Serum amyloid a antibody and use thereof in a test kit

Abstract

The application provides a serum amyloid A (SAA) antibody selected from an antibody a or an antibody b. The application also provides application of the SAA antibody and an SAA detection kit. The SAA antibody provided by the application has high specificity and high affinity, can effectively avoid non-specific cross-reaction, and can be used for preparing a magnetic microparticle chemiluminescence method detection product for detecting SAA. The SAA detection kit provided by the application realizes automatic detection and has the advantages of high sensitivity, high specificity, large flux and the like.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to serum amyloid A antibodies and their applications, as well as serum amyloid A detection kits. Background Technology

[0002] Serum amyloid A (SAA) is a key acute-phase reactant protein synthesized and secreted in large quantities by hepatocytes into the peripheral blood during acute-phase responses such as inflammation, infection, and tissue damage. It belongs to the apolipoprotein family. Under physiological conditions, the SAA content in human blood is extremely low. However, when the body is attacked by pathogens such as bacteria and viruses, or when pathological conditions such as autoimmune diseases, transplant rejection, or secondary amyloidosis occur, the SAA concentration can rapidly increase to 100-1000 times the normal level within 3-6 hours, with a half-life of only about 50 minutes. It can dynamically and sensitively reflect the degree of inflammation and the trend of disease progression.

[0003] Compared to the traditional inflammatory marker C-reactive protein (CRP), which is only sensitive to bacterial infections, SAA is significantly elevated in both bacterial and viral infections, effectively compensating for the shortcomings of CRP in viral infection detection. It has irreplaceable clinical value in the early differential diagnosis, disease monitoring, efficacy evaluation, and prognosis of infectious diseases, and has become a core indicator for clinical inflammation detection. It is widely used in many clinical departments, and there is also a huge clinical demand in primary healthcare and point-of-care testing (POCT).

[0004] Currently, clinical SAA testing primarily employs immunological methods, including enzyme-linked immunosorbent assay (ELISA), latex-enhanced immunoturbidimetry, colloidal gold immunochromatography, fluorescence immunochromatography, and chemiluminescence immunoassay. Specific SAA monoclonal antibodies are the core bioactive materials for these detection technologies, and their affinity, specificity, and stability directly determine the performance of the reagent kits. However, existing SAA detection technologies and antibody reagents still have many technical shortcomings, failing to meet the clinical demands for accurate, rapid, and stable testing. Summary of the Invention

[0005] The purpose of this invention is to provide a serum amyloid A antibody, which targets a specific antigenic epitope of human SAA protein, exhibiting high specificity and high affinity, thereby improving the sensitivity of detection products; the serum amyloid A antibody specifically recognizes serum amyloid A, avoiding cross-reaction with common blood proteins such as CRP and serum proteins.

[0006] The present invention also provides the application of the serum amyloid A antibody in the preparation of serum amyloid A magnetic particle chemiluminescence detection products.

[0007] The present invention also provides a product for the detection of serum amyloid A by magnetic microparticle chemiluminescence, including streptavidin magnetic microparticle solution, acridine sulfonamide labeled antibody solution, biotin labeled antibody solution, calibrators and quality control products, to solve the problems in the prior art.

[0008] To achieve the above objectives, the present invention provides serum amyloid A antibodies, including antibody a or antibody b. The antibody a includes the heavy chain complementarity-determining regions as described below: amino acid sequences as shown in SEQ ID NO.1 CDR1-VH, amino acid sequences as shown in SEQ ID NO.2 CDR2-VH, and amino acid sequences as shown in SEQ ID NO.3 CDR3-VH; The antibody a includes the following light chain complementarity-determining regions: CDR1-VL with amino acid sequences as shown in SEQ ID NO.4, CDR2-VL with amino acid sequences of TAVS, and CDR3-VL with amino acid sequences as shown in SEQ ID NO.5; The antibody b includes the heavy chain complementarity-determining regions as follows: amino acid sequences as shown in SEQ ID NO.6 CDR1-VH, amino acid sequences as shown in SEQ ID NO.7 CDR2-VH, and amino acid sequences as shown in SEQ ID NO.8 CDR3-VH; The antibody b includes the following light chain complementarity-determining regions: CDR1-VL with amino acid sequences as shown in SEQ ID NO.9, CDR2-VL with amino acid sequences of AAVTS, and CDR3-VL with amino acid sequences as shown in SEQ ID NO.10.

[0009] Preferably, the amino acid sequence of the heavy chain variable region of antibody a is shown in SEQ ID NO.11.

[0010] Preferably, the amino acid sequence of the light chain variable region of antibody a is as shown in SEQ ID NO.12.

[0011] Preferably, the amino acid sequence of the heavy chain variable region of antibody b is as shown in SEQ ID NO.13.

[0012] Preferably, the amino acid sequence of the light chain variable region of antibody b is as shown in SEQ ID NO.14.

[0013] Preferably, the amino acid sequence of the light chain constant region of antibody a or antibody b is as shown in SEQ ID NO. 15, and the amino acid sequence of the heavy chain constant region of antibody a or antibody b is as shown in SEQ ID NO. 16.

[0014] Preferably, antibody a is serum amyloid A monoclonal antibody 1, and antibody b is serum amyloid A monoclonal antibody 2; further, antibody a or antibody b is rabbit-derived.

[0015] The present invention also provides the coding sequence of the serum amyloid A antibody described in any of the preceding claims.

[0016] Preferably, the coding sequence of the variable region of the antibody a heavy chain is nucleotides 1 to 348 of the nucleotide sequence shown in SEQ ID NO.17.

[0017] Preferably, the coding sequence of the variable region of the antibody a light chain is nucleotides 1 to 324 of the nucleotide sequence shown in SEQ ID NO.18.

