Reverse serum amyloid a immunogens and methods of making and using the same

Abstract

The application discloses a reverse serum amyloid A immunogen and a preparation method and application thereof, and belongs to the technical field of biotechnology. In view of the technical problem that natural positive serum amyloid A cannot effectively capture SAA antibodies in human serum, the amino acid sequence of natural serum amyloid A is reversely arranged to obtain a reverse serum amyloid A immunogen; the immunogen is prepared by using a recombinant expression system, and is coupled to a solid-phase carrier; and human serum amyloid A antibodies are captured from a sample to be detected by using an affinity chromatography method. The captured antibodies can be used in immunodetection methods such as immunoblotting, immunoprecipitation, immunofluorescence, immunohistochemistry, flow cytometry and enzyme-linked immunosorbent assay, and can be used for preparing diagnostic reagents or diagnostic kits.

Description

Technical Field

[0001] This invention relates to the field of biotechnology. More specifically, this invention relates to a reverse serum amyloid A immunogen, its preparation method, and its uses. Background Technology

[0002] Serum amyloid A (SAA) is an acute-phase reactive protein, mainly synthesized by the liver. Its serum concentration can rise sharply in a short period of time when the body experiences infection, trauma, or inflammation. It is an important biomarker reflecting early inflammation in infectious diseases. SAA protein exists in the human body mainly in two subtypes, SAA1 and SAA2, which play similar biological functions in the acute phase response. SAA antibodies are important tools for detecting SAA protein and studying its biological functions, and are widely used in immunoassay methods such as immunoblotting, immunoprecipitation, immunohistochemistry, and enzyme-linked immunosorbent assay.

[0003] In existing technologies, the main approach to obtaining SAA antibodies is to use SAA protein as an immunogen to immunize animals, thereby generating polyclonal or monoclonal antibodies through the host's immune response. However, prokaryotically expressed recombinant SAA protein has poor stability in vitro, easily forming aggregates and degrading, resulting in insufficient immunogenicity and making it difficult to effectively stimulate the host to produce high-titer, high-specificity antibodies. To address this issue, Chinese patent CN109851668B, entitled "An SAA Protein Immunogen and Its Preparation Method and Anti-Human Serum Amyloid A Polyclonal Antibody," discloses a method for treating SAA protein with a protein cross-linking agent to form SAA polymers. The polymers, to a certain extent, mimic the hexamer structure of natural SAA protein, improving the stability and immunogenicity of SAA protein, thereby improving the antibody preparation effect. By adding an external cross-linking agent to chemically modify SAA protein to form polymers, it is then used to immunize animals.

[0004] There are also reports on the application of protein or peptide sequence orientation modification in the field of immunology. European patent EP0667786B1, entitled Synthetic Peptide Antigen Analogs, discloses a strategy for reverse modification of natural peptide antigens, namely, obtaining synthetic peptide antigen analogs by sequence inversion and amino acid configuration inversion of short peptides, which can induce the production of antibodies that cross-react with natural peptide antigens. This mainly focuses on the chemical synthesis modification of short peptide antigens.

[0005] None of the aforementioned existing technologies involve methods for directly capturing naturally occurring SAA antibodies in human serum using SAA proteins. In practical applications, human serum itself contains a certain level of natural SAA antibodies. If these antibodies could be captured and purified directly from serum samples, it would provide a more convenient way to obtain SAA antibodies. However, studies have found that when recombinant SAA proteins expressed in the natural forward sequence are used as capture ligands, they are difficult to bind effectively to SAA antibodies in human serum, resulting in low capture efficiency or even failure to capture. Currently, the specific mechanism by which the forward SAA protein, i.e., natural serum amyloid A, cannot effectively bind to SAA antibodies in serum is not fully understood. Therefore, how to obtain a ligand that can effectively capture SAA antibodies in human serum has become a technical problem to be solved in this field. Summary of the Invention

[0006] One object of the present invention is to solve at least the above-mentioned problems and to provide at least the advantages that will be described later.

[0007] To achieve these objectives and other advantages according to the invention, a reverse serum amyloid A immunogen is provided, comprising an amino acid sequence that is reversed in sequence with respect to the amino acid sequence of native serum amyloid A.

[0008] Preferably, the amino acid sequence of reverse serum amyloid A is shown in SEQ ID NO:1.

