Alkaline phosphatase antibody marker, preparation method thereof, detection reagent and kit

Abstract

The invention discloses an alkaline phosphatase antibody marker, a preparation method thereof, a detection reagent and a kit, and relates to the technical field of protein coupling. The alkaline phosphatase antibody marker provided by the technical scheme of the invention is prepared by jointly activating alkaline phosphatase through a polyethylene glycol compound containing a succinimide ester group and SMCC, or is prepared by modifying alkaline phosphatase through a polyethylene glycol molecule with an amino group and then activating the modified alkaline phosphatase with SMCC. Compared with a traditional Traut's-SMCC method, the method has the advantages that the hydrophilicity of the alkaline phosphatase antibody marker can be improved, the solubility of the alkaline phosphatase antibody marker can be improved, and the alkaline phosphatase antibody marker has certain steric hindrance, so that the possibility of continuous reaction of redundant active sites on alkaline phosphatase can be reduced, and the stability of the alkaline phosphatase antibody marker is improved. Under the condition that the crosslinking efficiency is not influenced, the crosslinking aggregation strength of the alkaline phosphatase and the antibody is slowed down, so that the stability and the yield of the alkaline phosphatase antibody marker are improved, and the batch difference is reduced.

Description

Technical Field

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

[0002] Enzymatic chemiluminescence is a widely used immunodiagnostic technology, in which alkaline phosphatase-labeled antibodies and their labeling process are an important part, affecting the stability, batch difference, signal-to-noise ratio and other detection performance of the luminescence kit. The commonly used processes for alkaline phosphatase labeling include glutaraldehyde method, carbodiimide method, sodium periodate oxidation method and 2-iminothiolane hydrochloride (Traut's) and 4-(N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester (SMCC) combination method. The first two labeling methods have no specific cross-linking, which easily causes alkaline phosphatase and / or antibodies to self-cross-link, reducing the detection efficiency; the sodium periodate oxidation method is used to treat natural alkaline phosphatase with less than 5% sugar groups, and the oxidation efficiency is low, and the aldehyde group formed is unstable, and there is a chance that it will react with its own amino group to produce self-polymerization.

[0003] The combined method of 2-iminothiolane hydrochloride (Traut's) and 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimidyl ester (SMCC) utilizes the heterofunctional groups of SMCC to connect alkaline phosphatase and antibodies respectively, so the coupling efficiency is high, but the solubility and stability of the alkaline phosphatase labeled antibody obtained by this method are poor, which ultimately leads to a reduced enzyme-labeled antibody yield and large batch-to-batch differences. At present, some researchers use coupling agents containing polyethylene glycol groups instead of SMCC reagents to make the coupled alkaline phosphatase antibody markers have the characteristics of high stability, high sensitivity, and low nonspecific reaction, but the heterobifunctional coupling agent containing ethylene glycol is costly, and the higher the degree of polymerization of ethylene glycol, the more expensive it is. In addition, the use of heterobifunctional coupling agents containing ethylene glycol cannot slow down the reaction of redundant active sites.

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

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

[0006] To achieve the above object, the present invention 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 respectively through a chemical bond, and an antibody connected to the first activator through a chemical bond;

[0007] Wherein, the first activator comprises 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester;

[0008] The second activator includes a polyethylene glycol compound containing a succinimide ester group.

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

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

[0011] The mass ratio of the second activator to 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 invention provides a method for preparing an alkaline phosphatase antibody marker, wherein the alkaline phosphatase antibody marker comprises a first activator, a second activator, an alkaline phosphatase connected to the first activator and the second activator respectively through a chemical bond, and an antibody connected to the first activator through a chemical bond; the method for preparing the alkaline phosphatase antibody marker comprises the following steps:

[0014] SA1, mixing alkaline phosphatase, a first activator and a second activator, performing activation treatment and desalting and purification treatment to obtain pretreated alkaline phosphatase; performing activation treatment and desalting and purification treatment on the antibody to obtain the pretreated antibody;

[0015] SA2. The pretreated alkaline phosphatase and the pretreated antibody are mixed and subjected to coupling reaction, blocking treatment, and purification treatment in sequence to obtain an alkaline phosphatase antibody marker.

[0016] In one embodiment, in step SA1, the steps of mixing alkaline phosphatase, a first activator, and a second activator, performing activation treatment, and desalting and purifying treatment to obtain pretreated alkaline phosphatase; and performing activation treatment, desalting and purifying treatment on the antibody to obtain the pretreated antibody include:

[0017] Alkaline phosphatase, enzyme activation buffer, a first activator and a second activator are mixed, and activation treatment and desalting and purification treatment are performed to obtain pretreated alkaline phosphatase; antibodies, antibody activation buffer and 2-iminothiolane hydrochloride are mixed, and activation treatment and desalting and purification treatment are performed to obtain pretreated antibodies.

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

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

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

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

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

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

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

[0025] Wherein, the activator includes 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester;

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

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

[0028] The mass ratio of the activator to 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 present invention provides a method for preparing an alkaline phosphatase antibody marker, wherein the alkaline phosphatase antibody marker comprises an activator, a modifier, an alkaline phosphatase connected to the activator and the modifier respectively through chemical bonds, and an antibody connected to the activator through a chemical bond; the method for preparing the alkaline phosphatase antibody marker comprises the following steps:

[0031] SB1, mixing alkaline phosphatase and a modifier for reaction to obtain pre-modified alkaline phosphatase;

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

[0033] SB3. The pretreated alkaline phosphatase and the pretreated antibody are mixed and subjected to coupling reaction, blocking treatment, and purification treatment in sequence to obtain an alkaline phosphatase antibody marker.

[0034] In one embodiment, in step SB1, the step of mixing alkaline phosphatase and a modifier for reaction to obtain pre-modified alkaline phosphatase comprises:

[0035] Alkaline phosphatase, carboxyl activation buffer, a modifier, N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (Suflo-NHS) are mixed for reaction to obtain pre-modified alkaline phosphatase.

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

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

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

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

[0040] In one embodiment, in step SB2, the steps of mixing the pre-modified alkaline phosphatase and the activator, performing activation treatment, desalting and purifying treatment to obtain the pre-treated alkaline phosphatase; and performing activation treatment, desalting and purifying treatment on the antibody to obtain the pre-treated antibody include:

[0041] The pre-modified alkaline phosphatase, enzyme activation buffer and activator are mixed, and activation treatment, desalting and purification treatment are carried out to obtain the pre-treated alkaline phosphatase; the antibody, antibody activation buffer and 2-iminothiolane hydrochloride are mixed, and activation treatment, desalting and purification treatment are carried out to obtain the pre-treated antibody.

