Preparation method and application of an antibody labeled with acridine

Abstract

The present application relates to a method for preparing an antibody labeled with an acridine substance and its application. The method comprises the following steps: S1, mixing the antibody to be labeled with a reducing agent and performing a reduction reaction, removing the unreacted reducing agent after the reaction is completed, and obtaining a solution containing thiol-containing antibodies; S2, mixing the prepared solution containing thiol-containing antibodies with NSP‑SA‑N‑Mal working solution for the first labeling reaction, and desalting the product after the reaction to obtain a solution containing NSP‑SA‑N‑Mal labeled antibodies; S3, diluting the solution containing NSP‑SA‑N‑Mal labeled antibodies and mixing it with NSP‑AS‑NHS working solution for the second labeling reaction, and desalting the product after the reaction to obtain antibodies labeled with acridine ester substances. The antibodies labeled with acridine substances prepared by the method described in the present application have high sensitivity, good accuracy, high signal-to-noise ratio and good reproducibility when detected.

Description

Technical Field

[0001] The present application relates to the technical field of chemiluminescent immunoassay, and in particular to a method for preparing an antibody labeled with an acridine substance and its application. Background Art

[0002] Chemiluminescence immunoassay (CLIA) technology began in the early 1980s and rapidly developed in the 1990s, becoming an emerging immunoassay technology following fluorescent immunoassay, radioimmunoassay, and enzyme-linked immunosorbent assay. Chemiluminescence immunoassay utilizes the free energy released by a chemical reaction to excite an intermediate, causing it to return from an excited state to a ground state. When the intermediate returns from the excited state to the ground state, it releases photons of equal energy, which are then measured for quantitative analysis. It is a combination of chemiluminescence and immunoassay. It combines the high sensitivity of chemiluminescence with the high selectivity of immunoassay. With advantages such as high sensitivity, strong specificity, good reagent stability, a wide detection range, and a high degree of automation, this technology has become the mainstream immunoassay method and is widely used in various fields such as clinical testing and food inspection.

[0003] Chemiluminescent immunoassays can be divided into three categories based on their labeling: chemiluminescent enzyme immunoassays, chemiluminescent immunoassays, and electrochemical luminescence (ECL) immunoassays. Commonly used luminescent substances in chemiluminescent immunoassay systems include luminol, isoluminol, 3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoinoyl)-phenyl-1,2-dioxetane disodium salt (AMPPD), ruthenium terpyridine, and acridine compounds. Isoluminol, ruthenium terpyridine, and acridine can all be used as tracer molecules and are flash-type luminescent substances. Luminol and AMPPD rely on peroxidase and alkaline phosphatase, respectively, as tracer molecules and are glow-type luminescent substances. Acridinium esters, a class of chemical substances among acridinium compounds, are used as chemiluminescent markers. In an alkaline H₂O₂ solution, when the acridinium ester molecule is attacked by hydrogen peroxide ions, the substituents on the acridinium ring react with C-9 on the acridinium ring and H₂O₂ (hydrogen peroxide) to form unstable ethylene dioxide. This ethylene dioxide decomposes into CO₂ and electronically excited N-methylacridone. Acridinium esters or acridinium sulfonamides emit light in dilute H₂O₂ solutions, eliminating the need for a catalyst and simplifying the luminescence system. Furthermore, these compounds exhibit flash-like luminescence, reaching maximum emission intensity approximately 0.4 seconds after addition of a luminescence initiation reagent, with a half-life of approximately 0.9 seconds. This makes them ideal luminescent materials, saving significant time.

[0004] Immunoglobulin monomers possess two antigen-binding sites located at the N-terminus of their hypervariable regions, which are crucial components of their activity. The binding of immunoglobulins to antigens is initiated by non-covalent bonds involving discrete amino acids in the heavy and light chains. In other words, the binding sites are not strictly linear amino acid sequences but rather formed by their unique tertiary structures. These binding sites bind to specific antigens with high affinity through van der Waals forces, ionic bonds, hydrophobic bonds, and hydrogen bonds. Antibodies possess a variety of functional groups suitable for conjugation, such as the ε-amino group or terminal amino group of lysine and the free carboxyl groups of aspartic acid and glutamic acid. These conjugation groups can bind to acridinium esters, forming acridinium ester-labeled antibodies. The uniform distribution of these functional groups within the antibody tertiary structure allows the conjugation reaction to occur randomly anywhere within the antibody structure. Randomly oriented conjugation of labels to antibodies can result in hidden or even occupied antigen-binding sites, thereby reducing the antibody's antigen-binding activity.

