Aptamer and kit for detecting serum amyloid a

Abstract

The application provides a nucleic acid aptamer for detecting serum amyloid A and a kit, and the nucleotide sequence of the nucleic acid aptamer for detecting serum amyloid A is shown in SEQ ID NO. 1. The kit for detecting serum amyloid A comprises the nucleic acid aptamer. The nucleic acid aptamer for serum amyloid A screened by SELEX technology has high specificity and affinity, and has the advantages of non-immunogenicity, small molecule, easy modification, easy synthesis, non-degradation, non-toxicity and the like. The kit has the characteristics of simple operation, rapid reaction, high accuracy and high sensitivity, and can realize accurate detection of the target protein in combination with a detection device such as an enzyme label instrument. The kit has high sensitivity, good specificity, wide measurement range, simple operation, and has a good market prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a nucleic acid aptamer and kit for detecting serum amyloid A. Background Technology

[0002] Infectious diseases are common clinical conditions. The pathogens causing these diseases are numerous, the routes of infection are diverse, and symptoms and signs vary from person to person. Failure to make a timely and accurate diagnosis and provide effective treatment can lead to serious consequences. Therefore, early diagnosis and differentiation of infectious diseases is a pressing clinical problem. Serum amyloid A is an acute-phase reactive protein encoded by multiple genes. During the acute phase of infection, serum amyloid A is rapidly synthesized and released into the bloodstream by activated macrophages and fibroblasts. It begins to rise 3-6 hours after infection and rapidly decreases to normal levels after pathogen clearance. Changes in serum amyloid A levels are of significant clinical value for the early diagnosis, risk assessment, monitoring of treatment efficacy, and prognostic evaluation of infectious diseases. Besides being elevated in bacterial infections, serum amyloid A is also significantly elevated in viral infections. International literature has reported significant increases in serum amyloid A during the acute phase of various viral infections, such as influenza virus, respiratory syncytial virus, and adenovirus. The degree of elevation or its combination with other indicators can suggest bacterial or viral infection.

[0003] Currently, commercially available test kits for serum amyloid A are all antigen-based, primarily using single-channel detection. Furthermore, these commercially available antigen test kits use antibody detection. Antibodies, as protein components, have drawbacks such as difficulty in expression and synthesis, easy degradation, and non-specific immunogenicity.

[0004] Therefore, it is necessary to develop a detection method with high specificity and high sensitivity. Summary of the Invention

[0005] The purpose of this invention is to provide a nucleic acid aptamer and kit for detecting serum amyloid A, which has high sensitivity, good specificity, wide measurement range, simple operation, can rapidly detect target protein, is suitable for large-scale promotion, and has good market prospects.

[0006] In a first aspect of the invention, a nucleic acid aptamer for detecting serum amyloid A is provided, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0007] Furthermore, the nucleic acid aptamer for detecting serum amyloid A further includes: a fluorescent substance, a nanoluminescent material, biotin, digoxigenin, and an enzyme labeling agent bound to the nucleotide sequence of the nucleic acid aptamer.

[0008] Furthermore, the fluorescent substance is a FAM fluorescent group or a Cy5 fluorescent group; the nanoluminescent material is a quantum dot or an upconversion nanoparticle; and the enzyme label is horseradish peroxidase or sucrase.

[0009] Furthermore, the nucleic acid aptamer for detecting serum amyloid A further includes a nucleic acid aptamer whose nucleotide sequence is phosphorylated, methylated, aminated, thiolated, or isotopically modified at a certain position.

[0010] In a second aspect of the invention, a kit for detecting serum amyloid A is provided, comprising the aforementioned nucleic acid aptamer.

[0011] Furthermore, the kit for detecting serum amyloid A also includes:

[0012] (A) Protein-coated plate for immobilizing sample solutions;

[0013] (B) Protein coating solution;

[0014] (C) Sealing solution;

[0015] (D) Rinse solution;

[0016] (E) Serum amyloid A calibrator.

[0017] Furthermore, the protein coating solution is formulated as follows: 35mM NaHCO3, 15mM Na2CO3, pH 9.3-pH 9.9.

