Alkaline phosphatase labeled CTNI antibody preparation method

Abstract

The invention relates to the technical field of chemiluminescence of external diagnostic reagents, in particular to a preparation method of an alkaline phosphatase labeled CTNI antibody, which comprises the following steps: reacting a hetero-terminal cross-linking agent with the CTNI antibody for 0.5-4 hours, terminating the reaction by using a reaction terminator, and removing a free reagent to obtain a first reactant; reacting alkaline phosphatase with a 2-IT reagent for 0.5-4 hours, terminating the reaction by using a reaction terminator, and removing a free reagent to obtain a second reactant; mixing the first reactant and the second reactant according to a preset proportion to obtain a mixture; diluting the reduced BSA to 5-10mg / mL by using 10mM of PBS (Phosphate Buffer Solution), and preparing a labeled sealing agent; and mixing the marked sealing agent and the mixture according to a preset proportion, and reacting for 5-20 minutes. According to the invention, the background is reduced, the signal-to-noise is optimized, the false positive rate is improved, the stability of the alkaline phosphatase labeled cTnI antibody labeled by the labeling method is improved, and the problems of low titer, poor signal-to-noise and instability of the existing alkaline phosphatase labeled CTNI antibody preparation method are solved.

Description

Technical Field

[0001] The invention relates to the technical field of chemiluminescence of external diagnostic reagents, and in particular to a method for preparing an alkaline phosphatase-labeled CTNI antibody. Background Art

[0002] According to survey data, the incidence of diseases such as myocardial infarction has risen sharply and has become the leading cause of death from non-communicable diseases worldwide. Myocardial infarction, as a common cardiovascular disease in clinical practice, is one of the main causes of chronic heart failure, and cardiac microvascular damage is a key factor in the occurrence and development of myocardial infarction. Studies have shown that cardiac microvascular damage can lead to insufficient blood supply to myocardial cells, which in turn triggers local acute thrombosis, causing myocardial infarction. Therefore, the prevention and treatment of myocardial infarction is extremely important for improving the quality of life and survival rate of patients.

[0003] Troponin is a group of structural proteins associated with striated muscle cells, consisting of three specific subunits: troponin C, troponin T, and troponin I. Studies have found that cTnI is one of the markers with the highest specificity and sensitivity for the diagnosis of myocardial injury. Compared with commonly used indicators such as CK, CK-MB, and CTNT, cTnI is not easily interfered by skeletal muscle injury and has no cross-reaction with skeletal muscle TnI and other myocardial proteins. After myocardial damage, the concentration of cTnI in serum increases significantly, lasts for a long time, and is easy to detect. Its specificity can reach 99% and its sensitivity exceeds 96%.

[0004] cTnI also shows many advantages in combined detection. The study by Gao Wen et al. showed that cTnI and DD and their ratio had high sensitivity and specificity in the differential diagnosis of acute myocardial infarction and aortic dissection, while the ratio of cTnI to DD and the combined detection of the two had higher value in differential diagnosis. Lan Huiqin explored the role of combined detection of N-terminal pro-B-type natriuretic peptide (NT-proBNP) and cTnI in the diagnosis of heart failure and the prediction of cardiac function classification in patients with myocardial infarction. The results showed that this combined detection had important clinical significance and the difference was statistically significant. Therefore, cTnI has far-reaching significance in the detection of diseases such as myocardial infarction, and how to detect it efficiently and accurately has become an urgent problem to be solved. Chemiluminescence detection has significant advantages in early detection of diseases due to its high efficiency, high sensitivity and high specificity. This method links small molecules or macromolecules through cross-linking agents or chemical substances, and links luminescent substances to antibodies. However, the primary cross-linking method has the problem of intramolecular cross-linking, which leads to low cross-linking efficiency and weakened product activity, thereby affecting the potency, signal-to-noise ratio and stability. The subsequent development of hetero-bifunctional cross-linkers improved the selectivity of cross-linking and achieved the uniformity and efficiency of cross-linked products.

[0005] At present, although there are many methods for labeling cTnI antibodies, there are not many methods that can label effectively and efficiently, and have excellent performance in various indicators. For example, patent CN107918022A uses TCEP to link antibodies and Sulfo-SMCC to link alkaline phosphatase, but the reaction time is too long, which is not conducive to production. Patent CN114994304B focuses on the production and coating of magnetic particles, and has less description of luminescent objects. Summary of the invention

[0006] The object of the present invention is to provide a method for preparing an alkaline phosphatase-labeled CTNI antibody, aiming to solve the problems of low titer, poor signal-to-noise ratio and instability in the existing method for preparing an alkaline phosphatase-labeled CTNI antibody.

