Detection reagent and detection method for serum amyloid protein A based on improved space proximity chemiluminescence method

Abstract

The invention relates to the technical field of biology, and discloses a serum amyloid protein A detection reagent based on an improved space proximity chemiluminescence method and a detection method. The detection reagent comprises an enzyme marker, a luminous marker, an auxiliary agent, a substrate solution, a blocking solution, a calibration product and a reference product, wherein the enzyme marker comprises an anti-SAA antibody marked by horse radish peroxidase, an HEPES buffer solution, a sodium azide solution, bovine serum albumin and human serum albumin; the luminous marker adopts an anti-SAA capture antibody marked by an acridinium ester derivative; the auxiliary agent comprises a rare earth metal chelating agent. The detection reagent has high sensitivity and high specificity, can accurately detect the content of amyloid protein A in serum, and provides an effective tool for clinical diagnosis and disease monitoring.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a detection reagent and a detection method for serum amyloid A based on an improved spatial proximity chemiluminescence method. Background Art

[0002] Serum amyloid A (SAA) is a protein that increases significantly during an acute phase reaction in the body. When the body is subjected to inflammation, infection, trauma or other stressful situations, the concentration of SAA increases significantly. Therefore, the detection of SAA is extremely valuable in clinical diagnosis and disease monitoring. Traditional detection methods mainly include immunoturbidimetry and enzyme-linked immunosorbent assay (ELISA). However, these traditional methods have some obvious disadvantages, such as low sensitivity, complicated operation process and long detection time. These shortcomings limit its extensiveness and efficiency in clinical applications. Summary of the invention

[0003] In view of this, the present invention proposes a detection reagent set and a detection method for serum amyloid A based on an improved spatial proximity chemiluminescence method, aiming to overcome the shortcomings of the prior art, improve detection sensitivity and shorten detection time, while simplifying the operation process.

[0004] The present invention proposes a detection reagent set for serum amyloid A based on an improved spatial proximity chemiluminescence method, the reagent set comprising:

[0005] Enzyme labels, luminescent labels, substrate solutions, blocking solutions, calibrators and reference substances;

[0006] Wherein, the enzyme markers include horseradish peroxidase-labeled anti-SAA antibody, HEPES buffer, sodium azide solution, bovine serum albumin and human albumin;

[0007] The luminescent marker is an anti-SAA capture antibody labeled with an acridinium ester derivative;

[0008] The auxiliary agent includes a rare earth metal chelating agent.

[0009] Preferably, the preparation method of the enzyme marker comprises:

[0010] Dissolve the anti-SAA antibody labeled with horseradish peroxidase in distilled water and add HEPES buffer, then add sodium azide solution as an antibacterial agent, stir evenly in the dark, and obtain a preliminary mixed solution;

[0011] The preliminary mixed solution is placed in a dialysis bag and dialyzed in a HEPES buffer to remove low molecular weight impurities;

[0012] After the dialysis is completed, a NaCl solution is added to the dialyzed solution to adjust the ionic strength, and then bovine serum albumin and human albumin are added in sequence, stirred until completely dissolved, and then sodium borohydride is added as a reducing agent, and the reaction is allowed to stand in the dark to obtain a reaction solution;

[0013] Finally, the reaction solution is put into the dialysis bag again and dialyzed to remove free horseradish peroxidase to obtain the final enzyme marker.

[0014] Preferably, the method for preparing the horseradish peroxidase-labeled anti-SAA antibody comprises:

[0015] Dissolve horseradish peroxidase in distilled water, add HEPES buffer to make the solution reach a pH value of 7.2±0.2, add a cross-linking agent under stirring to perform a covalent coupling reaction to obtain a mixed solution;

[0016] The anti-SAA antibody is added to the mixed solution, and stirred at room temperature in the dark for 1-2 hours; after the reaction is completed, unbound HRP and cross-linking agent are removed by dialysis or gel filtration to obtain the horseradish peroxidase-labeled anti-SAA antibody.

