds respectively, and an antibody connected to the activator by a chemical bond;

[0025] The activator comprises 4-(N-maleimido methyl) cyclohexane-1-carboxylic acid succinimidyl ester.

[0026] The modifier comprises at least one of CE210 and CE510.

[0027] In an embodiment, the mass ratio of the modifier to the alkaline phosphatase is 1:(30-40); and / or,

[0028] The mass ratio of the activator to the alkaline phosphatase is 1:(25-30); and / or,

[0029] The mass ratio of the alkaline phosphatase to the antibody is (1-1.4):1.

[0030] The application provides a preparation method of the alkaline phosphatase antibody marker, the alkaline phosphatase antibody marker comprising an activator, a modifier, alkaline phosphatase connected with the activator and the modifier respectively through chemical bonds and an antibody connected with the activator through a chemical bond; and the preparation method comprises the following steps:

[0031] SB1, mixing and reacting the alkaline phosphatase and the modifier to obtain pre-modified alkaline phosphatase;

[0032] SB2, mixing the pre-modified alkaline phosphatase and the activator, performing activation treatment and desalting purification treatment to obtain pre-processed alkaline phosphatase; and performing activation treatment and desalting purification treatment on the antibody to obtain pre-processed antibody;

[0033] SB3, mixing the pre-processed alkaline phosphatase and the pre-processed antibody to sequentially perform coupling reaction, blocking treatment and purification treatment to obtain the alkaline phosphatase antibody marker.

[0034] In an embodiment, in the step SB1, the step of mixing and reacting the alkaline phosphatase and the modifier to obtain pre-modified alkaline phosphatase comprises:

[0035] mixing and reacting the alkaline phosphatase, the carboxyl activation buffer, the modifier, N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (Suflo-NHS) to obtain pre-modified alkaline phosphatase.

[0036] In an embodiment, in the step SB1, the carboxyl activation buffer comprises a MES solution with a molar concentration of 10-100 mM; and / or,

[0037] the pH value of the carboxyl activation buffer is 5.0-6.5; and / or,

[0038] the mass ratio of the N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride to the alkaline phosphatase is 1:(10-20); and / or,

[0039] the mass ratio of the N-hydroxysulfosuccinimide to the alkaline phosphatase is 1:(5-10).

[0040] In an embodiment, in the step SB2, the steps of mixing the pre-modified alkaline phosphatase and the activator, performing activation treatment and desalting purification treatment to obtain pre-processed alkaline phosphatase; and performing activation treatment and desalting purification treatment on the antibody to obtain pre-processed antibody comprise:

[0041] The pre-modified alkaline phosphatase, the enzyme activation buffer and the activator are mixed, and then activation treatment and desalination purification treatment are performed to obtain the pre-processed alkaline phosphatase; the antibody, the antibody activation buffer and 2-iminothiolane hydrochloride are mixed, and then activation treatment and desalination purification treatment are performed to obtain the pre-processed antibody.

[0042] In an embodiment, in the SB2 step, the enzyme activation buffer comprises a mixed solution of PBS with a molar concentration of 10-100 mM and EDTA-2Na with a molar concentration of 1-5 mM; and / or,

[0043] The pH value of the enzyme activation buffer is 6.5-7.5; and / or,

[0044] The antibody activation buffer comprises a mixed solution of PBS with a molar concentration of 50-100 mM and EDTA-2Na with a molar concentration of 2-5 mM; and / or,

[0045] The pH value of the antibody activation buffer is 7.5-8.0; and / or,

[0046] The mass ratio of the pre-modified alkaline phosphatase and the activator is (10-20):1; and / or,

[0047] The mass ratio of the antibody and 2-iminothiolane hydrochloride is (35-45):1.

[0048] In an embodiment, in the SB3 step, the mass ratio of the pre-processed alkaline phosphatase and the pre-processed antibody is (1-1.4):1.

[0049] The present application provides a detection reagent, which comprises the alkaline phosphatase antibody label or is prepared according to the preparation method of the alkaline phosphatase antibody label.

[0050] The present application provides a kit, which comprises the detection reagent.

[0051] The present application has the following beneficial effects:

[0052] (1) The alkaline phosphatase is co-modified by SMCC and a polyethylene glycol compound containing a succinimidyl ester group, and then the co-modified alkaline phosphatase is reacted with an antibody to form an alkaline phosphatase antibody marker. The polyethylene glycol compound containing a succinimidyl ester group contains a polyethylene glycol group, has hydrophilicity and a certain steric hindrance, and thus can be used to improve the hydrophilicity of the periphery of the alkaline phosphatase, improve the hydrophilicity of the alkaline phosphatase antibody marker after the alkaline phosphatase antibody marker is formed, and improve the solubility of the alkaline phosphatase antibody marker. In addition, the certain steric hindrance can avoid the excess active sites on the alkaline phosphatase antibody marker from continuing to react to form a hydrophobic polymer, thus slowing down the intensity of the cross-linking and aggregation of the alkaline phosphatase and the antibody without affecting the cross-linking efficiency, improving the stability and yield of the final alkaline phosphatase antibody marker, reducing the batch-to-batch difference of the alkaline phosphatase antibody marker, and thus obtaining an alkaline phosphatase antibody marker with high stability and high sensitivity.

[0053] (2) The alkaline phosphatase is modified by using CE210 or CE510 as a modifier, and then the modified alkaline phosphatase, an activator SMCC and an antibody are reacted to form an alkaline phosphatase antibody marker. CE210 or CE510 is a polyethylene glycol molecule with an amino terminal, and the polyethylene glycol group can improve the hydrophilicity and has a certain steric hindrance. Therefore, after the alkaline phosphatase is modified by using CE210 or CE510, the hydrophilicity of the alkaline phosphatase antibody marker can be improved, the solubility of the alkaline phosphatase antibody marker can be improved, and the possibility of the excess maleimide active sites on the alkaline phosphatase antibody marker from continuing to react can be reduced. Thus, the intensity of the cross-linking and aggregation of the alkaline phosphatase and the antibody can be slowed down without affecting the cross-linking efficiency, the stability and yield of the final alkaline phosphatase antibody marker can be improved, the batch-to-batch difference of the alkaline phosphatase antibody marker can be reduced, and thus an alkaline phosphatase antibody marker with high stability and high sensitivity can be obtained. BRIEF DESCRIPTION OF DRAWINGS

[0054] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without any creative effort on the basis of the drawings shown.

[0055] Figure 1 The schematic diagram of the alkaline phosphatase antibody marker provided for Example 1, Example 8 and Comparative Example 1 of the present application.

[0056] The implementation, functional features and advantages of the present application will be further described with reference to the drawings. DETAILED DESCRIPTION

[0057] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort belong to the scope of protection of the present application.

[0058] It should be noted that if the embodiments of the present application involve directional indications (such as up, down, left, right, front, back, etc.), the directional indications are only used to explain the relative positional relationship, movement condition, etc. between components in a certain posture, and if the certain posture changes, the directional indications will also change accordingly.

[0059] In addition, if the embodiments of the present application involve descriptions such as "first", "second", etc., the descriptions of "first", "second", etc. are only for description purposes and cannot be understood as indicating or implying the relative importance of the indicated technical features or implicitly indicating the number of the indicated technical features. Therefore, the features limited by "first" and "second" can explicitly or implicitly include at least one of the features. In addition, "and / or" or "and / or" appearing throughout the text means that the three parallel schemes are included, for example, "A and / or B" includes A scheme, or B scheme, or A and B are satisfied at the same time. In addition, the technical solutions of each embodiment can be combined with each other, but it must be based on the fact that a person of ordinary skill in the art can realize it, and when the combination of technical solutions contradicts each other or cannot be realized, it should be considered that the combination of technical solutions does not exist and is not within the scope of protection required by the present application.

