A method for preparing an enzyme marker

Abstract

The present application discloses a method for preparing an enzyme marker, comprising the following steps: reacting a labeling enzyme with a first activator to obtain an activated labeling enzyme; detecting the actual concentration of the activated labeling enzyme in a solution as a first measured concentration; detecting the actual concentration of the labeled substance in the solution as a second measured concentration; determining the dosage of the reaction between the activated labeling enzyme and the labeled substance based on the first measured concentration, the second measured concentration, and the required dosage of the enzyme marker; coupling the activated labeling enzyme of the determined dosage with the labeled substance, and terminating the coupling reaction to obtain the enzyme marker. The method provided in the present application controls the coupling ratio of the labeling enzyme to the labeled substance by measuring the actual concentrations of the labeling enzyme and the labeled substance and fixing the molar ratio based on the actual concentrations, thereby achieving the effect of improving the batch-to-batch variation of the enzyme marker.

Description

Technical Field

[0001] The present application relates to the field of biochemical detection technology, and in particular to a method for preparing an enzyme marker. Background Art

[0002] EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) is a zero-length coupling agent. EDC allows carboxylic acid (-COOH) groups to directly conjugate with amino groups (-NH2) without becoming part of the final amide bond coupling between target molecules. Alkaline phosphatase contains both amino and carboxyl groups, as do labeled species such as antibodies, antigens, or antigens conjugated to macromolecules such as BSA (hereinafter referred to as antigen-BSA). The carboxyl groups on alkaline phosphatase react with EDC to form an o-acylisourea intermediate, which is easily replaced by amino groups. The amino groups on the labeled species, such as the antibody, antigen, or antigen-BSA, form amide bonds with the original carboxyl groups on the o-acylisourea intermediate, thereby conjugating alkaline phosphatase to the conjugates. Furthermore, because o-acylisourea intermediates are unstable and easily hydrolyzed, NHS or Sulfo-NHS is often added during the EDC coupling process to significantly improve the solubility and stability of the active intermediate.

[0003] There are generally two methods for labeling antibodies with HRP (horseradish peroxidase): glutaraldehyde coupling and periodate oxidation. The principle of the periodate oxidation method is that sodium periodate oxidizes the polysaccharide on the surface of the HRP molecule into aldehyde groups. These aldehyde groups form Schiff bases with amino groups on the antibody, resulting in a stable enzyme label. The conjugate can be further reduced with sodium bihydrogen hydride (NaBH4) or ethanolamine to produce a stable enzyme label. The principle of the glutaraldehyde coupling method is that glutaraldehyde is a homobifunctional coupling reagent with two aldehyde reactive groups. These two aldehyde groups form Schiff bases with amino groups on the antibody and alkaline phosphatase, respectively, linking the two molecules with a five-carbon bridge. The conjugate can be further reduced with NaBH4 or ethanolamine to produce a stable enzyme label. Sulfo-SMCC is a water-soluble amino-sulfhydryl heterobifunctional coupling reagent containing a Sulfo-NHS ester and a maleimido reactive group at each end. The NHS ester reacts with primary amine groups under appropriate conditions to form a stable amide bond, while the maleimido reactive group reacts with sulfhydryl groups under appropriate conditions to form a stable thioether bond. Traut's Reagent can introduce terminal sulfhydryl groups into amino-containing substances through a ring-opening reaction. Alternatively, reducing agents such as dithiothreitol (DTT) and tris(2-carboxyethyl)phosphine (TCEP) can also reduce amino-containing substances to sulfhydryl groups.

[0004] Alkaline phosphatase contains amino groups, and antibodies and other conjugates also contain amino groups. Therefore, the Sulfo-NHS ester group on Sulfo-SMCC can react with the amino groups on alkaline phosphatase or the labeled substance. The maleimide reactive group on the other end of Sulfo-SMCC can react with the labeled substance or alkaline phosphatase with a sulfhydryl group introduced by Traut's Reagent ring opening, thereby forming an alkaline phosphatase label coupled with Sulfo-SMCC.