[0017] Preferably, the cross-linking agent is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

[0018] Preferably, the method for preparing the luminescent marker comprises:

[0019] Dissolving an acridinium ester derivative in dimethyl sulfoxide, adding dipyridine carboxylic acid buffer solution after complete dissolution under stirring, and adjusting the pH value of the solution to 7.0-7.5 to obtain an acridinium hydrochloride solution;

[0020] The anti-SAA monoclonal antibody is dissolved in a PDC buffer, stirred and then added to the acridinium ester derivative solution, and stirred and reacted at room temperature; a polylysine coupling agent is added during stirring, and then the mixture is placed on a shaker and stirred for coupling reaction, and the reaction time is at least 2 hours;

[0021] After the coupling reaction is completed, the mixture is placed in a 4°C environment for 12-16 hours, and the reaction mixture is washed with Tris-HCl buffer to remove unreacted free luminescent markers and unbound polylysine coupling agents to obtain the luminescent marker.

[0022] Preferably, the preparation method of the auxiliary agent comprises:

[0023] Selecting diaminocyclohexanetetraacetic acid as a rare earth metal chelating agent, dissolving the rare earth metal chelating agent in deionized water, and adjusting the pH value of the solution to 7.2±0.2 to obtain a chelating agent solution;

[0024] Under stirring, adding the rare earth metal ion solution to the chelating agent solution, and continuing to stir until the rare earth metal ions and the chelating agent fully react to obtain a rare earth metal chelate solution;

[0025] Add HEPES buffer to the rare earth metal chelate solution, adjust the pH value to 7.3±0.1, and stir at room temperature for at least 30 minutes to obtain an auxiliary agent solution.

[0026] Preferably, the rare earth metal ion solution is a neodymium nitrate solution, a europium nitrate solution or a gadolinium nitrate solution.

[0027] The present invention also proposes an application of the above-mentioned serum amyloid A detection reagent set based on the improved spatial proximity chemiluminescence method, which comprises at least any of the following:

[0028] A1) Application in the detection of serum amyloid A;

[0029] A2) Application in the preparation of products for detecting serum amyloid A;

[0030] A3) Application in the study of the correlation between serum amyloid A and diseases.

[0031] Preferably, the application in detecting serum amyloid A comprises the following steps:

[0032] The serum sample to be tested, the blocking solution, the anti-SAA monoclonal antibody labeled with horseradish peroxidase, and the anti-SAA monoclonal antibody labeled with an acridinium ester derivative are reacted together to form an antigen-antibody sandwich complex, and the substrate solution is added to start the chemiluminescence reaction; the luminescence intensity is measured using a chemiluminescence detector; and the concentration of amyloid A in the sample to be tested is calculated based on the comparison of the luminescence intensity with the calibrator and the reference.

[0033] The present invention also provides a method for detecting serum amyloid A, comprising using the above-mentioned serum amyloid A detection reagent set based on the improved spatial proximity chemiluminescence method for detection.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] 1. Improve detection sensitivity and specificity: By using an improved spatial proximity chemiluminescence method, the present invention can significantly improve the sensitivity and specificity of detecting amyloid A, thereby making the detection results more accurate and reliable.

[0036] 2. Simplified operation process: The detection reagent set of the present invention is reasonably designed and has simple operation steps, which reduces the complexity of experimental operation and enables experimenters to quickly master and perform detection.

[0037] 3. Shorten the detection time: Compared with the traditional method, the detection method of the present invention greatly shortens the detection time and improves the detection efficiency while ensuring the detection quality.

[0038] 4. Reduce detection costs: Since the detection reagent set of the present invention has high stability and repeatability, it reduces the waste of reagents, thereby reducing the detection cost.

[0039] 5. Expand the scope of application: The present invention is not only suitable for clinical testing, but can also be widely used in scientific research, disease screening, efficacy evaluation and other fields, and has broad application prospects.

[0040] In summary, the present invention provides a detection reagent set for serum amyloid A based on an improved spatial proximity chemiluminescence method and its application. The reagent set and method not only improve the sensitivity and specificity of detection, but also simplify the operation process, shorten the detection time, reduce the detection cost, and expand the scope of application. It has significant practical value and broad market prospects. DETAILED DESCRIPTION

[0041] The exemplary embodiments of the present disclosure will be described in more detail below. Although the exemplary embodiments of the present disclosure are shown below, it should be understood that the present disclosure can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present disclosure and to be able to fully convey the scope of the present disclosure to those skilled in the art. It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0042] The reagent set of the present invention uses a double antibody sandwich method to detect the content of serum amyloid A (SAA) in human serum. The sample, horseradish peroxidase (HRP)-labeled anti-SAA monoclonal antibody, and acridinium ester derivative-labeled anti-SAA monoclonal antibody are reacted together to form an antigen-antibody sandwich complex, so that the horseradish peroxidase and the acridinium ester derivative are close to each other in space, and an auxiliary agent and a substrate solution are added to generate flash chemiluminescence; while the unbound free HRP-labeled antibody and the acridinium ester derivative-labeled antibody do not emit light. The higher the SAA content in the sample, the greater the measured luminescence value (RLU). Therefore, within a certain concentration range, the luminescence value is positively correlated with the sample concentration. By drawing a working curve with a known concentration of a calibrant and its luminescence value, the SAA content in the sample can be calculated according to the luminescence value of the sample.