[0060] Enzymatic chemiluminescence is a widely used immunodiagnostic technique, in which alkaline phosphatase-labeled antibody and its labeling process as an important link, affects the stability of the luminescent kit, batch difference, signal-to-noise ratio and other detection performance. The processes commonly used for alkaline phosphatase labeling include glutaraldehyde method, carbodiimide method, sodium periodate oxidation method and 2-iminothiolane hydrochloride (Traut's) combined with 4-(N-maleimidomethyl) cyclohexane-1-carboxylate succinimidyl ester (SMCC) method. Among them, using Traut's-SMCC method, although this bifunctional coupling agent can avoid protein cross-linking and improve labeling efficiency, but the cross-linking agent will reduce the solubility of the protein, and the complex formed by the alkaline phosphatase-labeled antibody will present a hydrophobic state in the periphery, and the excess active sites continue to react, and the hydrophobic polymer formed will precipitate in the solution, ultimately leading to low yield and large batch difference. Therefore, it is still of great significance to develop an alkaline phosphatase-labeled antibody with high stability and high sensitivity.

[0061] In view of this, the alkaline phosphatase antibody label provided by the present application comprises a first activating agent, a second activating agent, an alkaline phosphatase connected to the first activating agent and the second activating agent through chemical bonds respectively, and an antibody connected to the first activating agent through a chemical bond respectively;

[0062] The first activating agent comprises 4-(N-maleimide methyl) cyclohexane-1-carboxylic acid succinimidyl ester (SMCC).

[0063] The second activating agent comprises a polyethylene glycol compound containing a succinimidyl ester group.

[0064] The present application activates the alkaline phosphatase by using the first activating agent and the second activating agent, and then reacts the activated alkaline phosphatase with the antibody to form the alkaline phosphatase antibody label. The first activating agent is SMCC, which contains a maleimide group and a succinimidyl ester group. The second activating agent is a polyethylene glycol compound containing a succinimidyl ester group, which contains a polyethylene glycol group and a succinimidyl ester group. When the first activating agent and the second activating agent activate the alkaline phosphatase, the first activating agent and the second activating agent react with the primary amine (-NH2) on the alkaline phosphatase through the succinimidyl ester group to form an amide bond, thereby being combined to the alkaline phosphatase. The first activating agent also specifically reacts with the antibody activated by Traut's reagent through the maleimide group to form a stable carbon-sulfur (C-S) covalent bond, thereby being combined to the antibody.

[0065] Compared with the traditional Traut's-SMCC combination method for preparing the alkaline phosphatase antibody label, the present application activates the alkaline phosphatase by using SMCC and a polyethylene glycol compound containing a succinimidyl ester group. Since the polyethylene glycol compound containing a succinimidyl ester group can provide hydrophilicity and certain steric hindrance effect, it can improve the hydrophilicity of the periphery of the alkaline phosphatase, and improve the hydrophilicity of the alkaline phosphatase antibody label after forming the alkaline phosphatase antibody label. In addition, the certain steric hindrance effect can avoid the excess maleimide active sites from continuing to react to form hydrophobic polymers, slow down the strength of the cross-linking aggregation of the alkaline phosphatase antibody and the antibody without affecting the cross-linking efficiency, and finally improve the stability and yield of the alkaline phosphatase antibody label, reduce the batch-to-batch difference of the alkaline phosphatase antibody label, and be conducive to preparing the alkaline phosphatase antibody label with high stability and high sensitivity.

[0066] In the embodiments of the present application, the polyethylene glycol succinimidyl ester group-containing polyethylene glycol compound includes at least one of methoxypolyethylene glycol succinimidyl valerate (mPEG-SVA), methoxypolyethylene glycol succinimidyl carbonate (mPEG-SC), methoxypolyethylene glycol succinimidyl glutarate (mPEG-SG), methoxypolyethylene glycol succinimidyl succinate (mPEG-SS), methoxypolyethylene glycol succinimidyl carboxymethyl ester (mPEG-SCM), methoxypolyethylene glycol succinimidyl butyrate (mPEG-SBA), methoxypolyethylene glycol succinimidyl succinamide (mPEG-SSA) and methoxypolyethylene glycol succinimidyl propionate (mPEG-SPA). It should be noted that, considering that mPEG-SC, mPEG-SG, mPEG-SS, mPEG-SCM, mPEG-SBA, mPEG-SSA, mPEG-SPA are more prone to hydrolysis than mPEG-SVA, and the half-life of succinimidyl valerate (SVA) is longer, the present application preferably uses mPEG-SVA to prepare alkaline phosphatase antibody markers, which is conducive to obtaining alkaline phosphatase antibody markers with high stability.

[0067] In the embodiments of the present application, the mPEG-SVA includes mPEG-SVA containing polyethylene glycol groups with different molecular weights, and the molecular weight of the polyethylene glycol group is 1-10K. Using mPEG-SVA to co-activate alkaline phosphatase can improve the hydrophilicity of alkaline phosphatase after pretreatment, but the molecular weight of the polyethylene glycol group in mPEG-SVA will affect the activity of alkaline phosphatase and antibody binding after pretreatment. The larger the molecular weight of the polyethylene glycol, the more obvious the steric hindrance effect provided, which will increase the difficulty of alkaline phosphatase and antibody binding.

[0068] In the embodiments of the present application, the mass ratio of the first activating agent to alkaline phosphatase is 1:(25-30). It should be noted that, if the amount of the first activating agent is too large, it will lead to low or no coupling efficiency of mPEG-SVA, and reduce the solubility of the alkaline phosphatase antibody marker; if the amount of the first activating agent is too small, it will slow down the cross-linking efficiency of alkaline phosphatase and antibody. Setting the mass ratio of the first activating agent to alkaline phosphatase in the above range is conducive to obtaining alkaline phosphatase antibody markers with high stability and high sensitivity.

[0069] In the embodiment of the present application, the mass ratio of the second activating agent to the alkaline phosphatase is 1:(30-40). It should be noted that too much second activating agent will increase the production cost, and too little second activating agent will reduce the effect of slowing down the cross-linking and aggregation of the alkaline phosphatase antibody and the antibody. Setting the mass ratio of the second activating agent to the alkaline phosphatase in the above range is conducive to obtaining an alkaline phosphatase antibody marker with high stability and high sensitivity, and controlling the production cost.

[0070] In the embodiment of the present application, the mass ratio of the alkaline phosphatase to the antibody is (1-1.4):1. It should be noted that too little antibody will reduce the reactivity of the alkaline phosphatase antibody marker, and too much antibody will cause free antibody, reducing the possibility of binding of the alkaline phosphatase antibody marker to the antigen, thereby reducing the reactivity. When the mass ratio of the alkaline phosphatase to the antibody is (1-1.4):1, the alkaline phosphatase antibody marker has better reactivity with the subsequent antigen.

[0071] The present application provides a preparation method of the alkaline phosphatase antibody marker, which comprises a first activating agent, a second activating agent, alkaline phosphatase connected to the first activating agent and the second activating agent through chemical bonds, and an antibody connected to the first activating agent through a chemical bond; the preparation method comprises the following steps:

[0072] SA1, mixing the alkaline phosphatase, the first activating agent and the second activating agent, performing activation treatment and desalination purification treatment to obtain pretreated alkaline phosphatase; performing activation treatment and desalination purification treatment on the antibody to obtain pretreated antibody;

[0073] SA2, mixing the pretreated alkaline phosphatase and the pretreated antibody to sequentially perform coupling reaction, blocking treatment and purification treatment to obtain the alkaline phosphatase antibody marker.