[0005] In addition, traditional acridinium ester luminescent substrates mainly use hydrogen peroxide in combination with acidic and alkaline solutions as the luminescent substrate, and traditional enhancers such as Tween 20 and triton X-100 are added to the luminescent substrate to enhance luminescence. However, the luminescence enhancement effect after the addition of the enhancer is still not obvious, and the sensitivity of certain projects with high sensitivity requirements still needs to be improved.

[0006] Therefore, it is necessary to provide a method for labeling antibodies with acridinium ester substances that can directionally couple acridinium ester substances and effectively enhance the luminescence intensity of the luminescence system. Summary of the Invention

[0007] In order to address the shortcomings of the existing technology, the present application provides a new method for preparing antibodies labeled with acridine substances, which can effectively enhance the luminescence intensity of the luminescence system. The luminescence signal intensity is significantly stronger than the current mainstream method of acridinium ester-labeled antibodies, and has low background and high signal-to-noise ratio and good reproducibility.

[0008] To this end, the first aspect of the present application provides a method for preparing an antibody labeled with an acridine substance, the method comprising the following steps:

[0009] S1, mixing the labeled antibody with a reducing agent and performing a reduction reaction, and removing the unreacted reducing agent after the reaction to obtain a solution containing the thiol-containing antibody;

[0010] S2, mixing the prepared solution containing the thiol-containing antibody with an acridine sulfonamide-N-ethylaminomaleimide working solution, and performing a first labeling reaction. After the reaction, the product is desalted to obtain a solution containing the acridine sulfonamide-N-ethylaminomaleimide-labeled antibody;

[0011] S3, diluting the solution containing the acridinium sulfonamide salt-N-ethylaminomaleimide labeled antibody and mixing it with the acridinium sulfonamide working solution to perform a second labeling reaction. After the reaction is completed, the product is desalted to obtain an antibody labeled with an acridinium ester substance.

[0012] Antibodies are composed of two light chains and a heavy chain, and each chain is linked by a disulfide bond. The method described in this application first uses a reducing agent to reduce the disulfide bonds between the cysteines in the antibody to sulfhydryl groups, and then introduces sulfhydryl groups into the antibody. At the same time, the ε-amino group of the antibody lysine side chain and the -NH2 (α-amino group) at the N-terminus of the peptide chain retain their biological activity, and then different acridine substances can be coupled to the amino group and sulfhydryl group of the antibody respectively, so that an antibody molecule is connected to more acridine substances, thereby greatly improving the luminous intensity. In addition, the introduced sulfhydryl group is located in the hinge region of the antibody, and the binding site is far away from the antigen binding site. Therefore, the introduction of acridine substances on the sulfhydryl group can maintain the activity of the antibody and achieve directional coupling of the antibody.

[0013] In some embodiments, in step S1, the molar ratio of the antibody to be labeled to the reducing agent is 1:(40-50).

[0014] In some embodiments, in step S1, the molar ratio of the antibody to be labeled to the reducing agent is 1:45.

[0015] The present application controls the amount of the antibody to be labeled and the reducing agent within the above range, thereby better reducing the antibody to be labeled and adding a sulfhydryl group to the antibody to be labeled.

[0016] In some preferred embodiments, in step S1, the reduction reaction is carried out in a PBS buffer solution with a pH value of 7.1 to 8.0, the reaction temperature is 20 to 25° C., and the reaction time is 30 to 45 min.

[0017] In some specific embodiments, the reduction reaction is carried out in 80-100 mM PBS buffer with a pH of 7.4, at a reaction temperature of 25° C. (room temperature), and for 30 min.

[0018] In the present application, by carrying out a reduction reaction under the above conditions, the disulfide bonds between the heavy chains of the antibody can be selectively opened, while the disulfide bonds between the heavy chain and the light chain are not destroyed, so that the antigen binding site on the antibody is not destroyed while the sulfhydryl group is introduced into the antibody, thereby retaining the activity of the antibody as much as possible.