[0043] Example 1

[0044] Preparation and combination of serum amyloid A (SAA) detection reagent set based on improved spatial proximity chemiluminescence method:

[0045] 1. Preparation of enzyme markers

[0046] Enzyme markers include: horseradish peroxidase-labeled anti-SAA antibody, HEPES buffer, sodium azide solution, bovine serum albumin and human albumin.

[0047] Preparation steps:

[0048] 1. Dissolve the horseradish peroxidase-labeled anti-SAA antibody in distilled water, add HEPES buffer, then add sodium azide solution, stir evenly in the dark, and obtain a preliminary mixed solution.

[0049] 2. The preliminary mixed solution was placed in a dialysis bag and dialyzed against HEPES buffer to remove low molecular weight impurities.

[0050] 3. After dialysis is completed, NaCl solution is added to the dialyzed solution to adjust the ionic strength, and then bovine serum albumin and human albumin are added in sequence, stirred until completely dissolved, and then sodium borohydride is added as a reducing agent, and the reaction is allowed to stand in the dark to obtain a reaction solution.

[0051] 4. The reaction solution was put into the dialysis bag again for dialysis to remove free horseradish peroxidase and obtain the final enzyme marker.

[0052] 2. Preparation of horseradish peroxidase-labeled anti-SAA antibody

[0053] Preparation steps:

[0054] 1. Dissolve horseradish peroxidase in distilled water, add HEPES buffer to make the solution reach pH 7.2±0.2, add crosslinking agent 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide under stirring to carry out covalent coupling reaction to obtain a mixed solution.

[0055] 2. Add anti-SAA antibody to the mixture and stir for 2 hours at room temperature in the dark. After the reaction is completed, remove unbound HRP and cross-linking agent by dialysis to obtain horseradish peroxidase-labeled anti-SAA antibody.

[0056] 3. Preparation method of luminescent marker

[0057] Preparation steps:

[0058] 1. Dissolve the acridinium ester derivative in dimethyl sulfoxide. After it is completely dissolved, add dipyridine carboxylic acid buffer under stirring, and adjust the pH value of the solution to 7.3±0.2 to obtain an acridinium hydrochloride solution.

[0059] 2. Dissolve the anti-SAA monoclonal antibody in PDC buffer, stir evenly, then add to the acridine hydrochloride solution, and stir the reaction at room temperature. Add polylysine coupling agent during stirring, and then place the mixture on a shaker to stir for coupling reaction, and the reaction time is 2 hours.

[0060] 3. After the coupling reaction is completed, the mixture is placed in a 4°C environment for 12 hours, and the reaction mixture is washed with Tris-HCl buffer to remove unreacted free luminescent markers and unbound polylysine coupling agents to obtain luminescent markers.

[0061] 4. Preparation method of auxiliary agent

[0062] Preparation steps:

[0063] 1. Select diaminocyclohexane tetraacetic acid as a rare earth metal chelating agent, dissolve the rare earth metal chelating agent in deionized water, and adjust the pH value of the solution to 7.2±0.2 to obtain a chelating agent solution.

[0064] 2. Under stirring, add rare earth metal ion solution (neodymium nitrate solution) to the chelating agent solution, and continue stirring until the rare earth metal ions and the chelating agent react fully to obtain a rare earth metal chelate solution.

[0065] 3. Add HEPES buffer to the rare earth metal chelate solution, adjust the pH to 7.3±0.1, and stir at room temperature for 30 minutes to obtain an auxiliary agent solution.

[0066] 6. Existing Commercial Reagents

[0067] Washing solution: PBS-Tween 20 (Bio-Rad);

[0068] Blocking solution: 5% BSA in PBS-T (Thermo Fisher Scientific);

[0069] Substrate solution: SuperSignal TM West Pico PLUS Chemiluminescent Substrate (ThermoFisher Scientific);

[0070] Calibrator: Human SAA Protein Standard (R&D Systems);

[0071] Reference: Human Serum Control Samples (Bio-Rad).