[0074] The preparation method provided by the technical scheme of the present application first activates the alkaline phosphatase with the first activating agent and the second activating agent, after the activation treatment, the first activating agent and the second activating agent react with the amino groups on the alkaline phosphatase respectively, and are combined with the alkaline phosphatase by forming amide bonds to form pretreated alkaline phosphatase; at the same time, the antibody is activated to introduce sulfhydryl groups (-SH) on the antibody; finally, the pretreated alkaline phosphatase and the antibody with sulfhydryl groups are mixed to perform coupling reaction, the pretreated alkaline phosphatase is covalently connected to the sulfhydryl groups on the antibody through the maleimide group to form the alkaline phosphatase antibody marker.

[0075] The first activator and the second activator are used to activate the alkaline phosphatase together, compared with the prior art in which the first activator or the second activator is used alone and then the second activator or the first activator is used, the alkaline phosphatase antibody label with high stability and high sensitivity can be prepared.

[0076] In the embodiment of the present application, in the SA1 step, the alkaline phosphatase, the first activator and the second activator are mixed, and activation treatment and desalination purification treatment are performed to obtain the pretreated alkaline phosphatase; the antibody is subjected to activation treatment and desalination purification treatment to obtain the pretreated antibody.

[0077] The alkaline phosphatase, the enzyme activation buffer, the first activator and the second activator are mixed, and activation treatment and desalination purification treatment are performed to obtain the pretreated alkaline phosphatase; the antibody, the antibody activation buffer and 2-iminothiolane hydrochloride are mixed, and activation treatment and desalination purification treatment are performed to obtain the pretreated antibody.

[0078] The enzyme activation buffer comprises a mixed solution of PBS with a molar concentration of 10-100 mM and EDTA-2Na with a molar concentration of 1-5 mM; and the pH value of the enzyme activation buffer is 6.5-7.5.

[0079] The antibody activation buffer comprises a mixed solution of PBS with a molar concentration of 50-100 mM and EDTA-2Na with a molar concentration of 2-5 mM; and the pH value of the antibody activation buffer is 7.5-8.0.

[0080] The mass ratio of the antibody to 2-iminothiolane hydrochloride is (35-45):1.

[0081] The mass ratio of the pretreated alkaline phosphatase to the pretreated antibody is (1-1.4):1.

[0082] In the technical scheme of the present application, 2-iminothiolane hydrochloride (Traut’s) is used to activate the antibody to introduce a thiol group (-SH) on the antibody, so that the antibody is covalently connected to a substance containing a maleimide group through the newly introduced thiol group, thereby forming a label. The mass ratio of the antibody to 2-iminothiolane hydrochloride is set to (35-45):1, so that the surface of the protein is not excessively modified, and the structure and function of the protein can be maintained.

[0083] The mass ratio of the pre-processed alkaline phosphatase and the pre-processed antibody after the pre-processing is (1-1.4):1. Within this range, the mass ratio of the pre-processed alkaline phosphatase and the pre-processed antibody after the pre-processing can be 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, the mass ratio of the pre-processed alkaline phosphatase and the pre-processed antibody after the pre-processing within the above range can reduce free antibodies and improve reactivity.

[0084] In an embodiment of the present application, in the SA1 step, the step of desalting and purifying the activated alkaline phosphatase and the activated antibody includes: respectively adding the activated alkaline phosphatase and the activated antibody into a desalting column, and centrifuging (1000xg, 4°C) for 2 min in a centrifuge to remove unreacted first activator, second activator and Traut's reagent.

[0085] In an embodiment of the present application, in the SA2 step, the step of mixing the pre-processed alkaline phosphatase and the pre-processed antibody for coupling reaction includes: mixing the pre-processed alkaline phosphatase and the pre-processed antibody in a mass ratio of (1-1.4):1, and placing them in a constant temperature and humidity incubator at 25°C for 1h of light-free reaction.

[0086] In an embodiment of the present application, in the SA2 step, the step of blocking includes: after the coupling reaction of the pre-processed alkaline phosphatase and the pre-processed antibody is completed, adding 2-mercaptoethanesulfonic acid sodium and oscillating for 1 min, and blocking for 10 min in the dark. Then, N-ethylmaleimide is added and oscillated for 1 min, and blocked for 10 min in the dark.

[0087] In an embodiment of the present application, in the SA2 step, the purification step after the blocking includes: adding the blocked alkaline phosphatase antibody label into a desalting column, and centrifuging (1000xg, 4°C) for 2 min in a centrifuge to remove unreacted blocking agent.

[0088] In an embodiment of the present application, the SA2 step is followed by: testing the concentration of the enzyme-labeled complex by NanoDrop A280 method, and diluting the alkaline phosphatase antibody label to 0.5 mg / mL with an enzyme binding stabilizer, and storing at 4°C.

[0089] The present application provides an alkaline phosphatase antibody label, which comprises an activator, a modifier, an alkaline phosphatase connected to the activator and the modifier by chemical bonds respectively, and an antibody connected to the activator by a chemical bond.

[0090] The activating agent comprises 4-(N-maleimido methyl) cyclohexane-1-carboxylic acid succinimidyl ester.

[0091] The modifying agent comprises at least one of CE210 and CE510.

[0092] The alkaline phosphatase is modified by the modifying agent, and the modified alkaline phosphatase, the activating agent and the antibody are combined to form an alkaline phosphatase antibody marker, wherein the modifying agent is selected from CE210 or CE510, and the activating agent is selected from 4-(N-maleimido methyl) cyclohexane-1-carboxylic acid succinimidyl ester (SMCC). CE210 and CE510 are PEG molecules with an oligoamino short chain connected at the terminal end, and the short chain contains an amino terminal end which can be coupled with a carboxyl terminal end of the alkaline phosphatase to be combined with the alkaline phosphatase; the activating agent is specifically reacted with the antibody containing sulfydryl (-SH) through a maleimide group to form a stable carbon-sulfur (C-S) covalent bond, thereby being combined with the antibody.

[0093] Compared with the traditional Traut's-SMCC combination method, the technical scheme of the present application selects CE210 or CE510 as the modifying agent, pre-modifies the alkaline phosphatase, and then activates the alkaline phosphatase by using SMCC. Since the alkaline phosphatase is activated by EDC / Suflo-NHS to form a negatively charged sulfonyl group, the modifying agent with a -NH + , can more effectively approach the periphery of the alkaline phosphatase, thereby promoting the reaction of the modifying agent with the alkaline phosphatase. Since the modifying agent also has a hydrophilic polyethylene glycol group and has a certain steric repulsion, after the alkaline phosphatase is modified, not only the hydrophilicity of the alkaline phosphatase antibody marker can be improved, but also a certain steric distance exists between the modified alkaline phosphatase molecules. After the modified alkaline phosphatase, the activating agent and the antibody are combined to form the alkaline phosphatase antibody marker, the possibility of the alkaline phosphatase antibody marker continuing to react to produce polymers can be reduced, the strength of the alkaline phosphatase and the antibody cross-linking aggregation can be slowed down without affecting the cross-linking efficiency, thereby improving the stability and yield of the final alkaline phosphatase antibody marker, reducing the batch-to-batch difference of the alkaline phosphatase antibody marker, and being conducive to preparing the alkaline phosphatase antibody marker with high stability and high sensitivity.

[0094] In the embodiments of the present application, the mass ratio of the modifying agent to the alkaline phosphatase is 1:(5-10), the mass ratio of the activating agent to the alkaline phosphatase is 1:(10-20), and the mass ratio of the alkaline phosphatase to the antibody is (1-1.4):1. The amounts of the activating agent, the modifying agent, the alkaline phosphatase and the antibody are set in the above range, the reaction efficiency is high, and the toxic effect of the reagents on the antibody can be reduced.