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

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

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

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

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

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

[0048] In one embodiment, in step SB3, the mass ratio of the pretreated alkaline phosphatase to the pretreated antibody is (1-1.4):1.

[0049] The present invention provides a detection reagent, which includes the alkaline phosphatase antibody marker, or includes the alkaline phosphatase antibody marker prepared according to the preparation method of the alkaline phosphatase antibody marker.

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

[0051] The beneficial effects of the technical solution of the present invention are as follows:

[0052] (1) Alkaline phosphatase is co-modified with SMCC and polyethylene glycol compounds containing succinimidyl ester groups, and then the co-modified alkaline phosphatase reacts with antibodies to form alkaline phosphatase antibody markers. Since polyethylene glycol compounds containing succinimidyl ester groups contain polyethylene glycol groups, they are hydrophilic and have certain spatial repulsion effects. Therefore, they can be used to increase the hydrophilicity of the periphery of alkaline phosphatase. After the alkaline phosphatase antibody marker is formed, the hydrophilicity of the alkaline phosphatase antibody marker is increased, and the solubility of the alkaline phosphatase antibody marker is increased. In addition, a certain spatial repulsion effect can prevent the redundant active sites on the alkaline phosphatase antibody marker from continuing to react to form hydrophobic polymers. Therefore, the intensity of cross-linking aggregation between alkaline phosphatase and antibodies is slowed down without affecting the cross-linking efficiency, which can improve the stability and yield of the final alkaline phosphatase antibody marker and reduce the batch-to-batch difference of the alkaline phosphatase antibody marker, thereby preparing a highly stable and highly sensitive alkaline phosphatase antibody marker.

[0053] (2) CE210 or CE510 is selected as a modifier to modify alkaline phosphatase, and then the modified alkaline phosphatase, activator SMCC and antibody are reacted to form an alkaline phosphatase antibody marker. CE210 or CE510 is a polyethylene glycol molecule with an amino terminal. The polyethylene glycol group can improve hydrophilicity and has a certain spatial repulsion. Therefore, after using CE210 or CE510 to modify alkaline phosphatase, not only 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 continuing to react can be reduced. Therefore, without affecting the cross-linking efficiency, the intensity of cross-linking aggregation between alkaline phosphatase and antibody is slowed down, the stability and yield of the final alkaline phosphatase antibody marker can be improved, and the batch difference of the alkaline phosphatase antibody marker can be reduced, thereby preparing a highly stable and highly sensitive alkaline phosphatase antibody marker. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying creative work.

[0055] Figure 1 Schematic diagram of the alkaline phosphatase antibody marker provided in Example 1, Example 8 and Comparative Example 1 of the present invention.

[0056] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0057] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

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

[0059] In addition, if there are descriptions involving "first", "second", etc. in the embodiments of the present invention, the descriptions of "first", "second", etc. are only used for descriptive purposes and cannot be understood as indicating or implying their relative importance or implicitly indicating the number of technical features indicated. Therefore, the features limited to "first" and "second" may explicitly or implicitly include at least one of the features. In addition, if "and / or" or "and / or" appears in the full text, its meaning includes three parallel solutions. Taking "A and / or B" as an example, it includes solution A, solution B, or solutions that satisfy both A and B. In addition, the technical solutions between the various embodiments can be combined with each other, but it must be based on the ability of ordinary technicians in this field to implement. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0060] Enzymatic chemiluminescence is a widely used immunodiagnostic technology, in which alkaline phosphatase labeled antibodies and their labeling process are an important part, affecting the stability, batch difference, signal-to-noise ratio and other detection performance of the luminescence kit. The processes commonly used for alkaline phosphatase labeling include glutaraldehyde method, carbodiimide method, sodium periodate oxidation method and 2-iminothiolane hydrochloride (Traut's) and 4-(N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester (SMCC) combination method. Among them, the Traut's-SMCC method is used. Although the bifunctional coupling agent can avoid protein self-crosslinking and improve labeling efficiency, the crosslinking agent will reduce the solubility of the protein, making the periphery of the complex formed by the alkaline phosphatase labeled antibody present a hydrophobic state, and at the same time, the excess active sites continue to react, and the hydrophobic polymer formed will precipitate in the solution, which ultimately leads to a decrease in yield and a large batch difference. Therefore, it is still of great significance to develop a highly stable and sensitive alkaline phosphatase labeled antibody.

[0061] In view of this, the present invention provides an alkaline phosphatase antibody marker, which includes a first activator, a second activator, an alkaline phosphatase connected to the first activator and the second activator respectively through a chemical bond, and an antibody connected to the first activator respectively through a chemical bond;

[0062] wherein the first activator comprises succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC);

[0063] The second activator includes a polyethylene glycol compound containing a succinimide ester group.

[0064] The technical solution of the present invention uses a first activator and a second activator to jointly activate alkaline phosphatase, and then the jointly activated alkaline phosphatase reacts with an antibody to form an alkaline phosphatase antibody marker, wherein the first activator is SMCC, which contains a maleimide group and a succinimide ester group; the second activator is a polyethylene glycol compound containing a succinimide ester group, which contains a polyethylene glycol group and a succinimide ester group. When the first activator and the second activator activate alkaline phosphatase, the first activator and the second activator both react with the primary amine (-NH2) on the alkaline phosphatase through the succinimide ester group to form an amide bond, thereby binding to the alkaline phosphatase; the first activator also specifically reacts with the antibody activated by Traut's reagent through the maleimide group to form a stable carbon-sulfur (CS) covalent bond, thereby binding to the antibody.

[0065] The traditional Traut's-SMCC combination method for preparing alkaline phosphatase antibody markers uses SMCC to activate alkaline phosphatase. Compared with the traditional Traut's-SMCC combination method, the technical solution of the present invention uses SMCC and polyethylene glycol compounds containing succinimide ester groups to jointly activate alkaline phosphatase. Since the polyethylene glycol compounds containing succinimide ester groups can provide hydrophilicity and a certain steric hindrance, the hydrophilicity of the periphery of alkaline phosphatase can be improved. After the alkaline phosphatase antibody marker is formed, the hydrophilicity of the alkaline phosphatase antibody marker is improved; in addition, a certain steric repulsion can prevent the redundant maleimide active sites from continuing to react to form a hydrophobic polymer, and the strength of the cross-linking aggregation between the alkaline phosphatase antibody and the antibody is slowed down without affecting the cross-linking efficiency. Finally, the stability and yield of the alkaline phosphatase antibody marker can be improved, and the batch difference of the alkaline phosphatase antibody marker can be reduced, which is conducive to the preparation of a highly stable and highly sensitive alkaline phosphatase antibody marker.