[0018] Furthermore, the blocking solution is formulated as follows: 136 mM NaCl, 2.6 mM KCl, 2 mM KH2PO4, 8 mM Na2HPO4, 0.05% Tween-20, 1% BSA, pH 7.1-pH 7.7.

[0019] Furthermore, the rinsing solution is formulated with 136mM NaCl, 2.6mM KCl, 2mM KH2PO4, 8mM Na2HPO4, 0.05% Tween-20, and pH 7.1-pH 7.7.

[0020] Furthermore, the serum amyloid A calibrator has five concentrations: 6.25 μg / L, 12.5 μg / L, 25 μg / L, 50 μg / L, and 100 μg / L.

[0021] In a fourth aspect of the invention, the use of the nucleic acid aptamer for detecting serum amyloid A and / or the detection kit described herein in detecting the content of serum amyloid A in infected samples is provided.

[0022] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0023] This invention provides a nucleic acid aptamer and kit for detecting serum amyloid A. The nucleic acid aptamer for serum amyloid A screened by this invention using SELEX technology has high specificity and affinity, and also has advantages such as non-immunogenicity, small molecular size, modifiability, easy synthesis, non-degradability, and non-toxicity. The nucleic acid aptamer sequences used in this invention are obtained through SELEX technology and are completely original.

[0024] The kit of this invention has high sensitivity, good specificity, wide measurement range, simple operation, and can rapidly detect target proteins. It is suitable for large-scale promotion and has good market prospects. Specifically, the kit of this invention has high precision in the detection of serum amyloid A, with a coefficient of variation of 2.54%; in the anti-interference test, the relative error for various interfering substances is within 5%.

[0025] The kit of this invention uses nucleic acid aptamers as the detection substance, which can effectively overcome the problems of difficulty in expression and synthesis, easy degradation, and non-specific immunogenicity. It has the advantages of small molecule size, modifiability, easy synthesis, non-degradability, non-toxicity, and non-immunogenicity. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 This is a secondary structure diagram of the nucleotide sequence of serum amyloid A-aptamer, the nucleic acid aptamer of this invention;

[0028] Figure 2 The standard curve of fluorescence intensity versus target protein concentration is provided for embodiments of the present invention. Detailed Implementation

[0029] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0030] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0031] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be obtained by purchasing them from the market or by existing methods.

[0032] The technical solution of this application embodiment is to solve the above-mentioned technical problems, and the general idea is as follows:

[0033] This application utilizes serum amyloid A antigenic epitopes with the amino acid sequence shown in SEQ ID NO.2. Nucleic acid aptamers for serum amyloid A obtained through SELEX technology are then linked to fluorescent groups for observation and identification. Finally, these nucleic acid aptamers are used to prepare a detection kit, specifically comprising:

[0034] 1. Identify the antigenic epitopes of the target protein to be tested.

[0035] Through literature review and bioinformatics analysis, the inventors determined the amino acid sequence of the serum amyloid A antigenic epitope as shown in SEQ ID NO.2.

[0036] 2. Screening for specifically binding nucleic acid aptamer sequences

[0037] A single-stranded DNA library containing 42 random sequences was synthesized in vitro. Using specific regions of the target protein of serum amyloid A as screening criteria, the SELEX technology was used to screen for DNA aptamer sequences with high specificity and high affinity. The nucleic acid aptamer of serum amyloid A has the nucleotide sequence shown in SEQ ID NO.1.

[0038] 3. Synthesize nucleic acid aptamers labeled with fluorescent groups.

[0039] The nucleic acid aptamer sequences obtained from screening were synthesized directly at a routine biotechnology service company. During synthesis, a fluorescent group was covalently attached to the 5' end of the nucleic acid aptamer. The nucleic acid aptamer for detecting serum amyloid A has the sequence shown in SEQ ID No. 1, and the attached fluorescent group is FAM.

[0040] 4. Prepare the reagent kit

[0041] The kit includes the following components: A) a protein-coated plate for fixing sample solutions, B) a protein coating solution, C) a blocking solution, D) a washing solution, E) a nucleic acid aptamer labeled with a fluorescent group, and F) a serum amyloid A calibrator.