[0095] The application provides a preparation method of the alkaline phosphatase antibody marker, the alkaline phosphatase antibody marker comprising an activator, a modifier, alkaline phosphatase connected with the activator and the modifier respectively through chemical bonds and an antibody connected with the activator through a chemical bond; and the preparation method of the alkaline phosphatase antibody marker comprises the following steps:

[0096] SB1, mixing and reacting the alkaline phosphatase and the modifier to obtain pre-modified alkaline phosphatase;

[0097] SB2, mixing the pre-modified alkaline phosphatase and the activator, performing activation treatment and desalting purification treatment to obtain pre-processed alkaline phosphatase; and performing activation treatment and desalting purification treatment on the antibody to obtain pre-processed antibody;

[0098] SB3, mixing the pre-processed alkaline phosphatase and the pre-processed antibody to sequentially perform coupling reaction, blocking treatment and purification treatment to obtain the alkaline phosphatase antibody marker.

[0099] The technical scheme of the application first modifies the alkaline phosphatase by using the modifier, the amino group of the modifier is combined with the carboxylic acid of the alkaline phosphatase, after pre-modification treatment, the hydrophilicity of the alkaline phosphatase is improved and a certain spatial distance exists between the alkaline phosphatase molecules; then, the pre-modified alkaline phosphatase is activated by using the activator, the activator reacts with the amino group on the alkaline phosphatase to combine with the alkaline phosphatase by forming an amide bond to form the pre-processed alkaline phosphatase, and the antibody is activated at the same time to introduce the sulfhydryl group (-SH) into the antibody to form the pre-processed antibody; then, the pre-processed alkaline phosphatase and the pre-processed antibody are mixed, the pre-processed alkaline phosphatase is covalently connected with the sulfhydryl group on the antibody through the maleimide group to form the alkaline phosphatase antibody marker.

[0100] It should be noted that the adding sequence of the modifier affects the combination efficiency of the activator and the alkaline phosphatase, the technical scheme of the application selects to modify the alkaline phosphatase by using the modifier first and then adding the activator to activate, which is beneficial to improving the hydrophilicity of the alkaline phosphatase antibody marker and slowing down the crosslinking and aggregation strength of the alkaline phosphatase and the antibody, so that the alkaline phosphatase antibody marker with high stability and high sensitivity is prepared.

[0101] In the embodiment of the application, in the step SB1, the step of mixing and reacting the alkaline phosphatase and the modifier to obtain pre-modified alkaline phosphatase comprises the following steps:

[0102] Mixing alkaline phosphatase, carboxyl activation buffer, modifier, N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (Sulfo-NHS) to react to obtain pre-modified alkaline phosphatase;

[0103] The carboxyl activation buffer comprises a MES solution with a molar concentration of 10-100 mM;

[0104] The carboxyl activation buffer has a pH value of 5.0-6.5.

[0105] The mass ratio of the N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride to the alkaline phosphatase is 1:(10-20);

[0106] The mass ratio of the N-hydroxysulfosuccinimide to the alkaline phosphatase is 1:(5-10).

[0107] The modifier has an amino group, and the modifier is connected to the alkaline phosphatase by reacting with the carboxyl terminal of the alkaline phosphatase. The addition of EDC can activate the carboxyl group (-COOH) to react with the amino group (-NH2) to form a stable amide bond. EDC is used together with Sulfo-NHS, and Sulfo-NHS can stabilize the carboxyl intermediate activated by EDC to prevent its hydrolysis and promote its effective reaction with the amino group. The amount of N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysulfosuccinimide is set in the above range, which can effectively control the binding degree of the modifier and reduce the self-crosslinking of the alkaline phosphatase.

[0108] In the embodiment of the present application, in the SB2 step, the step of mixing the pre-modified alkaline phosphatase and the activation agent to perform activation treatment and desalination purification treatment to obtain the pre-processed alkaline phosphatase, and the step of performing activation treatment and desalination purification treatment on the antibody to obtain the pre-processed antibody comprise:

[0109] Mixing the pre-modified alkaline phosphatase, enzyme activation buffer and activation agent to perform activation treatment and desalination purification treatment to obtain the pre-processed alkaline phosphatase, and mixing the antibody, antibody activation buffer and 2-iminothiolane salt to perform activation treatment and desalination purification treatment to obtain the pre-processed antibody;

[0110] The enzyme activation buffer comprises a mixed solution of PBS with a molar concentration of 10-100 mM and EDTA-2Na with a molar concentration of 1-5 mM;

[0111] The enzyme activation buffer has a pH value of 6.5-7.5;

[0112] The antibody activation buffer comprises a mixed solution of PBS with a molar concentration of 50-100 mM and EDTA-2Na with a molar concentration of 2-5 mM;

[0113] The pH value of the antibody activation buffer is 7.5-8.0;

[0114] The mass ratio of the pre-modified alkaline phosphatase and the activator is (10-20):1;

[0115] The mass ratio of the antibody and 2-iminothiolane hydrochloride is (35-45):1;

[0116] The mass ratio of the pretreated alkaline phosphatase and the pretreated antibody is (1-1.4):1.

[0117] The activator, modifier and alkaline phosphatase are set in the above range, the reaction efficiency is high, and the toxic effect of the reagent on the antibody can be reduced. The mass ratio of the pretreated alkaline phosphatase and the pretreated antibody is set in the above range, the free antibody can be reduced, and the reactivity can be improved. The mass ratio of the antibody and 2-iminothiolane hydrochloride is set in the above range, the protein surface is not excessively modified, and the structure and function of the protein can be maintained.

[0118] In the embodiment of the present application, in the SB2 step, the step of desalting and purifying the activated alkaline phosphatase and the activated antibody comprises: respectively adding the activated alkaline phosphatase and the activated antibody into a desalting column, and centrifuging (1000xg, 4℃) for 2 min in a centrifuge to remove unreacted SMCC activator and Traut's reagent.

[0119] In the embodiment of the present application, in the SB3 step, the step of mixing the pretreated alkaline phosphatase and the pretreated antibody for coupling reaction comprises: mixing the pretreated alkaline phosphatase and the pretreated antibody at a mass ratio of (1-1.4):1, and placing them in a constant temperature and humidity incubator at 25℃ for dark reaction for 1 h.

[0120] In the embodiment of the present application, in the SB3 step, the step of blocking treatment comprises: after the coupling reaction of the pretreated alkaline phosphatase and the pretreated antibody is completed, adding 2-mercaptoethanesulfonic acid sodium and oscillating for 1 min, and blocking in the dark for 10 min. Then, N-ethylmaleimide is added and oscillated for 1 min, and blocked in the dark for 10 min.

[0121] In the embodiment of the present application, in the SB3 step, the desalination purification step after the blocking treatment comprises: adding the blocked alkaline phosphatase antibody marker into a desalination column, and centrifuging (1000xg, 4℃) for 2min to remove the unreacted blocking agent.

[0122] In the embodiment of the present application, the SB3 step is followed by: testing the concentration of the enzyme-labeled complex by NanoDrop A280 method, and diluting the alkaline phosphatase antibody marker to 0.5mg / mL with an enzyme binding stabilizer, and storing at 4℃.

[0123] The present application provides a detection reagent comprising the alkaline phosphatase antibody marker, or comprising the alkaline phosphatase antibody marker prepared according to the preparation method of the alkaline phosphatase antibody marker. The kit comprises all the technical solutions of the alkaline phosphatase antibody marker, thus having all the beneficial effects of the alkaline phosphatase antibody marker, which will not be repeated here.