[0005] The aforementioned coupling method involves multiple chemical reactions, such as the reaction of NHS ester groups with amino groups, the reaction of Traut's Reagent with amino groups, and the reaction of maleimido-reactive groups with sulfhydryl groups, making it relatively complex. Current production practices have shown that batches of enzyme-labeled labeled substances can vary significantly, leading to increased bias in subsequent test results and reduced accuracy. To minimize batch variability, verification is required with each change, which is labor-intensive and inefficient. Summary of the Invention

[0006] In order to solve the above technical problems, the first object of the present invention is to provide a method for preparing an enzyme marker; the method provided in this application controls the coupling ratio of the labeling enzyme and the labeled substance by measuring the actual concentrations of the labeling enzyme and the labeled substance and fixing the molar ratio according to the actual concentrations, thereby achieving the effect of improving the batch-to-batch difference of the enzyme marker.

[0007] The technical solutions provided by the present invention are as follows:

[0008] A method for preparing an enzyme marker comprises the following steps:

[0009] reacting the labeling enzyme with a first activator to obtain an activated labeling enzyme;

[0010] The actual concentration of the activated labeling enzyme in the detection solution is used as the first measured concentration;

[0011] detecting the actual concentration of the labeled substance in the solution as the second measured concentration;

[0012] Determining the dosage of the activated labeling enzyme for reacting with the labeled substance based on the first measured concentration, the second measured concentration, and the required dosage of the enzyme label;

[0013] The activated labeling enzyme of the determined dosage is coupled with the labeled substance, and the enzyme labeling substance is obtained after terminating the coupling reaction.

[0014] Preferably, the method further comprises the following steps:

[0015] The labeled substance reacts with the second activator to obtain an activated labeled substance; the second measured concentration is the actual concentration of the activated labeled substance.

[0016] Preferably, the method further comprises the following steps:

[0017] detecting an actual concentration of the labeling enzyme as a third measured concentration, and then reacting the labeling enzyme with the first activator based on the third measured concentration;

[0018] The actual concentration of the labeled substance is detected as a fourth measured concentration, and then the labeled substance is reacted with the second activating agent based on the fourth measured concentration.

[0019] Preferably, the dosage of the activated labeling enzyme and the labeled substance is determined based on the ratio of the first measured concentration to the second measured concentration, the required dosage of the enzyme label, and the correspondence between the coupling groups of the labeling enzyme and the labeled substance.

[0020] Preferably, the labeling enzyme is any one of alkaline phosphatase and horseradish peroxidase;

[0021] The labeled substance is any one of an antibody, an antigen, or an antigen conjugated with BSA.

[0022] Preferably, the first activator and the second activator are respectively selected from any one of EDC / NHS, glutaraldehyde, Sulfo-SMCC, and Traut's Reagent.

[0023] Preferably, the activated labeling enzyme is desalted and purified using a desalting column, and then the first measured concentration is detected; and / or,

[0024] The activated labeled substance is desalted and purified using a desalting column, and then the second measured concentration is detected.

[0025] Preferably, the desalting column is washed with a buffer and centrifuged before use; and / or,

[0026] The molecular weight cut-off of the desalting column is 7-7.5k.

[0027] Preferably, the reaction is terminated using a buffer containing an amino group.

[0028] Preferably, after terminating the reaction, the method further includes purifying the product and adding a preservation solution for preservation.

[0029] Starting with alkaline phosphatase-labeled antibodies, the applicants studied their preparation methods and batch-to-batch variability. They found that the three chemical reactions—the reaction between NHS ester groups and amino groups, the reaction between Traut's Reagent and amino groups, and the reaction between maleimido-reactive groups and sulfhydryl groups—all require appropriate reaction conditions to proceed smoothly, and that the ratio of reactants is a key factor in coupling efficiency. Further research revealed that the ratio of activator to activated species (antibody or enzyme) in these three chemical reactions affects the activation results, and the ratio of the two activated species with maleimido-reactive groups to sulfhydryl groups in the coupling step also affects the coupling results.

[0030] The current standard practice in the industry is to calculate the mass ratio based on the theoretical concentrations of alkaline phosphatase and the conjugate, without monitoring the real-time concentration during the activator conjugation process. This method has two potential drawbacks: first, inaccurate theoretical concentrations of alkaline phosphatase and the conjugate between batches can lead to large batch variability; second, the lack of monitoring concentration changes before and after conjugation can lead to inconsistent actual conjugation ratios within each batch, resulting in significant batch variability.