[0009] Preferably, the reverse serum amyloid A immunogen is coupled to a solid-phase support, which includes an affinity chromatography packing material.

[0010] This invention provides a method for preparing reverse serum amyloid A immunogen, comprising the following steps:

[0011] S1. Construct a recombinant expression vector encoding reverse serum amyloid A;

[0012] S2. Transform the recombinant expression vector obtained in step S1 into host cells to obtain a recombinant expression strain;

[0013] S3. The recombinant expression strain obtained in step S2 is induced to express reverse serum amyloid A in the host cells.

[0014] S4. Isolate and purify reverse serum amyloid A from the host cells induced and cultured in step S3 to obtain reverse serum amyloid A immunogen.

[0015] Preferably, in step S1, the recombinant expression vector is the pQE80L plasmid vector, and in step S2, the host cell is Escherichia coli BL21-DE3.

[0016] Preferably, in step S4, the separation and purification includes the following steps:

[0017] The bacterial cells were collected, resuspended in lysis buffer, and then sonicated after the addition of protease inhibitors. The supernatant was collected by centrifugation and filtered through a 0.45 μm filter membrane.

[0018] Preferably, the method further includes step S5, in which the reverse serum amyloid A immunogen obtained in step S4 is dissolved in a coupling buffer and mixed with the activated solid-phase carrier to carry out a coupling reaction, so that the reverse serum amyloid A immunogen is covalently fixed on the solid-phase carrier. After the coupling reaction is completed, the residual active groups on the solid-phase carrier are blocked, and after washing, a solid-phase carrier coupled with the reverse serum amyloid A immunogen is obtained.

[0019] This invention provides the use of a reverse serum amyloid A immunogen in capturing human serum amyloid A antibodies.

[0020] Preferably, the capture is performed by affinity chromatography, comprising: pretreating the sample to be tested, contacting it with a solid-phase carrier conjugated with reverse serum amyloid A immunogen, washing the solid-phase carrier with equilibration buffer, and eluting the bound human serum amyloid A antibody with elution buffer.

[0021] Preferably, the captured human serum amyloid A antibody is used in an immunoassay method selected from at least one of immunoblotting, immunoprecipitation, immunofluorescence, immunohistochemistry, flow cytometry, and enzyme-linked immunosorbent assay; or, the reverse serum amyloid A immunogen is used to prepare diagnostic reagents or diagnostic kits.

[0022] The present invention has at least the following beneficial effects:

[0023] First, the reverse serum amyloid A immunogen provided by this invention obtains a protein structure with a spatial conformation completely different from that of natural SAA by reversing the amino acid sequence of natural SAA. Based on the conformational difference, the reverse SAA immunogen can specifically bind to naturally occurring SAA antibodies in human serum, solving the technical problem that natural serum amyloid A cannot effectively capture serum SAA antibodies. This provides a novel affinity ligand for the acquisition of SAA antibodies. At the same time, the immunogen can be coupled to solid-phase carriers such as affinity chromatography packing materials or enzyme-linked immunosorbent assay (ELISA) plates, which facilitates subsequent antibody capture and detection applications and has good scenario adaptability.

[0024] Secondly, the preparation method of this invention adopts a recombinant expression strategy. By constructing a recombinant expression vector encoding reverse SAA and inducing expression in Escherichia coli, the reverse SAA immunogen can be obtained stably and efficiently, avoiding the problems of high cost and poor uniformity of chemically synthesized long-chain peptides. The process conditions are clear and controllable. The obtained protein is verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to have the expected molecular weight and purity, with good batch-to-batch consistency. Furthermore, after the reverse SAA immunogen is coupled to an activated solid-phase carrier, a reusable immobilized affinity ligand can be obtained, which is convenient for subsequent large-scale production and application.

[0025] Third, when the reverse SAA immunogen provided by this invention is used to capture SAA antibodies from human serum, it can specifically enrich and purify SAA antibodies from human serum samples. The obtained antibodies have good activity retention capacity as shown by thermal stability test and freeze-thaw stability test, making them suitable as the core raw material for immunoassay reagents. The captured SAA antibodies can be applied to various immunoassay methods such as immunoblotting, immunoprecipitation, immunofluorescence, immunohistochemistry, flow cytometry, and enzyme-linked immunosorbent assay. They can also be further used to prepare diagnostic reagents or kits related to SAA antibody detection, and have application value in the clinical auxiliary diagnosis and monitoring of diseases such as inflammation and infection.

