Monoclonal antibodies against human soluble scavenger receptor a and uses thereof

Abstract

The application discloses a monoclonal antibody for identifying human soluble scavenger receptor A, and the amino acid sequences of the heavy chain and the light chain variable region are shown as SEQ ID NO. 1 and SEQ ID NO. 5. A double antibody sandwich detection method is established by using the monoclonal antibody as a capture antibody, the whole operation time is shortened from the conventional 4 hours to 1.5 hours, the detection P / N value is significantly improved, the minimum detection limit reaches 15 pg / mL, and the detectable range is 15-7680 ng / mL. The method is suitable for human soluble scavenger receptor A detection.

Description

Technical Field

[0001] This invention relates to the field of medical immunoassay technology, specifically to a high-affinity monoclonal antibody that recognizes human soluble scavenger receptor A (SR-A) and its application in medical immunoassay. Background Technology

[0002] Rheumatoid arthritis (RA) is a chronic, progressive, and erosive systemic autoimmune disease characterized by symmetrical polyarthritis. In my country, the prevalence is 0.24%–0.4%, with a 3-year disability rate as high as 75% in untreated patients. The "China Rheumatoid Arthritis Development Report 2020" shows that in my country, it takes an average of more than two years from the onset of symptoms to diagnosis for RA patients, and approximately 40% of patients are already in a high-disease-activity state at the time of diagnosis, missing the optimal treatment window of 6 months to 1 year. Early intervention can effectively block disease progression, prevent joint damage and other complications, and improve patients' quality of life; therefore, early and accurate diagnosis of RA is crucial. According to the classification criteria for RA diagnosis established by the American College of Rheumatology and the European League Against Rheumatism in 2010, the main serological markers are rheumatoid factor (RF) and anti-cyclic citrullinated peptide antibodies (anti-CCP antibodies). However, RF and anti-CCP antibodies have low sensitivity for detecting early RA. The sensitivity and specificity of anti-CCP antibodies are 67% and 95%, respectively, while the sensitivity and specificity of RF are 69% and 85%, respectively. Approximately 30-40% of patients who are negative for RF and anti-CCP antibodies cannot be diagnosed and treated in a timely manner due to the lack of effective diagnostic markers.

[0003] Scavenger receptor A (SR-A), also known as SCARA1, macrophage scavenger receptor 1 (MSR-1), or CD204, is a natural immune pattern recognition receptor widely expressed on the surface of macrophages. It plays an important role in many macrophage-related biological processes such as adhesion and phagocytosis, as well as in the pathological mechanisms caused by non-infectious diseases, and is associated with a variety of autoimmune diseases such as rheumatoid arthritis (RA), systemic lupus erythematosus, inflammatory bowel disease, and multiple sclerosis. A study by the team of Li Zhanguo and Hu Fanlei at Peking University People's Hospital found that soluble SR-A exists in the serum of RA patients, which has important value in the diagnosis of RF and anti-CCP antibody negative RA and early RA, and can effectively make up for the shortcomings of existing biomarkers. Their large-scale multicenter clinical study confirmed that soluble SR-A significantly improves the diagnostic efficiency of RF / CCP negative RA patients (Hu F, Jiang X, Guo C, et al. Scavenger receptor-A is a biomarker and effector of rheumatoid arthritis: A large-scale multicenter study. Nat Commun, 2020, 11(1):1911.;Application of SR-A as a diagnostic biomarker and intervention target for rheumatoid arthritis, CN 106526196 A;Multi-serum biomarker combination for the diagnosis of rheumatoid arthritis, CN202310436248).

[0004] Currently, the determination of human soluble scavenger receptor A (SR-A) levels suffers from low sensitivity and long reaction times due to the low affinity of antibodies recognizing SR-A. For example, in commercially available ELISA methods for human soluble scavenger receptor A, the incubation time after adding serum samples is 2 hours, and the incubation time after adding the detection antibody is 1 hour, with the entire assay process exceeding 4 hours. This hinders the clinical application and promotion of this method and prevents timely report generation, thus delaying disease diagnosis. Therefore, to overcome these shortcomings, this invention prepares a high-affinity monoclonal antibody that recognizes human soluble scavenger receptor A, achieving the beneficial effects of significantly improving sensitivity and shortening detection time. Summary of the Invention

[0005] Therefore, the purpose of this invention is to provide a monoclonal antibody prepared using mouse hybridoma technology that can recognize human soluble scavenger receptor A with high affinity. The monoclonal antibody, as a capture antibody, achieves specific detection of human soluble scavenger receptor A through a double antibody sandwich method. Due to the high affinity of the monoclonal antibody of this invention, the detection time is significantly shortened to 1.5 hours.