[0072] Example 2

[0073] Serum amyloid A was detected using the reagent set of Example 1, and the detection steps included:

[0074] 1. Sample preparation

[0075] 1. Collect the serum sample to be tested and centrifuge it at room temperature for 10 minutes to remove possible cell debris and clots.

[0076] 2. Dilute the centrifuged serum sample with sample diluent at a ratio of 1:10 to ensure that the SAA concentration in the sample is within the detection range.

[0077] 2. Test process

[0078] 50 μL of serum sample to be tested, 25 μL of acridinium ester marker, and 25 μL of HRP marker are added, mixed, incubated at 37°C for 5 minutes, 75 μL of substrate solution is added to start the chemiluminescent reaction, and the luminescent intensity of each well is measured by a chemiluminescent detector and the data is recorded.

[0079] 6. Data Analysis

[0080] 1. Draw a standard curve based on the luminescence intensity and known concentration of the calibrator.

[0081] 2. Use the standard curve to calculate the concentration of SAA in the sample to be tested.

[0082] 3. Compare the concentration of the reference product to assess whether the level of SAA in the sample is normal.

[0083] VII. Quality Control

[0084] 1. Perform quality control on each step in the experimental process to ensure the accuracy of the experimental results.

[0085] 2. Perform statistical analysis on the experimental data and evaluate the repeatability and reliability of the reagent set.

[0086] Through the above steps, the content of amyloid A in serum can be accurately detected, providing an important reference for clinical diagnosis and disease monitoring.

[0087] Comparative Example 1

[0088] CN109142337A was used as the comparison method of the present application to conduct a comparison experiment.

[0089] The experimental results are shown in Table 1.

[0090] Table 1

[0091] Test Parameters Example 2 Comparative Example 1 Sensitivity 0.5ng / mL 1.0ng / mL Specificity High (no significant cross-reaction) Moderate (slight cross-reactivity) accuracy High (high consistency between standards and samples) Medium (low consistency between standard and sample) Repeatability CV<5% CV<10%

[0092] As can be seen from Table 1, the method of Example 2 is superior to the method of Comparative Example 1 in terms of sensitivity, specificity, accuracy and repeatability. Specifically, the detection sensitivity of Example 2 reaches 0.5 ng / mL, which is much higher than 1.0 ng / mL of Comparative Example 1, indicating that Example 2 can detect lower concentrations of SAA, thereby providing the possibility for early diagnosis. In terms of specificity, Example 2 shows high specificity and no obvious cross-reaction, while Comparative Example 1 shows moderate specificity and slight cross-reaction, which may affect the accuracy of the test results. The accuracy assessment shows that the test results of Example 2 are highly consistent with the standards and samples, while the consistency of Comparative Example 1 is low, which further confirms the reliability of the method of Example 2. In terms of repeatability, the coefficient of variation (CV) of Example 2 is less than 5%, while the CV of Comparative Example 1 is less than 10%, indicating that the method of Example 2 has better repeatability and the experimental results are more stable and reliable.

[0093] In summary, the method of Example 2 has significant advantages in detecting serum amyloid A, and can provide more accurate, reliable and highly reproducible test results. This has important practical application value for clinical diagnosis, disease monitoring and evaluation of treatment effects. Therefore, the reagent set and detection method of the present invention have broad application prospects and promotion value in related fields.

[0094] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in the relevant field should understand that the specific implementation methods of the present invention can still be modified or replaced by equivalents. Any modification or equivalent replacement that does not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A detection reagent set for serum amyloid A based on improved spatial proximity chemiluminescence method, characterized in that: The reagent set includes: Enzyme labels, luminescent labels, auxiliary agents, substrate solutions, blocking solutions, calibrators and reference substances; Wherein, the enzyme markers include horseradish peroxidase-labeled anti-SAA antibody, HEPES buffer, sodium azide solution, bovine serum albumin and human albumin; The luminescent marker is an anti-SAA capture antibody labeled with an acridinium ester derivative; The auxiliary agent includes a rare earth metal chelating agent.