[0026] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0027] Figure 1 The image shows the SDS-PAGE results of the original protein, where M is the protein molecular weight marker, lane 1 is the precipitate after cell lysis, lane 2 is the supernatant after cell lysis, lanes 3-4 are the affinity chromatography elution samples, lane 5 is the elution sample, and the original protein is the natural serum amyloid A.

[0028] Figure 2 The image shows the results of reverse protein SDS-PAGE detection, where M is the protein molecular weight marker, lane 1 is the precipitate after bacterial lysis, lane 2 is the supernatant after bacterial lysis, lanes 3-4 are the affinity chromatography elution samples, and lane 5 is the elution sample. The reverse protein is the reverse serum amyloid A immunogen.

[0029] Figure 3 This is a comparison chart showing the antibody capture effects of the original protein and the reverse protein.

[0030] Figure 4 This is a comparison diagram of the crystal structures of the original protein and the reverse protein. Detailed Implementation

[0031] The present invention will now be described in further detail with reference to examples, so that those skilled in the art can implement it based on the description.

[0032] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0033] Example 1

[0034] Preparation of reverse serum amyloid A immunogen

[0035] Based on the amino acid sequence of natural human serum amyloid A, the amino acid sequence from its C-terminus to its N-terminus was completely reversed to obtain the reverse sequence from the N-terminus to the C-terminus, i.e., the amino acid sequence shown in SEQ ID NO:1:

[0036] SEQ ID NO:1:YKEPLGARFHNPDKGSRGWENAAQDALSDEAGHFFRQINERADSIAEAAWVGGPGRKAADYNGRAHFKYKDSGYNAEARMDSYARWMDRAGDFAEGLFSFFAGSSRMHSGRPVLGLSS

[0037] The coding nucleotide sequence was obtained by reverse translation of SEQ ID NO:1, and the whole genome was synthesized by a gene synthesis company. Figure 4 The figure shows a comparison of the three-dimensional spatial conformation of the reverse SAA protein obtained by the reverse arrangement strategy and the natural (forward) SAA protein. As can be seen from the figure, although they are composed of the same amino acids, they exhibit completely different spatial folding modes due to the complete reversal of the sequence direction. The significant structural difference is the structural basis for the reverse SAA immunogen to specifically capture SAA antibodies in human serum, while the natural forward SAA cannot achieve this function.

[0038] Construction of the recombinant expression vector: The synthesized gene fragment encoding inverse serum amyloid A was digested with enzymes and inserted into the multiple cloning site of the pQE80L plasmid vector to construct the recombinant expression plasmid pQE80L-RevSAA. The recombinant plasmid was transformed into Escherichia coli BL21-DE3 competent cells and plated on LB agar plates containing 100 μg / mL ampicillin (Amp) and incubated overnight at 37°C. Single colonies were picked for colony PCR verification, and positive clones were screened to obtain the recombinant expression strain.

[0039] Protein expression induction: Select a single colony of the validated recombinant expression strain and inoculate it into 5 mL of LB liquid medium (containing 100 μg / mL Amp), and culture overnight at 37°C with shaking at 180 rpm to serve as seed culture;

[0040] The overnight seed culture was transferred at a volume ratio of 1:100 to 100 mL of fresh LB liquid medium (containing 100 μg / mL LAmp), and cultured at 37°C with shaking at 200 rpm until the bacterial culture OD... 600 When the concentration reached 0.6-0.8 (approximately 3 hours), isopropyl-β-D-thiogalactoside (IPTG) was added to the culture medium to a final concentration of 0.2 mM, and expression was induced at 16°C and 160 rpm for 16 hours.

[0041] After induction, the bacterial culture was centrifuged at 4°C and 8000 rpm for 10 minutes. The supernatant was discarded, and the bacterial pellet was collected. The bacterial pellet was resuspended in 10 mL of pre-cooled lysis buffer (20 mM Tris, 300 mM NaCl, pH adjusted to 8.0 with HCl). Benzyl sulfonyl fluoride (PMSF) was added to a final concentration of 1 mM as a protease inhibitor. The procedure was performed on ice.

[0042] Protein separation and purification: The above resuspended bacterial solution was sonicated. The sonication conditions were: power 15%, sonication time 9 seconds, interval 9 seconds, total duration 30 minutes, and ice bath operation throughout. The lysate after sonication was centrifuged at 4℃ and 12000rpm for 30 minutes. The supernatant was collected and filtered through a 0.45μm microporous membrane to obtain a crude extract containing reverse serum amyloid A immunogen.