[0019] In some embodiments, the reducing agent is selected from any one of DTT (dithiothreitol), TCEP (tris(2-carboxyethyl)phosphine) and MEA (ethanolamine). In some preferred embodiments, the reducing agent is DTT.

[0020] In the present application, using DTT as a reducing agent is more conducive to the reduction of the labeled antibody, thereby improving the performance of the final acridine-labeled antibody.

[0021] In this application, unreacted DTT can be removed using an ultrafiltration tube. Specifically, the product after the reduction reaction can be transferred to a 0.5 mL 10 kDa ultrafiltration tube and centrifuged at 5000 g for 1 to 5 minutes. The purpose of removing unreacted DTT is to remove extraneous thiols and prevent them from competing with the antibody to bind to acridine sulfonamide-N-ethylaminomaleimide, resulting in reduced antibody coupling efficiency.

[0022] In some embodiments, in step S2, during the first labeling reaction, the pH value of the reaction system is 6.5-7.5.

[0023] In some preferred embodiments, in step S2, during the first labeling reaction, the pH value of the reaction system is 7.4.

[0024] In the present application, at the above pH value, it is more conducive to the specific reaction of acridine sulfonamide salt-N-ethylamino maleimide with the sulfhydryl group (-SH) on the antibody to form a stable thioether bond. Therefore, the pH value of the corresponding reaction system can be adjusted to 6.5-7.5 before the first labeling reaction. If the pH value of the reaction system of the first labeling reaction is higher (pH>8.5), the reaction will support primary amine coupling and will also increase the rate at which the maleimide group is hydrolyzed into non-reactive maleamic acid, thereby reducing the labeling effect.

[0025] In some embodiments, in step S2, the concentration of acridinium sulfonamide-N-ethylaminomaleimide in the acridinium sulfonamide-N-ethylaminomaleimide working solution is 0.1-0.5 mg / mL, and the mass ratio of acridinium sulfonamide-N-ethylaminomaleimide to the antibody to be labeled is 1:(15-20).

[0026] In some preferred embodiments, the concentration of acridinium sulfonamide-N-ethylaminomaleimide in the acridinium sulfonamide-N-ethylaminomaleimide working solution is 0.25 mg / mL, and the mass ratio of acridinium sulfonamide-N-ethylaminomaleimide to the antibody to be labeled is 1:17.5.

[0027] In the present application, the acridinium sulfonamide-N-ethylaminomaleimide working solution is prepared by preparing NSP-SA-N-Mal with DMF. In the present application, by controlling the concentration of acridinium sulfonamide-N-ethylaminomaleimide in the acridinium sulfonamide-N-ethylaminomaleimide working solution and the mass ratio of acridinium sulfonamide-N-ethylaminomaleimide to the antibody to be labeled within the above range, it is more conducive to coupling acridinium sulfonamide-N-ethylaminomaleimide to the antibody to be labeled, providing the luminous intensity of the luminescent system, and thus improving the sensitivity and accuracy of the detection.

[0028] In some embodiments, in step S3, the dilution factor of the solution containing acridine sulfonamide-N-ethylaminomaleimide-labeled antibody is 5 to 10 times, and the diluent used for dilution is NaHCO3 solution, the concentration of NaHCO3 in the NaHCO3 solution is 0.15 to 0.25 M, and the pH value is 8.5 to 9.0.

[0029] In some preferred embodiments, in step S3, the dilution factor of the solution containing acridine sulfonamide-N-ethylaminomaleimide-labeled antibody is 10 times, and the diluent used for dilution is NaHCO3 solution, the concentration of NaHCO3 in the NaHCO3 solution is 0.2M, and the pH value is 9.0.

[0030] In this application, diluting the solution containing the acridinium sulfonamide salt-N-ethylaminomaleimide-labeled antibody with the above-mentioned diluent is more conducive to the subsequent second labeling reaction of the acridinium sulfonamide salt-N-ethylaminomaleimide-labeled antibody with acridinium sulfonamide. This is because under alkaline conditions of pH 8.5-9.0, NHS will leave and the acridinium ester will bind to the protein via a stable amide bond to form an acridinium compound.

[0031] In some embodiments, in step S3, the concentration of the acridinium sulfonamide working solution is 0.1-0.5 mg / mL, and the mass ratio of the acridinium sulfonamide to the antibody to be labeled is 1:(15-20).

