Signal amplification system based on acridinium ester chemiluminescence and kit

Abstract

The invention provides a signal amplification system based on acridinium ester chemiluminescence and a kit, belongs to the technical field of in-vitro diagnostic reagents, and particularly provides a signal amplification system based on acridinium ester chemiluminescence, which comprises a streptavidin polymer and a luminescent compound, the luminous compound is a biotin-carrier protein-acridinium ester compound, and the biotin-carrier protein-acridinium ester compound and the streptavidin polymer are in bridge connection through specific binding of biotin and streptavidin. The signal amplification system provided by the invention can realize the detection of the subpictogram-level antigen, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of in vitro diagnostic reagent technology, and more specifically, to a signal amplification system and reagent kit based on acridinium ester chemiluminescence. Background Technology

[0002] Currently, chemiluminescent in vitro diagnostic reagents typically consist of three main components: magnetic beads coated with capture antibodies, detection antibodies for specific binding of antigens, and luminescent reagents for generating chemiluminescence. Taking acridine ester chemiluminescent kits as an example, in the earliest acridine ester chemiluminescent kits, acridine ester was labeled on the detection antibody. Therefore, when using this type of kit, only two sample additions were required to detect the target substance. However, in this system, the detection antibody participates in both the immune reaction and the signal amplification reaction. Both reactions depend on the detection antibody, and the reactions are highly interdependent. Therefore, when the concentration of the target substrate is too low, the overall reaction signal will also decrease to a low level. When detecting some picogram- or femtogram-level antigens, this type of acridine ester chemiluminescent kit is difficult to meet the detection requirements.

[0003] To reduce the detection limit of acridinium ester chemiluminescence reagent kits, Chinese invention patent application CN112098651A discloses a high-sensitivity chemiluminescence immunoassay reagent. This high-sensitivity chemiluminescence immunoassay reagent mainly consists of three parts: magnetic beads coated with a first antibody, a biotin-labeled second antibody, and a streptavidin-labeled luminescent reagent. Compared with traditional chemiluminescence reagent kits, this immunoassay reagent effectively improves detection sensitivity through the stepwise amplification effect of biotin-streptavidin. However, the applicant found that in actual use, the signal amplification capability of this system is still limited for subpick-level substrates, and it cannot achieve effective detection of subpick-level substrates.

[0004] In chemiluminescence diagnostic kits, signal amplification methods for low-concentration target substances include increasing the molar ratio of the detection antibody to acridine ester, i.e., using more acridine ester to enhance the signal, or changing the labeling process to increase the detection signal value to amplify the signal. However, increasing the molar ratio of antibody to acridine ester can easily lead to increased background, decreased signal-to-noise ratio, and affect the sensitivity of the detection reagent; while changing the labeling process, such as the buffer during labeling, has a relatively limited effect on the signal value improvement over time. Summary of the Invention

[0005] The technical problem to be solved by this invention is: how to use a chemiluminescence reagent kit to achieve effective detection of picogram and subpicogram level antigens.

[0006] To address the aforementioned technical problems, the first aspect of this invention provides a signal amplification system based on acrid ester chemiluminescence, the signal amplification system comprising: Streptavidin polymers, and The luminescent complex is a biotin-carrier protein-acrididine ester complex. The biotin-carrier protein-acridone ester complex and the streptavidin polymer are bridged through the specific binding of biotin and streptavidin.

[0007] Preferably, each streptavidin polymer molecule is bridged to 1 to (n-1) luminescent complex molecules, where n is the degree of polymerization of the streptavidin polymer and n is not less than 4.

[0008] The role of streptavidin polymers is to connect biotin in the detection antibody and biotin in the luminescent complex. Theoretically, the degree of polymerization of streptavidin polymers is at least 4, which means that after streptavidin polymers bind to the second antibody, each streptavidin polymer molecule can bind 3 luminescent complex molecules. Therefore, compared with acridine ester labeled second antibody or traditional streptavidin-acrididine ester luminescence, the signal amplification system provided by this invention can significantly improve the signal intensity of chemiluminescence.

[0009] Preferably, the signal amplification system further includes a trapping magnetic bead.

[0010] Preferably, the capturing magnetic beads are selected from any one of carboxyl magnetic beads, toluenesulfonyl magnetic beads, and amino magnetic beads.

[0011] Preferably, the surface of the capturing magnetic beads is immobilized with a first antibody.