[0066] In an embodiment of the present invention, the polyethylene glycol compound containing a succinimide ester group includes at least one of methoxy polyethylene glycol succinimidyl valerate (mPEG-SVA), methoxy polyethylene glycol succinimidyl carbonate (mPEG-SC), methoxy polyethylene glycol succinimidyl glutarate (mPEG-SG), methoxy polyethylene glycol succinimidyl succinate (mPEG-SS), methoxy polyethylene glycol succinimidyl carboxymethyl ester (mPEG-SCM), methoxy polyethylene glycol succinimidyl butyrate (mPEG-SBA), methoxy polyethylene glycol succinimidyl succinamide (mPEG-SSA) and methoxy polyethylene glycol succinimidyl propionate (mPEG-SPA). It should be noted that, considering that mPEG-SC, mPEG-SG, mPEG-SS, mPEG-SCM, mPEG-SBA, mPEG-SSA, and mPEG-SPA are more susceptible to hydrolysis than mPEG-SVA, and succinimidyl valerate (SVA) has a longer half-life, the technical solution of the present invention preferably uses mPEG-SVA to prepare alkaline phosphatase antibody markers, which is conducive to obtaining highly stable alkaline phosphatase antibody markers.

[0067] In an embodiment of the present invention, the mPEG-SVA includes mPEG-SVA containing polyethylene glycol groups of different molecular weights, and the molecular weight of the polyethylene glycol group is 1 to 10K. Using mPEG-SVA and SMCC to activate alkaline phosphatase together 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 binding to antibodies after pretreatment. The larger the molecular weight of polyethylene glycol, the more obvious the steric hindrance provided, which will increase the difficulty of binding between alkaline phosphatase and antibodies.

[0068] In an embodiment of the present invention, the mass ratio of the first activator to alkaline phosphatase is 1: (25-30). It should be noted that excessive use of the first activator will result in low mPEG-SVA coupling efficiency or failure to couple, reducing the solubility of the alkaline phosphatase antibody marker; when the first activator is used in too little amount, the cross-linking efficiency of alkaline phosphatase and antibody is slowed down. Setting the mass ratio of the first activator to alkaline phosphatase in the above range is conducive to obtaining a highly stable and sensitive alkaline phosphatase antibody marker.

[0069] In an embodiment of the present invention, the mass ratio of the second activator to alkaline phosphatase is 1:(30-40). It should be noted that too much second activator will increase production costs, and too little second activator will reduce the effect of slowing down the cross-linking aggregation of alkaline phosphatase antibodies and antibodies. Setting the mass ratio of the second activator to alkaline phosphatase in the above range is conducive to obtaining a highly stable and sensitive alkaline phosphatase antibody marker and controlling production costs.

[0070] In an embodiment of the present invention, the mass ratio of alkaline phosphatase to antibody is (1-1.4):1. It should be noted that too low an amount of antibody will reduce the reactivity of the alkaline phosphatase antibody marker; too high an amount of antibody will result in the presence of free antibodies, which will reduce the possibility of binding of the alkaline phosphatase antibody marker to the antigen, thereby reducing the reactivity. When the mass ratio of alkaline phosphatase to antibody is set at (1-1.4):1, the reactivity of the alkaline phosphatase antibody marker with the subsequent antigen is better.

[0071] The present invention provides a method for preparing an alkaline phosphatase antibody marker, wherein the alkaline phosphatase antibody marker comprises a first activator, a second activator, an alkaline phosphatase connected to the first activator and the second activator respectively through a chemical bond, and an antibody connected to the first activator through a chemical bond; the method for preparing the alkaline phosphatase antibody marker comprises the following steps:

[0072] SA1, mixing alkaline phosphatase, a first activator and a second activator, performing activation treatment and desalting and purification treatment to obtain pretreated alkaline phosphatase; performing activation treatment and desalting and purification treatment on the antibody to obtain the pretreated antibody;

[0073] SA2. The pretreated alkaline phosphatase and the pretreated antibody are mixed and subjected to coupling reaction, blocking treatment, and purification treatment in sequence to obtain an alkaline phosphatase antibody marker.

[0074] The preparation method provided by the technical solution of the present invention first activates alkaline phosphatase with a first activator and a second activator. After the activation treatment, the first activator and the second activator react with the amino group on the alkaline phosphatase respectively, and combine with the alkaline phosphatase by forming an amide bond to form a pre-treated alkaline phosphatase; at the same time, the antibody is activated to introduce a thiol (-SH) group on the antibody; finally, the pre-treated alkaline phosphatase and the antibody with a thiol group are mixed for a coupling reaction, and the pre-treated alkaline phosphatase is covalently linked to the thiol group on the antibody through a maleimide group to form an alkaline phosphatase antibody marker.

[0075] The technical solution of the present invention uses the first activator and the second activator to jointly activate alkaline phosphatase, which is conducive to preparing a highly stable and highly sensitive alkaline phosphatase antibody marker compared with first using the first activator or the second activator alone and then using the second activator or the first activator.

[0076] In an embodiment of the present invention, in step SA1, the steps of mixing alkaline phosphatase, a first activator and a second activator, performing activation treatment and desalting and purification treatment to obtain pretreated alkaline phosphatase; and performing activation treatment and desalting and purification treatment on the antibody to obtain the pretreated antibody include:

[0077] Alkaline phosphatase, enzyme activation buffer, a first activator and a second activator are mixed, and activation treatment and desalting and purification treatment are performed to obtain pretreated alkaline phosphatase; antibodies, antibody activation buffer and 2-iminothiolane hydrochloride are mixed, and activation treatment and desalting and purification treatment are performed to obtain pretreated antibodies;

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

[0079] The antibody activation buffer comprises a mixed solution of PBS with a molar concentration of 50 to 100 mM and EDTA-2Na with a molar concentration of 2 to 5 mM; the pH value of the antibody activation buffer is 7.5 to 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 solution of the present invention, 2-iminothiolane hydrochloride (Traut's) is used to activate the antibody, so that a thiol group (-SH) is introduced into the antibody, so that the antibody can be covalently linked to a substance containing a maleimide group through the newly introduced thiol group, thereby forming a marker. The mass ratio of the antibody to 2-iminothiolane hydrochloride is set to (35-45):1, so as to avoid excessive modification of the protein surface and maintain the structure and function of the protein.