[0018] Preferably, the coding sequence of the variable region of the antibody b heavy chain is nucleotides 1 to 363 of the nucleotide sequence shown in SEQ ID NO.19.

[0019] Preferably, the coding sequence of the variable region of the antibody b light chain is nucleotides 1 to 327 of the nucleotide sequence shown in SEQ ID NO.20.

[0020] Preferably, the coding sequence of the antibody a heavy chain is the nucleotide sequence shown in SEQ ID NO.17.

[0021] Preferably, the coding sequence of the antibody a light chain is the nucleotide sequence shown in SEQ ID NO.18.

[0022] Preferably, the coding sequence of the antibody b heavy chain is the nucleotide sequence shown in SEQ ID NO.19.

[0023] Preferably, the coding sequence of the antibody b light chain is the nucleotide sequence shown in SEQ ID NO.20.

[0024] The present invention also provides a recombinant expression vector comprising any of the coding sequences described above. The recombinant expression vector comprises the coding sequence of antibody a and / or the coding sequence of antibody b.

[0025] Preferably, the recombinant expression vector contains the coding sequence for the variable region of the antibody α light chain.

[0026] Preferably, the recombinant expression vector contains the coding sequence of the variable region of the antibody α heavy chain.

[0027] Preferably, the recombinant expression vector contains a coding sequence for the variable region of the antibody b light chain.

[0028] Preferably, the recombinant expression vector contains the coding sequence of the antibody b heavy chain variable region.

[0029] Preferably, the recombinant expression vector contains the coding sequence of the antibody α light chain.

[0030] Preferably, the recombinant expression vector contains an antibody α heavy chain coding sequence.

[0031] Preferably, the recombinant expression vector contains the coding sequence of the antibody b light chain.

[0032] Preferably, the recombinant expression vector contains an antibody b heavy chain coding sequence.

[0033] Preferably, the recombinant expression vector is constructed by linking the coding sequence provided by the present invention to various expression vectors using conventional methods in the art. The expression vector can be any conventional vector in the art, as long as it can accommodate nucleic acid molecules containing the coding sequence, it is suitable for the present invention. The expression vector preferably includes various plasmids, granules, bacteriophages, or viral vectors, such as pCDNA3.4, pCDNA3.1 series vectors, etc. The expression vector is preferably a eukaryotic expression vector, and more preferably an expression vector suitable for mammalian cells.

[0034] The present invention also provides a host cell comprising any of the recombinant expression vectors described above or having the coding sequences described above integrated into the genome of the host cell. The host cell is preferably a mammalian cell.

[0035] In a preferred embodiment of the present invention, the recombinant expression vector is preferably a eukaryotic expression vector, and the host cell is preferably a mammalian cell. Further, the recombinant expression vector is preferably pCDNA3.4, and the host cell is preferably a 293T cell.

[0036] The serum amyloid A antibody described in this invention can be a monoclonal antibody prepared via hybridoma, or an antibody obtained using any of the recombinant expression vectors or host cells described above. A recombinant expression vector containing the antibody a heavy chain coding sequence and a recombinant expression vector containing the antibody a light chain coding sequence are co-transfected into host cells (preferably mammalian cells) in equal proportions. The recombinant expression vectors containing the antibody a heavy chain coding sequence and the antibody a light chain coding sequence undergo transcription, translation, and various modifications within the host cells to form an antibody, which is then purified to obtain antibody a. Similarly, a recombinant expression vector containing the antibody b heavy chain coding sequence and a recombinant expression vector containing the antibody b light chain coding sequence are co-transfected into host cells (preferably mammalian cells) in equal proportions. The recombinant expression vectors containing the antibody b heavy chain coding sequence and the antibody b light chain coding sequence undergo transcription, translation, and various modifications within the cells to form an antibody, which is then purified to obtain antibody b. In this invention, both the monoclonal antibody obtained via hybridoma and the antibody obtained via recombinant expression can specifically recognize serum amyloid A, while avoiding cross-contamination with CRP and serum proteins.

[0037] The present invention provides the application of the serum amyloid A antibody, the coding sequence of the serum amyloid A antibody, the recombinant expression vector, or the host cell described above in any of the above-described methods in the preparation of a serum amyloid A magnetic microparticle chemiluminescence detection product.

[0038] This invention provides a serum amyloid A detection kit, which includes streptavidin magnetic microparticle solution, acridine sulfonamide labeled antibody solution, biotin labeled antibody solution, calibrators, and quality control products.

[0039] The kit provided by this invention can effectively detect serum amyloid A at concentrations of 1–200 mg / L. Further, the detection range is preferably 1–20 mg / L.

[0040] Preferably, the acridine sulfonamide-labeled antibody solution contains the serum amyloid A antibody described in any of the preceding claims, wherein the serum amyloid A antibody is labeled with acridine sulfonamide.

[0041] Preferably, the biotin-labeled antibody solution contains the serum amyloid A antibody described in any of the preceding claims, wherein the serum amyloid A antibody is labeled with biotin.

[0042] Preferably, the acridine sulfonamide-labeled antibody solution and the biotin-labeled antibody solution contain different serum amyloid A antibodies.

[0043] In a preferred embodiment of the present invention, the serum amyloid A antibody on the acridine sulfonamide-labeled antibody solution is antibody a, and the serum amyloid A antibody on the biotin-labeled antibody solution is antibody b; in a preferred embodiment of the present invention, the serum amyloid A antibody on the acridine sulfonamide-labeled antibody solution is antibody b, and the serum amyloid A antibody on the biotin-labeled antibody solution is antibody a.

[0044] Preferably, the biotin-labeled antibody solution contains 0.5 to 1.5 μg / mL of the serum amyloid A antibody; more preferably, it contains 0.5, 1.0, 1.5 μg / mL or a range thereof.