[0012] During testing, the capture magnetic beads immobilized with the primary antibody can first bind to the analyte to form an antibody-antigen complex.

[0013] Preferably, the signal amplification system further includes a second antibody.

[0014] Preferably, the second antibody is a biotin-labeled antibody.

[0015] The biotin-labeled secondary antibody can bind to the antibody-antigen complex to form an antibody-antigen-antibody complex. This antibody-antigen-antibody complex can then link to the streptavidin multimer in the aforementioned signal amplification system, thereby achieving signal amplification.

[0016] A second aspect of the present invention also provides a reagent kit comprising the signal amplification system based on acrid ester chemiluminescence described in the first aspect.

[0017] Compared with the prior art, the present invention has the following beneficial effects: 1. The signal amplification system provided by this invention uses streptavidin polymer to bridge biotin-carrier protein-acridoid ester complex to form a multi-level signal amplification structure. Compared with traditional acridinoid ester directly labeled detection antibody or single biotin labeled detection antibody, the signal amplification factor is effectively improved. This enables the signal amplification system provided by this invention to achieve sub-picometer level antigen detection, with the advantages of high sensitivity and strong specificity. It effectively solves the problem that chemiluminescent reagents in the prior art cannot achieve ultra-low concentration antigen detection. 2. This invention uses a carrier protein as an intermediary and a biotin-carrier protein-acrididine ester complex as a luminescent group. By changing the molar ratio of the carrier protein and acridinium ester, the labeling amount of acridinium ester can be changed, resulting in stronger luminescence intensity. This avoids the problems of non-specific binding and increased background caused by changing the molar ratio of the detection antibody to acridinium ester in the prior art, effectively improving the signal-to-noise ratio of the detection and ensuring the accuracy and reliability of the detection results. 3. The signal amplification system based on acridinium ester chemiluminescence provided by this invention is compatible with existing dual-antibody sandwich chemiluminescence reagent kits and can be directly integrated into diagnostic reagents, making it suitable for large-scale promotion. Attached Figure Description

[0018] Figure 1 This refers to the signal amplification system in the existing technology; Figure 2 The signal amplification system provided by this invention. Detailed Implementation

[0019] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention are described in detail below. It should be noted that the following embodiments are only used to illustrate the implementation methods and typical parameters of the present invention, and are not intended to limit the parameter range described in the present invention. Reasonable variations derived therefrom are still within the protection scope of the present invention.

[0020] It should be noted that the endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0021] As described in the background section, current chemiluminescence reagent kits cannot accurately detect low concentrations of antigen substrates; therefore, there is a need to develop a chemiluminescence signal amplification scheme.

[0022] See Figure 1 , Figure 1The diagram shows a signal amplification scheme based on a double-antibody sandwich immunoreaction, which mainly includes magnetic beads coated with capture antibodies and biotin-labeled detection antibodies. Based on the traditional antibody-antigen-antibody complex, this signal amplification scheme uses acridinium ester-streptavidin as the signal amplification system. However, this signal amplification system cannot achieve effective signal amplification for subpicoscale antigen substrates.

[0023] See Figure 2 The present invention provides a signal amplification system based on acridinium ester chemiluminescence. The signal amplification system includes streptavidin polymer and a luminescent complex. The luminescent complex is a biotin-carrier protein-acriddinium ester complex. The biotin-carrier protein-acriddinium ester complex and the streptavidin polymer are bridged through the specific binding of biotin and streptavidin.

[0024] In the above embodiments, each streptavidin polymer molecule is bridged to 1 to (n-1) luminescent complex molecules, where n is the degree of polymerization of the streptavidin polymer and n is not less than 4.

[0025] In the above embodiments, the signal amplification system also includes a trapping magnetic bead.

[0026] In the above embodiments, the capturing magnetic beads are selected from any one of carboxyl magnetic beads, toluenesulfonyl magnetic beads, and amino magnetic beads.

[0027] In the above embodiments, a first antibody is immobilized on the surface of the capturing magnetic beads.

[0028] In the above embodiments, the amplification system also includes a second antibody.

[0029] In the above embodiments, the second antibody is a biotin-labeled antibody.

[0030] More specifically, in the above embodiments, the acridine ester is selected from at least one of NSP-DMAE-NHS, NSP-DMAE-HEG-NHS, and NSP-SA-NHS.

[0031] More specifically, in the above embodiments, a carrier protein can link 1 to 20 acrid ester molecules, thereby effectively improving the luminescence intensity of the final chemiluminescence.