[0042] The following will provide a detailed description of a nucleic acid aptamer and kit for detecting serum amyloid A, based on embodiments and experimental data.

[0043] Example 1: Nucleic acid aptamers for detecting serum amyloid A

[0044] 1. Identify serum amyloid A antigenic epitopes

[0045] Based on literature review and bioinformatics analysis, the specific antigenic epitope regions on the target proteins that are suitable as nucleic acid aptamer detection targets were identified.

[0046] (1) The full-length mature peptide of serum amyloid A was selected as the aptamer for screening target proteins, having the sequence shown in SEQ ID NO.2:

[0047] RSFFSFLGEAFDGARDMWRAYSDMREANYIGSDKYFHARGNYDAAKRGPGGVWAAEAISDARENIQRFFGHGAEDSLADQAANEWGRSGKDPNHFRPAGLPEKY(SEQ ID NO.2)

[0048] (2) Based on the determined amino acid sequence, the nucleotide sequence was synthesized directly at the company and the prokaryotic codon was optimized. The synthesized gene fragment (nucleotide sequence as shown in SEQ ID NO.5) was inserted into the BamHI / HindIII restriction site of the prokaryotic expression vector pET32a to induce the expression of the corresponding target protein for aptamer screening.

[0049] 2. Construction of aptamer library and primer design

[0050] A single-stranded DNA library containing 42 random sequences was synthesized in vitro using the gene synthesis services of a conventional biotechnology service company. Its nucleotide sequence is as follows:

[0051] 5'-GATGACATTGCACAAGTCAGG-(N42)-GAGTGAATCCTGCTGTTCGA-3'

[0052] (2) An upstream primer P1 was designed and synthesized targeting the fixed 5' end sequence of the nucleic acid aptamer, with the sequence shown in SEQ ID NO.3: 5'-GATGACATTGCACAAGTCAGG-3' (SEQ ID NO.3)

[0053] (3) A downstream primer P2 was designed and synthesized targeting the fixed sequence at the 3' end of the nucleic acid aptamer. This primer has the sequence shown in SEQ ID NO..4, and a biotin group is attached to the 5' end of the primer. Specifically:

[0054] 5'-biotin-TCGAACAGCAGGATTCACTC-3'(SEQ ID NO.4)

[0055] 3. Using SELEX screening technology, nucleic acid aptamer sequences that can effectively bind to target proteins were screened from the library.

[0056] (1) Dissolve 10 nmol of the initial single-stranded DNA random library in 500 μl of PBS solution, incubate at 92°C for 5 min, then immediately place on ice for 10 min. Incubate the treated initial single-stranded DNA random library with the corresponding target protein on ice for 1 h, then add Ni-NTA Magnetic Agarose Beads and continue incubation for 1 h. After incubation, remove the supernatant using a magnetic separator, and wash the beads with 2 ml of PBS. Finally, resuspend the beads in 10 ml of pre-cooled PBS, mix thoroughly by pipetting, incubate at 92°C for 10 min, centrifuge at 13000 g, and collect the supernatant. This is the single-stranded DNA library that specifically recognizes the target protein after the first round of screening.

[0057] (2) The screened single-stranded DNA library was subjected to PCR amplification to prepare a secondary library. The PCR reaction system was as follows: 25 μl Premix Tap Mix, 8 μl of the screened single-stranded DNA library, 2.5 μl of upstream primer, 2.5 μl of downstream primer, 12 μl of ddH2O, and a total volume of 50 μl. PCR amplification was performed according to the following program: denaturation at 95℃ for 3 min, followed by 35 cycles of amplification at 95℃ for 30 s, 55℃ for 30 s, and 72℃ for 30 s, with a final extension at 72℃ for 10 min, and the reaction was terminated at 16℃. The PCR-amplified library was then recovered by agarose gel electrophoresis to obtain pure library fragments.