[0083] The mass ratio of the pretreated alkaline phosphatase to the pretreated antibody is set to (1-1.4): 1. Within this range, the mass ratio of the pretreated alkaline phosphatase to the pretreated antibody can be 1: 1, 1.1: 1, 1.2: 1, 1.3: 1, 1.4: 1. When the mass ratio of the pretreated alkaline phosphatase to the pretreated antibody is within the above range, free antibodies can be reduced and reactivity can be improved.

[0084] In an embodiment of the present invention, in the SA1 step, the step of desalting and purifying the activated alkaline phosphatase and the activated antibody comprises: adding the activated alkaline phosphatase and the activated antibody to desalting columns respectively, and centrifuging them in a centrifuge (1000×g, 4°C) for 2 minutes to remove the unreacted first activator, the second activator and Traut's reagent.

[0085] In an embodiment of the present invention, in step SA2, the step of mixing the pretreated alkaline phosphatase and the pretreated antibody for coupling reaction includes: mixing the pretreated alkaline phosphatase and the pretreated antibody in a mass ratio of (1 to 1.4): 1, and placing them in a constant temperature and humidity incubator at 25°C, and reacting in the dark for 1 hour.

[0086] In an embodiment of the present invention, in step SA2, the blocking treatment step includes: after the coupling reaction of the pretreated alkaline phosphatase and the pretreated antibody is completed, sodium 2-mercaptoethane sulfonate is added and shaken for 1 minute, and the mixture is blocked in a dark place for 10 minutes. Then, N-ethylmaleimide is added and shaken for 1 minute, and the mixture is blocked in a dark place for 10 minutes.

[0087] In an embodiment of the present invention, in step SA2, the purification step after the blocking treatment includes: adding the blocked alkaline phosphatase antibody marker to a desalting column and centrifuging it in a centrifuge (1000×g, 4°C) for 2 minutes to remove unreacted blocking agent.

[0088] In an embodiment of the present invention, the SA2 step includes: testing the concentration of the enzyme-labeled complex using the NanoDrop A280 method, and diluting the alkaline phosphatase antibody marker to 0.5 mg / mL using an enzyme-binding stabilizer and storing it at 4°C.

[0089] The present invention provides an alkaline phosphatase antibody marker, which comprises an activator, a modifier, an alkaline phosphatase connected to the activator and the modifier respectively through chemical bonds, and an antibody connected to the activator through a chemical bond;

[0090] Wherein, the activator includes 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester;

[0091] The modifier includes at least one of CE210 and CE510.

[0092] Alkaline phosphatase is modified by a modifier, and then the modified alkaline phosphatase, an activator and an antibody are reacted to form an alkaline phosphatase antibody marker, wherein the modifier is CE210 or CE510, and the activator is 4-(N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester (SMCC). CE210 and CE510 are PEG molecules with oligoamine short chains connected at the ends, and the short chains contain amino terminals that can be coupled with the carboxyl terminals of alkaline phosphatase, thereby binding to alkaline phosphatase; the activator reacts specifically with the sulfhydryl (-SH)-containing antibody through the maleimide group to form a stable carbon-sulfur (CS) covalent bond, thereby binding to the antibody.

[0093] Compared with the traditional Traut's-SMCC combination method, the technical scheme of the present invention uses CE210 or CE510 as a modifier, first pre-modifies alkaline phosphatase, and then uses SMCC to activate alkaline phosphatase. Since alkaline phosphatase forms a negatively charged sulfonyl group after being activated by EDC / Suflo-NHS, the modifier ends with -NH + , can more effectively approach the periphery of alkaline phosphatase, thereby promoting the reaction between the modifier and alkaline phosphatase. Since the modifier also carries a hydrophilic polyethylene glycol group and has a certain spatial repulsion, after modifying alkaline phosphatase, it can not only improve the hydrophilicity of the alkaline phosphatase antibody marker, but also make a certain spatial distance between the modified alkaline phosphatase molecules. After combining with the activator and the antibody to form an alkaline phosphatase antibody marker, it can reduce the possibility of the redundant active sites on the alkaline phosphatase antibody marker continuing to react and produce polymers. Without affecting the cross-linking efficiency, it can slow down the intensity of cross-linking aggregation between alkaline phosphatase and antibodies, thereby improving the stability and yield of the final alkaline phosphatase antibody marker, reducing the batch difference of the alkaline phosphatase antibody marker, and is conducive to the preparation of a highly stable and sensitive alkaline phosphatase antibody marker.

[0094] In an embodiment of the present invention, the mass ratio of the modifier to alkaline phosphatase is 1: (5-10); the mass ratio of the activator to alkaline phosphatase is 1: (10-20); the mass ratio of the alkaline phosphatase to the antibody is (1-1.4): 1. When the amounts of the activator, modifier, alkaline phosphatase and antibody are set within the above ranges, the reaction efficiency is high and the toxic effect of the reagents on the antibody can be reduced.

[0095] The present invention provides a method for preparing an alkaline phosphatase antibody marker, wherein the alkaline phosphatase antibody marker comprises an activator, a modifier, an alkaline phosphatase connected to the activator and the modifier respectively through chemical bonds, and an antibody connected to the activator through a chemical bond; the method for preparing the alkaline phosphatase antibody marker comprises the following steps:

[0096] SB1, mixing alkaline phosphatase and a modifier for reaction to obtain pre-modified alkaline phosphatase;

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

[0098] SB3. The pretreated alkaline phosphatase and the pretreated antibody are mixed and subjected to coupling reaction, blocking treatment, and purification treatment in sequence to obtain an alkaline phosphatase antibody marker.