[0032] In some preferred embodiments, in step S3, the concentration of the acridinium sulfonamide working solution is 0.25 mg / mL, and the mass ratio of the acridinium sulfonamide to the antibody to be labeled is 1:15.

[0033] In this application, an acridine sulfonamide working solution is prepared by preparing NSP-AS-NHS with DMF. By controlling the concentration of NSP-AS-NHS in the acridine sulfonamide working solution and the mass ratio of NSP-AS-NHS to the antibody to be labeled within the aforementioned ranges, this application can facilitate the coupling of NSP-AS-NHS to the antibody to be labeled, increase the luminescence intensity of the luminescent system, and thereby improve the sensitivity and accuracy of detection.

[0034] In some embodiments, the conditions for the first labeling reaction and the second labeling reaction are: reacting at 20-25° C. for 1-2 hours in the dark, for example, reacting at 37° C. for 2 hours in the dark.

[0035] In some specific embodiments, the reaction is carried out under rotating conditions, which is more conducive to maintaining the reaction system in a uniform mixing state, allowing the antibody to be labeled to effectively contact the acridine substance, and improving the labeling efficiency.

[0036] By carrying out the labeling reaction under the above conditions, the present application can achieve a good coupling effect between the antibody to be labeled and the acridine substance, thereby improving the sensitivity and repeatability of the detection reagent.

[0037] In the present application, after the first labeling reaction and the second labeling reaction are completed, the product needs to be desalted to remove the by-products produced during the labeling process and the unlabeled acridine substances. If the above substances cannot be effectively removed, it will lead to problems such as high background values ​​and poor repeatability caused by uncertain nonspecific adsorption. The present application uses desalting treatment to enable the antibody labeled with acridine substances prepared by the method described in the present application to be tested with a lower test background value, a high signal-to-noise ratio and better repeatability. The desalting treatment can be carried out, for example, by a desalting column (7KD MWCO) or an ultrafiltration tube.

[0038] In the present application, the prepared acridinium ester-labeled antibody can be mixed with an equal volume of glycerol and stored for later use.

[0039] The antibodies to be labeled in this application are conventional antibodies in the art, and those skilled in the art can select them as needed. For example, the antibodies to be labeled can be PCT (procalcitonin) antibodies, HIV antibodies, CT-1 (anti-cardiotrophic factor) antibodies, and cTnI (human cardiac troponin I).

[0040] The second aspect of the present application provides a use of an antibody labeled with an acridine substance prepared by the method described in the first aspect of the present application in the preparation of a chemiluminescent immunoassay detection reagent.

[0041] The acridine-labeled antibody prepared by the method described in the present application has a good luminescent signal intensity, a high signal-to-noise ratio and better repeatability when detected using it. Therefore, it can be well used in the preparation of chemiluminescent immunoassay detection reagents and has good application prospects.

[0042] The beneficial technical effects of the present application are as follows: the antibody labeled with acridine substances prepared by the method described in the present application has a good luminescent signal intensity and high antibody activity. Therefore, when using the labeled antibody for detection, the sensitivity and accuracy are high, and the signal-to-noise ratio is high and the reproducibility is good. It can be well used in the preparation of chemiluminescent immunoassay detection reagents and has good application prospects. DETAILED DESCRIPTION

[0043] To make this application easier to understand, the following examples will be used to further illustrate this application. These examples are for illustrative purposes only and are not intended to limit the scope of application of this application. Unless otherwise specified, the raw materials or components used in this application can be obtained through commercial channels or conventional methods.

[0044] Example 1: Preparation of cTnI Antibodies Labeled with Acridine

[0045] cTnI antibody was dissolved in 100 mM PBS (pH 7.4) buffer, followed by the addition of the reducing agent DTT. After mixing thoroughly, the solution was reduced at room temperature (25°C) for 30 minutes. The reduced product was transferred to a 0.5 mL 10 kDa ultrafiltration tube and centrifuged at 5000 g for 3 minutes to remove unreacted DTT, yielding a solution containing thiolated cTnI antibody. The concentration of cTnI antibody in the reduction system was 100 nM, the concentration of DTT was 5 mM, and the molar ratio of labeled antibody to reducing agent was 1:50.

[0046] Preparation of acridine sulfonamide (NSP-AS-NHS) and acridine sulfonamide salt-N-ethylaminomaleimide (NSP-SA-N-Mal) working solutions: NSP-AS-NHS and NSP-SA-N-Mal were prepared in DMF to a concentration of 0.25 mg / mL each.