[0032] A further embodiment of the present invention provides a kit comprising the aforementioned signal amplification system based on acridinium ester chemiluminescence.

[0033] More specifically, in the above embodiments, the method of using the reagent kit includes the following steps: S1: Mix the analyte with capture magnetic beads coated with the first antibody and react. After the reaction, wash away the unbound analyte by magnetic separation to obtain the antibody-antigen complex. S2: Mix the antibody-antigen complex and the biotin-labeled second antibody, and react them. After the reaction is complete, wash away the unbound second antibody by magnetic separation to obtain the antibody-antigen-antibody complex. S3: A mixture of antibody-antigen-antibody complex and streptavidin polymer-biotin-carrier protein-acridoid ester complex is reacted. After the reaction, unbound streptavidin polymer-biotin-carrier protein-acridoid ester complex is washed away by magnetic separation. Then, HNO3-H2O2 solution and NaOH solution are added to the magnetic separation product. The luminescence intensity is measured by instruments such as an enzyme-linked immunosorbent assay (ELISA) reader and a chemiluminescence immunoassay analyzer. The content of the target antigen in the analyte can be calculated from this.

[0034] More specifically, in the above embodiments, if a greater signal amplification intensity is required, the coupling amount of acridine ester can be increased.

[0035] In some specific implementations, capture magnetic beads coated with a first antibody and a second antibody labeled with biotin can be added simultaneously, mixed with the analyte, and then unbound components are washed away by magnetic separation, thereby shortening the detection time and improving the detection efficiency.

[0036] In the above embodiments, since one carrier protein can couple multiple acrid ester molecules, and the degree of polymerization of the streptavidin polymer is at least 4, theoretically, one streptavidin polymer can bridge up to 3 luminescent complexes, and each luminescent complex may include 1 to 20 acrid ester molecules. Therefore, compared with the detection antibody directly coupling acrid ester, the technical solution provided by the above embodiments can amplify the reaction signal by several to tens of times.

[0037] The technical solutions of the present invention are further described below through specific embodiments. Unless otherwise defined, all terms, symbols, and other scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art. In some cases, terms with conventional meanings are limited herein for clarification or ease of reference, and such limitations should not be construed as indicating a significant difference from conventional understanding in the art. The technical methods described or referenced herein are generally well understood by those skilled in the art and have been employed by conventional methods. Unless otherwise stated, the use of commercially available kits, reagents, and instruments shall be performed according to the manufacturer's instructions and parameters.

[0038] Example 1 Reagent preparation of acridinium ester-based chemiluminescence signal amplification system In this embodiment, acridine ester was prepared using NSP-SA-NHS (purchased from Herlisen Biotechnology Co., Ltd.). 5 mg of NSP-SA-NHS was added to 2.5 mL of N,N-dimethylformamide to prepare a 2 mg / mL acridine ester solution. The solution was vortexed for 5 min until the solution was clear and free of precipitate.

[0039] In this embodiment, bovine serum albumin (BSA, purchased from Bovostar) was used as the carrier protein. 1 g of BSA was weighed and added to 10 mL of purified water. The mixture was stirred using a roller mixer for 30 minutes to ensure complete dissolution. Then, 1 μL of BSA was diluted 1000 times with 0.02 M PBS solution. Biotin and acridine ester were added, making the molar ratio of biotin to BSA 1:(5~15) and the molar ratio of acridine ester to BSA 1:(10~30). The mixture was stirred in the dark for 30 minutes. After the reaction was complete, 1% Tris (by total volume) was added, and the mixture was stirred in the dark for 15 minutes to obtain the biotin-bovine serum albumin-acrididine ester complex.

[0040] Streptavidin polymer (purchased from BioTez) was diluted 10-fold with 0.02M PBS solution and then mixed with a biotin-bovine serum albumin-acrididine ester complex. The volume ratio of streptavidin polymer to biotin-bovine serum albumin-acrididine ester complex was (1~10):1000. The streptavidin polymer-biotin-bovine serum albumin-acrididine ester complex was obtained.

[0041] Streptavidin (purchased from Feipeng Biotechnology) was diluted 10-fold with 0.02M PBS solution and then mixed with a biotin-bovine serum albumin-acrididine ester complex. The volume ratio of streptavidin to biotin-bovine serum albumin-acrididine ester complex was (1~10):1000. The streptavidin-biotin-bovine serum albumin-acrididine ester complex was obtained.