[0007] To achieve the above object, the present invention provides a method for preparing an alkaline phosphatase-labeled CTNI antibody, comprising the following steps:

[0008] After reacting the hetero-crosslinking agent with the CTNI antibody for 0.5-4 hours, the reaction is terminated with a reaction terminator, and the free reagent is removed to obtain a first reactant;

[0009] After alkaline phosphatase and 2-IT reagent are reacted for 0.5-4 hours, the reaction is terminated using the reaction terminator, and the free reagent is removed to obtain a second reactant;

[0010] Mixing the first reactant and the second reactant in a preset ratio to obtain a mixture;

[0011] Dilute the reduced BSA to 5-10 mg / mL with 10 mM PBS to prepare the labeling blocking agent;

[0012] The label blocking agent and the mixture are mixed according to a preset ratio and reacted for 5 minutes to 20 minutes.

[0013] Among them, in “after reacting the CTNI antibody with a heteroterpene cross-linking agent for 0.5-4 hours, using a reaction terminator to terminate the reaction, and removing the free reagent to obtain a first reactant”, the heteroterpene cross-linking agent is SMCC or SPDP amino-thiol coupling reagent.

[0014] Among them, in “after reacting the heteroterminal cross-linking agent with the CTNI antibody for 0.5-4 hours, using a reaction terminator to terminate the reaction, and removing the free reagent to obtain a first reactant”, the reactant is an ammonia solution, such as lysine, glycine or TRIS.

[0015] Among them, the method for preparing an alkaline phosphatase-labeled CTNI antibody as described in claim 3 is characterized in that, in "mixing the first reactant and the second reactant in a preset ratio to obtain a mixture", the preset ratio is 1:0.1-1:0.8.

[0016] Among them, the method for preparing an alkaline phosphatase-labeled CTNI antibody as described in claim 4 is characterized in that in "mixing the labeling blocking agent and the mixture in a preset ratio for 5 minutes to 20 minutes", the preset ratio is 1:0.5-1:1.

[0017] The present invention discloses a method for preparing an alkaline phosphatase-labeled CTNI antibody, comprising the following steps: using a heteroterminal cross-linking agent to react with the CTNI antibody for 0.5-4h, then using a reaction terminator to terminate the reaction, and removing the free reagent to obtain a first reactant; using the reaction terminator to terminate the reaction after alkaline phosphatase reacts with a 2-IT reagent for 0.5-4h, and removing the free reagent to obtain a second reactant; mixing the first reactant and the second reactant according to a preset ratio to obtain a mixture; diluting the reduced BSA to 5-10mg / mL with 10mM PBS to prepare a labeling sealer; mixing the labeling sealer and the mixture according to a preset ratio for 5min-20min. The present invention reduces the background, optimizes its signal-to-noise, improves the false positive rate, and improves the stability of the alkaline phosphatase-labeled cTnI antibody labeled by the labeling method, solving the problems of low titer, poor signal-to-noise and instability in the existing alkaline phosphatase-labeled CTNI antibody preparation method. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] 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 these drawings without paying creative work.

[0019] Figure 1 The present invention provides a flow chart of a method for preparing an alkaline phosphatase-labeled CTNI antibody. DETAILED DESCRIPTION

[0020] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and should not be construed as limiting the present invention.

[0021] See also Figure 1 The present invention provides a method for preparing an alkaline phosphatase-labeled CTNI antibody, comprising the following steps:

[0022] S1 uses a heteroterminal cross-linking agent to react with the CTNI antibody for 0.5-4 hours, then uses a reaction terminator to terminate the reaction and remove the free reagent to obtain the first reactant;

[0023] The heteroterminal cross-linking agent is SMCC or SPDP amino-thiol coupling reagent. The reactant is an ammonia solution, such as lysine, glycine or TRIS.

[0024] Specifically, a heteroterminal cross-linking agent is used to react with the CTNI antibody for 0.5 to 4 hours, such as SMCC, SPDP and other amino-thiol coupling reagents; a reaction terminator is used to terminate the reaction, generally an ammonia solution such as lysine, glycine, TRIS, etc. After the reaction is completed, the free reagent is removed.

[0025] S2: reacting alkaline phosphatase with 2-IT reagent for 0.5-4h, then using the reaction terminator to terminate the reaction, and removing the free reagent to obtain a second reactant;

[0026] Specifically, alkaline phosphatase and 2-IT reagent are reacted for 0.5 to 4 hours, and the reaction is terminated using the above-mentioned reaction terminator. After the reaction is completed, the free reagent is removed.