[0031] Through experiments, the applicant verified that before coupling alkaline phosphatase and antibodies, the actual concentrations of the two were tested separately, and activation and coupling were performed according to the molar ratio converted according to the actual concentrations. The batch differences of the obtained products were smaller than the conventional method of calculating the ratio based on the theoretical concentration. This method can improve batch differences and improve product quality.

[0032] Further research by the applicants revealed that the above method is applicable to reactions involving various labeling enzymes, including alkaline phosphatase and horseradish peroxidase, with labeled targets such as antibodies, antigens, or antigens conjugated to BSA. Conjugation with different antibodies and labeling enzymes differs in the number of conjugated groups and molecular weights of the antibodies and enzymes, respectively. However, these are known issues. Substituting different antibodies or labeling enzymes for conjugation using the technical solution of the present invention can achieve the desired results.

[0033] Alkaline phosphatase or other labeling enzymes require activation before use. The first measured concentration refers to the concentration of the labeling enzyme after activation. More preferably, the actual concentration of the labeling enzyme is also measured before activation, and this is used as the third measured concentration. When the labeling enzyme reacts with the first activator, the reaction ratio is calculated based on the third measured concentration. This results in more complete activation of the labeling enzyme, reduced free activator residue after the reaction, easier purification, and reduced reagent usage.

[0034] The actual concentration can be detected using different protein concentration testing instruments, preferably an enzyme-labeled instrument with a trace nucleic acid quantification module, thermo NanoDrop Lite, etc., which can accurately measure the concentration of 1-3ul trace proteins.

[0035] In this application, the labeled substance can be used without activation or after activation with a second activating agent. When the labeled substance is activated, the second measured concentration refers to the value obtained by measuring the actual concentration of the activated labeled substance. More preferably, the concentration is also measured before the activation reaction of the labeled substance, and the fourth measured concentration is used as the reaction ratio when the labeled substance reacts with the second activating agent.

[0036] After obtaining the measured concentrations of the labeling enzyme and the labeled substance, the dosage of the activated labeling enzyme and the labeled substance is determined based on the ratio of the first measured concentration to the second measured concentration, the required dosage of the enzyme label, and the correspondence between the coupling groups of the labeling enzyme and the labeled substance. The dosage of the activated labeling enzyme and the labeled substance is determined using conventional methods in the art, such as making a one-to-one correspondence between the activated labeling enzyme and the labeled substance; or determining the ratio according to the number of groups of the labeling enzyme and the labeled substance; or the ratio can be made based on the priority of satisfying the purpose of completely labeling the labeled substance. The present application improves batch differences and improves product quality, and has no direct relationship with the chemical reaction performance of the antibody itself. The innovation of the present application is that when converting the dosage, it is converted based on the first measured concentration and the second measured concentration. Replacing the antibody does not change the technical effect of the present invention.

[0037] Conjugates such as antibodies and antigens are usually stored in solutions containing protective agent components. The components in these solutions may interfere with whether the labeled substance can participate in the activation reaction or coupling reaction. Whether to perform actual concentration detection before activation can be determined based on the difference between the theoretical concentration and the measured concentration of the labeled substance or the labeling enzyme. When the difference between the theoretical concentration and the actual concentration is usually large, performing actual concentration detection before activation can ensure that the concentration of the resulting activated substance is within the target range. The labeled substance or the labeling enzyme needs to undergo a coupling reaction as soon as possible after activation (usually within 20-30 minutes), and the activation reaction process usually takes about 1 hour. If the difference between the activated substance and the target range is too large, the coupling reaction will not proceed smoothly, or reagents will be wasted.

[0038] The first activating agent and the second activating agent used are each selected from any one of EDC / NHS, glutaraldehyde, Sulfo-SMCC, and Traut's Reagent. For example, when the antibody and alkaline phosphatase are activated separately, Sulfo-SMCC and Traut's Reagent can be used as activating agents. When Sulfo-SMCC reacts with the antibody, alkaline phosphatase reacts with Traut's Reagent for activation; when Sulfo-SMCC reacts with alkaline phosphatase, the antibody reacts with Traut's Reagent for activation.

[0039] Preferably, the activated labeling enzyme or the activated labeled substance is desalted and purified using a desalting column, and then the actual concentration is measured before coupling. Preferably, the desalting column is washed with buffer and centrifuged before use. The molecular weight cut-off of the desalting column is preferably 7-7.5k.

[0040] The reaction is preferably terminated using a buffer containing an amino group, such as N-ethylmaleimide.