[0042] Example 2 Luminescence Experiment In this embodiment, the first antibody is antiphosphorylated tau protein 217 (p-tau-217), and the first antibody is coated on the surface of carboxyl magnetic beads.

[0043] In this embodiment, the second antibody is a biotin-labeled anti-tau protein antibody.

[0044] In this embodiment, ptau217 antigen controls and clinical samples at different concentrations were used as analytes.

[0045] Take 100 μL of each analyte. First, add 50 μL of carboxyl magnetic beads coated with the primary antibody at a concentration of 0.4 mg / mL. Incubate at 37°C for 15 min, followed by magnetic separation and washing 4 times. Then, add 50 μL of the secondary antibody at a concentration of 1 μg / mL. Incubate at 37°C for 10 min, followed by magnetic separation and washing 4 times. Then, divide the analytes into 3 groups. Add 50 μL of streptavidin labeled with acridin ester at a concentration of 1 μg / mL to control group 1. Add 50 μL of the streptavidin-biotin-bovine serum albumin-acriddin ester complex prepared in Example 1 at a concentration of 1 μg / mL to control group 2. Add 50 μL of the streptavidin polymer-biotin-bovine serum albumin-acriddin ester complex prepared in Example 1 at a concentration of 1 μg / mL to the experimental group. Incubate at 37°C for 5 min, followed by magnetic separation and washing 4 times. Add 100 μL of the secondary antibody to each group. The luminescence values ​​of each group were measured using a SMART500S chemiluminescence immunoassay analyzer (Clarice Biotechnology) with HNO3-H2O2 solution and 100 μL sodium hydroxide solution. The final signal intensities for each group are shown in Table 1. Table 1. Luminescence values ​​of control group 1, control group 2, and experimental group .

[0046] As shown in Table 1, compared with control group 1 and control group 2, the streptavidin polymer-biotin-bovine serum albumin-acridone ester complex provided by the specific embodiment of the present invention can effectively amplify the chemiluminescence signal, and its luminescence intensity is more than 10 times higher than that of the existing antibody-directly coupled acridinone ester.

[0047] Example 3 Stability test The streptavidin polymer-biotin-bovine serum albumin-acridone ester complex prepared in Example 1 was divided into two groups. One group was placed in a refrigerator at 4°C, and the other group was placed in an oven at 37°C. After standing for 7 days, the analytes in each group were subjected to luminescence detection according to the steps in Example 2. The detection results are shown in Table 2. Table 2 Stability Test Results .

[0048] As shown in Table 2, the streptavidin polymer-biotin-bovine serum albumin-acridone ester complex provided in the specific embodiments of the present invention exhibits excellent open-end stability at 4°C and 37°C, making it suitable for use in immunoassay analyzer platforms.

[0049] While the disclosure is as stated above, its scope of protection is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of this disclosure, and all such changes and modifications will fall within the protection scope of this invention.

Claims

1. A signal amplification system based on acridine ester chemiluminescence, characterized in that, The signal amplification system includes Streptavidin polymer and a luminescent complex, wherein the luminescent complex is a biotin-carrier protein-acrididine ester complex. The biotin-carrier protein-acridone ester complex and the streptavidin polymer are bridged through the specific binding of biotin and streptavidin.

2. The signal amplification system based on acridinium ester chemiluminescence as described in claim 1, characterized in that, Each streptavidin polymer molecule is bridged to 1~(n-1) luminescent complex molecules, where n is the degree of polymerization of the streptavidin polymer and n is not less than 4.

3. The signal amplification system based on acridinium ester chemiluminescence as described in claim 1, characterized in that, The signal amplification system also includes a trapping magnetic bead.

4. The signal amplification system based on acridinium ester chemiluminescence as described in claim 3, characterized in that, The capturing magnetic beads are selected from any one of carboxyl magnetic beads, toluenesulfonyl magnetic beads, and amino magnetic beads.

5. The signal amplification system based on acridinium ester chemiluminescence as described in claim 3, characterized in that, The surface of the capturing magnetic beads is immobilized with a first antibody.

6. The signal amplification system based on acridinium ester chemiluminescence as described in claim 1, characterized in that, The signal amplification system also includes a second antibody.

7. The signal amplification system based on acridinium ester chemiluminescence as described in claim 6, characterized in that, The second antibody is a biotin-labeled antibody.

8. A reagent kit, characterized in that, The kit includes the signal amplification system based on acridine ester chemiluminescence as described in any one of claims 1 to 7.

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