[0099] The technical solution of the present invention first modifies alkaline phosphatase with a modifier, and the amino group of the modifier is combined with the carboxyl acid of the alkaline phosphatase. After pre-modification treatment, the hydrophilicity of the alkaline phosphatase is improved and a certain spatial distance exists between alkaline phosphatase molecules; then, the pre-modified alkaline phosphatase is activated with an activator, the activator reacts with the amino group on the alkaline phosphatase, and combines with the alkaline phosphatase by forming an amide bond to form the alkaline phosphatase after pretreatment, and at the same time, the antibody is activated to introduce a thiol group (-SH) into the antibody to form the pre-treated antibody; then, after the pre-treated alkaline phosphatase and the pre-treated antibody are mixed, the pre-treated alkaline phosphatase is covalently linked to the thiol group on the antibody through a maleimide group to form an alkaline phosphatase antibody marker.

[0100] It should be noted that the order of adding the modifiers will affect the binding efficiency of the activator and alkaline phosphatase. The technical solution of the present invention selects to modify the alkaline phosphatase with the modifier first, and then add the activator for activation, which is beneficial to improve the hydrophilicity of the alkaline phosphatase antibody marker and slow down the intensity of cross-linking aggregation between alkaline phosphatase and antibody, thereby obtaining a highly stable and highly sensitive alkaline phosphatase antibody marker.

[0101] In an embodiment of the present invention, in step SB1, the step of mixing alkaline phosphatase and a modifier for reaction to obtain pre-modified alkaline phosphatase comprises:

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

[0103] Wherein, the carboxyl activation buffer comprises a MES solution having a molar concentration of 10 to 100 mM;

[0104] The pH value of the carboxyl activation buffer is 5.0-6.5.

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

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

[0107] The modifier has an amino group, and the modifier reacts with the carboxyl end of alkaline phosphatase through the amino group, thereby connecting to the alkaline phosphatase. Adding EDC can activate the carboxyl group (-COOH), so that the carboxyl group reacts with the amino group (-NH2) to form a stable amide bond. When EDC is used together with Sulfo-NHS, Sulfo-NHS can stabilize the carboxyl intermediate activated by EDC, prevent its hydrolysis, and promote its effective reaction with the amino group. Setting the amount of N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysulfosuccinimide within the above range can effectively control the degree of binding of the modifier and reduce the self-crosslinking of alkaline phosphatase.

[0108] In an embodiment of the present invention, in step SB2, the steps of mixing the pre-modified alkaline phosphatase and the activator, performing activation treatment and desalting and purification treatment to obtain the pre-treated alkaline phosphatase; and performing activation treatment and desalting and purification treatment on the antibody to obtain the pre-treated antibody include:

[0109] The pre-modified alkaline phosphatase, enzyme activation buffer and activator are mixed, and activation treatment and desalting and purification treatment are performed to obtain the pre-treated alkaline phosphatase; the antibody, antibody activation buffer and 2-iminothiolane salt are mixed, and activation treatment and desalting and purification treatment are performed to obtain the pre-treated antibody;

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

[0111] The pH value of the enzyme activation buffer is 6.5 to 7.5;

[0112] The antibody activation buffer comprises a mixed solution of PBS with a molar concentration of 50 to 100 mM and EDTA-2Na with a molar concentration of 2 to 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 to the activator is (10-20):1;

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

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

[0117] Setting the activator, modifier and alkaline phosphatase within the above ranges can improve the reaction efficiency and reduce the toxic effects of the reagents on the antibody. Setting the mass ratio of the pre-treated alkaline phosphatase to the pre-treated antibody within the above range can reduce free antibodies and improve reactivity. Setting the mass ratio of the antibody to 2-iminothiolane hydrochloride within the above range can avoid excessive modification of the protein surface and maintain the structure and function of the protein.

[0118] In an embodiment of the present invention, in the SB2 step, the step of desalting and purifying the activated alkaline phosphatase and the activated antibody comprises: adding the activated alkaline phosphatase and the activated antibody to desalting columns respectively, and centrifuging them in a centrifuge (1000×g, 4° C.) for 2 min to remove unreacted SMCC activator and Traut's reagent.

[0119] In an embodiment of the present invention, in step SB3, the step of mixing the pretreated alkaline phosphatase and the pretreated antibody for coupling reaction includes: mixing the pretreated alkaline phosphatase and the pretreated antibody in a mass ratio of (1 to 1.4): 1, and placing them in a constant temperature and humidity incubator at 25°C, and reacting in the dark for 1 hour.

[0120] In an embodiment of the present invention, in step SB3, the blocking treatment step includes: after the coupling reaction of the pretreated alkaline phosphatase and the pretreated antibody is completed, sodium 2-mercaptoethane sulfonate is added and shaken for 1 minute, and the mixture is blocked in a dark place for 10 minutes. Then, N-ethylmaleimide is added and shaken for 1 minute, and the mixture is blocked in a dark place for 10 minutes.

[0121] In an embodiment of the present invention, in step SB3, the desalting and purification step after the blocking treatment includes: adding the blocked alkaline phosphatase antibody marker to a desalting column and centrifuging it in a centrifuge (1000×g, 4° C.) for 2 minutes to remove unreacted blocking agent.

[0122] In an embodiment of the present invention, the SB3 step includes: testing the concentration of the enzyme-labeled complex using the NanoDrop A280 method, and diluting the alkaline phosphatase antibody marker to 0.5 mg / mL using an enzyme-binding stabilizer and storing it at 4°C.

[0123] The present invention provides a detection reagent, which includes the alkaline phosphatase antibody marker, or includes the alkaline phosphatase antibody marker prepared according to the preparation method of the alkaline phosphatase antibody marker. The kit includes all technical solutions of the alkaline phosphatase antibody marker, and thus has all the beneficial effects of the alkaline phosphatase antibody marker, which will not be described in detail in the present invention.

[0124] The present invention provides a kit, which includes the detection reagent. The kit includes all technical solutions of the detection reagent, and thus has all the beneficial effects of the detection reagent, which will not be described in detail in the present invention.

[0125] Example 1

[0126] An alkaline phosphatase antibody marker, comprising a first activator, a second activator, an alkaline phosphatase connected to the first activator and the second activator respectively through a chemical bond, and an antibody connected to the first activator through a chemical bond;

[0127] Among them, the first activator is 4-(N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester (SMCC); the second activator is methoxy polyethylene glycol succinimidyl valerate (mPEG-SVA); the antibody is N-terminal B-type natriuretic peptide original antibody (NT-proBNP102, purchased from Boyue Bio); the mass ratio of the first activator to alkaline phosphatase is 1:25; the mass ratio of the second activator to alkaline phosphatase is 1:30; the mass ratio of alkaline phosphatase to antibody is 1:1.