[0124] The present application provides a kit comprising the detection reagent. The kit comprises all the technical solutions of the detection reagent, thus having all the beneficial effects of the detection reagent, which will not be repeated here.

[0125] Embodiment 1

[0126] An alkaline phosphatase antibody marker comprising a first activating agent, a second activating agent, an alkaline phosphatase and an antibody connected to the first activating agent and the second activating agent by chemical bonds respectively, and the antibody connected to the first activating agent by a chemical bond;

[0127] The first activating agent is 4-(N-maleimide methyl) cyclohexane-1-carboxylic acid succinimidyl ester (SMCC); the second activating agent is methoxy polyethylene glycol succinimidyl valerate (mPEG-SVA); the antibody is N-terminal B-type natriuretic peptide precursor antibody (NT-proBNP102, purchased from Bioyue Biotech); the mass ratio of the first activating agent to the alkaline phosphatase is 1:25; the mass ratio of the second activating agent to the alkaline phosphatase is 1:30; and the mass ratio of the alkaline phosphatase to the antibody is 1:1.

[0128] Embodiment 2

[0129] Compared with embodiment 1, the difference is that the second activating agent is polyethylene glycol monomethyl ether succinimidyl carbonate (mPEG-SC); the mass ratio of the first activating agent to the alkaline phosphatase is 1:28; the mass ratio of the second activating agent to the alkaline phosphatase is 1:35; and the mass ratio of the alkaline phosphatase to the antibody is 1.2:1.

[0130] Example 3

[0131] The difference compared with Example 1 is that the second activating agent is methoxy polyethylene glycol succinimidyl glutarate (mPEG-SG); the mass ratio of the first activating agent to alkaline phosphatase is 1:30; the mass ratio of the second activating agent to alkaline phosphatase is 1:40; and the mass ratio of the alkaline phosphatase to the antibody is 1.4:1.

[0132] Comparative Example 1

[0133] Compared with Example 3, mPEG-SVA is not used, and only SMCC is used as an activating agent to activate alkaline phosphatase.

[0134] Example 4

[0135] A preparation method of an alkaline phosphatase antibody marker, and the preparation steps are as follows:

[0136] (1) Alkaline phosphatase activation: Take 1.4 mg of alkaline phosphatase (product number P5512, purchased from Sigma-Aldrich, hereinafter also referred to as AP), and reconstitute with 350 μL of enzyme activation buffer (50 mM PBS, 2 mM EDTA·2Na, pH 7.2), and mix well. Add 10 μL of 5 mg / mL SMCC (product number 22360, purchased from Thermo Fisher) and 10 μL of 20 mg / mL mPEG-SVA (purchased from Huabio, PEG molecular weight is 10K), shake and mix well for 1 min, and place in a 25°C constant temperature and humidity incubator, avoid light and static reaction for 1 h, to obtain activated AP.

[0137] (2) Antibody activation: Take 1 mg of antibody (NT-proBNP102, purchased from Boyue Bio, hereinafter also referred to as Ab), and reconstitute with 250 μL of antibody activation buffer (50 mM PBS, 2 mM EDTA·2Na, pH 8.0), and mix well. Add 5 μL of 5 mg / mL Traut’s reagent (product number 26101, purchased from Thermo Fisher), shake and mix well for 1 min, and place in a 25°C constant temperature and humidity incubator, avoid light and static reaction for 1 h, to obtain activated Ab. This step is carried out at the same time as the above-mentioned AP activation.

[0138] (3) Desalting purification after activation: The activated AP and the activated Ab are added to the desalting column respectively, and centrifuged in a centrifuge (1000 x g, 4°C) for 2 min, to remove the unreacted SMCC, mPEG-SVA and Traut’s reagent, to obtain pretreated AP and pretreated Ab.

[0139] (4) Coupling reaction of AP and Ab: the pretreated AP and the pretreated Ab were mixed in a mass ratio of 1.4:1, and placed in a 25°C constant temperature and humidity incubator, and reacted in the dark for 1 h to obtain the alkaline phosphatase antibody marker to be blocked.

[0140] (5) Blocking: after the coupling reaction was completed, 3 μL of 10 mg / mL 2-mercaptoethanesulfonic acid sodium (M407758, purchased from Aladdin) was added to the alkaline phosphatase antibody marker to be blocked, and shaken for 1 min, and blocked in the dark for 10 min. Then 4 μL of 10 mg / mL N-ethylmaleimide (HY-D0843, purchased from MCE) was added and shaken for 1 min, and blocked in the dark for 10 min to obtain the blocked alkaline phosphatase antibody marker.

[0141] (6) Desalting purification after blocking: the blocked alkaline phosphatase antibody marker was added to a desalting column, and centrifuged (1000 x g, 4°C) for 2 min to remove unreacted blocking agent, and the alkaline phosphatase antibody marker was obtained.

[0142] (7) Storage of alkaline phosphatase antibody marker: the concentration of the alkaline phosphatase antibody marker was tested by NanoDrop A280 method, and diluted to 0.5 mg / mL with enzyme binding stabilizer (SA01-1000, purchased from SurModics), and stored at 4°C for standby.

[0143] Example 5

[0144] Compared with Example 4, the difference is that the PEG molecular weight in mPEG-SVA is 5K.

[0145] Example 6

[0146] Compared with Example 4, the difference is that the PEG molecular weight in mPEG-SVA is 2K.

[0147] Example 7

[0148] Compared with Example 4, the difference is that the PEG molecular weight in mPEG-SVA is 1K.

[0149] Comparative Example 2

[0150] Compared with Example 4, the difference is that in step (1), the AP is first activated with SMCC, and then the AP is activated with mPEG-SVA, and the specific steps of step (1) are as follows:

[0151] Take 1.4 mg of AP, reconstitute with 350 μL of enzyme activation buffer, mix well. Add 10 μL of 5 mg / mL SMCC, shake well for 1 min, and place in a 25°C constant temperature and humidity incubator, avoid light and static reaction for 1 h. After the reaction is completed, add to the desalting column, centrifuge in the centrifuge (1000 x g, 4°C) for 2 min, remove the unreacted SMCC.

[0152] Mix the SMCC-activated AP with 10 μL of 20 mg / mL mPEG-SVA (PEG molecular weight is 10K), shake well for 1 min, and place in a 25°C constant temperature and humidity incubator, avoid light and static reaction for 1 h.

[0153] Comparative Example 3

[0154] Compared with Example 4, the difference is that in step (1), the AP is first activated with mPEG-SVA, and then activated with SMCC. The specific steps of step (1) are as follows:

[0155] Take 1.4 mg of AP, reconstitute with 350 μL of enzyme activation buffer, mix well. Add 10 μL of 20 mg / mL mPEG-SVA (PEG molecular weight is 10K), shake well for 1 min, and place in a 25°C constant temperature and humidity incubator, avoid light and static reaction for 1 h. After the reaction is completed, add to the desalting column, centrifuge in the centrifuge (1000 x g, 4°C) for 2 min, remove the unreacted SMCC.

[0156] Mix the mPEG-SVA-activated AP with 10 μL of 5 mg / mL SMCC, shake well for 1 min, and place in a 25°C constant temperature and humidity incubator, avoid light and static reaction for 1 h.

[0157] Example 8

[0158] A basic phosphatase antibody marker, comprising an activator, a modifier, a basic phosphatase (AP) connected to the activator and the modifier by a chemical bond respectively, and an antibody (Ab) connected to the activator by a chemical bond;

[0159] Wherein, the activator is SMCC; the modifier is CE210 (purchased from JSR); the mass ratio of SMCC to AP is 1:25; the mass ratio of CE210 to AP is 1:30; the mass ratio of AP to Ab is 1:1.