[0006] Therefore, a first aspect of the present invention relates to a high-affinity monoclonal antibody or an antigen-binding fragment thereof that recognizes human soluble scavenger receptor A, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, and the light chain variable region comprises light chain CDR1, light chain CDR2, and light chain CDR3, wherein, The amino acid sequence of the heavy chain CDR1 is the sequence shown in SEQ ID NO.2; The amino acid sequence of the heavy chain CDR2 is the sequence shown in SEQ ID NO.3; The amino acid sequence of the heavy chain CDR3 is the sequence shown in SEQ ID NO.4; The amino acid sequence of the light chain CDR1 is the sequence shown in SEQ ID NO.6; The amino acid sequence of the light chain CDR2 is LVS; The amino acid sequence of the light chain CDR3 is the sequence shown in SEQ ID NO.7.

[0007] Furthermore, the present invention also relates to the above-mentioned monoclonal antibody or its antigen-binding fragment, wherein the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO.5.

[0008] Furthermore, the present invention also relates to the above-mentioned monoclonal antibody or its antigen-binding fragment, wherein the antibody or antigen-binding fragment is a Fab fragment, a Fab' fragment, an F(ab')2 fragment, a single-chain antibody or a humanized antibody, which, because they retain the variable regions of the light chain and the heavy chain, or only retain the variable region of the heavy chain, are able to recognize human soluble scavenger receptor A with high affinity.

[0009] A second aspect of the present invention relates to a nucleic acid molecule comprising a nucleic acid encoding the above-described monoclonal antibody or an antigen-binding fragment thereof.

[0010] A third aspect of the present invention relates to an expression vector comprising the above-described nucleic acid molecules, said expression vector being capable of expressing the above-described monoclonal antibody or its antigen-binding fragment.

[0011] The fourth aspect of the present invention relates to a recombinant comprising the above-mentioned nucleic acid molecule or the above-mentioned expression vector, which can produce the above-mentioned monoclonal antibody or its antigen-binding fragment, and further, it can be a mammalian cell recombinant, an insect cell recombinant, a yeast cell recombinant or a bacterial cell recombinant.

[0012] The fifth aspect of this invention relates to a mouse hybridoma cell line that secretes the aforementioned monoclonal antibody capable of recognizing human soluble scavenger receptor A with high affinity. Further, the mouse hybridoma cell line is mouse hybridoma cell line 5B41 with accession number CGMCC No. 46796.

[0013] The sixth aspect of this invention relates to the use of the above-mentioned monoclonal antibody or its antigen-binding fragment in the preparation of a kit for detecting human soluble scavenger receptor A.

[0014] The seventh aspect of the present invention relates to a method for detecting human soluble scavenger receptor A, wherein the method utilizes the above-mentioned monoclonal antibody or its antigen-binding fragment as a capture antibody to achieve specific detection of human soluble scavenger receptor A.

[0015] An eighth aspect of the present invention relates to a kit for detecting human soluble scavenger receptor A, the kit comprising the aforementioned monoclonal antibody or its antigen-binding fragment. Further, the kit is a double-antibody sandwich type kit, wherein the monoclonal antibody or its antigen-binding fragment is used as a capture antibody to capture human soluble scavenger receptor A.

[0016] Instructions for the Preservation of Biological Materials

[0017] The mouse hybridoma cell line 5B41 of this invention has been deposited at the China General Microbiological Culture Collection Center (CGMCC), with registration number CGMCC No. 46796, deposit date of April 1, 2026, and classified as: Mouse Hybridoma Cell Line. The address of the China General Microbiological Culture Collection Center is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China. Attached Figure Description

[0018] Figure 1 This is a diagram showing the distribution of human scavenger receptor A protein B cell epitopes.

[0019] Figure 2 This is an SDS-PAGE electrophoresis image showing prokaryotic expression of soluble human SR-A protein, where M is the molecular weight standard.

[0020] Figure 3 This is the result of the titer assay for rabbit anti-human soluble SR-A protein polyclonal antibody.

[0021] Figure 4 The antibody recognition specificity was identified by Western blotting, where 1 is the anti-human soluble SR-A protein monoclonal antibody 5B41 prepared in this invention, 2 is the rabbit anti-SR-A protein polyclonal antibody prepared in this invention, and 3 is the commercially available antibody MM03.

[0022] Figure 5 This is a specific assay result of human soluble SR-A protein in an in vitro biological sample.