[0045] Preferably, the acridine sulfonamide-labeled antibody solution contains 0.5 to 1.5 μg / mL of the serum amyloid A antibody. More preferably, the concentration is 0.5, 1.0, 1.5 μg / mL or a range thereof.

[0046] Preferably, the serum amyloid A antibody diluent is a 10-50 mM Tris-HCl buffer containing BSA, with each 100 mL of the diluent containing 0.1-1.0 g of BSA. More preferably, the concentration of the Tris-HCl buffer is 10, 20, 30, 40, 50 mM or a range thereof; more preferably, each 100 mL of the diluent contains 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 g of BSA or a range thereof. The serum amyloid A antibody diluent is either the diluent for the acridine sulfonamide-labeled antibody solution or the diluent for the biotin-labeled antibody solution.

[0047] Preferably, the concentration of the streptavidin magnetic microparticle solution is 0.1~0.5 mg / mL, and more preferably 0.1, 0.3, 0.5 mg / mL or a range thereof.

[0048] Preferably, the calibrator is a lyophilized serum amyloid A product, and preferably also includes lyophilized excipients. Preferably, it is reconstituted with pure water before use.

[0049] Preferably, the quality control product is a lyophilized serum amyloid A product, and preferably also includes lyophilized excipients. Preferably, it is reconstituted with pure water before use.

[0050] Preferably, the serum amyloid A lyophilized product is serum amyloid A prepared using existing technology, or a commercially available serum amyloid A protein product. This invention utilizes serum amyloid A from Beijing Baixinyi Biotechnology Co., Ltd., but it should be noted that this invention is not limited to the source and preparation method of serum amyloid A.

[0051] Preferably, the freeze-drying excipients are conventional freeze-drying excipients in the prior art, preferably including sugars such as trehalose, sucrose, and lactose; preferably including polyols such as mannitol and sorbitol; preferably including amino acids such as glycine and histidine; preferably including proteins such as bovine serum albumin (BSA); preferably including gelatin; preferably including buffer systems such as phosphate-buffered saline (PBS) and Tris-HCl; preferably including surfactants such as Tween-20 and polyvinylpyrrolidone (PVP); preferably including preservatives such as Proclin 300 and sodium azide.

[0052] Compared with the prior art, the present invention has the following beneficial effects: The serum amyloid A antibody provided by this invention has good specificity and high affinity for serum amyloid A, avoiding cross-contamination with common blood proteins such as CRP and serum proteins. It can be used to prepare magnetic particle chemiluminescence detection products for detecting serum amyloid A.

[0053] The serum amyloid A detection kit provided by this invention has the advantages of automated detection, high sensitivity, strong specificity, and high throughput. Attached Figure Description

[0054] Figure 1 This is an electrophoresis diagram of serum amyloid A monoclonal antibody in a preferred embodiment of the present invention.

[0055] Figure 2 The serum amyloid A detection kits in preferred embodiments 2 and 3 of this invention are examples of the present invention.

[0056] Figure 3 The serum amyloid A detection reagents in preferred embodiments 2 and 3 of the present invention are as follows. Detailed Implementation

[0057] The present invention will be further described in detail below through specific embodiments.

[0058] It should be noted that the "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of either the heavy or light chain. The variable domain of the heavy chain can be referred to as "VH," and the variable domain of the light chain as "VL." These domains are typically the most variable parts of the antibody and contain antigen-binding sites. The variable region of the light or heavy chain consists of a framework region interrupted by three hypervariable regions called "complementarity-determining regions" or "CDRs." The framework region of the antibody, that is, the framework region of the combination of the light and heavy chains, plays a role in locating and aligning the CDRs, which are primarily responsible for binding to the antigen.

[0059] The “framework” or “FR” region refers to the region outside of those defined as CDRs of the antibody variable domain. Each antibody variable domain framework can be further subdivided into adjacent regions separated by CDRs (FR1, FR2, FR3, and FR4).

[0060] Typically, the variable regions VL / VH of heavy and light chains can be obtained by connecting the following numbered CDRs with FRs in the following combination: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0061] In this invention, CDR1-VH, CDR2-VH and CDR3-VH refer to the three highly variable regions of the heavy chain variable region, and correspondingly, CDR1-VL, CDR2-VL and CDR3-VL refer to the three highly variable regions of the light chain variable region.

[0062] In a first aspect, the present invention provides a serum amyloid A antibody, selected from antibody a and / or antibody b; The antibody a includes heavy chain complementarity-determining regions CDR1-VH, CDR2-VH, and CDR3-VH with amino acid sequences as shown in SEQ ID NO.1 to SEQ ID NO.3, and light chain complementarity-determining regions CDR1-VL, CDR2-VL with amino acid sequence TAVS, and CDR3-VL with amino acid sequence as shown in SEQ ID NO.4. The antibody b includes heavy chain complementarity-determining regions CDR1-VH, CDR2-VH, and CDR3-VH with amino acid sequences as shown in SEQ ID NO. 6 to SEQ ID NO. 8, light chain complementarity-determining regions CDR1-VL with amino acid sequences as shown in SEQ ID NO. 9, light chain complementarity-determining regions CDR2-VL with amino acid sequences of AAVTS, and light chain complementarity-determining regions CDR3-VL with amino acid sequences as shown in SEQ ID NO. 10.

[0063] The amino acid sequences shown in SEQ ID NO.1 to SEQ ID NO.10 are shown in Table 1.

[0064] Table 1

[0065] The serum amyloid A antibody provided by this invention has good specificity, high biological activity, and high affinity for serum amyloid A, and can be used to prepare products for detecting serum amyloid A.