[0047] A solution containing thiolated cTnI antibody was mixed with NSP-SA-N-Mal working solution at a mass ratio of 1:17.5 (NSP-SA-N-Mal:cTnI antibody) to form a reaction system. The pH of the reaction system was adjusted to 7.4, and the reaction was incubated at room temperature (25°C) in the dark for 2 hours with rotation for the first labeling reaction. The reaction product was then desalted on a desalting column (7 kDa MWCO) to obtain a solution containing acridine sulfonamide-N-ethylaminomaleimide-labeled cTnI antibody.

[0048] A solution containing acridine sulfonamide-N-ethylaminomaleimide-labeled cTnI antibody was diluted 10-fold with 0.2 M NaHCO₃ solution (pH = 9.0). The NSP-AS-NHS working solution was then added to the dilution solution at a mass ratio of NSP-AS-NHS:cTnI antibody = 1:15. The reaction was allowed to react with rotation at room temperature (25°C) in the dark for 2 hours for a second labeling reaction. The reaction product was then desalted on a desalting column (7 kD MWCO) to obtain a solution containing acridine-labeled cTnI antibody.

[0049] The solution containing acridine-labeled cTnI antibodies was added to an equal volume of glycerol and mixed thoroughly for later use.

[0050] Example 2: Preparation of cTnI Antibodies Labeled with Acridine

[0051] The preparation process was basically the same as that of Example 1, except that the cTnI antibody was dissolved in 100 mM PBS (pH 7.0) buffer solution.

[0052] Example 3: Preparation of cTnI Antibodies Labeled with Acridine

[0053] The preparation process was basically the same as that of Example 1, except that the cTnI antibody was dissolved in 100 mM PBS (pH 7.8) buffer solution.

[0054] Example 4: Preparation of cTnI Antibodies Labeled with Acridine

[0055] The preparation process was basically the same as that of Example 1, except that the concentration of cTnI antibody in the reduction treatment system was 100 nM, the concentration of DTT was 4.5 mM, and the molar ratio of the antibody to be labeled to the reducing agent was 1:45.

[0056] Example 5: Preparation of cTnI Antibodies Labeled with Acridine

[0057] The preparation process was basically the same as that of Example 1, except that the concentration of cTnI antibody in the reduction treatment system was 100 nM, the concentration of DTT was 3.5 mM, and the molar ratio of the antibody to be labeled to the reducing agent was 1:35.

[0058] Example 6: Preparation of cTnI Antibodies Labeled with Acridine

[0059] The preparation process was basically the same as that in Example 1, except that the solution containing the thiolated cTnI antibody was mixed with the NSP-SA-N-Mal working solution at a mass ratio of NSP-SA-N-Mal:cTnI antibody = 1:15 to form a reaction system.

[0060] Example 7: Preparation of cTnI Antibodies Labeled with Acridine

[0061] The preparation process was basically the same as that in Example 1, except that the solution containing the thiolated cTnI antibody was mixed with the NSP-SA-N-Mal working solution at a mass ratio of NSP-SA-N-Mal:cTnI antibody = 1:20 to form a reaction system.

[0062] Example 8: Preparation of cTnI Antibodies Labeled with Acridine

[0063] The preparation process was basically the same as that of Example 1, except that the pH of the reaction system was adjusted to 6.8, and the first labeling reaction was carried out at room temperature (25° C.) in the dark with rotation for 2 hours.

[0064] Example 9: Preparation of cTnI Antibodies Labeled with Acridine

[0065] The preparation process was basically the same as that of Example 1, except that the pH of the reaction system was adjusted to 8.5, and the first labeling reaction was carried out at room temperature (25° C.) in the dark with rotation for 2 hours.

[0066] Example 10: Preparation of cTnI Antibodies Labeled with Acridine

[0067] The preparation process was basically the same as in Example 1, except that the NSP-AS-NHS working solution was added to the diluent at a mass ratio of NSP-AS-NHS:cTnI antibody = 1:20 and the reaction was carried out at room temperature (25° C.) in the dark for 2 hours with rotation for the second labeling reaction.