[0023] Figure 6 This is a diagram showing the identification results of the 5B41 subtype of the anti-human soluble SR-A high-affinity monoclonal antibody. Detailed Implementation

[0024] The purpose of this invention is to provide a monoclonal antibody prepared using mouse hybridoma technology that can recognize human soluble scavenger receptor A with high affinity. The monoclonal antibody serves as a capture antibody and achieves specific detection of human soluble scavenger receptor A through a double antibody sandwich method.

[0025] The specific preparation process is as follows: First, the dominant antigenic epitope segment of the human soluble scavenger receptor A protein is identified through bioinformatics analysis. Then, this antigenic segment is expressed in prokaryotes and used as an immunogen to immunize mice to prepare monoclonal antibodies. The monoclonal antibody with the highest affinity for recognizing the human soluble scavenger receptor A protein is selected. The mouse hybridoma cell line secreting this monoclonal antibody is named mouse hybridoma cell line 5B41 or 5B41 in this patent application. The inventors have deposited this mouse hybridoma cell line at the China General Microbiological Culture Collection Center on April 1, 2026, with accession number CGMCC No. 46796, classified and named it mouse hybridoma cell line, and the monoclonal antibody secreted by it is named monoclonal antibody 5B41.

[0026] Next, the inventors used the prepared high-affinity monoclonal antibody 5B41, which recognizes human soluble scavenger receptor A protein, as a capture antibody, and established a specific detection method for human soluble scavenger receptor A based on the double antibody sandwich principle.

[0027] The detection method for human soluble scavenger receptor A established in this invention reduces the overall operation time from the conventional 4 hours to 1.5 hours. Specifically, the reaction time after sample addition is reduced from 2 hours to 30 minutes, the reaction time after adding the detection antibody is reduced from 1 hour to 30 minutes, and the color development time is reduced from 20 minutes to 10 minutes. Furthermore, the detection P / N ratio is significantly improved. The method of this invention achieves a limit of detection of 15 pg / mL for human soluble scavenger receptor A, with a detectable range of 15–7680 ng / mL, significantly superior to comparative reagents.

[0028] The inventors amplified and sequenced the gene sequence of the monoclonal antibody 5B41 secreted by the mouse hybridoma cell line CGMCC No. 46796. Then, using public software from the National Center for Biotechnology Information (NCBI) website, they analyzed the immunoglobulin domain sequence of this monoclonal antibody and found that its heavy chain variable region has 125 amino acids, specifically: QVQLEESGADLVRPGTSVKVSCKAS GYAFTNYL IEWVKQRPGQGLEWIGV INPGSGIT HYNEKFKGKATLTADKSSSTAYMQLSSLTSDDSAVYFC ARWLYYGNSRDDYYAMDY WGQGTSVTVSS (SEQ ID NO.1) has three CDR regions indicated by underscores: CDR1 is located at 26-33 aa with the amino acid sequence GYAFTNYL (SEQ ID NO.2); CDR2 is located at 51-58 aa with the amino acid sequence INPGSGIT (SEQ ID NO.3); and CDR3 is located at 97-114 aa with the amino acid sequence ARWLYYGNSRDDYYAMDY (SEQ ID NO.4). The light chain variable region has 109 amino acids, and its sequence is as follows: DIVLTQSPASLAVSLGQRATISYRAS KSVSTSGYSY MHWNQQKPGQPPRLLIY LVS NLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYC QHIRELTR SEGAPSWKS (SEQ ID NO.5) has three CDR regions indicated by underscores. CDR1 is located at 27-36 aa and has the amino acid sequence KSVSTSGYSY (SEQ ID NO.6); CDR2 is located at 54-56 aa and has the amino acid sequence LVS; and CDR3 is located at 93-100 aa and has the amino acid sequence QHIRELTR (SEQ ID NO.7).

[0029] As is well known in the art, although the CDR regions of the antibody heavy chain and light chain are important amino acid sequence regions for recognizing and binding to corresponding antigens, conserved amino acid substitution is a biotechnological means in protein engineering to maintain the functional properties of proteins by replacing amino acid residues of the same family with similar physicochemical properties. This method mainly involves the directional substitution between amino acids of the same family, thereby ensuring that the binding affinity and specificity of the protein do not change significantly after substitution. In this patent application, the conserved amino acid substitution includes the substitution between aromatic amino acids Phe, Trp, and Tyr; the substitution between aliphatic amino acids Ala, Gly, Leu, Ile, and Val; the substitution between polar amino acids Gln and Asn; the substitution between basic amino acids Lys, Arg, and His; the substitution between acidic amino acids Asp and Glu; and the substitution between hydroxyl amino acids Ser and Thr. The conserved substitution of a single amino acid in the amino acid sequences of the heavy chain CDR region and the light chain CDR region should not change the structure of the protein. Therefore, the conserved substitution of a single amino acid in the above-mentioned regions may still have the property of binding to the corresponding antigen. Therefore, monoclonal antibodies or their antigen-binding fragments obtained by making a conserved substitution of one amino acid in heavy chain CDR1 and / or heavy chain CDR2 and / or heavy chain CDR3 and / or light chain CDR1 and / or light chain CDR2 and / or light chain CDR3 can still recognize human soluble scavenger receptor A protein with high affinity.