[0041] Preferably, after terminating the reaction, the method further comprises the steps of purifying the product (eg, using an ultrafiltration tube) and adding a preservation solution (eg, glycerol) for preservation. DETAILED DESCRIPTION

[0042] In order to help those skilled in the art better understand the technical solutions in this application, the technical solutions in the embodiments of this application will be clearly and completely described below. Obviously, the embodiments described are only part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without making any creative efforts shall fall within the scope of protection of this application.

[0043] The applicant selected the coupling experiment of PCT antibody and alkaline phosphatase for verification.

[0044] A total of 6 groups of experiments were designed, using Sulfo-SMCC to activate alkaline phosphatase and Traut's Reagent to activate antibodies.

[0045] Among them, Groups 1-3 are experimental groups (as Example 1). The actual concentrations of alkaline phosphatase and antibody are detected in the experimental groups before activation as the third measured concentration and the fourth measured concentration; and the concentrations of activated alkaline phosphatase and activated antibody are detected before coupling as the first measured concentration and the second measured concentration. Activation and coupling are performed according to the molar ratios converted from the above actual concentrations. Among them, the ratio of Sulfo-SMCC: antibody = 1:10, the ratio of Traut's Reagent: alkaline phosphatase = 1:10, and the coupling molar ratio = 1:1. Three batches of antibodies and three batches of alkaline phosphatase were selected from the experimental groups 1-3, and the inter-batch difference CV1 of groups 1-3 was tested after labeling was completed.

[0046] Groups 4-6 were three parallel control groups (as Comparative Example 1). Conventional industry procedures were used. The concentrations of alkaline phosphatase and antibody were not tested during the process. The mass ratios of each component were calculated according to the theoretical concentrations of antibody and alkaline phosphatase for activation and coupling (Sulfo-SMCC: antibody = 1:10; Traut's Reagent: alkaline phosphatase = 1:10; coupling mass ratio = 1:1). Three batches of antibody and three batches of alkaline phosphatase were used in the 4-6 control groups, respectively. After labeling, the inter-batch difference CV2 of groups 4-6 was tested.

[0047] Example 1: Alkaline phosphatase labeled PCT antibody - process control molar ratio labeling

[0048] Test the concentration of alkaline phosphatase and antibody (as the third measured concentration and the fourth measured concentration)

[0049] 1) The concentrations of three batches of alkaline phosphatase P1, P2, and P3, and three batches of antibody Q1, Q2, and Q3 were measured using the plate nanoquant module of the Tecan Spark microplate reader.

[0050]

[0051] Antibody activation

[0052] 2) Based on the fourth measured concentration, 0.1 mg of each of the antibodies Q1, Q2, and Q3 were taken, with 30.28 μl of Q1, 18.15 μl of Q2, and 18.74 μl of Q3 (volume = required antibody mass / measured antibody concentration. For example, 30.28 μl = 0.1 mg / 3.303 mg / ml). Then, 0.01 mg (1 mg / mL) of Traut's Reagent solution was added to each of the solutions. After vortexing, the mixture was allowed to react at room temperature for 60 min.

[0053] 3) Wash 75 μl of a 7K molecular weight cut-off desalting column three times with 0.01 M PBS: add 50 μl of 0.01 M PBS and centrifuge at 1500 g for 1 min.

[0054] 4) The PCT antibody after the reaction in step 2) was added to three washed desalting columns, respectively, and centrifuged at 1500 g for 2 minutes to obtain three activated PCT antibodies, which were respectively designated as Ab-1, Ab-2, and Ab-3.

[0055] 5) Take 2 μl of the activated antibody prepared in step 4) and dilute it to 10 μl with 0.01 M PBS. Measure the antibody concentration using the plate nanoquant module of the Tecan Spark microplate reader, loading 2 μl at a time. Measure the same sample three times, multiplying by a factor of 5 to obtain three concentration results. The average of these three concentrations is the final concentration of the same sample (used as the second measured concentration).

[0056]

[0057] Activation and purification of alkaline phosphatase (concurrent with antibody activation)

[0058] 6) Based on the third measured concentration, 0.1 mg of alkaline phosphatase (P1, P2, and P3) was taken (7.79 μl of P1, 8.28 μl of P2, and 7.92 μl of P3). 0.01 mg (2 mg / mL) of sulfo-SMCC stock solution was added to each of the three solutions. The mixture was vortexed and allowed to react at room temperature for 60 min.