[0043] SDS-PAGE analysis of the crude extract confirmed the presence of a distinct protein band at the target molecular weight position, indicating that the reverse serum amyloid A immunogen was successfully expressed in soluble form in Escherichia coli. Figure 1 The figure shows the SDS-PAGE analysis results of recombinant expression and purification of natural (positive) serum amyloid A. Lane M is the protein molecular weight marker; lane 1 is the precipitate after cell lysis; lane 2 is the supernatant after cell lysis; lanes 3-4 are the affinity chromatography elution samples; and lane 5 is the elution sample. As can be seen from the figure, the positive SAA protein, i.e., natural serum amyloid A, was successfully expressed in soluble form in Escherichia coli, and the target protein band appeared at the expected molecular weight position after purification. Figure 2 The figure shows the SDS-PAGE analysis results of the recombinant expression and purification of reverse serum amyloid A immunogen. In the figure, lane M is the protein molecular weight marker; lane 1 is the precipitate after cell lysis; lane 2 is the supernatant after cell lysis; lanes 3-4 are affinity chromatography elution samples; and lane 5 is the elution sample. As can be seen from the figure, the reverse SAA protein was also successfully expressed in soluble form in E. coli. After purification, the target protein band appeared at the expected molecular weight position, and the expression level and purity were comparable to those of the forward protein, indicating that the reverse sequence design did not affect the soluble expression efficiency of the protein.

[0044] Solid-phase carrier coupling: Weigh an appropriate amount of agarose gel filler activated with cyanogen bromide (CNBr) (CNBr Activated Seplife 4FF), pretreat it according to the manufacturer's instructions, dissolve the obtained reverse serum amyloid A immunogen at a concentration of 2 mg / mL in coupling buffer (0.1 mol / L NaHCO3, 0.5 mol / L NaCl, pH 8.3), mix it with the pretreated activated filler, stir and react at room temperature for 2-4 hours, or react overnight at 4°C, so that the reverse serum amyloid A immunogen is covalently fixed on the filler;

[0045] After the coupling reaction was completed, the packing material was first washed with coupling buffer to remove uncoupled excess ligands, and then washed with pure water. The washed gel was then suspended in pure water, and 20 mM Tris-HCl (pH 8.0) buffer was added. The mixture was stirred at room temperature to block the residual active groups on the packing material. After blocking, the packing material was washed three times alternately with acetate buffer containing NaCl (0.1 mol / L acetate, 0.5 mol / L NaCl, pH 4.0) and Tris-HCl buffer containing NaCl (0.1 mol / L Tris-HCl, 0.5 mol / L NaCl, pH 8.0). Finally, the packing material was washed thoroughly with PBS buffer to obtain affinity chromatography packing material coupled with reverse serum amyloid A immunogen. The packing material was stored at 4°C for later use.

[0046] In other embodiments, reverse serum amyloid A immunogen can also be immobilized on other solid-phase carriers such as magnetic beads by physical adsorption or covalent coupling to achieve different capture or detection application scenarios.

[0047] Example 2

[0048] Capture human serum amyloid A antibody

[0049] Sample pretreatment: Take a human serum sample, centrifuge at 4°C and 8000 rpm for 30 minutes, discard the supernatant, collect the precipitate, and take 1 / 5 of the original serum sample volume of buffer solution (20mM Tris-HCl, 150mM NaCl, pH 8.0). Resuspend the precipitate thoroughly to obtain the pretreated sample to be tested. In this embodiment, human serum is used as an example for illustration. The capture method of this invention is also applicable to other biological samples containing SAA antibodies, such as human plasma.

[0050] Affinity chromatography capture: The affinity chromatography packing material prepared in Example 1 and coupled with reverse serum amyloid A immunogen was packed into the chromatography column. After the chromatography column was fully equilibrated with equilibration buffer (20 mM PBS, pH 7.4), the pretreated test sample was loaded onto the column.

[0051] After loading the sample, the chromatography column was washed with equilibration buffer (20mM PBS, pH 7.4) to remove unbound contaminating proteins and other components. After washing until the baseline stabilized, the column was eluted with elution buffer (20mM Tris-HCl, pH 8.0), and the elution peak was collected to obtain purified human serum amyloid A antibody.