[0128] Example 2

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

[0130] Example 3

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

[0132] Comparative Example 1

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

[0134] Example 4

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

[0136] (1) Activation of alkaline phosphatase: 1.4 mg of alkaline phosphatase (product number P5512, purchased from Sigma-Aldrich, hereinafter referred to as AP) was re-dissolved in 350 μL of enzyme activation buffer (50 mM PBS, 2 mM EDTA·2Na, pH 7.2) and mixed. 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 Weihua Biotechnology, PEG molecular weight is 10K) were added, and the mixture was shaken and mixed for 1 min, and then placed in a constant temperature and humidity incubator at 25°C, protected from light, and allowed to react for 1 h to obtain activated AP.

[0137] (2) Antibody activation: Take 1 mg of antibody (NT-proBNP102, purchased from Boyue Biotechnology, hereinafter referred to as Ab), re-dissolve it in 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, away from light, and react for 1 h to obtain activated Ab. This step is carried out simultaneously with the above-mentioned AP activation.

[0138] (3) Post-activation desalting and purification: The activated AP and activated Ab were added to desalting columns, respectively, and centrifuged (1000×g, 4°C) for 2 min to remove unreacted SMCC, mPEG-SVA and Traut's reagent to obtain pretreated AP and pretreated Ab.

[0139] (4) AP and Ab coupling reaction: The pretreated AP and the pretreated Ab were mixed at a mass ratio of 1.4:1, and placed in a constant temperature and humidity incubator at 25°C, protected from light, for 1 hour to obtain the alkaline phosphatase antibody marker to be blocked.

[0140] (5) Blocking: After the coupling reaction is completed, add 3 μL of 10 mg / mL sodium 2-mercaptoethane sulfonate (M407758, purchased from Aladdin) to the alkaline phosphatase antibody marker to be blocked, shake and mix for 1 min, and block for 10 min in the dark. Then add 4 μL of 10 mg / mL N-ethylmaleimide (HY-D0843, purchased from MCE), shake and mix for 1 min, and block for 10 min in the dark to obtain a blocked alkaline phosphatase antibody marker.

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

[0142] (7) Storage of alkaline phosphatase antibody marker: The concentration of 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 future use.

[0143] Example 5

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

[0145] Example 6

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

[0147] Example 7

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

[0149] Comparative Example 2

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

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

[0152] The AP activated by SMCC was mixed with 10 μL of 20 mg / mL mPEG-SVA (PEG molecular weight was 10K), oscillated for 1 min, and placed in a constant temperature and humidity incubator at 25° C. to react for 1 h in the dark.

[0153] Comparative Example 3

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

[0155] Take 1.4 mg of alkaline phosphatase, re-dissolve it with 350 μL of enzyme activation buffer, and mix it. Add 10 μL of 20 mg / mL mPEG-SVA (PEG molecular weight is 10K), shake and mix for 1 min, and place it in a 25°C constant temperature and humidity incubator, away from light, and react for 1 hour. After the reaction is completed, add it to the desalting column and centrifuge it (1000×g, 4°C) for 2 min to remove the unreacted mPEG-SVA.

[0156] The AP activated by mPEG-SVA was mixed with 10 μL 5 mg / mL SMCC, shaken and mixed for 1 min, and placed in a constant temperature and humidity incubator at 25°C to react for 1 h in the dark.

[0157] Example 8

[0158] An alkaline phosphatase antibody marker, comprising an activator, a modifier, alkaline phosphatase (AP) connected to the activator and the modifier respectively through chemical bonds, and an antibody (Ab) connected to the activator through a chemical bond;

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

[0160] Example 9

[0161] Compared with Example 8, the differences are 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] Compared with Example 8, the differences are 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] Embodiment 11

[0165] A method for preparing an alkaline phosphatase antibody marker, the preparation steps are as follows:

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

[0167] (2) AP activation: Take 1.4 mg of pre-modified AP, re-dissolve it 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 for 1 min, and place in a 25°C constant temperature and humidity incubator, away from light, and let it react for 1 h.

[0168] (3) Antibody activation: Take 1 mg Ab and re-dissolve it in 250 μL antibody activation buffer (50 mM PBS, 2 M EDTA·2Na, pH 8.0) and mix well. Add 5 μL 5 mg / mL Traut's reagent, shake and mix for 1 min, and place in a 25°C constant temperature and humidity incubator, away from light, and react for 1 h. This step is carried out simultaneously with the above AP activation.

[0169] (4) Post-activation desalting and purification: The activated AP and activated Ab were added to desalting columns, respectively, and centrifuged (1000×g, 4°C) for 2 min to remove unreacted SMCC and Traut's reagent, thereby obtaining pretreated AP and pretreated Ab.

[0170] (5) AP and Ab coupling reaction: The pretreated AP and the pretreated Ab were mixed at a mass ratio of 1.4:1 and placed in a constant temperature and humidity incubator at 25°C in the dark for 1 h.

[0171] (6) Blocking: After the labeling reaction is completed, add 3 μL of 10 mg / mL sodium 2-mercaptoethane sulfonate and shake and mix for 1 min, and block for 10 min in the dark. Then add 4 μL of 10 mg / mL N-ethylmaleimide and shake and mix for 1 min, and block for 10 min in the dark to obtain a blocked alkaline phosphatase antibody marker.

[0172] (7) Post-blocking desalting and purification: Add the blocked enzyme-labeled antibody complex to a desalting column and centrifuge it (1000×g, 4°C) for 2 min to remove the unreacted blocking agent and obtain the alkaline phosphatase antibody marker.

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

[0174] Example 12

[0175] Compared with Example 11, the difference is that CE210 is replaced by CE510.

[0176] Comparative Example 4

[0177] Compared with Example 11, the difference is that step (1) and step (2) are different, SMCC activation is performed first and then CE210 is added for modification. The specific steps of step (1) and step (2) are:

[0178] (1) AP activation: Take 1.4 mg of pre-modified AP, re-dissolve it 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 for 1 min, and place in a 25°C constant temperature and humidity incubator, away from light, and let it react for 1 h.

[0179] (2) AP modification: Take 1.4 mg of AP activated in step (1), re-dissolve it with 350 μL carboxyl activation buffer (50 mM MES, pH 6.0), and mix well. Add 2 μL 2 mg / mL CE210 (purchased from JSR), shake and mix for 1 min, then add 10 μL 10 mg / mL EDC and 20 μL 10 mg / mL Sulfo-NHS activator and place in a 25°C constant temperature and humidity incubator, avoid light and react for 1 h. Then place the modified AP in a desalting column for purification to remove excess SMCC, CE210, EDC and Sulfo-NHS.