[0059] 7) Take three 75 μl desalting columns with a molecular weight cut-off of 7K and wash them three times with 0.01 M PBS: add 50 μl of 0.01 M PBS and centrifuge at 1500 g for 1 min.

[0060] 8) The alkaline phosphatase after the reaction in step 6) was added to a desalting column for desalting and purification to obtain three activated alkaline phosphatases, which were respectively designated as AP-1, AP-2, and AP-3.

[0061] 9) Take 2 μl of the activated alkaline phosphatase from step 8) and dilute it to 10 μl with 0.01 M PBS (the dilution is only used for concentration determination of the activated enzyme; use the undiluted activated enzyme for coupling). Measure the alkaline phosphatase concentration using the plate nanoquant module of the Tecan Spark microplate reader, loading 2 μl at a time. Measure the same sample three times, multiplying by a factor of 5 to obtain three concentration results. The average of these three concentrations is the final concentration of the sample (as the first measured concentration).

[0062]

[0063] Activated alkaline phosphatase and antibody conjugation

[0064] 10) Determine the amount of alkaline phosphatase added based on the number of amino groups in each batch of activated antibodies. Assuming that the free amino groups per mol of antibody are X and the free amino groups of alkaline phosphatase are Y, the volume of alkaline phosphatase added to couple the activated antibody to V2 is calculated as follows:

[0065]

[0066] Where:

[0067] V1: measured volume of antibody after activation;

[0068] C1: measured concentration of antibody after activation;

[0069] C2: measured concentration of alkaline phosphatase after activation (not the concentration after dilution, which is the mean concentration in the above table);

[0070] X: Concentration of free amino groups in the antibody (or the number of groups available for conjugation);

[0071] Y: concentration of free amino groups on alkaline phosphatase (or the number of groups available for coupling);

[0072] M1: Antibody molecular weight, for example, the PCT antibody used in this example has a molecular weight of 150,000;

[0073] M1: molecular weight of alkaline phosphatase, for example, the molecular weight of the alkaline phosphatase used in this example is 124,000.

[0074] When coupling, the ratio is determined according to the corresponding relationship between the number of groups of the two, as calculated by the above formula; there are several ratio methods as needed:

[0075] If the labeling enzyme and the labeled substance have a one-to-one correspondence, then X = 1, Y = 1, that is, the number of labeling enzymes and antibodies corresponds one to one;

[0076] If the ratio is determined based on the number of groups in the labeling enzyme and antibody, for the PCT antibody in this example, X = 5. In this case, if the labeling enzyme is paired with the antibody, for example, alkaline phosphatase, which has 5-13 mol / mol free amino groups, and a minimum concentration of 5 mol / mol is used, then Y = 5. Alternatively, a maximum concentration of 13 mol / mol can be used, resulting in Y = 13. This value can be adjusted based on actual coupling requirements; for example, Y can be any number between 1 and 13, preferably between 1 and 5, and more preferably 1, 5, or 13.

[0077] Furthermore, the ratio can be determined based on the free groups on the antibody, giving priority to ensuring that the antibody is completely labeled. Based on the above calculation formula, Y = 1, that is, each group on each antibody is matched with a labeling enzyme. When using PCT antibodies, X = 5.

[0078] In this example, the antibodies and alkaline phosphatase (undiluted after activation) obtained in step 5) and step 9) were converted according to the first measured concentration and the second measured concentration, and a coupling mode of X=4, Y=5 was selected. They were mixed in pairs: Ab-1 was mixed with AP-1, Ab-2 was mixed with AP-2, and Ab-3 was mixed with AP-3. After vortex mixing, the mixture was allowed to react at room temperature for 60 minutes.

[0079]

[0080] 11) To each of the three mixed solutions in step 10), 0.05 mg (5 mg / mL) of N-ethylmaleimide was added, vortexed to mix, and allowed to react at room temperature for 30 min.

[0081] 12) The three alkaline phosphatase-labeled antibodies from step 11) were purified using a 30K ultrafiltration tube, diluted to a concentration of 1 mg / mL with 50 mM Tris solution, and then an equal volume of glycerol was added for storage, resulting in a final concentration of 0.5 mg / mL.