[0052] SDS-PAGE verification: The eluted sample was analyzed by SDS-PAGE. The results showed that under reducing conditions, the reverse serum amyloid A immunogen-coupled filler successfully captured human serum SAA antibody, and clear heavy chain and light chain bands were displayed on the gel.

[0053] In contrast, affinity chromatography packing material conjugated with natural (positive) serum amyloid A was prepared using the same method and the same serum sample was treated under the same conditions. SDS-PAGE results showed that the positive SAA conjugated packing material failed to effectively capture SAA antibodies. The comparative results confirmed that the reverse alignment strategy is the key to achieving efficient capture of SAA antibodies.

[0054] Example 3

[0055] Stability test of capture antibody

[0056] Accelerated aging stability at 37°C: The human serum amyloid A antibody captured in Example 2 was prepared into high-value and low-value samples, respectively, and placed at 37°C for 14 days. Samples were taken on days 0, 3, 5, 7 and 14, and the antibody reactivity was detected using a Hitachi 7180 biochemical analyzer (the average value was taken from three repeated tests).

[0057] The test results are shown in Table 1. After 14 days of accelerated aging at 37℃, the deviation of the reactivity of the high-value sample from that on day 0 was -2.3%, and the deviation of the low-value sample was 3.86%. Both deviations were less than 10%, indicating that the antibody captured by the reverse serum amyloid A immunogen has good thermal stability.

[0058] Table 1. Stability of human serum amyloid A antibody at 37℃ during accelerated aging.

[0059]

[0060] Freeze-thaw stability: The human serum amyloid A antibody captured in Example 2 was prepared into high-value and low-value samples, respectively. After being frozen at -20°C, it was completely thawed at room temperature. The above freeze-thaw process was repeated 5 times. Samples were taken after the 1st, 2nd, 3rd, 4th and 5th freeze-thaw tests to detect the reactivity (the average value of three repeated tests was taken).

[0061] The test results are shown in Table 2. After 5 freeze-thaw cycles, the deviation of the high-value sample reactivity from the first freeze-thaw cycle was -7.5%, and the deviation of the low-value sample was -7.1%. Both deviations were less than 10%, indicating that the freeze-thaw stability of the antibody captured by the reverse serum amyloid A immunogen was qualified.

[0062] Table 2 Freeze-thaw stability of human serum amyloid A antibody

[0063]

[0064] Example 4

[0065] Immunoassay Applications

[0066] The human serum amyloid A antibody captured in Example 2 was applied to at least one of the following immunoassay methods: Western blotting (WB), using the captured SAA antibody as the primary antibody to detect SAA antigen in cell or tissue lysates; Immunoprecipitation (IP), using the captured SAA antibody coupled with Protein A / G magnetic beads to enrich SAA protein in the sample to be tested; Immunofluorescence (IF), using the captured SAA antibody as the primary antibody, combined with a fluorescently labeled secondary antibody, to stain and observe cells or tissue sections.

[0067] Immunohistochemistry (IHC) was used to stain paraffin-embedded tissue sections with captured SAA antibodies.

[0068] Flow cytometry (FCM) uses captured SAA antibodies to label SAA proteins on the cell surface or inside the cell for flow cytometry analysis.

[0069] Enzyme-linked immunosorbent assay (ELISA) uses captured SAA antibodies as coating or detection antibodies to establish a double-antibody sandwich ELISA detection system for SAA antigen.

[0070] The reverse serum amyloid A immunogen prepared in Example 1 or the SAA antibody captured in Example 2 can be used as the core raw material and combined with supporting components such as buffer, enzyme-labeled secondary antibody, and substrate to prepare diagnostic reagents or diagnostic kits for detecting SAA antibodies. These kits are widely applicable to the clinical auxiliary diagnosis and monitoring of inflammation, infection and other related diseases.