[0058] (3) Further preparation of the amplified secondary library. The amplified double-stranded DNA library was incubated with 100 μl of streptavidin-labeled magnetic beads at room temperature for 20 min. The double-stranded DNA library was bound to the surface of the magnetic beads by the affinity between biotin and streptavidin. Then, the magnetic beads were adsorbed using a magnetic separator, the supernatant was removed, and the magnetic beads were washed with 2 ml of PBS. NaOH solution with a final concentration of 200 mM was added and reacted at room temperature for 10 min to denature the double-stranded DNA library into single strands. The biotin-labeled strand bound to the magnetic beads, and the unlabeled strand detached into the supernatant. The supernatant was collected, NaOH was removed using a desalting column, and finally 500 μl of PBS was added. The collected solution is the single-stranded DNA library required for the next round of screening.

[0059] (4) The positive screening process, PCR amplification, and secondary single-stranded DNA library preparation process were repeated 15 times. The final single-stranded DNA library obtained showed the strongest recognition ability for the target protein. After cloning and sequencing the obtained single-stranded DNA library, the nucleic acid aptamer for detecting serum amyloid A, selected in this invention, was finally obtained. Its DNA sequence is as follows: Serum Amyloid A-aptamer

[0060] GATGACATTGCACAAGTCAGGCCTCCATGATCGTCGAGACCGGACGAGGCCCCGACCTGATGGGAGTGAATCCTGCTGTTCGA;

[0061] (5) To enable sensitive detection of the reaction products within the reaction system, a fluorescent group (covalently bonded and synthesized directly at a routine biotechnology service company) was labeled at the 5' end of the aptamer. The final aptamer information in the kit is as follows: serum amyloid A-aptamer, with the sequence shown in SEQ ID NO.1:

[0062] 5'-FAM-GATGACATTGCACAAGTCAGGCCTCCATGATCGTCGAGACCGGACGAGGCCCCGACCTGATGGGAGTGAATCCTGCTGTTCGA-3' (SEQ ID NO. 1).

[0063] Example 2: Validation of nucleic acid aptamers

[0064] 1. The secondary structures of the screened nucleic acid aptamers were analyzed using the RNA Structure Program software. The results showed that these aptamers can form specific stem-loop and hairpin structures. Serum amyloid A-aptamer can form structures such as... Figure 1 The secondary structure shown.

[0065] 2. The binding affinity between nucleic acid aptamers and target proteins was detected using a ForteBio Octet RED96 interaction analyzer. The results are as follows:

[0066] Table 1

[0067] Serum amyloid A-aptamer 8.1 PBS control No combination

[0068] 3. Specificity detection was performed using human serum albumin, immune serum globulin, and nucleic acid aptamers, respectively. Binding experiments revealed that the aptamers did not bind to these proteins. Cross-reactivity detection was performed using target proteins against the aptamers, and the results showed that the aptamers only bound to their corresponding target proteins.

[0069] 4. Take 0.2 μg of aptamer and place it in room temperature serum or aqueous solution. After 4 weeks, RT-PCR detection showed that the placed nucleic acid aptamer had a stable structure and had not degraded.

[0070] Example 3: A kit for detecting serum amyloid A and its preparation method.

[0071] 1. The kit for detecting serum amyloid A contains the following formulation:

[0072] (A) Protein-coated plate with immobilized sample solution; as a specific implementation method, a 96-well protein-coated plate is used;

[0073] (B) Protein coating solution;

[0074] (C) Sealing solution;

[0075] (D) Rinse solution;

[0076] (E) Nucleic acid aptamers labeled with fluorescent groups (SEQ ID NO.1);

[0077] (F) Serum amyloid A calibrator.

[0078] 2. Preparation of a kit for detecting serum amyloid A

[0079] (A) 96-well protein-coated plate for immobilizing sample solutions: Use commercially available ELISA plates with protein adsorption capabilities directly.

[0080] (B) Protein coating solution: This is a routine molecular detection reagent with a formula of 35mM NaHCO3, 15mM Na2CO3, pH 9.3-pH 9.9, prepared using double-distilled water.

[0081] (C) Blocking solution: This is a standard molecular diagnostic reagent with the following formula: 136mM NaCl, 2.6mM KCl, 2mM KH2PO4, 8mM Na2HPO4, 0.05% Tween-20, 1% BSA, pH 7.1-pH 7.7, prepared with double-distilled water.