[0066] In a preferred embodiment of the present invention, the antibody a includes a heavy chain variable region and a light chain variable region, with amino acid sequences as shown in SEQ ID NO.11 and SEQ ID NO.12, respectively. The antibody b includes a heavy chain variable region and a light chain variable region, with amino acid sequences as shown in SEQ ID NO.13 and SEQ ID NO.14, respectively. Preferably, the serum amyloid A antibody is rabbit-derived.

[0067] The amino acid sequences shown in SEQ ID NO.11 to SEQ ID NO.14 are shown in Table 2.

[0068] Table 2

[0069] The heavy chain of antibody a, namely serum amyloid A monoclonal antibody 1, consists of the variable region and constant region of the heavy chain of serum amyloid A monoclonal antibody 1, and the light chain of serum amyloid A monoclonal antibody 1 consists of the variable region and constant region of the light chain of serum amyloid A monoclonal antibody 1. Similarly, the heavy chain of antibody b, namely serum amyloid A monoclonal antibody 2, consists of the variable region and constant region of the heavy chain of serum amyloid A monoclonal antibody 2, and the light chain of serum amyloid A monoclonal antibody 2 consists of the variable region and constant region of the light chain of serum amyloid A monoclonal antibody 2. The amino acid sequence of the constant region of the light chain of antibody a or antibody b is shown in SEQ ID NO. 15, and the amino acid sequence of the constant region of the heavy chain of antibody a or antibody b is shown in SEQ ID NO. 16, as shown in Table 3. Accordingly, the amino acid sequence of the heavy chain of antibody a is composed of the amino acid sequences shown in SEQ ID NO.11 and SEQ ID NO.16 in sequence, and the amino acid sequence of the light chain of antibody a is composed of the amino acid sequences shown in SEQ ID NO.12 and SEQ ID NO.15 in sequence; the amino acid sequence of the heavy chain of antibody b is composed of the amino acid sequences shown in SEQ ID NO.13 and SEQ ID NO.16 in sequence, and the amino acid sequence of the light chain of antibody b is composed of the amino acid sequences shown in SEQ ID NO.14 and SEQ ID NO.15 in sequence.

[0070] Table 3

[0071] Secondly, the present invention provides the application of the serum amyloid A antibody in the preparation of serum amyloid A magnetic particle chemiluminescence detection products.

[0072] Thirdly, the present invention provides a serum amyloid A detection kit, the detection kit comprising streptavidin magnetic microparticle solution, acridine sulfonamide labeled antibody solution, biotin labeled antibody solution, calibrators, and quality control products.

[0073] Preferably, the concentration of the streptavidin magnetic microparticle solution is 0.1~0.5 mg / mL.

[0074] Preferably, the acridine sulfonamide-labeled antibody solution contains the serum amyloid A antibody described in any one of the above-mentioned embodiments, and the serum amyloid A antibody is labeled with acridine sulfonamide, the concentration of which is identified by a detection instrument.

[0075] Preferably, the acridine sulfonamide-labeled antibody solution contains 0.5~1.5 μg / mL of the serum amyloid A antibody.

[0076] Preferably, the biotin-labeled antibody solution contains 0.5~1.5 μg / mL of the serum amyloid A antibody.

[0077] Preferably, the diluent for the biotin-labeled antibody solution and the acridine sulfonamide-labeled antibody solution is a 10-50 mM Tris-HCl buffer containing BSA, with each 100 mL of the diluent containing 0.1-1.0 g of BSA.

[0078] In a preferred embodiment of the present invention, the serum amyloid A antibody on the acridine sulfonamide-labeled antibody solution is different from the serum amyloid A antibody contained in the biotin-labeled antibody solution.

[0079] In a preferred embodiment of the present invention, the antibody in the acridine sulfonamide-labeled antibody solution is antibody a, which is serum amyloid A monoclonal antibody 1, and the antibody in the biotin-labeled antibody solution is antibody b, which is serum amyloid A monoclonal antibody 2.

[0080] In a preferred embodiment of the present invention, the antibody in the acridine sulfonamide-labeled antibody solution is antibody b, and the antibody in the biotin-labeled antibody solution is antibody a.

[0081] Preferably, the calibrator is a lyophilized serum amyloid A product, and preferably also includes lyophilized excipients. Preferably, it is reconstituted with pure water before use.

[0082] Preferably, the quality control product is a lyophilized serum amyloid A product, and preferably also includes lyophilized excipients. Preferably, it is reconstituted with pure water before use.

[0083] Preferably, the lyophilized serum amyloid A product is serum amyloid A prepared using existing technology, or a commercially available serum amyloid A protein product. This invention uses serum amyloid A from Beijing Baixinyi Biotechnology Co., Ltd., but this invention is not limited to the source and preparation method of serum amyloid A.

[0084] Preferably, the freeze-drying excipients are conventional freeze-drying excipients in the prior art, preferably including sugars such as trehalose, sucrose, and lactose; preferably including polyols such as mannitol and sorbitol; preferably including amino acids such as glycine and histidine; preferably including proteins such as bovine serum albumin (BSA); preferably including gelatin; preferably including buffer systems such as phosphate-buffered saline (PBS) and Tris-HCl; preferably including surfactants such as Tween-20 and polyvinylpyrrolidone (PVP); preferably including preservatives such as Proclin 300 and sodium azide.

[0085] The serum amyloid A detection kit provided by this invention has the advantages of automated operation, high sensitivity, strong specificity, and high throughput.

[0086] The reagent provided by this invention is based on the principle of antigen-antibody reaction. It involves labeling the serum amyloid A antibody with acridine sulfonamide, then preparing a sulfidine sulfonamide-labeled antibody solution using a diluent. The biotin-labeled antibody solution contains another biotin-labeled antibody against serum amyloid A. Based on the principle of antigen-antibody reaction, the reaction can be detected using a matching instrument. If serum amyloid A is present in the sample, a double-antibody sandwich structure is formed, and the instrument will detect a strong light signal. If serum amyloid A is not present in the sample, the instrument will detect a weaker light signal. The analyte loading is predicted based on the intensity of the light signal.