[0160] Example 9

[0161] Compared with Example 8, the difference is that the mass ratio of SMCC to AP is 1:28; the mass ratio of CE210 to AP is 1:35; and the mass ratio of AP to Ab is 1.2:1.

[0162] Example 10

[0163] The difference compared with Example 8 is that the mass ratio of SMCC to alkaline phosphatase is 1:30; the mass ratio of CE210 to AP is 1:40; and the mass ratio of AP to Ab is 1.4:1.

[0164] Example 11

[0165] A preparation method of an alkaline phosphatase antibody marker, the preparation steps being as follows:

[0166] (1) AP pre-modification: take 1.4 mg of AP, reconstitute with 350 μL of carboxyl activation buffer (50 mM MES, pH 6.0), and mix well. Add 2 μL of 2 mg / mL CE210 (purchased from JSR), shake and mix well for 1 min, then add 10 μL of 10 mg / mL EDC and 20 μL of 10 mg / mL Sulfo-NHS activator, and place in a 25°C constant temperature and humidity incubator, avoid light and static reaction for 1 h. Then purify the pre-modified AP in a desalting column to remove excess EDC, Sulfo-NHS and CE210.

[0167] (2) AP activation: take 1.4 mg of pre-modified AP, reconstitute with 350 μL of enzyme activation buffer (50 mM PBS, 2 mM EDTA·2Na, pH 7.2), and mix well. Add 10 μL of 5 mg / mL SMCC, shake and mix well for 1 min, and place in a 25°C constant temperature and humidity incubator, avoid light and static reaction for 1 h.

[0168] (3) Antibody activation: take 1 mg of Ab, reconstitute with 250 μL of antibody activation buffer (50 mM PBS, 2 M EDTA·2Na, pH 8.0), and mix well. Add 5 μL of 5 mg / mL Traut’s reagent, shake and mix well for 1 min, and place in a 25°C constant temperature and humidity incubator, avoid light and static reaction for 1 h. This step is carried out at the same time as the above-mentioned AP activation.

[0169] (4) Desalting purification after activation: add the activated AP and the activated Ab to a desalting column respectively, and centrifuge in a centrifuge (1000 x g, 4°C) for 2 min to remove unreacted SMCC and Traut’s reagent, to obtain pre-processed AP and pre-processed Ab.

[0170] (5) Coupling reaction of AP and Ab: mix the pre-processed AP and the pre-processed Ab in a mass ratio of 1.4:1, and place in a 25°C constant temperature and humidity incubator, avoid light and reaction for 1 h.

[0171] (6) Blocking: After the labeling reaction was completed, 3 μL of 10 mg / mL sodium 2-mercaptoethanesulfonate was added, mixed well for 1 min, and blocked in the dark for 10 min. Then 4 μL of 10 mg / mL N-ethylmaleimide was added, mixed well for 1 min, and blocked in the dark for 10 min, to obtain the blocked alkaline phosphatase antibody label.

[0172] (7) Purification after blocking: The blocked enzyme-labeled antibody complex was added to a desalting column and centrifuged (1000 x g, 4°C) for 2 min to remove unreacted blocking agent, to obtain the alkaline phosphatase antibody label.

[0173] (8) Storage of alkaline phosphatase antibody label: The concentration of the alkaline phosphatase antibody label was tested by the NanoDrop A280 method, and diluted to 0.5 mg / mL with enzyme binding stabilizer, and stored at 4°C for standby.

[0174] Example 12

[0175] The difference compared with Example 11 is that CE210 is replaced by CE510.

[0176] Comparative Example 4

[0177] The difference compared with Example 11 is that steps (1) and (2) are different, first activated by SMCC and then modified by CE210, and the specific steps of steps (1) and (2) are as follows:

[0178] (1) AP activation: 1.4 mg of pre-modified AP was reconstituted with 350 μL of enzyme activation buffer (50 mM PBS, 2 mM EDTA·2Na, pH 7.2), mixed well. 10 μL of 5 mg / mL SMCC was added, mixed well for 1 min, and incubated in a 25°C constant temperature and humidity incubator in the dark for 1 h.

[0179] (2) AP modification: 1.4 mg of AP activated in step (1) was reconstituted with 350 μL of carboxyl activation buffer (50 mM MES, pH 6.0), mixed well. 2 μL of 2 mg / mL CE210 (purchased from JSR) was added, mixed well for 1 min, and then 10 μL of 10 mg / mL EDC and 20 μL of 10 mg / mL Sulfo-NHS activator were added and incubated in a 25°C constant temperature and humidity incubator in the dark for 1 h. Then the modified AP was purified in a desalting column to remove excess SMCC, CE210, EDC and Sulfo-NHS.

[0180] Example 13

[0181] A detection reagent comprising the alkaline phosphatase antibody label of Example 1; the antibody is N-terminal pro-B-type natriuretic peptide antibody (NT-proBNP102, purchased from Biouniquer).

[0182] Example 14

[0183] A detection reagent comprising the alkaline phosphatase antibody label of Example 8; the antibody is N-terminal pro-B-type natriuretic peptide antibody.

[0184] Example 15

[0185] A kit comprising a detection reagent, the detection reagent comprising a first reagent and a second reagent, the first reagent comprising the alkaline phosphatase antibody label of Example 1, and the second reagent comprising N-terminal pro-B-type natriuretic peptide antibody (NT-proBNP McAb) coupled to p-toluenesulfonyl (Tosyl) magnetic beads, wherein the magnetic beads are Tosyl superparamagnetic magnetic beads (MC160, purchased from JSR), and the antibody is N-terminal pro-B-type natriuretic peptide antibody (NT-proBNP101, purchased from Biouniquer).

[0186] Example 16

[0187] A kit comprising a detection reagent, the detection reagent comprising a first reagent and a second reagent, the first reagent comprising the alkaline phosphatase antibody label of Example 8, and the second reagent comprising N-terminal pro-B-type natriuretic peptide antibody coupled to p-toluenesulfonyl magnetic beads, wherein the magnetic beads are Tosyl superparamagnetic magnetic beads, and the antibody is N-terminal pro-B-type natriuretic peptide antibody.

[0188] Performance test

[0189] The alkaline phosphatase antibody labels of Examples 1-12 and Comparative Examples 1-4 were evaluated for signal-to-noise ratio, yield, solution state, and batch-to-batch difference. The detection method was chemiluminescent immunoassay, which used a double antibody sandwich principle for concentration determination, and the test instrument was a fully automatic chemiluminescent immunoassay analyzer, model MT60 (purchased from Shenzhen Jinrui Biological Technology Co., Ltd.). The principle is as follows:

[0190] Serum sample, superparamagnetic magnetic beads coated with N-terminal B-type natriuretic peptide probody and alkaline phosphatase antibody labeled marker of examples 1-12 and comparative examples 1-4 were added into the reaction tube, after incubation, N-terminal brain natriuretic peptide precursor in serum sample combined with anti-N-terminal brain natriuretic peptide precursor antibody coated on magnetic beads, at the same time, alkaline phosphatase antibody labeled marker (anti-N-terminal brain natriuretic peptide precursor antibody-alkaline phosphatase labeled marker) combined with N-terminal brain natriuretic peptide precursor in serum sample. Double antibody sandwich immune complex was formed, after reaction, magnetic field attracted magnetic beads, and uncombined substances were washed away. Chemiluminescence substrate solution was added into the reaction tube, and unstable excited state product was generated under the catalysis of alkaline phosphatase. When the product in excited state returned from excited state to ground state, chemical luminescence was generated. The luminescence value generated by reaction was detected by photomultiplier tube, and the luminescence value was proportional to the concentration of N-terminal brain natriuretic peptide precursor in sample.