[0027] S3: mixing the first reactant and the second reactant in a preset ratio to obtain a mixture;

[0028] The preset ratio is 1:0.1-1:0.8.

[0029] Specifically, the reaction products in step S1 and step S2 are mixed and reacted in a mass ratio of 1:0.1 to 1:0.8.

[0030] S4 dilute the reduced BSA to 5-10 mg / mL with 10 mM PBS to prepare the labeling blocking agent;

[0031] Specifically, a disulfide bond reducing agent is used to reduce BSA to release free thiol groups, such as TCEP. The reduced BSA is diluted to 5-10 mg / mL with 10 mM PBS to prepare a labeling blocking agent.

[0032] S5: mixing the label blocking agent and the mixture according to a preset ratio for reaction for 5 min to 20 min.

[0033] The preset ratio is 1:0.5-1:1.

[0034] Specifically, the blocking agent obtained in step S4 is added to the product of step 3 at a volume ratio of 1:0.5 to 1:1, and the mixture is reacted for 5 to 20 minutes.

[0035] Example 1

[0036] Comparison marking method

[0037] The coupling buffer was prepared as follows:

[0038] BSA and NaN3 were added to a Tris-HCl buffer solution with a concentration of 0.01Mol / L and a pH value of 8.0, and the mixture was stirred evenly to prepare an analytical buffer solution. The mass percentage concentration of BSA in the analytical buffer solution was 1%, and the mass percentage concentration of NaN3 was 0.2%.

[0039] Take 2 mg of alkaline phosphatase and 4 mg of cTnI antibody, add them into 10 mM PBS buffer and stir evenly.

[0040] 50 μL of glutaraldehyde was added to 10 mM PBS buffer to prepare a mixed solution with a volume of 0.5 mL. The solution was stirred for 15 minutes and then reacted for 4 hours.

[0041] Ethanolamine was added to the mixed solution at a rate of 0.1 mol / L, stirred for 2 hours, and dialyzed against PBS overnight.

[0042] Add an equal volume of glycerol and mix well; add 0.1% NaN3 by mass percentage and store at 4°C for later use.

[0043] The ALP-labeled cTnI antibody obtained by the above labeling process was diluted to 1 μg / mL working solution with coupling buffer, and the test sample was tested for potency. The sample was placed in a 37°C oven for accelerated experiment, and combined with the cTnI antibody magnetic particle working solution produced by our company to form a kit to measure the same group of samples, with 4°C as the control group, and the signal retention rate at different time points was calculated. The comparison reagent was used as the control group to calculate the false positive rate.

[0044] Table 1cTnI-ALP titer test

[0045]

[0046] It can be seen from the above table that the signal-to-noise ratio brought by this process is only 1.41, and the gradient ratio of 24.64 is smaller than the concentration ratio of 234.34.

[0047] Table 2 cTnI-ALP accelerated stability test

[0048]

[0049] As can be seen from the above table, after the cTnI-ALP obtained by the labeling process was accelerated, the signal loss rate was 40% after 3 days and 50% after 6 days.

[0050] Table 3cTnI-ALP false positive rate test

[0051] Reagents Number of tests / cases Positive rate / % False positive rate / % Product 1 200 20 15 Comparison reagents 200 5 /

[0052] From the above table, we can see that the positive rate of the comparison labeling process is 5%, the positive rate of the comparison reagent is 20%, and the false positive rate of the comprehensive comparison is 15%.

[0053] Example 2

[0054] The marking method is as follows:

[0055] The cTnI antibody was diluted to a concentration of 1 mg / mL using 10 mM PBS buffer, pH=8.0.

[0056] SM(PEG)4 was dissolved and diluted with N,N-dimethylformamide, and then added to the antibody at a 1:1 mol ratio and reacted for 30 min.

[0057] 2-IT was dissolved and diluted with 10 mM PBSE buffer at pH = 7.5, ALP was diluted to a working solution with a reaction concentration of 1 mg / mL using 10 mM PBSE buffer at pH = 7.5, and 2-IT was added to ALP at a 1:1 mol ratio for reaction for 30 min.

[0058] The cTnI antibody coupled with SM(PEG)4 and the ALP coupled with 2-IT were mixed at a mass ratio of 1:0.5 and reacted for 30 minutes.

[0059] The reaction solution was purified by desalting column.

[0060] An equal volume of glycerol was added and stored at 4°C until use.

[0061] The ALP-labeled cTnI antibody obtained by the above labeling process was diluted to 1 μg / mL working solution with coupling buffer, and the test sample was tested for potency. The sample was placed in a 37°C oven for accelerated experiment, and combined with the cTnI antibody magnetic particle working solution produced by our company to form a kit to measure the same group of samples, with 4°C as the control group, and the signal retention rate at different time points was calculated. The comparison reagent was used as the control group to calculate the false positive rate.