[0087] Using the reagent kit provided by this invention, the entire process can yield test results in 30 minutes, which is fast and efficient. This helps medical personnel obtain test results in a timely manner, make comprehensive judgments based on the results, and take timely actions.

[0088] It should be noted that the diluent for the serum amyloid A antibody described in this invention is a 10-50 mM Tris-HCl buffer containing BSA, with each 100 mL of the diluent containing 0.1-1.0 g of BSA. Unless otherwise specified in the following embodiments, preferably, the diluent for the serum amyloid A antibody is a 20 mM Tris-HCl buffer containing BSA, with each 100 mL of the diluent containing 0.5 g of BSA.

[0089] The methods for preparing serum amyloid A monoclonal antibody 1 (as shown in antibody a) and serum amyloid A monoclonal antibody 2 (as shown in antibody b) of this invention are conventional methods for preparing monoclonal antibodies in the prior art, and are briefly described below: (1) Animal immunization: A: Serum amyloid A purchased from Beijing Baixinyi Biotechnology Co., Ltd. was mixed with Freund's adjuvant in equal volumes to a suitable volume and completely emulsified. New Zealand white rabbits were immunized by subcutaneous multi-point injection. Each New Zealand white rabbit was injected with 200 μg of immunogen, once every two weeks. B: After 6 immunizations, serum amyloid A was coated using the indirect ELISA method, and the antibody titer in the serum was tested. Rabbits with an OD value greater than 1.0 in a 100,000-fold serum dilution were selected.

[0090] (2) Preparation of monoclonal antibodies: A: Spleen cells from New Zealand white rabbits were fused with myeloma cells, and the fused cells were plated and cultured using the limiting dilution method. B: Screening and culturing monoclonal cell wells, the monoclonal cell wells with the highest OD values ​​detected in the cell culture supernatant are selected as target hybridoma cells, cultured and expanded to obtain hybridoma cell lines and cryopreserved. C: Isolation of antibody variable region gene from hybridoma cells using RT-PCR: After homogenizing hybridoma cells, add cell lysis buffer for RNA extraction, precipitate RNA from the aqueous phase with isopropanol, wash the precipitated RNA after centrifugation to remove impurities, resuspend and reverse transcribe to obtain cDNA. D: PCR was performed using known New Zealand white rabbit-specific primers. Hybridoma cell cDNA was used as a template to amplify the variable regions of the antibody's heavy and light chains. A 50 μL system contained 5 μL cDNA, HotStarTaqPlus enzyme, dNTPs, and 0.5 μM specific primers. PCR amplification was performed under the following conditions: pre-denaturation at 94℃ for 5 min; 35 cycles of 94℃ for 30 s, 55℃ for 30 s, and 72℃ for 50 s; followed by 72℃ for 7 min. The obtained PCR products were identified by 1% agarose gel electrophoresis. The target fragment was recovered and sequenced. Based on the sequenced antibody gene sequence, the amino acid sequences of the antibody variable regions, as shown in SEQ ID NO: 11-14, were further obtained. Specifically, the amino acid sequence of the light chain variable region of antibody a is shown in SEQ ID NO: 12, the amino acid sequence of the heavy chain variable region of antibody a is shown in SEQ ID NO: 11, the amino acid sequence of the light chain variable region of antibody b is shown in SEQ ID NO: 14, and the amino acid sequence of the heavy chain variable region of antibody b is shown in SEQ ID NO: 14. As shown in NO.13. The amino acid sequence of the light chain constant region of antibody a or antibody b is shown in SEQ ID NO.15, and the amino acid sequence of the heavy chain constant region of antibody a or antibody b is shown in SEQ ID NO.16.

[0091] E: Constructing expression vectors for monoclonal antibodies: Based on the antibody gene sequence obtained from step D, an expression vector for monoclonal antibodies was constructed using conventional methods in the art. Homologous recombination arms were added to both ends of the variable region gene of the antibody heavy chain and the variable region gene of the light chain, respectively, using homologous recombination primers. The expression plasmid containing the constant regions of the rabbit antibody heavy and light chains IgG1 was linearized using a dual-enzyme approach to generate homologous recombination arms. The variable region gene fragment with added homologous recombination arms and the linearized plasmid were ligated together via homologous recombination to form a complete expression vector, pCDNA3.4. The recombination product was transformed into TOP10 E. coli competent cells to amplify the plasmid, resulting in pCDNA3.4-antibody a heavy chain plasmid, pCDNA3.4-antibody a light chain plasmid, pCDNA3.4-antibody b heavy chain plasmid, and pCDNA3.4-antibody b light chain plasmid.

[0092] The gene fragment encoding the antibody a heavy chain inserted into the pCDNA3.4 vector is the nucleotide sequence shown in SEQ ID NO.17, wherein nucleotides 1 to 348 are the coding sequence for the variable region of the antibody a heavy chain, and nucleotides 349 to 1317 are the coding sequence for the constant region of the antibody a heavy chain.

[0093] The gene fragment encoding the antibody a light chain inserted into the pCDNA3.4 vector is the nucleotide sequence shown in SEQ ID NO.18, wherein nucleotides 1 to 324 are the coding sequence for the variable region of the antibody a light chain, and nucleotides 325 to 636 are the coding sequence for the constant region of the antibody a light chain.