[0191] When detection was performed, the preparation steps of the N-terminal B-type natriuretic peptide probody antibody (NT-proBNP McAb) coupled with p-toluenesulfonyl (Tosyl) magnetic beads were as follows:

[0192] (1) Coated antibody: 10 mg of Tosyl superparamagnetic magnetic beads (MC160, purchased from JSR) were taken in a centrifuge tube, and washed with reaction buffer (0.1M BB, pH 9.5) twice, and 0.5 mL of reaction buffer was added for reconstitution. Then 80 μL of 2.51 mg / mL NT-proBNP McAb (NT-proBNP101, purchased from Bi Yue Biology) was added, and mixed for 1 min. 300 μL of catalyst (3M (NH4)2SO4, pH 9.5) was added, mixed for 1 min, and the centrifuge tube was placed in a horizontal mixer, 1500 rpm, 37°C, and reacted for 24 h.

[0193] (2) Magnetic bead blocking: after the above reaction was completed, the supernatant was removed by magnetic separation, and the unreacted antibody was removed. Then 0.5 mL of blocking solution (0.05% Tween-20, 0.5% BSA, 0.05% Proclin300, 50 mM Tris, pH 8.5) was added, mixed for 1 min, the centrifuge tube was placed in a horizontal mixer, 1500 rpm, 37°C, and reacted for 24 h.

[0194] (3) Magnetic bead preservation: after the magnetic bead blocking was completed, the magnetic beads were washed with magnetic bead preservation solution (0.05% Tween-20, 0.5% BSA, 0.05% Proclin300, 50 mM Tris, pH 7.4) for 3 times, and finally 0.5 mL of preservation solution was added and stored at 4°C for standby use.

[0195] 1. The evaluation method and evaluation results of signal-to-noise ratio are as follows:

[0196] The alkaline phosphatase antibody markers of Examples 1-12 and Comparative Examples 1-4 were tested with NT-proBNP antigen (NT-proBNP302, purchased from Bioawes) at different concentrations of 0, 5, 15, 100, 1000, 1700, 3000 pg / mL, and each NT-proBNP antigen concentration was recorded as C0, C1, C2, C3, C4, C5, C6, respectively. C1, C2, C3, C4, C5, C6 were divided by C0, respectively, and the obtained ratio was recorded as signal-to-noise ratio. The higher the signal-to-noise ratio value, the better the quality and the higher the accuracy of the reagent. The test results are shown in Tables 1 and 2.

[0197] Table 1 Signal-to-noise ratio results of Examples 1-7 and Comparative Examples 1-3

[0198]

[0199] As can be seen from Table 1, compared with Comparative Example 1, the C0 value of Examples 1-7 decreased, significantly reducing the non-specific adsorption of alkaline phosphatase; the signal-to-noise ratio results of the alkaline phosphatase antibody markers of Examples 1-7 were also better than those of Comparative Example 1, indicating that using SMCC and a reagent with a polyethylene glycol group and a succinimidyl ester group to co-activate alkaline phosphatase can improve the accuracy and quality of the reagent. In addition, as can be seen from the test results of Examples 4-7, with the decrease of the PEG molecular weight in mPEG-SVA, the signal-to-noise ratio first increased and then decreased, and the test results of Example 6 were the best.

[0200] As can be seen from the test results of Examples 4-7 and Comparative Examples 2-3, the signal-to-noise ratio results of Examples 4-7 were better than those of Comparative Examples 2-3, indicating that using SMCC and mPEG-SVA to co-activate alkaline phosphatase simultaneously is better than using a single SMCC or mPEG-SVA separately.

[0201] Table 2 Signal-to-noise ratio results of Examples 8-12 and Comparative Example 4

[0202]

[0203] As can be seen from Tables 1 and 2, compared with Comparative Example 1, the C0 value of Examples 8-12 decreased, significantly reducing the non-specific adsorption of alkaline phosphatase; the signal-to-noise ratio results of the alkaline phosphatase antibody markers of Examples 8-12 were also better than those of Comparative Example 1, indicating that using CE210 or CE510 to modify alkaline phosphatase can improve the accuracy and quality. As can also be seen from Table 2, the accuracy results of using CE210 to modify alkaline phosphatase are better than those of using CE510 to modify.

[0204] From the test results of Examples 11-12 and Comparative Example 4, it can be seen that the effect of first modifying the alkaline phosphatase with CE210 and then activating the alkaline phosphatase with SMCC is better than that of first modifying the alkaline phosphatase with SMCC and then modifying the alkaline phosphatase with CE210.

[0205] 2. The evaluation method and evaluation results of yield and solution state are as follows:

[0206] The yield and solution state of Examples 4-7, Examples 11-12 and Comparative Example 1 were evaluated, wherein the yield was calculated by the following formula: (A 280 × volume after desalination) / (extinction coefficient × total amount of feed) × 100%, and the test results are shown in Table 3;

[0207] The evaluation method of solution state: the solubility of the labeled process complex affects the uniformity of the reagent, and the physical state is observed by visual observation, and the test results are shown in Table 3.

[0208] Table 3 Yield results of Examples 4-7, Examples 11-12 and Comparative Example 1

[0209]

[0210] In Table 3, the total amount of feed is the amount of antibody and alkaline phosphatase; since the complex of antibody and phosphatase is in multiple complex states, the extinction coefficient is set to 1 when calculating the yield.

[0211] From Table 3, it can be seen that modifying mPEG-SVA, CE210 and CE510 on alkaline phosphatase can effectively avoid the precipitation of antibody-alkaline phosphatase complex. Compared with Comparative Example 1, the solution state of Examples 4-7 and Examples 7-11 is clear and transparent, indicating that modifying mPEG-SVA, CE210 and CE510 on alkaline phosphatase increases the solubility of the alkaline phosphatase antibody label, and the final yield is increased by 10.3-23.0%, indicating that the preparation method of Examples 4-7 and Examples 11-12 can effectively reduce the production cost.

[0212] 3. The evaluation method and evaluation results of batch difference are as follows:

[0213] Examples 6 and 11 were taken as examples and compared with Comparative Example 1, and the batch difference of the prepared alkaline phosphatase antibody label was evaluated, and the evaluation method and evaluation results of batch difference are as follows:

[0214] By repeated preparation, the coefficients of variation (CV) of C0, C1, C4 and C6 NT-proBNP antigen concentrations were calculated, and the lower the CV value, the smaller the batch difference. The test results are shown in Table 4.

[0215] Table 4 Batch to batch difference results of Example 6, Example 11 and Comparative Example 1

[0216]

[0217] From the data in Table 4, the CV of each concentration point in the test results of Example 6 and Example 11 are all less than 10%, while the CV of low value (C1) and high value (C6) in Comparative Example 1 are both greater than 10%, indicating that the modification of mPEG-SVA, CE210 on alkaline phosphatase effectively reduces the batch to batch difference caused by the aggregation of the label.

[0218] The above merely describes exemplary embodiments of the present application, and is not intended to limit the patent scope of the present application. Any equivalent structural transformation made according to the technical concept of the present application, or direct / indirect application in other related technical fields, is included in the patent protection scope of the present application.