[0082] (D) Rinsing solution: It is a conventional molecular diagnostic reagent with the following formula: 136mM NaCl, 2.6mM KCl, 2mM KH2PO4, 8mM Na2HPO4, 0.05% Tween-20, pH 7.1-pH 7.7, prepared with double-distilled water.

[0083] (E) Nucleic acid aptamers labeled with fluorescent groups: synthesized directly through the gene synthesis service of a routine biotechnology service company, dissolved in TE buffer and prepared to working concentration (200 nM).

[0084] (F) Serum amyloid A calibrator: There are 5 concentrations of serum amyloid A calibrator, namely 6.25 μg / L, 12.5 μg / L, 25 μg / L, 50 μg / L and 100 μg / L, which are prepared by dissolving recombinant serum amyloid A of different concentrations in coating solution.

[0085] Example 4: Detection of serum amyloid A content in samples

[0086] The kit for detecting serum amyloid A described in Example 3 was used to detect the serum amyloid A content in infected samples, including:

[0087] 1. Before use, allow all reagents and samples to return to room temperature, and dilute the sample to be tested to an appropriate concentration with the coating solution.

[0088] 2. Add 100 μL of standard and test sample to the corresponding well and incubate at room temperature for 2 hours. The coating time can be optimized and adjusted according to the actual situation.

[0089] 3. Discard the solution and wash twice with rinsing solution, adding 200 μl of rinsing solution to the sample well each time. After the final wash, remove any remaining rinsing solution by aspiration or pouring.

[0090] 4. Add 200 μL of blocking solution to the sample well and incubate at room temperature for 2 hours.

[0091] 5. Discard the solution. Repeat the washing steps in step 3.

[0092] 6. Add 100 μL of fluorescently labeled nucleic acid aptamers to the sample wells and incubate at room temperature in the dark for 2 hours. The incubation time can be optimized and adjusted according to the actual fluorescence signal intensity detected.

[0093] 7. Discard the solution. Repeat step 3 for a total of 4 washes.

[0094] 8. Use a full-band laser microplate reader to detect the sample wells, with the following settings: excitation wavelength 494nm and detection wavelength 522nm.

[0095] 9. Obtain a standard curve of fluorescence intensity versus target protein concentration using calibrators (specifically as follows). Figure 2 (As shown), the concentration of the target protein in the sample is then calculated based on the fluorescence intensity of the sample well and the sample dilution factor.

[0096] Example 5: Detection of the coefficient of variation of the reagent kit

[0097] Using the kit from Example 3 and the detection method from Example 4, a single clinical serum sample was randomly tested 10 times. The results are as follows:

[0098] Table 2

[0099] Detected concentration (mg / L) 43 42 42 41 44 43 42 43 44 41

[0100] As shown in Table 2, based on the results of 10 repeated tests, the coefficient of variation was calculated to be 2.54%, which is less than 5%, proving that the kit of the present invention has high precision.

[0101] Example 6: Interference Resistance Test of the Reagent Kit

[0102] The kit from Example 3 and the detection method from Example 4 were used, along with a commercially available serum amyloid A antibody detection kit, to perform an anti-interference test. A random clinical serum sample was selected, sterile water or other interfering agents were added, and the target protein concentration was measured. The results are as follows:

[0103] Table 3

[0104]

[0105] As shown in Table 3, after adding interfering substances, the relative error of the kit of this invention to various interfering substances is within 5%, while the relative error of the control kit to various interfering substances is mostly above 10%. The experimental results prove that even if a certain concentration of interfering substances (such as hemoglobin, triglycerides, or bilirubin) is present in the test sample of this kit, it will not have a significant impact on the test results.