[0094] The gene fragment encoding the antibody b heavy chain inserted into the pCDNA3.4 vector is the nucleotide sequence shown in SEQ ID NO.19, wherein nucleotides 1 to 363 are the coding sequence for the variable region of the antibody b heavy chain, and nucleotides 364 to 1332 are the coding sequence for the constant region of the antibody b heavy chain.

[0095] The gene fragment encoding the antibody b light chain inserted into the pCDNA3.4 vector is the nucleotide sequence shown in SEQ ID NO.20, wherein nucleotides 1 to 327 are the coding sequence for the variable region of the antibody b light chain, and nucleotides 328 to 639 are the coding sequence for the constant region of the antibody b light chain.

[0096] F: Expression and purification of monoclonal antibodies: The monoclonal antibody heavy and light chain expression plasmids obtained in step E were added to Opti-Mem transfection medium at a 1:1 ratio. After thorough mixing, PEI transfection reagent (4 times the mass of DNA) was added. The mixture was then incubated at room temperature in the dark for 10 min, followed by incubation in 293T cells. After 6 h of incubation, the transfection system was removed, and FreeStyle™ 293 expression medium was added. After 5 days of culture, the cell culture supernatant was collected. The expressed cell culture supernatant was purified using affinity purification (Protein A) to obtain the monoclonal antibody. The specific steps are as follows: (1) Centrifuge the expressed antibody supernatant at 2500×g at room temperature for 10 min to remove the precipitate; (2) Wash the affinity purification column containing Protein A thoroughly with 10 volumes of binding buffer; (3) Pass the expression supernatant through the purification column at a flow rate of 5 mL / min; (4) Wash the purification column thoroughly with 20 times the column volume of binding buffer; (5) Elute the purification column with 0.1 M citrate buffer (pH=3.0-3.5) until the elution peak reaches equilibrium, and adjust the pH to 7.0 with 1 M Tris-HCl buffer (pH=9.0). (6) The purified monoclonal antibody was concentrated using a concentrated centrifuge column, PBS was used as the antibody preservation buffer, and finally the concentration of the concentrated antibody was determined using an ultra-micro UV spectrophotometer.

[0097] In a preferred embodiment of the present invention, pCDNA3.4-antibody a heavy chain plasmid and pCDNA3.4-antibody a light chain plasmid are co-transfected at a 1:1 ratio and expressed and purified according to the method in step F to obtain antibody a. pCDNA3.4-antibody b heavy chain plasmid and pCDNA3.4-antibody b light chain plasmid are expressed and purified according to the method in step F to obtain antibody b. The obtained antibodies a and b are verified by SDS-PAGE and ELISA respectively using the methods described in Example 1. Both antibodies a and b show two characteristic bands of approximately 25 KD and 50 KD, representing the light and heavy chains of IgG, respectively. The titer determination by indirect ELISA confirms that both antibodies a and b can specifically recognize serum amyloid A.

[0098] The methods used in this invention for antibody preparation, antibody gene sequencing, construction and identification of monoclonal antibody expression vectors are all conventional methods in molecular biology. The primers and other related sequences involved can be obtained from existing technologies such as gene databases or existing literature, or from the heavy chain or light chain target gene fragments of antibody a or b, such as those shown in SEQ ID NO.17 to SEQ ID NO.20, which will not be elaborated here.

[0099] Example 1 The serum amyloid A antibodies include: serum amyloid A monoclonal antibody 1 (antibody a) and serum amyloid A monoclonal antibody 2 (antibody b). The variable region sequences of the above antibodies are shown in Table 2, and the constant region sequences are shown in Table 3.

[0100] A 12% SDS-PAGE gel was prepared using standard methods. The above-mentioned antibodies were loaded at 5 μg, and electrophoresis was performed using protein molecular weight standards as a reference. The results showed that the two serum amyloid A antibodies exhibited two characteristic bands of approximately 25 KD and 50 KD, representing the light and heavy chains of IgG, respectively. Figure 1 The antibody content of all bands was above 90% after scanning and analysis.

[0101] Figure 1 In the diagram, lane 1 is the marker; lane 2 is the serum amyloid A monoclonal antibody 1; and lane 3 is the serum amyloid A monoclonal antibody 2.

[0102] The antibodies used in the following examples are the same as those in Example 1.

[0103] Verification of the specificity of serum amyloid A monoclonal antibody: The interference of hemoglobin, bilirubin, and triglycerides on the detection results was investigated. The method is as follows: Samples with detection results of 5 mg / L, 20 mg / L, and 100 mg / L were selected respectively, and hemoglobin, bilirubin, and triglycerides of different final concentrations were added to them respectively. The reagents prepared by the antibody of this invention were used for detection, and the absolute value of the relative deviation of concentration >10% was used as the criterion for the existence of interference.

[0104] Table 4

[0105] The results showed that, under the conditions of a final hemoglobin concentration of 7 mg / mL, a final bilirubin concentration of 300 mg / L, and a final triglyceride concentration of 7.5 mmol / L, the absolute value of the relative deviation of the concentration of each sample did not exceed 10%, indicating that hemoglobin, bilirubin, and triglycerides did not significantly interfere with the detection of serum amyloid A mediated by this antibody.

[0106] Example 2 A serum amyloid A detection kit, such as Figure 2 As shown, BAM is the main reagent, CAL is the calibrator, and QC is the quality control reagent. The main reagent is as follows: Figure 3 As shown, M is a streptavidin magnetic microparticle solution, B is a biotin-labeled antibody solution, and A is an acridine sulfonamide-labeled antibody solution. The biotin-labeled antibody solution comprises biotin-labeled antibody a and a diluent. The acridine sulfonamide-labeled antibody solution comprises acridine sulfonamide-labeled antibody b and a diluent. The calibrators and quality control samples are lyophilized products, comprising serum amyloid A and lyophilized excipients.