[0082] Comparative Example 1: Alkaline phosphatase labeled PCT antibody - theoretical concentration mass ratio

[0083] Antibody activation

[0084] 1) According to the theoretical concentration, 0.1 mg of PCT antibody from batches Q1, Q2, and Q3 was taken, including 22.22 μl of Q1, 21.28 μl of Q2, and 22.22 μl of Q3. 0.01 mg (1 mg / mL) of Traut's Reagent solution was added to each aliquot. After vortexing, the mixture was allowed to react at room temperature for 60 min.

[0085] 2) Wash 75 μl of a 7K molecular weight cut-off desalting column three times with 0.01 M PBS: add 50 μl of 0.01 M PBS and centrifuge at 1500 g for 1 min.

[0086] 3) The three PCT antibodies after the reaction in step 1) were added to three washed desalting columns respectively, and centrifuged at 1500g for 2 minutes to obtain three activated PCT antibodies, which were recorded as Ab-4, Ab-5, and Ab-6.

[0087] Activation and purification of alkaline phosphatase (concurrent with antibody activation)

[0088] 4) According to the theoretical concentration, 0.1 mg of alkaline phosphatase from batches P1, P2, and P3 was added to 0.01 mg (2 mg / mL) of SMCC stock solution, vortexed to mix, and allowed to react at room temperature for 60 min.

[0089] 5) Take three 75 μl desalting columns with a molecular weight cut-off of 7K and wash them three times with 0.01 M PBS: add 50 μl of 0.01 M PBS and centrifuge at 1500 g for 1 min.

[0090] 6) The alkaline phosphatase after the reaction in step 4) was added to a desalting column for desalting and purification to obtain three activated alkaline phosphatases, which were respectively designated as AP-4, AP-5, and AP-6.

[0091] Alkaline phosphatase and antibody conjugate

[0092] 7) All volumes of antibodies and alkaline phosphatase obtained in steps 3) and 6) were mixed in pairs, Ab-4 was mixed with AP-4, Ab-5 was mixed with AP-5, and Ab-6 was mixed with AP-6. After vortex mixing, the mixture was allowed to react at room temperature for 60 minutes.

[0093] 8) To each of the three mixed solutions in step 7), 0.05 mg (5 mg / mL) of N-ethylmaleimide was added, vortexed to mix, and allowed to react at room temperature for 30 min.

[0094] 9) The alkaline phosphatase-labeled antibodies from the three reactions in step 8) were purified using a 30K ultrafiltration tube, diluted to 100 μL with 50 mM Tris solution, and then 100 μL of glycerol was added for storage, resulting in a final volume of 200 μL. The theoretical antibody concentration was 0.5 mg / mL.

[0095] Comparative experiment

[0096] 1) The six alkaline phosphatase-labeled antibodies labeled in two ways in Example 1 and Comparative Example 1 were diluted 1000-fold with a buffer containing 50 mM Tris, 0.9% NaCl, 0.5% BSA, 1.5 mM MgCl2, 0.2 mM ZnCl2, 0.05% proclin300, pH 7.4, and were respectively labeled Rb1, Rb2, Rb3, Rb4, Rb5, and Rb6.

[0097] 2) The Ra component (another PCT monoclonal antibody coated on magnetic beads) was diluted 100-fold with a buffer solution containing 50 mM Tris, 0.9% NaCl, 0.5% BSA, 0.05% proclin 300, pH 7.4.

[0098] 3) Divide Ra into 6 parts and match them with Rb1, Rb2, Rb3, Rb4, Rb5, and Rb6 respectively to form 6 PCT kits.

[0099] 4) Establish a master calibration curve for each set of reagents, which is a functional relationship between the concentration of the PCT working calibrator and its corresponding luminescence value obtained by four-parameter fitting.

[0100] 5) The six PCT kits mentioned above were tested with the PCT enterprise repeatability reference materials of 0.542 ng / mL (level 1) and 10.277 ng / mL (level 2), respectively, and the measurements were repeated 10 times. The mean (M) and standard deviation (SD) of the results of 30 measurements of the same enterprise reference material for Rb1, Rb2, and Rb3 were calculated. The mean (M) and standard deviation (SD) of the results of 30 measurements of the same enterprise reference material for Rb4, Rb5, and Rb6 were calculated. The coefficient of variation (CV) was obtained according to Formula 1.