[0106] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0107] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0108] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations. sequence list <110> Wuhan Qianmai Medical Laboratory Co., Ltd. <120> A nucleic acid aptamer and kit for detecting serum amyloid A <160> 5 <170> SIPOSequenceListing 1.0 <210> 1 <211> 83 <212> DNA <213> Artificial Sequence <400> 1 gatgacattg cacaagtcag gcctccatga tcgtcgagac cggacgaggc cccgacctga 60 tgggagtgaa tcctgctgtt cga 83 <210> 2 <211> 104 <212> PRT <213> Artificial Sequence <400> 2 Arg Ser Phe Phe Ser Phe Leu Gly Glu Ala Phe Asp Gly Ala Arg Asp 1 5 10 15 Met Trp Arg Ala Tyr Ser Asp Met Arg Glu Ala Asn Tyr Ile Gly Ser 20 25 30 Asp Lys Tyr Phe His Ala Arg Gly Asn Tyr Asp Ala Ala Lys Arg Gly 35 40 45 Pro Gly Gly Val Trp Ala Ala Glu Ala Ile Ser Asp Ala Arg Glu Asn 50 55 60 Ile Gln Arg Phe Phe Gly His Gly Ala Glu Asp Ser Leu Ala Asp Gln 65 70 75 80 Ala Ala Asn Glu Trp Gly Arg Ser Gly Lys Asp Pro Asn His Phe Arg 85 90 95 Pro Ala Gly Leu Pro Glu Lys Tyr 100 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <400> 3 gatgacattg cacaagtcag g 21 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <400> 4 tcgaacagca ggattcactc 20 <210> 5 <211> 315 <212> DNA <213> Artificial Sequence <400> 5 cgctcctttt tctctttcct gggcgaagcg tttgatggtg cacgtgatat gtggcgtgct 60 tatagcgata tgcgtgaagc taactacatc ggctctgaca aatacttcca cgcacgtggt 120 aactacgatg cggcaaaacg tggtccgggt ggtgtttggg cagcagaagc tattagcgat 180 gcacgtgaaa atattcagcg tttctttggt cacggtgcag aagatagcct ggctgatcag 240 gctgcgaacg aatggggtcg ttctggcaaa gacccgaatc atttccgtcc tgcaggtctg 300 ccggaaaaat attaa 315

Claims

1. A nucleic acid aptamer for detecting serum amyloid A, characterized in that, The nucleotide sequence of the nucleic acid aptamer used to detect serum amyloid A is shown in SEQ ID NO.

1.

2. The nucleic acid aptamer according to claim 1, characterized in that, The nucleic acid aptamer for detecting serum amyloid A further includes one of the following: a fluorescent substance, a nanoluminescent material, biotin, digoxigenin, and an enzyme labeling, which are bound to the nucleotide sequence of the nucleic acid aptamer.

3. The nucleic acid aptamer according to claim 2, characterized in that, The fluorescent substance is a FAM fluorescent group or a Cy5 fluorescent group; the nanoluminescent material is a quantum dot or an upconversion nanoparticle; the enzyme label is horseradish peroxidase or sucrase.

4. A kit for detecting serum amyloid A, characterized in that, Includes the nucleic acid aptamer as described in any one of claims 1-3.

5. A kit for detecting serum amyloid A according to claim 4, characterized in that, The kit for detecting serum amyloid A also includes: (A) Protein-coated plate with immobilized sample solution; (B) Protein coating solution; (C) Sealing liquid; (D) Rinse solution; (E) Serum amyloid A calibrator.

6. The kit for detecting serum amyloid A according to claim 5, characterized in that, The protein coating solution was formulated as follows: 35mM NaHCO3, 15mM Na2CO3, pH 9.3-pH 9.

9.

7. A kit for detecting serum amyloid A according to claim 5, characterized in that, The blocking solution is formulated as follows: 136mM NaCl, 2.6mM KCl, 2mM KH2PO4, 8mM Na2HPO4, 0.05% Tween-20, 1% BSA, pH 7.1-pH 7.

7.

8. A kit for detecting serum amyloid A according to claim 5, characterized in that, The rinsing solution is formulated with 136mM NaCl, 2.6mM KCl, 2mM KH2PO4, 8mM Na2HPO4, 0.05% Tween-20, and pH 7.1-pH 7.

7.

9. A kit for detecting serum amyloid A according to claim 5, characterized in that, The serum amyloid A calibrator has five concentrations: 6.25 μg / L, 12.5 μg / L, 25 μg / L, 50 μg / L, and 100 μg / L.

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