[0107] The diluent for antibody a or antibody b is a 10-50 mM Tris-HCl buffer containing BSA, with each 100 mL of the diluent containing 0.1-1.0 g of BSA.

[0108] Example 3 Example 3 provides a serum amyloid A detection kit.

[0109] 1. Main Materials 1.1 Antibodies: The monoclonal antibodies are all serum amyloid A antibodies (antibody a and antibody b) as described in this invention. They are rabbit-derived monoclonal antibodies and are used to label biotin and acridine sulfonamide, respectively.

[0110] 1.2 Streptavidin magnetic microparticles: JSR.

[0111] 1.3 Acridine sulfonamide NSP-SA-NHS: Herlison (Xiamen) Biotechnology Co., Ltd.

[0112] 1.4 Sulfo-NHS-LC-Biotin: Thermo.

[0113] 1.5 Other consumables: The chemiluminescence reagent rack kit is provided by Kelais Biotechnology (Chongqing) Co., Ltd.; all commonly used reagents are analytical grade reagents.

[0114] 1.6 Serum amyloid A: Purchased from Beijing Baixinyi Biotechnology Co., Ltd., catalog number A10E03.

[0115] 2 Methods 2.1 Preparation of biotin-labeled antibody solution: The steps for preparing biotin-labeled antibody solution are as follows: (1) Take 1 mg of the antibody to be labeled and place it in a glass bottle. Dilute the antibody to be labeled to 1 mg / mL with 0.01 M PBS (pH 7.2) buffer. (2) Take 18.5 μL of 10 mg / mL Sulfo-NHS-LC-Biotin solution and add it to the above antibody-containing PBS buffer. Mix well and keep at room temperature for 2 hours in the dark. Dialyze with 0.01 M PBS (pH 7.2) buffer at 2-8°C in the dark for 5-10 times (2 h interval between each time). (3) Dilute the labeled product to 0.5~1.5 μg / mL with a diluent; the diluent is a 10~50mM Tris-HCl buffer containing BSA, and each 100 mL of the diluent contains 0.1~1.0 g of BSA.

[0116] (4) Dispense the contents at 4 mL / bottle.

[0117] 2.2 Preparation of acridine sulfonamide-labeled antibody solution: The preparation steps for acridine sulfonamide-labeled antibody solution are as follows: (1) Take 1 mg of the antibody to be labeled and place it in a glass bottle. Dilute the antibody to be labeled to 1 mg / mL with 0.1 M CBS (pH 9.0); (2) Take 91 μL of 2 mg / mL acridine sulfonamide NSP-SA-NHS and add it to the above-mentioned antibody-containing CBS buffer. Mix well and keep at room temperature for 2 hours in the dark. Dialyze with 0.01 M PBS (pH 7.2) buffer at 2-8°C in the dark for 5-10 times (2 h apart each time). (3) Dilute the labeled product to 0.5~1.5 μg / mL with a diluent; the diluent is a 10~50mM Tris-HCl buffer containing BSA, and each 100 mL of the diluent contains 0.1~1.0 g of BSA.

[0118] (4) Dispense the contents at 4 mL / bottle.

[0119] 2.3 Preparation of streptavidin magnetic microparticle solution: Dilute streptavidin magnetic microparticles to 0.1–0.5 mg / mL using a diluent; dispense in 6 mL vials. The diluent is a 10–50 mM Tris-HCl buffer containing BSA, with each 100 mL of diluent containing 0.1–1.0 g of BSA.

[0120] 2.4 Preparation of calibrators and quality control samples: Serum amyloid A was diluted to two concentrations, 5 mg / L and 100 mg / L, using lyophilization solution. The solutions were then aliquoted into 0.6 mL vials and lyophilized. The 5 mg / L sample served as a low-value calibrator and a low-value quality control, while the 100 mg / L sample served as a high-value calibrator and a high-value quality control.

[0121] 2.5 Assembly of the reagent kit: refer to Figure 2 The layout involves placing the biotin-labeled antibody solution, acridine sulfonamide-labeled antibody solution, streptavidin magnetic microparticle solution, calibrators, and quality control materials in their respective locations.

[0122] 2.6 Testing: Step 1: Take out the sample to be tested and allow it to equilibrate to room temperature; preferably, the sample of the present invention is serum.

[0123] Step 2: Remove the refrigerated reagent kit and... Figure 3 The reagent shown is installed in the reagent compartment of the analyzer; Step 3: Reconstitute the calibrator and quality control sample with 600 μL of pure water, and then take 60 μL for a 10-fold dilution; Step 4: Place the reconstituted and diluted calibrators, quality control samples, and test samples onto the instrument for testing; Step 5: Pipette 50-100 μL of calibrator, quality control sample or test sample into a reaction vessel, then add 10-30 μL of biotin-labeled antibody solution and 10-30 μL of acridine sulfonamide-labeled antibody, mix well and incubate at 37°C for 5-25 min. Step 6: Add 10-50 μL of streptavidin magnetic microparticle solution, mix well, and incubate at 37°C for 2-10 min; Step 7: After washing, add pre-excitation solution and excitation solution, and the analyzer detects the luminescence value.

[0124] 3. Results: The analyzer automatically calibrates its built-in curve based on the luminescence value detected by the calibrator to obtain the calibration curve; the concentration of serum amyloid A in the quality control sample and the sample to be tested is calculated through the calibration curve.

[0125] In Example 3, the diluent was a 10-50 mM Tris-HCl buffer containing BSA, with each 100 mL of the diluent containing 0.1-1.0 g of BSA; the lyophilized solution was a 10-50 mM Tris-HCl buffer containing mannitol, BSA, and PVP, with each 100 mL of the diluent containing 1-10 g of mannitol, 0.1-1.0 g of BSA, and 0.02-0.5 g of PVP.