[0062] Table 4 cTnI-ALP titer test

[0063]

[0064] It can be seen from the above table that the signal-to-noise ratio brought by this process is only 1.41, and the gradient ratio of 113.97 is smaller than the concentration ratio of 234.34.

[0065] Table 5 cTnI-ALP accelerated stability test

[0066]

[0067] As can be seen from the above table, the cTnI-ALP obtained by the labeling process in this example was accelerated, and the signal loss rate after 3 days was 15%, and the signal loss rate after 6 days was 20%, which is better than the comparative labeling method.

[0068] Table 6cTnI-ALP false positive rate test

[0069] Reagents Number of tests / cases Positive rate / % False positive rate / % Product 1 200 13 8 Comparison reagents 200 5 /

[0070] Example 3

[0071] The marking method is as follows:

[0072] The labeling buffer was prepared as follows:

[0073] The cTnI antibody was diluted to a concentration of 1 mg / mL using 10 mM PBS buffer, pH=8.0.

[0074] SM(PEG)4 was dissolved and diluted with N,N-dimethylformamide, and then added to the antibody at a 1:1 mol ratio and reacted for 30 min.

[0075] 2-IT was dissolved and diluted with 10 mM PbSE buffer at pH 7.5, and ALP was diluted to a working solution with a reaction concentration of 1 mg / mL using 10 mM PbSE buffer at pH 7.5. 2-IT was added to ALP at a 1:1 mol ratio and reacted for 30 min.

[0076] The cTnI antibody coupled with SM(PEG)4 and the ALP coupled with 2-IT were mixed at a mass ratio of 1:0.5 and reacted for 30 minutes.

[0077] The blocking agent iodoacetamide was dissolved and diluted in 10 mM PBS at pH 6.8, and added to the reaction solution at a mass ratio of 1:1, and reacted for 1 hour.

[0078] The reaction solution was purified by desalting column.

[0079] An equal volume of glycerol was added and stored at 4°C until use.

[0080] The ALP-labeled cTnI antibody obtained by the above labeling process was diluted to 1 μg / mL working solution with coupling buffer, and the test sample was tested for potency. The sample was placed in a 37°C oven for accelerated experiment, and combined with the cTnI antibody magnetic particle working solution produced by our company to form a kit to measure the same group of samples, with 4°C as the control group, and the signal retention rate at different time points was calculated. The comparison reagent was used as the control group to calculate the false positive rate.

[0081] Table 7cTnI-ALP titer test

[0082]

[0083] As can be seen from the above table, in this example, the signal-to-noise ratio brought by the labeling process is 3.14, and the gradient ratio is 271.97, which is greater than the concentration ratio of 234.34. The potency has been improved, with better signal-to-noise ratio and gradient ratio.

[0084] Table 8 cTnI-ALP accelerated stability test

[0085]

[0086]

[0087] As can be seen from the above table, the cTnI-ALP obtained by the labeling process in this example was accelerated, and the signal loss rate at 3 days and 6 days was about 10%, which was better than the comparative labeling method and Example 2.

[0088] Table 9cTnI-ALP false positive rate test

[0089] Reagents Number of tests / cases Positive rate / % False positive rate / % Product 1 200 10 5 Comparison reagents 200 5 /

[0090] From the table above, we can see that the positive rate of the comparison labeling process is 5%, the positive rate of the comparison reagent is 10%, and the false positive rate of the comprehensive comparison is 8%.

[0091] Example 4

[0092] The cTnI antibody was diluted to a concentration of 1 mg / mL using 10 mM PBS buffer (pH=8.0).

[0093] Dissolve and dilute SM(PEG)4 with N,N-dimethylformamide, add SM(PEG)4 to the eluent at a 1:1 mol ratio, and react for 30 minutes.

[0094] 2-IT was dissolved and diluted with 10 mM PbSE buffer at pH 7.5, and ALP was diluted to a working solution with a reaction concentration of 1 mg / mL using 10 mM PbSE buffer at pH 7.5. 2-IT was added to ALP at a 1:1 mol ratio and reacted for 30 min.

[0095] The cTnI antibody coupled with SM(PEG)4 and the ALP coupled with 2-IT were mixed at a mass ratio of 1:0.5 and reacted for 30 minutes. After the reaction was completed, the reaction solution was purified with a desalting column.