[0068] Example 11: Preparation of cTnI Antibodies Labeled with Acridine

[0069] The preparation process is basically the same as that of Example 1, except that after the reduction treatment, the step of removing the unreacted DTT in the product after the reduction treatment is not performed.

[0070] Example 12: Preparation of cTnI Antibodies Labeled with Acridine

[0071] cTnI antibody was dissolved in 100 mM PBS (pH 7.4) buffer, followed by the addition of the reducing agent DTT. After mixing thoroughly, the solution was reduced at room temperature (25°C) for 30 minutes. The reduced product was transferred to a 0.5 mL 10 kDa ultrafiltration tube and centrifuged at 5000 g for 3 minutes to remove unreacted DTT, yielding a solution containing thiolated cTnI antibody. The concentration of cTnI antibody in the reduction system was 100 nM, the concentration of DTT was 5 mM, and the molar ratio of labeled antibody to reducing agent was 1:50.

[0072] Preparation of acridine sulfonamide (NSP-AS-NHS) and acridine sulfonamide salt-N-ethylaminomaleimide (NSP-SA-N-Mal) working solutions: NSP-AS-NHS and NSP-SA-N-Mal were prepared in DMF to a concentration of 0.25 mg / mL each.

[0073] NSP-AS-NHS working solution and NSP-SA-N-Mal working solution were added to a solution containing thiolated cTnI antibody at a mass ratio of NSP-AS-NHS:cTnI antibody = 1:17.5 and NSP-SA-N-Mal:cTnI antibody = 1:15 to form a reaction system. The pH of the reaction system was adjusted to 7.4, and the reaction was incubated at room temperature (25°C) in the dark for 2 hours with rotation to perform the labeling reaction. The reaction product was then desalted on a desalting column (7 kD MWCO) to obtain a solution containing acridine-labeled cTnI antibody. The acridine-labeled cTnI antibody solution was added to an equal volume of glycerol, mixed, and stored for later use.

[0074] Example 13: Preparation of cTnI Antibodies Labeled with Acridine

[0075] cTnI antibody was dissolved in 100 mM PBS (pH 7.4) buffer, followed by the addition of the reducing agent DTT. After mixing thoroughly, the solution was reduced at room temperature (25°C) for 30 minutes. The reduced product was transferred to a 0.5 mL 10 kDa ultrafiltration tube and centrifuged at 5000 g for 3 minutes to remove unreacted DTT, yielding a solution containing thiolated cTnI antibody. The concentration of cTnI antibody in the reduction system was 100 nM, the concentration of DTT was 5 mM, and the molar ratio of labeled antibody to reducing agent was 1:50.

[0076] Preparation of acridine sulfonamide (NSP-AS-NHS) and acridine sulfonamide salt-N-ethylaminomaleimide (NSP-SA-N-Mal) working solutions: NSP-AS-NHS and NSP-SA-N-Mal were prepared in DMF to a concentration of 0.25 mg / mL each.

[0077] The solution containing the thiolated cTnI antibody was diluted 10-fold with 0.2 M NaHCO₃ solution (pH 9.0). The NSP-AS-NHS working solution was then added to the dilution solution at a mass ratio of 1:15 (NSP-AS-NHS:cTnI antibody). The reaction was allowed to incubate at room temperature (25°C) in the dark for 2 hours with rotation. The reaction product was then desalted on a desalting column (7 kDa MWCO) to obtain a solution containing the acridine sulfonamide-labeled cTnI antibody.

[0078] Mix the NSP-SA-N-Mal working solution with a solution containing acridine sulfonamide-labeled cTnI antibody at a mass ratio of 1:17.5 (NSP-SA-N-Mal:cTnI antibody) to form a reaction system. Adjust the pH of the reaction system to 7.4 and incubate the mixture in the dark at room temperature (25°C) for 2 hours with rotation for a second labeling reaction. Desalt the reaction product on a desalting column (7 kDa MWCO) to obtain a solution containing acridine-labeled cTnI antibody. Add an equal volume of glycerol to the solution, mix thoroughly, and store for later use.