[0126] In Example 3, the preferred instrument used is the fully automated chemiluminescence immunoassay analyzer from CLASS Biotechnology (Chongqing) Co., Ltd., model: Venus 100S.

[0127] Example 4 Example 4 provides the kit detection process.

[0128] Sample preparation: Serum amyloid A was diluted to 500, 200, 100, 50, 10, 5.0, and 1.0 mg / L with 0.01 M PBS containing 1% BSA.

[0129] Sample testing: The reconstituted calibrators, quality control samples and test samples were tested using an analyzer according to the method described in Example 3.

[0130] Example 4 used a fully automated chemiluminescence immunoassay analyzer, model: Venus 100S, from Claris Biotechnology (Chongqing) Co., Ltd. Calculations were performed using the instrument's built-in program. The built-in curve fitting method was a double logarithmic four-parameter curve fitting (automatic fitting by the device software system); the calibration method was a two-point segmented calibration; and the sample concentration was calculated by substituting the sample detection light signal into the calibration curve. Specifically, the built-in curve was imported and calibrated to obtain the calibration curve. The concentration values ​​of serum amyloid A in the quality control sample and the test sample were calculated using the calibration curve. The detection results are shown in Table 5.

[0131] Table 5. Serum amyloid A test results

[0132] The above embodiments are merely illustrative examples and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A serum amyloid A antibody, including antibody a or antibody b, characterized in that, The antibody a includes the heavy chain complementarity-determining regions as described below: amino acid sequences as shown in SEQ ID NO.1 (CDR1-VH), amino acid sequences as shown in SEQ ID NO.2 (CDR2-VH), and amino acid sequences as shown in SEQ ID NO.3 (CDR3-VH). The antibody a includes the following light chain complementarity-determining regions: CDR1-VL with amino acid sequences as shown in SEQ ID NO.4, CDR2-VL with amino acid sequences of TAVS, and CDR3-VL with amino acid sequences as shown in SEQ ID NO.5; The antibody b includes the heavy chain complementarity-determining regions as follows: amino acid sequences as shown in SEQ ID NO.6 CDR1-VH, amino acid sequences as shown in SEQ ID NO.7 CDR2-VH, and amino acid sequences as shown in SEQ ID NO.8 CDR3-VH; The antibody b includes the following light chain complementarity-determining regions: CDR1-VL with amino acid sequences as shown in SEQ ID NO.9, CDR2-VL with amino acid sequences of AAVTS, and CDR3-VL with amino acid sequences as shown in SEQ ID NO.

10.

2. The serum amyloid A antibody as described in claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of antibody a is shown in SEQ ID NO.11; the amino acid sequence of the light chain variable region of antibody a is shown in SEQ ID NO.

12.

3. The serum amyloid A antibody as described in claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of antibody b is shown in SEQ ID NO.13; the amino acid sequence of the light chain variable region of antibody b is shown in SEQ ID NO.

14.

4. The coding sequence of the serum amyloid A antibody according to any one of claims 1 to 3, characterized in that, The coding sequence of the variable region of the heavy chain of antibody a is nucleotides 1 to 348 of the nucleotide sequence shown in SEQ ID NO.17; the coding sequence of the variable region of the light chain of antibody a is nucleotides 1 to 324 of the nucleotide sequence shown in SEQ ID NO.18; the coding sequence of the variable region of the heavy chain of antibody b is nucleotides 1 to 363 of the nucleotide sequence shown in SEQ ID NO.19; and the coding sequence of the variable region of the light chain of antibody b is nucleotides 1 to 327 of the nucleotide sequence shown in SEQ ID NO.

20.

5. The encoded sequence as described in claim 4, characterized in that, The coding sequence of the antibody a heavy chain is the nucleotide sequence shown in SEQ ID NO. 17; the coding sequence of the antibody a light chain is the nucleotide sequence shown in SEQ ID NO. 18; the coding sequence of the antibody b heavy chain is the nucleotide sequence shown in SEQ ID NO. 19; and the coding sequence of the antibody b light chain is the nucleotide sequence shown in SEQ ID NO.

20.

6. A recombinant expression vector comprising the coding sequence of claim 4 or 5.

7. A host cell, characterized in that, The host cell comprises the recombinant expression vector of claim 6 or the genome of the host cell integrates the coding sequence of claim 4 or 5.

8. The application of the serum amyloid A antibody according to any one of claims 1 to 3, or the coding sequence of the serum amyloid A antibody according to claim 4 or 5, or the recombinant expression vector according to claim 6, or the host cell according to claim 7, in the preparation of a serum amyloid A magnetic microparticle chemiluminescence detection product.

9. A serum amyloid A detection kit, the kit comprising streptavidin magnetic microparticle solution, acridine sulfonamide-labeled antibody solution, biotin-labeled antibody solution, calibrators, and quality control materials, characterized in that, The acridine sulfonamide-labeled antibody solution contains the serum amyloid A antibody according to any one of claims 1 to 3, wherein the serum amyloid A antibody is labeled with acridine sulfonamide; the biotin-labeled antibody solution contains the serum amyloid A antibody according to any one of claims 1 to 3, wherein the serum amyloid A antibody is labeled with biotin; the serum amyloid A antibody contained in the acridine sulfonamide-labeled antibody solution and the biotin-labeled antibody solution are different.

10. The kit according to claim 9, characterized in that, The serum amyloid A antibody diluent is a 10-50 mM Tris-HCl buffer containing BSA, with each 100 mL of diluent containing 0.1-1.0 g of BSA; the biotin-labeled antibody solution contains 0.5-1.5 μg / mL of the serum amyloid A antibody; the acridine sulfonamide-labeled antibody solution contains 0.5-1.5 μg / mL of the serum amyloid A antibody.

Images