[0096] A 10 mM TCEP solution and a 2 mg / mL BSA solution were reacted at a volume ratio of 1:10 at 37°C for 5 min to prepare a labeling blocking agent. The obtained blocking agent was added to the product at a volume ratio of 1:0.5 and reacted for 10 min.

[0097] The reaction solution was purified by desalting column.

[0098] An equal volume of glycerol was added and stored at 4°C until use.

[0099] The ALP-labeled cTnI antibody obtained by the above labeling process was diluted to 1 μg / mL working solution with coupling buffer, and the test sample was tested for potency. The sample was placed in a 37°C oven for accelerated experiment, and combined with the cTnI antibody magnetic particle working solution produced by our company to form a kit to measure the same group of samples, with 4°C as the control group, and the signal retention rate at different time points was calculated. The comparison reagent was used as the control group to calculate the false positive rate.

[0100] Table 10 cTnI-ALP titer test

[0101]

[0102]

[0103] It can be seen from the above table that in this example, the signal-to-noise ratio brought by the labeling process is 5.75, and the gradient ratio is 2447.96, which is greater than the concentration ratio of 234.34. The potency has been improved, with better signal-to-noise ratio and gradient ratio.

[0104] Table 11cTnI-ALP accelerated stability test

[0105]

[0106] As can be seen from the above table, the cTnI-ALP obtained by the labeling process in this example was accelerated, and the signal loss rate at 3 days and 6 days was less than 5%, which was better than the comparative labeling method and Example 2.

[0107] Table 12cTnI-ALP false positive rate test

[0108] Reagents Number of tests / cases Positive rate / % False positive rate / % Product 1 200 5 0 Comparison reagents 200 5 /

[0109] From the above table, we can see that the positive rate of the comparison labeling process is 5%, the positive rate of the comparison reagent is 5%, and the false positive rate of the comprehensive comparison is 0%.

[0110] The method for preparing an alkaline phosphatase-labeled CTNI antibody provided by the present invention has the following beneficial effects:

[0111] 1. A method for preparing an alkaline phosphatase-labeled CTNI antibody reduces background, optimizes its signal-to-noise, and improves the false positive rate.

[0112] 2. A method for preparing an alkaline phosphatase-labeled CTNI antibody improves the stability of the alkaline phosphatase-labeled cTnI antibody labeled by the labeling method.

[0113] The above disclosure is only a preferred embodiment of the method for preparing an alkaline phosphatase-labeled CTNI antibody of the present invention, and it is certainly not intended to limit the scope of the present invention. A person skilled in the art can understand that all or part of the processes of the above embodiment and equivalent changes made according to the claims of the present invention still fall within the scope of the invention.

Claims

1. A method for preparing an alkaline phosphatase-labeled CTNI antibody, characterized in that: The following steps are involved: After reacting the hetero-crosslinking agent with the CTNI antibody for 0.5-4 hours, the reaction is terminated with a reaction terminator, and the free reagent is removed to obtain a first reactant; After alkaline phosphatase and 2-IT reagent are reacted for 0.5-4 hours, the reaction is terminated using the reaction terminator, and the free reagent is removed to obtain a second reactant; Mixing the first reactant and the second reactant in a preset ratio to obtain a mixture; Dilute the reduced BSA to 5-10 mg / mL with 10 mM PBS to prepare the labeling blocking agent; The label blocking agent and the mixture are mixed according to a preset ratio and reacted for 5 minutes to 20 minutes.

2. The method for preparing an alkaline phosphatase-labeled CTNI antibody according to claim 1, characterized in that: In the step of “after reacting the CTNI antibody with a heteroterpene crosslinker for 0.5-4 h, using a reaction terminator to terminate the reaction, and removing the free reagent to obtain a first reactant”, the heteroterpene crosslinker is an SMCC or SPDP amino-thiol coupling reagent.

3. The method for preparing an alkaline phosphatase-labeled CTNI antibody according to claim 1, characterized in that: In "after reacting the CTNI antibody with a heteroterminal cross-linking agent for 0.5-4 hours, the reaction is terminated with a reaction terminator, and the free reagent is removed to obtain a first reactant", the reactant is an ammonia solution, such as lysine, glycine or TRIS.

4. The method for preparing an alkaline phosphatase-labeled CTNI antibody according to claim 1, characterized in that: In “mixing the first reactant and the second reactant in a preset ratio to obtain a mixture”, the preset ratio is 1:0.1-1:0.

8.

5. The method for preparing an alkaline phosphatase-labeled CTNI antibody according to claim 1, characterized in that: In “mixing the label blocking agent and the mixture in a preset ratio for 5 min-20 min”, the preset ratio is 1:0.5-1:1.

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