[0180] Embodiment 13

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

[0182] Embodiment 14

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

[0184] Embodiment 15

[0185] A kit, the detection kit comprising a detection reagent, the detection reagent comprising a first reagent and a second reagent, the first reagent comprising the alkaline phosphatase antibody marker of Example 1, the second reagent comprising tosyl magnetic beads coupled to N-terminal pro-B-type natriuretic peptide antibody (NT-proBNP McAb), 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 Boyue Biotechnology).

[0186] Example 16

[0187] A test kit comprises a detection reagent, wherein the detection reagent comprises a first reagent and a second reagent, wherein the first reagent comprises the alkaline phosphatase antibody marker of Example 8, and the second reagent comprises tosyl magnetic beads coupled to an N-terminal B-type natriuretic peptide original antibody, wherein the magnetic beads are Tosyl superparamagnetic magnetic beads, and the antibody is an N-terminal B-type natriuretic peptide original antibody.

[0188] Performance Testing

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

[0190] Serum sample, superparamagnetic magnetic beads coated with N-terminal B-type natriuretic peptide original antibody, and alkaline phosphatase antibody markers of Examples 1 to 12 and Comparative Examples 1-4 are added to a reaction tube. After incubation, the N-terminal pro-brain natriuretic peptide in the serum sample binds to the anti-N-terminal pro-brain natriuretic peptide antibody coated on the magnetic beads, and the alkaline phosphatase antibody marker (anti-N-terminal pro-brain natriuretic peptide antibody-alkaline phosphatase marker) binds to the N-terminal pro-brain natriuretic peptide in the serum sample. A double antibody sandwich immune complex is formed. After the reaction is completed, the magnetic beads are attracted by the magnetic field and the unbound substances are washed away. A chemiluminescent substrate solution is added to the reaction tube. The luminescent substrate generates an unstable excited state product under the catalysis of alkaline phosphatase. When the product in the excited state returns to the ground state from the excited state, chemiluminescence is generated. The luminescence value generated by the reaction is proportional to the concentration of the N-terminal pro-brain natriuretic peptide in the sample by the photomultiplier tube.

[0191] When performing the detection, the preparation steps of the tosyl magnetic beads coupled with N-terminal pro-B-type natriuretic peptide antibody (NT-proBNP McAb) are as follows:

[0192] (1) Antibody coating: 10 mg of Tosyl superparamagnetic beads (MC160, purchased from JSR) were placed in a centrifuge tube, washed twice with reaction buffer (0.1 M BB, pH 9.5), and re-dissolved with 0.5 mL of reaction buffer. Then, 80 μL of 2.51 mg / mL NT-proBNP McAb (NT-proBNP101, purchased from Boyue Bio) was added and mixed for 1 min. 300 μL of catalyst (3 M (NH4)2SO4, pH 9.5) was added and mixed for 1 min. The centrifuge tube was placed in a horizontal mixer at 1500 rpm and 37°C for 24 h.

[0193] (2) Magnetic bead blocking: After the above reaction is completed, the supernatant is aspirated by magnetic separation to remove unreacted antibodies, and then 0.5 mL of blocking solution (0.05% Tween-20, 0.5% BSA, 0.05% Proclin 300, 50 mM Tris, pH 8.5) is added and mixed for 1 min. The centrifuge tube is placed in a horizontal mixer at 1500 rpm and 37°C for reaction for 24 h.

[0194] (3) Magnetic bead storage: After the magnetic beads are blocked, wash them three times with magnetic bead storage solution (0.05% Tween-20, 0.5% BSA, 0.05% Proclin 300, 50 mM Tris, pH 7.4), and finally add 0.5 mL of storage solution and store at 4°C for later use.

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

[0196] The alkaline phosphatase antibody markers of Examples 1 to 12 and Comparative Examples 1-4 were tested with NT-proBNP antigen (NT-proBNP302, purchased from Boyue Bio) 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. C1, C2, C3, C4, C5, C6 were divided by C0, and the obtained ratio was recorded as the signal-to-noise ratio. The higher the signal-to-noise ratio, the better the quality of the reagent and the higher the accuracy. 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 shown in Table 1, compared with Comparative Example 1, the C0 values ​​of Examples 1-7 decreased, which significantly reduced the nonspecific 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 the use of SMCC and reagents with polyethylene glycol groups and succinimidyl ester groups to co-activate alkaline phosphatase can improve the accuracy and quality of the reagents. In addition, it can be seen from the test results of Examples 4-7 that as the molecular weight of PEG in mPEG-SVA decreases, the signal-to-noise ratio first increases and then decreases, and the test result of Example 6 is the best.

[0200] From the test results of Example 4-7 and Comparative Example 2-3, it can be seen that the signal-to-noise ratio result of Example 4-7 is better than that of Comparative Example 2-3, indicating that the simultaneous use of SMCC and mPEG-SVA to activate alkaline phosphatase is more effective than the separate use of single SMCC or mPEG-SVA.

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

[0202]

[0203] As shown in Table 1 and Table 2, compared with Comparative Example 1, the C0 values ​​of Examples 8-12 decreased, which significantly reduced the nonspecific 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 the use of CE210 or CE510 to modify alkaline phosphatase can improve accuracy and quality. It can also be seen from Table 2 that the accuracy results of alkaline phosphatase modified with CE210 are better than those modified with CE510.

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

[0205] 2. The evaluation methods and 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 desalting) / (extinction coefficient × total number of materials fed) × 100%, the test results are shown in Table 3;

[0207] Evaluation method of solution state: The solubility of the labeling process complex affects the uniformity of the reagents. Its physical state is observed by visual inspection. 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 feed amount is the amount of antibody and alkaline phosphatase used; since the complex of antibody and phosphatase is in multiple complex states, the extinction coefficient is set to 1 when calculating the yield.

[0211] As shown in Table 3, modification of 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 states of Examples 4-7 and Examples 7-11 are clear and transparent, indicating that the solubility of the alkaline phosphatase antibody marker is increased after modification of mPEG-SVA, CE210, and CE510 on alkaline phosphatase, and the final yield is calculated to be increased by 10.3 to 23.0%, indicating that the preparation methods of Examples 4-7 and Examples 11-12 can effectively reduce production costs.