[0079] Test Example 1

[0080] The acridine-labeled cTnI antibodies prepared in Examples 1-13 were used to detect cTnI-containing samples. The specific detection process was as follows: the coated magnetic bead marker (magnetic bead-labeled cTnI antibody) was diluted to 0.2 mg / mL to form a magnetic bead working solution; the acridine labeling solution (a solution containing acridine-labeled cTnI antibody) was diluted to 0.15 μg / mL to form an acridine working solution. The same clinical sample was tested using a fully automatic chemiluminescence immunoassay using the following reaction mode: 50 μL of magnetic bead working solution + 20 μL of sample + 50 μL of acridine working solution, incubation time 10 minutes. 100 μL of HNO₃-H₂O₂ solution and 100 μL of sodium hydroxide solution were added sequentially, and the luminescence value was measured using the chemiluminescence immunoassay. Three measurements were performed in parallel, and the average value was calculated. The results are shown in Table 1.

[0081] Table 1: Comparison of the detection results of acridine-labeled cTnI antibodies prepared in Examples 1-13

[0082]

[0083]

[0084] As can be seen from the test results in Table 1 above, the luminescence intensity of the test results of Example 11 is significantly reduced compared to that of Example 1. This indicates that when the reduction treatment is performed, if the unreacted DTT is not removed, the extraneous thiol will reduce the coupling effect between the acridine substance and the antibody, thereby reducing the luminescence intensity and detection sensitivity of the antibody labeled with the acridine substance. At the same time, the luminescence intensity of the test results of Examples 12-13 is significantly reduced compared to that of Example 1, indicating that when the antibody is labeled with acridine sulfonamide and acridine sulfonamide salt-N-ethylaminomaleimide at the same time, or when the labeling order of acridine sulfonamide and acridine sulfonamide salt-N-ethylaminomaleimide is changed, the labeling effect will be deteriorated, thereby reducing the luminescence intensity and detection sensitivity of the antibody labeled with the acridine substance.

[0085] From the test results of Examples 1-3, it can be seen that when performing the reduction reaction, a pH value of 7.4 in the system is more favorable for the introduction of thiol groups on the antibody, and ultimately improves the luminescence intensity and detection sensitivity of the test results. From the test results of Examples 1 and 4-5, it can be seen that when performing the reduction reaction, a molar ratio of the labeled antibody to the reducing agent of 1: (40-50), especially 1:45, is more favorable for the introduction of thiol groups on the antibody, and ultimately improves the luminescence intensity and detection sensitivity of the test results.

[0086] From the detection results of Examples 1, 6-7 and 10, it can be seen that during the labeling process, adjusting the mass ratio of acridine sulfonamide to the antibody to be labeled, and the mass ratio of acridine sulfonamide salt-N-ethylaminomaleimide to the antibody to be labeled will affect the luminescence intensity and detection sensitivity of the final acridine-labeled antibody.

[0087] From the test results of Examples 1 and 8-9, it can be seen that when the pH value of the reaction system is controlled at 7.4 during the first labeling, it is more conducive to the coupling reaction between acridine sulfonamide-N-ethylaminomaleimide and the antibody.

[0088] Comparative Example 1: Preparation of cTnI Antibody Labeled with Acridine

[0089] Preparation of acridine sulfonamide (NSP-AS-NHS) working solution: NSP-AS-NHS was prepared into a solution with a concentration of 0.25 mg / mL in DMF.

[0090] The cTnI antibody was diluted with 0.2 M NaHCO₃ solution (pH 9.0) to a concentration of 150 μg / mL. The NSP-AS-NHS working solution was then added to the antibody dilution solution at a mass ratio of 1:17.5 (NSP-AS-NHS:cTnI antibody). The labeling reaction was carried out at room temperature (25°C) with rotation for 2 hours in the dark. The reaction product was then desalted on a desalting column (7 kDa MWCO) to obtain a solution containing acridine sulfonamide-labeled cTnI antibody.

[0091] The solution containing acridine sulfonamide-labeled cTnI antibody was added to an equal volume of glycerol and mixed thoroughly for later use.

[0092] Test Example 2

[0093] The acridine-labeled cTnI antibodies prepared in Example 4 and Comparative Example 1 were diluted to the same antibody working concentration and tested with cTnI antibody-coated magnetic beads in the same 8 samples. The specific detection process is as follows: the coated magnetic bead marker (magnetic bead-labeled cTnI antibody) is diluted to 0.2 mg / mL to form a magnetic bead working solution; the acridine labeling solution (a solution containing acridine-labeled cTnI antibody) is diluted to 0.15 ug / mL to form an acridine working solution; the same clinical sample is taken and tested using a fully automatic chemiluminescence immunoassay using the reaction mode: 50 uL of magnetic bead working solution + 20 uL of sample + 50 uL of acridine working solution, incubation time 10 minutes. 100 μL of HNO3-H2O2 solution and 100 μL of sodium hydroxide solution are added successively, and the luminescence value is measured using the chemiluminescence immunoassay. The results are shown in Table 2.