[0030] Those skilled in the art can also use existing techniques to prepare various antibody fragments, i.e., antigen-binding fragments, capable of recognizing human soluble SR-A protein with high affinity from the monoclonal antibodies of the present invention. These fragments include, but are not limited to, Fab, Fab', and F(ab')2. The Fab fragment is the region in the antibody structure that can bind to the antigen. It consists of a complete light chain and a variable region VH and a constant region CH1 domain (Fd segment) of the heavy chain. Both the light and heavy chains have a constant region and a variable region, and disulfide bonds link the light and heavy chains. The antigen-binding fragments can be prepared as follows: for example, after enzymatic digestion with papain, antibody IgG is degraded into two Fab fragments and one Fc fragment. Under the action of pepsin, antibody IgG is degraded into one F(ab')2 fragment and one Fc fragment. The F(ab')2 fragment is further reduced to form two Fab' fragments. Because the above antigen-binding fragments can still bind the corresponding antigen, they can be used to prepare kits for detecting human soluble scavenger receptor A.

[0031] Those skilled in the art can also prepare single-chain antibodies (scFv) from the monoclonal antibodies of the present invention using existing techniques. A single-chain antibody is an antibody composed of a heavy chain variable region and a light chain variable region linked by a short peptide linker of several amino acids; it has only one chain and is a synthetically produced antibody. A single-chain antibody may also contain only the heavy chain variable region. The length and amino acid composition of the short peptide linker are well known in the art, and usable short peptide linkers for the monoclonal antibodies of the present invention can be determined through simple repeatable experiments. The single-chain antibody can be expressed, for example, in *E. coli* using genetic engineering techniques. The single-chain antibody of the present invention prepared in this way has the property of recognizing human soluble scavenger receptor A protein with high affinity, and therefore can be used to prepare kits for detecting human soluble scavenger receptor A.

[0032] Those skilled in the art can design and synthesize nucleic acid molecules encoding the variable region of monoclonal antibodies that recognize human soluble scavenger receptor A protein with high affinity, based on the aforementioned amino acid sequence. They can also insert the synthesized nucleic acid molecules into nucleic acid vectors to construct expression vectors that can express monoclonal antibodies or their antigen-binding fragments that recognize human soluble scavenger receptor A protein with high affinity. Those skilled in the art can also introduce the synthesized nucleic acid molecules or constructed expression vectors into host cells such as mammalian cells, insect cells, yeast cells, or bacterial cells to obtain mammalian cell recombinants, insect cell recombinants, yeast cell recombinants, or bacterial cell recombinants, and express the antibodies or their antigen-binding fragments of the present invention through these recombinants. The antibodies or their antigen-binding fragments expressed in this way can recognize human soluble scavenger receptor A protein with high affinity; therefore, the aforementioned nucleic acid molecules, expression vectors, and mammalian cell recombinants, bacterial recombinants, or yeast recombinants are within the scope of protection of the claims of this invention. Furthermore, the above-described techniques are all well-known in the art and can be carried out by those skilled in the art without inventive effort.

[0033] As described above, the antibody or its antigen-binding fragment of the present invention can recognize human soluble scavenger receptor A protein with high affinity, and therefore can be used to prepare a kit for detecting human soluble scavenger receptor A. The kit can be any kit that utilizes the antibody or its antigen-binding fragment of the present invention to react with human soluble scavenger receptor A protein, such as, but not limited to, double antibody sandwich kits. Specific kits include, but are not limited to, kits using enzyme-linked immunosorbent assay (ELISA), chemiluminescence, fluorescence immunochromatography, colloidal gold immunochromatography, Western blotting, and immunohistochemistry.

[0034] To explain in detail the technical content, objectives, and effects of the technical solution, the following description is provided in conjunction with specific embodiments.