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

[0213] Taking Example 6 and Example 11 as examples, and comparing them with Comparative Example 1, the obtained alkaline phosphatase antibody markers were evaluated for batch differences. The evaluation method and evaluation results of the batch differences are as follows:

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

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

[0216]

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

[0218] The above description is only an exemplary embodiment of the present invention, and does not limit the patent scope of the present invention. All equivalent structural changes made by using the contents of the present invention and the drawings under the technical concept of the present invention, or directly / indirectly applied in other related technical fields are included in the patent protection scope of the present invention.

Claims

1. An alkaline phosphatase antibody marker, characterized in that The alkaline phosphatase antibody marker comprises a first activator, a second activator, an alkaline phosphatase connected to the first activator and the second activator respectively through a chemical bond, and an antibody connected to the first activator through a chemical bond; Wherein, the first activator comprises 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester; The second activator includes a polyethylene glycol compound containing a succinimide ester group.

2. The alkaline phosphatase antibody marker according to claim 1, characterized in that The polyethylene glycol compound containing a succinimide ester group includes at least one of methoxy polyethylene glycol succinimide valerate, methoxy polyethylene glycol succinimide carbonate, methoxy polyethylene glycol succinimide glutarate, methoxy polyethylene glycol succinimide succinate, methoxy polyethylene glycol succinimide carboxymethyl ester, methoxy polyethylene glycol succinimide butyrate, methoxy polyethylene glycol succinimide succinamide and methoxy polyethylene glycol succinimide propionate; and / or, The mass ratio of the first activator to alkaline phosphatase is 1:(25-30); and / or, The mass ratio of the second activator to 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 marker according to any one of claims 1 or 2, characterized in that: The following steps are involved: SA1, mixing alkaline phosphatase, a first activator and a second activator, performing activation treatment and desalting and purification treatment to obtain alkaline phosphatase after pretreatment; The antibody is activated, desalted and purified to obtain the pre-treated antibody; SA2. The pretreated alkaline phosphatase and the pretreated antibody are mixed and subjected to coupling reaction, blocking treatment, and purification treatment in sequence to obtain an alkaline phosphatase antibody marker.

4. The method for preparing an alkaline phosphatase antibody marker according to claim 3, characterized in that: In step SA1, alkaline phosphatase, a first activator and a second activator are mixed, and activation treatment and desalting and purification treatment are performed to obtain pretreated alkaline phosphatase; The steps of activating and desalting the antibody to obtain the pre-treated antibody include: Alkaline phosphatase, enzyme activation buffer, a first activator and a second activator are mixed, and activation treatment and desalting and purification treatment are performed to obtain pretreated alkaline phosphatase; The antibody, antibody activation buffer and 2-iminothiolane hydrochloride are mixed, and activation treatment and desalting and purification treatment are performed to obtain the pretreated antibody.

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

1.

6. An alkaline phosphatase antibody marker, characterized in that The alkaline phosphatase antibody marker comprises an activator, a modifier, an alkaline phosphatase connected to the activator and the modifier respectively through chemical bonds, and an antibody connected to the activator through a chemical bond; Wherein, the activator includes 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester; The modifier includes at least one of CE210 and CE510.

7. The alkaline phosphatase antibody marker according to claim 6, characterized in that The mass ratio of the modifier to alkaline phosphatase is 1:(30-40); and / or, The mass ratio of the activator to alkaline phosphatase is 1:(25-30); and / or, The mass ratio of the alkaline phosphatase to the antibody is (1-1.4):

1.

8. A method for preparing an alkaline phosphatase antibody marker as claimed in claim 6 or 7, characterized in that: The following steps are involved: SB1, mixing alkaline phosphatase and a modifier for reaction to obtain pre-modified alkaline phosphatase; SB2, mixing the pre-modified alkaline phosphatase and an activator, performing activation treatment, desalting and purification treatment, and obtaining the pre-treated alkaline phosphatase; The antibody is activated, desalted and purified to obtain the pre-treated antibody; SB3. The pretreated alkaline phosphatase and the pretreated antibody are mixed and subjected to coupling reaction, blocking treatment, and purification treatment in sequence to obtain an alkaline phosphatase antibody marker.

9. The method for preparing an alkaline phosphatase antibody marker according to claim 8, characterized in that: In step SB1, the step of mixing alkaline phosphatase and a modifier for reaction to obtain pre-modified alkaline phosphatase comprises: Alkaline phosphatase, carboxyl activation buffer, a modifier, N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysulfosuccinimide are mixed for reaction to obtain pre-modified alkaline phosphatase.

10. The method for preparing an alkaline phosphatase antibody marker according to claim 9, characterized in that: The carboxyl activation buffer comprises a MES solution having a molar concentration of 10 to 100 mM; and / or, The pH value of the carboxyl activation buffer is 5.0 to 6.5; and / or, The mass ratio of N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride to alkaline phosphatase is 1:(10-20); and / or, The mass ratio of the N-hydroxysulfosuccinimide to alkaline phosphatase is 1:(5-10).

11. The method for preparing an alkaline phosphatase antibody marker according to claim 8, characterized in that: In step SB2, the pre-modified alkaline phosphatase is mixed with an activator, and subjected to activation treatment, desalting and purification treatment to obtain pre-treated alkaline phosphatase; The steps of activating and desalting the antibody to obtain the pre-treated antibody include: The pre-modified alkaline phosphatase, enzyme activation buffer and activator are mixed, and activation treatment, desalting and purification treatment are performed to obtain the pre-treated alkaline phosphatase; The antibody, antibody activation buffer and 2-iminothiolane hydrochloride are mixed, and activation treatment and desalting and purification treatment are performed to obtain the pretreated antibody.

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

1.

13. A detection reagent, characterized in that: The detection reagent includes the alkaline phosphatase antibody marker described in 1 or 2, or includes the alkaline phosphatase antibody marker prepared according to the preparation method of the alkaline phosphatase antibody marker according to any one of claims 3 to 5.

14. A detection reagent, characterized in that: The detection reagent includes the alkaline phosphatase antibody marker described in 6 or 7, or includes the alkaline phosphatase antibody marker prepared by the preparation method of the alkaline phosphatase antibody marker according to any one of claims 8 to 12.

15. A kit, characterized in that: The kit comprises the detection reagent as claimed in claim 13.

16. A kit, characterized in that: The kit comprises the detection reagent as claimed in claim 14.

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