[0094] Table 2: Comparison of the detection results of cTnI antibodies labeled with acridine substances prepared in Example 4 and Comparative Example 1

[0095]

[0096] As shown in the above test results, compared to Comparative Example 1, the luminescence intensity of the cTnI antibody labeled with an acridine prepared in Example 4 of the present application was significantly enhanced by approximately 2-fold; the signal-to-noise ratio (S / N) was also significantly improved, significantly enhancing detection performance. This demonstrates that the acridine-labeled antibody prepared by the method described herein has excellent detection performance and is well suited for use in chemiluminescent immunoassays.

[0097] It should be noted that the embodiments described above are only used to explain the present application and do not constitute any limitation to the present application. The present application has been described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory words, rather than restrictive words. The present application may be modified as specified within the scope of the claims of the present application, and the invention may be revised without departing from the scope and spirit of the present application. Although the present application described therein relates to specific methods, materials and embodiments, it does not mean that the present application is limited to the specific examples disclosed therein. On the contrary, the present application can be extended to all other methods and applications with the same function.

Claims

1. A method for preparing an antibody labeled with an acridine substance, characterized in that: The method comprises the following steps: S1, mixing the antibody to be labeled with a reducing reagent and performing a reduction reaction, and removing the unreacted reducing reagent after the reaction is completed to obtain a solution containing the thiol-labeled antibody; S2, mixing the prepared solution containing the thiol-containing antibody with an acridine sulfonamide-N-ethylaminomaleimide working solution, and performing a first labeling reaction. After the reaction, the product is desalted to obtain a solution containing the acridine sulfonamide-N-ethylaminomaleimide-labeled antibody; S3, diluting the solution containing the acridinium sulfonamide salt-N-ethylaminomaleimide labeled antibody and mixing it with the acridinium sulfonamide working solution to perform a second labeling reaction. After the reaction is completed, the product is desalted to obtain an antibody labeled with an acridinium ester substance.

2. The method according to claim 1, characterized in that In step S1, the molar ratio of the antibody to be labeled to the reducing reagent is 1:(40-50).

3. The method according to claim 1 or 2, characterized in that In step S1, the reduction reaction is carried out in a PBS buffer solution with a pH value of 7.1-8.0, a reaction temperature of 20-25° C., and a reaction time of 30-45 min.

4. The method according to claim 1 or 2, characterized in that The reducing agent is selected from any one of DTT, TCEP and MEA.

5. The method according to claim 4, characterized in that The reducing agent is DTT.

6. The method according to claim 1 or 2, characterized in that In step S2, during the first labeling reaction, the pH value of the reaction system is 6.5-7.

5.

7. The method according to claim 1 or 2, characterized in that In step S2, the concentration of acridine sulfonamide-N-ethylaminomaleimide in the acridine sulfonamide-N-ethylaminomaleimide working solution is 0.1-0.5 mg / mL, and the mass ratio of acridine sulfonamide-N-ethylaminomaleimide to the antibody to be labeled is 1:(15-20).

8. The method according to claim 1 or 2, characterized in that In step S3, the dilution ratio of the solution containing acridine sulfonamide-N-ethylaminomaleimide labeled antibody is 5 to 10 times, and the diluent used for dilution is NaHCO3 solution, the concentration of NaHCO3 in the NaHCO3 solution is 0.15 to 0.25M, and the pH value is 8.5 to 9.

0.

9. The method according to claim 1 or 2, characterized in that In step S3, the concentration of the acridine sulfonamide working solution is 0.1-0.5 mg / mL, and the mass ratio of the acridine sulfonamide to the antibody to be labeled is 1:(15-20).

10. The method according to claim 1 or 2, characterized in that The conditions for the first labeling reaction and the second labeling reaction are: reacting at 20-25° C. for 1-2 hours in the dark.

11. Use of an antibody labeled with an acridine substance prepared by the method according to any one of claims 1 to 10 in the preparation of a chemiluminescent immunoassay detection reagent.

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