2. The detection reagent set for serum amyloid A based on improved spatial proximity chemiluminescence method according to claim 1, characterized in that: The preparation method of the enzyme marker comprises: Dissolve the anti-SAA antibody labeled with horseradish peroxidase in distilled water and add HEPES buffer, then add sodium azide solution as an antibacterial agent, stir evenly in the dark, and obtain a preliminary mixed solution; The preliminary mixed solution is placed in a dialysis bag and dialyzed in a HEPES buffer to remove low molecular weight impurities; After the dialysis is completed, a NaCl solution is added to the dialyzed solution to adjust the ionic strength, and then bovine serum albumin and human albumin are added in sequence, stirred until completely dissolved, and then sodium borohydride is added as a reducing agent, and the reaction is allowed to stand in the dark to obtain a reaction solution; Finally, the reaction solution is put into the dialysis bag again and dialyzed to remove free horseradish peroxidase to obtain the final enzyme marker.

3. The detection reagent set for serum amyloid A based on improved spatial proximity chemiluminescence method according to claim 2, characterized in that: The preparation method of the horseradish peroxidase-labeled anti-SAA antibody comprises: Dissolve horseradish peroxidase in distilled water, add HEPES buffer to make the solution reach a pH value of 7.2±0.2, add a cross-linking agent under stirring to perform a covalent coupling reaction to obtain a mixed solution; The anti-SAA antibody is added to the mixed solution, and stirred at room temperature in the dark for 1-2 hours; after the reaction is completed, unbound HRP and cross-linking agent are removed by dialysis or gel filtration to obtain the horseradish peroxidase-labeled anti-SAA antibody.

4. The detection reagent set for serum amyloid A based on improved spatial proximity chemiluminescence method according to claim 3, characterized in that: The cross-linking agent is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

5. The detection reagent set for serum amyloid A based on improved spatial proximity chemiluminescence method according to claim 1, characterized in that: The preparation method of the luminescent marker comprises: Dissolving an acridinium ester derivative in dimethyl sulfoxide, adding dipyridine carboxylic acid buffer solution after complete dissolution under stirring, and adjusting the pH value of the solution to 7.0-7.5 to obtain an acridinium hydrochloride solution; The anti-SAA monoclonal antibody is dissolved in a PDC buffer, stirred and then added to the acridinium ester derivative solution, and stirred and reacted at room temperature; a polylysine coupling agent is added during stirring, and then the mixture is placed on a shaker and stirred for coupling reaction, and the reaction time is at least 2 hours; After the coupling reaction is completed, the mixture is placed in a 4°C environment for 12-16 hours, and the reaction mixture is washed with Tris-HCl buffer to remove unreacted free luminescent markers and unbound polylysine coupling agents to obtain the luminescent marker.

6. The detection reagent set for serum amyloid A based on improved spatial proximity chemiluminescence method according to claim 1, characterized in that: The preparation method of the auxiliary agent comprises: Selecting diaminocyclohexanetetraacetic acid as a rare earth metal chelating agent, dissolving the rare earth metal chelating agent in deionized water, and adjusting the pH value of the solution to 7.2±0.2 to obtain a chelating agent solution; Under stirring, adding the rare earth metal ion solution to the chelating agent solution, and continuing to stir until the rare earth metal ions and the chelating agent fully react to obtain a rare earth metal chelate solution; Add HEPES buffer to the rare earth metal chelate solution, adjust the pH value to 7.3±0.1, and stir at room temperature for at least 30 minutes to obtain an auxiliary agent solution.

7. Use of a detection reagent set for serum amyloid A based on improved spatial proximity chemiluminescence method according to any one of claims 1 to 6, characterized in that: At least one of the following: A1) Application in the detection of serum amyloid A; A2) Application in the preparation of products for detecting serum amyloid A; A3) Application in the study of the correlation between serum amyloid A and diseases.

8. The use of the serum amyloid A detection reagent set based on improved spatial proximity chemiluminescence method according to claim 7, characterized in that: The application in detecting serum amyloid A comprises the following steps: The serum sample to be tested, the blocking solution, the anti-SAA monoclonal antibody labeled with horseradish peroxidase, and the anti-SAA monoclonal antibody labeled with an acridinium ester derivative are reacted together to form an antigen-antibody sandwich complex, and the substrate solution is added to start the chemiluminescence reaction; the luminescence intensity is measured using a chemiluminescence detector; and the concentration of amyloid A in the sample to be tested is calculated based on the comparison of the luminescence intensity with the calibrator and the reference.

9. A method for detecting serum amyloid A, characterized in that: The method comprises using the serum amyloid A detection reagent set based on the improved spatial proximity chemiluminescence method as described in any one of claims 1 to 6 for detection.