[0071] Comparative Example

[0072] To compare the capture effects of forward and reverse SAA, and to verify the key role of the reverse arrangement strategy, affinity chromatography packing material conjugated with natural (forward) serum amyloid A was prepared simultaneously. The amino acid sequence of the forward SAA is (reversely corresponding to SEQ ID NO:1), as shown in SEQ ID NO:2:

[0073] SEQ ID NO:2:SSGLVPRGSHMRSFFSFLGEAFDGARDMWRAYSDMREANYIGSDKYFHARGNYDAAKRGPGGVWAAEAISDARENIQRFFGHGAEDSLADQAANEWGRSGKDPNHFRPAGLPEKY

[0074] The preparation steps of the forward SAA-conjugated packing material were exactly the same as in Example 1, except that the reverse SAA was replaced with forward SAA. Antibody capture experiments were performed using the same serum samples and the same chromatographic conditions (same as in Example 2). The results showed that the forward SAA-conjugated packing material failed to capture SAA antibodies from human serum, while the reverse SAA-conjugated packing material successfully captured them. This comparative experiment fully demonstrates that the protein immunogen obtained by reversing the amino acid sequence of natural SAA possesses the ability to capture serum SAA antibodies, a capability that natural SAA does not have. Figure 3 The figure shows a comparison of the antibody capture performance of forward SAA-conjugated packing material and reverse SAA immunogen-conjugated packing material after treating the same human serum sample under the same conditions. As can be seen from the figure, the reverse SAA immunogen-conjugated packing material shows a clear antibody band at the target position, while the forward SAA-conjugated packing material does not show an obvious band at the corresponding position. This directly confirms that the reverse alignment strategy is a key technical means to achieve efficient capture of SAA antibodies from human serum.

[0075] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. Other modifications can be easily made by those skilled in the art. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and examples shown and described herein.

Claims

1. A reverse serum amyloid A immunogen, characterized in that, It contains an amino acid sequence that is reversed compared to the amino acid sequence of natural serum amyloid A.

2. The reverse serum amyloid A immunogen according to claim 1, characterized in that, The amino acid sequence of reverse serum amyloid A is shown in SEQ ID NO:

1.

3. The reverse serum amyloid A immunogen according to claim 1 or 2, characterized in that, The reverse serum amyloid A immunogen is coupled to a solid-phase support, which includes affinity chromatography packing material.

4. A method for preparing the reverse serum amyloid A immunogen according to any one of claims 1 to 3, characterized in that, Includes the following steps: S1. Construct a recombinant expression vector encoding reverse serum amyloid A; S2. Transform the recombinant expression vector obtained in step S1 into host cells to obtain a recombinant expression strain; S3. The recombinant expression strain obtained in step S2 is induced to express reverse serum amyloid A in the host cells. S4. Isolate and purify reverse serum amyloid A from the host cells induced and cultured in step S3 to obtain reverse serum amyloid A immunogen.

5. The method for preparing reverse serum amyloid A immunogen according to claim 4, characterized in that, In step S1, the recombinant expression vector is the pQE80L plasmid vector, and in step S2, the host cell is Escherichia coli BL21-DE3.

6. The method for preparing reverse serum amyloid A immunogen according to claim 4, characterized in that, In step S4, the separation and purification includes the following steps: The bacterial cells were collected, resuspended in lysis buffer, and then sonicated after the addition of protease inhibitors. The supernatant was collected by centrifugation and filtered through a 0.45 μm filter membrane.

7. The method for preparing reverse serum amyloid A immunogen according to any one of claims 4 to 6, characterized in that, The method also includes step S5, in which the reverse serum amyloid A immunogen obtained in step S4 is dissolved in a coupling buffer and mixed with the activated solid-phase carrier to carry out a coupling reaction, so that the reverse serum amyloid A immunogen is covalently fixed on the solid-phase carrier. After the coupling reaction is completed, the residual active groups on the solid-phase carrier are blocked, and after washing, a solid-phase carrier coupled with the reverse serum amyloid A immunogen is obtained.

8. Use of the reverse serum amyloid A immunogen according to any one of claims 1 to 3 in capturing human serum amyloid A antibodies.

9. The use according to claim 8, characterized in that, The capture was performed by affinity chromatography, which included: pretreating the sample to be tested, contacting it with a solid-phase carrier conjugated with reverse serum amyloid A immunogen, washing the solid-phase carrier with equilibration buffer, and eluting the bound human serum amyloid A antibody with elution buffer.

10. The use according to claim 8 or 9, characterized in that, The captured human serum amyloid A antibody is used in an immunoassay method selected from at least one of immunoblotting, immunoprecipitation, immunofluorescence, immunohistochemistry, flow cytometry, and enzyme-linked immunosorbent assay; or, the reverse serum amyloid A immunogen is used to prepare diagnostic reagents or diagnostic kits.

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