[0101] CV=SD / M×100%................(I)

[0102] Where:

[0103] CV – coefficient of variation;

[0104] SD - standard deviation of 30 measurement results;

[0105] M is the average value of 30 measurements.

[0106] 6) Experimental results

[0107]

[0108]

[0109] The experimental results show that the inter-batch differences of the three alkaline phosphatase-labeled PCT antibodies labeled in Example 1 are smaller than the inter-batch differences of the three alkaline phosphatase-labeled PCT antibodies labeled in Comparative Example 1.

[0110] The above experimental results indicate that by real-time monitoring of the concentrations of activated alkaline phosphatase and antibodies, and calculating the molar weight of the amino groups on the activated alkaline phosphatase relative to the theoretical molar weight of the amino groups on the antibody, the theoretical molar ratio of alkaline phosphatase to the conjugate can be controlled, thereby improving batch-to-batch variability in enzyme-labeled products. Furthermore, monitoring the concentration of PCT antibodies revealed a significant discrepancy between the manufacturer's reported concentration and the measured concentration, which may also contribute to the large batch-to-batch variability. Therefore, real-time monitoring of the concentrations of alkaline phosphatase and antibodies should be performed during enzyme labeling.

[0111] Additionally, before activation, alkaline phosphatase and antibody concentrations were monitored, and the amount of activator adjusted based on the results. However, in this example, no adjustment was made to the activators, Traut's Reagent and Sulfo-SMCC, as they were both in excess. Theoretically, the amount of activator can be adjusted for different antibody conjugates based on the project characteristics and monitored concentrations.

[0112] In the examples, N-ethylmaleimide was used to block excess sulfhydryl groups after activation. Theoretically, the molar amount of N-ethylmaleimide should be equal to or greater than the molar amount of amino groups on the antibody after activation with Traut's Reagent. Since the amount of N-ethylmaleimide was in excess in this example, no adjustment was made. Theoretically, the amount of blocking agent can be adjusted for different antibody conjugations based on the specific application and the concentration being monitored.

[0113] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing an enzyme marker, characterized in that: The following steps are involved: detecting an actual concentration of the labeling enzyme as a third measured concentration, and then reacting the labeling enzyme with a first activator based on the third measured concentration to obtain an activated labeling enzyme; The actual concentration of the activated labeling enzyme in the detection solution is used as the first measured concentration; detecting an actual concentration of the labeled substance as a fourth measured concentration, and then reacting the labeled substance with a second activator based on the fourth measured concentration to obtain an activated labeled substance; detecting the actual concentration of the activated labeled substance in the solution as the second measured concentration; Determining the dosage of the activated labeling enzyme for reacting with the labeled substance based on the first measured concentration, the second measured concentration, and the required dosage of the enzyme label; The activated labeling enzyme of the determined dosage is coupled with the labeled substance, and the enzyme labeling substance is obtained after terminating the coupling reaction.

2. The method for preparing an enzyme marker according to claim 1, wherein The dosages of the activated labeling enzyme and the labeled substance are determined according to the ratio of the first measured concentration to the second measured concentration, the required dosage of the enzyme label, and the correspondence between the coupling groups of the labeling enzyme and the labeled substance.

3. The method for preparing an enzyme marker according to claim 1, wherein The labeling enzyme is any one of alkaline phosphatase and horseradish peroxidase; The labeled substance is any one of an antibody, an antigen, or an antigen conjugated with BSA.

4. The method for preparing an enzyme marker according to claim 1, wherein The first activator and the second activator are each selected from any one of EDC / NHS, glutaraldehyde, Sulfo-SMCC, and Traut's Reagent.

5. The method for preparing an enzyme marker according to claim 1, wherein The activated labeling enzyme is desalted and purified using a desalting column, and then the first measured concentration is detected; and / or, The activated labeled substance is desalted and purified using a desalting column, and then the second measured concentration is detected.

6. The method for preparing an enzyme marker according to claim 5, wherein The desalting column is washed with buffer and centrifuged before use; and / or, The molecular weight cut-off of the desalting column is 7-7.5k.

7. The method for preparing an enzyme marker according to claim 1, wherein The reaction was terminated with an amino group-containing buffer.

8. The method for preparing an enzyme marker according to claim 7, wherein After the reaction is terminated, the method further includes the steps of purifying the product and adding a preservation solution for preservation.

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