Claims

1. An alkaline phosphatase antibody label, characterized in that, The alkaline phosphatase antibody marker comprises a first activating agent, a second activating agent, alkaline phosphatase connected with the first activating agent and the second activating agent respectively through chemical bonds, and an antibody connected with a third activating agent through a chemical bond; The first activating agent comprises 4-(N-maleimido methyl) cyclohexane-1-carboxylic acid succinimidyl ester; The second activating agent comprises a polyethylene glycol compound containing a succinimidyl ester group, and the polyethylene glycol compound containing a succinimidyl ester group comprises at least one of methoxypolyethylene glycol succinimidyl valerate, methoxypolyethylene glycol succinimidyl carbonate, methoxypolyethylene glycol succinimidyl glutarate, methoxypolyethylene glycol succinimidyl succinate, methoxypolyethylene glycol succinimidyl carboxymethyl ester, methoxypolyethylene glycol succinimidyl butyrate, methoxypolyethylene glycol succinimidyl succinamide and methoxypolyethylene glycol succinimidyl propionate; The third activating agent is 2-iminothiolane hydrochloride; The first activating agent and the second activating agent are used to co-activate the alkaline phosphatase, and the co-activated alkaline phosphatase is then reacted with the antibody to form the alkaline phosphatase antibody marker.

2. The alkaline phosphatase antibody label of claim 1, wherein, The mass ratio of the first activating agent to the alkaline phosphatase is 1:(25-30); and / or, The mass ratio of the second activating agent to the alkaline phosphatase is 1:(30-40); and / or, The mass ratio of the alkaline phosphatase to the antibody is (1-1.4):

1.

3. A method for preparing an alkaline phosphatase antibody label according to any one of claims 1 or 2, characterized in that, The method comprises the following steps: SA1, mixing the alkaline phosphatase, the first activating agent and the second activating agent, performing activation treatment and desalination purification treatment to obtain the alkaline phosphatase after pretreatment; activating the antibody, performing desalination purification treatment to obtain the antibody after pretreatment; SA2, mixing the alkaline phosphatase after pretreatment and the antibody after pretreatment to sequentially perform coupling reaction, blocking treatment and purification treatment to obtain the alkaline phosphatase antibody marker.

4. The method for preparing an alkaline phosphatase antibody label according to claim 3, wherein In the step SA1, the alkaline phosphatase, the first activating agent and the second activating agent are mixed, and activation treatment and desalination purification treatment are performed to obtain the alkaline phosphatase after pretreatment. The step of activating the antibody and performing desalination purification treatment to obtain the antibody after pretreatment comprises: The alkaline phosphatase, the enzyme activation buffer, the first activating agent and the second activating agent are mixed, and activation treatment and desalination purification treatment are performed to obtain the alkaline phosphatase after pretreatment. The antibody, the antibody activation buffer and 2-iminothiolane hydrochloride are mixed, and activation treatment and desalination purification treatment are performed to obtain the antibody after pretreatment.

5. The method for preparing alkaline phosphatase antibody markers as described in claim 4, characterized in that, The enzyme activation buffer comprises a mixed solution of PBS with a molar concentration of 10-100 mM and EDTA-2Na with a molar concentration of 1-5 mM; and / or, The pH value of the enzyme activation buffer is 6.5-7.5; and / or, The antibody activation buffer comprises a mixed solution of PBS with a molar concentration of 50-100 mM and EDTA-2Na with a molar concentration of 2-5 mM; and / or, The pH value of the antibody activation buffer is 7.5-8.0; and / or, The mass ratio of the antibody and 2-iminothiolane hydrochloride is (35-45):

1.

6. An alkaline phosphatase antibody label, characterized in that, The alkaline phosphatase antibody marker comprises a first activating agent, a modifying agent, alkaline phosphatase respectively connected with the first activating agent and the modifying agent through chemical bonds, and an antibody connected with a second activating agent through a chemical bond; The first activating agent comprises 4-(N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester. The modifying agent comprises at least one of CE210 and CE510. The second activating agent is 2-iminothiolane hydrochloride. The alkaline phosphatase is modified by selecting a modifying agent, and the modified alkaline phosphatase, the first activating agent and the antibody are combined to form the alkaline phosphatase antibody marker.

7. The alkaline phosphatase antibody label of claim 6, wherein, The mass ratio of the modifying agent and the alkaline phosphatase is 1:(30-40); and / or, The mass ratio of the first activating agent and the alkaline phosphatase is 1:(25-30); and / or, The mass ratio of the alkaline phosphatase and the antibody is (1-1.4):

1.

8. A method for preparing an alkaline phosphatase antibody label as claimed in claim 6 or 7, characterized in that, The method comprises the following steps: SB1, mixing and reacting the alkaline phosphatase and the modifying agent to obtain pre-modified alkaline phosphatase; SB2, mixing the pre-modified alkaline phosphatase and the first activating agent, and performing activation treatment and desalting purification treatment to obtain pre-treated alkaline phosphatase; The antibody is activated and desalted and purified to obtain pre-treated antibody. SB3, mixing the pre-treated alkaline phosphatase and the pre-treated antibody, and sequentially performing coupling reaction, blocking treatment and purification treatment to obtain the alkaline phosphatase antibody marker.

9. The method for preparing alkaline phosphatase antibody markers as described in claim 8, characterized in that, In the step SB1, the step of mixing and reacting the alkaline phosphatase and the modifying agent to obtain pre-modified alkaline phosphatase comprises: The alkaline phosphatase, a carboxyl activating buffer, the modifying agent, N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide are mixed and reacted to obtain pre-modified alkaline phosphatase.

10. The method of producing an alkaline phosphatase antibody label according to claim 9, wherein The carboxyl activating buffer comprises a MES solution with a molar concentration of 10-100 mM; and / or, The pH value of the carboxyl activating buffer is 5.0-6.5; and / or, The mass ratio of the N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride and the alkaline phosphatase is 1:(10-20); and / or, The mass ratio of the N-hydroxysuccinimide and the alkaline phosphatase is 1:(5-10).

11. The method for preparing alkaline phosphatase antibody markers as described in claim 8, characterized in that, In the step SB2, the step of mixing the pre-modified alkaline phosphatase and the first activating agent, and performing activation treatment and desalting purification treatment to obtain pre-treated alkaline phosphatase comprises: The step of activating and desalting and purifying the antibody to obtain pre-treated antibody comprises: The pre-modified alkaline phosphatase, an enzyme activation buffer and the first activating agent are mixed to perform activation treatment and desalting purification treatment to obtain pre-treated alkaline phosphatase; The antibody, an antibody activation buffer and 2-iminothiolane hydrochloride are mixed to perform activation treatment and desalting purification treatment to obtain pre-treated antibody.

12. The method for preparing alkaline phosphatase antibody markers as described in claim 11, characterized in that, The enzyme activation buffer comprises a mixed solution of PBS with a molar concentration of 10-100 mM and EDTA-2Na with a molar concentration of 1-5 mM; and / or, The enzyme activation buffer has a pH value of 6.5-7.5; and / or, The antibody activation buffer comprises a mixed solution of PBS with a molar concentration of 50-100 mM and EDTA-2Na with a molar concentration of 2-5 mM; and / or, The antibody activation buffer has a pH value of 7.5-8.0; and / or, The mass ratio of the pre-modified alkaline phosphatase and the first activator is (10-20):1; and / or, The mass ratio of the antibody and 2-iminothiolane hydrochloride is (35-45):

1.

13. A test reagent, characterized by, The detection reagent comprises the alkaline phosphatase antibody label of claim 1 or 2, or the alkaline phosphatase antibody label prepared by the preparation method of the alkaline phosphatase antibody label according to any one of claims 3 to 5.

14. A test reagent, characterized by, The detection reagent comprises the alkaline phosphatase antibody label of claim 6 or 7, or the alkaline phosphatase antibody label prepared by the preparation method of the alkaline phosphatase antibody label according to any one of claims 8 to 12.

15. A kit comprising, The kit comprises the detection reagent of claim 13.

16. A kit comprising, The kit comprises the detection reagent of claim 14.

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