[0035] Example 1: Determination of dominant epitope regions of human soluble SR-A antigens

[0036] First, the NCBI reference sequence for human scavenger receptor A protein, NP_619729.1, was found in the GenBank database of the National Center for Biotechnology Information (NCBI) (https: / / www.ncbi.nlm.nih.gov / ). The full-length human scavenger receptor A protein is 451 amino acids long, with the following sequence: MEQWDHFHNQQEDTDSCSESVKFDARSMTALLPPNPKNSPSLQEKLKSFKAALIALYLLVFAVLIPLIGIVAAQLLKWETKNCSVSSTNANDITQSLTGKGNDSEEEMRFQEVFMEHMSNMEKRIQHILDMEANLMDTEHFQNFSMTTDQRFNDILLQLSTLFSSVQGHGNAIDEISKSLISLNTTLLDLQLNIENLNGKIQENTFKQQEEISKLE ERVYNVSAEIMAMKEEQVHLEQEIKGEVKVLNNITNDLRLKDWEHSQTLRNITLIQGPPGPPGEKGDRGPTGESGPRGFPGPIGPPGLKGDRGAIGFPGSRGLPGYAGRPGNSGPKGQKGEKGSGNTLTPFTKVRLVGGSGPHEGRVEILHSGQWGTICDDRWEVRVGQVVCRSLGYPGVQAVHKAAHFGQGTGPIWLNEVFCFGRESSIEECKIRQWGTRACSHSEDAGVTCTL (SEQ ID NO.8), scavenger receptor A belongs to type I transmembrane glycoproteins and consists of 6 domains: N-terminal cytoplasmic region, transmembrane region, spacer region, α-helix coil region, collagen-like region, and C-terminal cysteine-rich region, of which the transmembrane region is 51-76 amino acids. Subsequently, the distribution of B-cell epitopes of human scavenger receptor A protein was analyzed using the bioinformatics analysis software BIOSUN. The predicted epitope distribution is as follows: Figure 1 As shown, dominant antigenic epitopes are mainly concentrated in the anterior and middle segments of the extracellular region, i.e. Figure 1 The antigenic epitope curve segment is located in the range of 80-350 aa.

[0037] To obtain a soluble scavenger receptor A protein that can be expressed efficiently, the extracellular region containing the dominant epitopes (350-450 aa) at the C-terminus, which is prone to protein instability, was selected for prokaryotic expression. This region, specifically the 77-349 aa dominant antigenic epitope segment, is 273 amino acids long and has the following amino acid sequence: KWETKNCSVSSTNANDITQSLTGKGNDSEEEMRFQEVFMEHMSNMEKRIQHILDMEANLMDTEHFQNFSMTTDQRFNDILL Question ID NO.9).

[0038] Example 2: Prokaryotic expression of the dominant human soluble SR-A antigenic epitope segment

[0039] The purpose of this embodiment is to prepare human soluble SR-A protein. For the 77-349aa dominant epitope antigen region identified above, an optimized nucleotide sequence suitable for expression in the *E. coli* expression system was first derived based on the genetic code preferences of *E. coli*. The optimized nucleotide sequence for the 77-349aa dominant epitope antigen region was ultimately determined to be (SEQ ID NO. 10). The optimized nucleotide sequence for the above-mentioned human soluble SR-A dominant epitope antigen region was synthesized by Beijing Qingke Biotechnology Co., Ltd.Then, using conventional molecular biology techniques, the gene was ligated into the pGEX-4T-2 vector to construct the recombinant expression vector pGEX-SR-A. The upstream primer restriction site was BamHI, and the downstream primer restriction site was EcoRI. The vector was then transformed into *E. coli* BL21(DE3) for culture, and positive bacteria with the inserted target gene were screened using conventional techniques. Single colonies were picked and cultured in 2 mL of LB broth containing ampicillin sodium at 37°C overnight with shaking. The next day, the colonies were inoculated into 200 mL of fresh LB broth and cultured at 37°C for 4 h until the OD600nm reached 0.4-0.6. 150 μL of 1M IPTG induction medium was added, and the cells were induced overnight at 16°C. The induced bacterial cells were collected by centrifugation at 6000 rpm for 10 min at 4°C. The cells were resuspended in 25 mM Tris-HCl (pH 8.5) and sonicated on ice. The supernatant was collected by centrifugation at 12000 rpm for 10 min at 4°C. The antigen expressed by the recombinant vector was purified by affinity chromatography using a GST column, and the results of SDS-PAGE gel electrophoresis are shown below. Figure 2 . Figure 2 The results showed that the prokaryotic expression system of this invention yielded human soluble SR-A protein expressed in a soluble form, with a single band without any extraneous bands, and a molecular weight of approximately 56 kDa. Figure 2 ).

[0040] Example 3: Preparation and screening of high-affinity monoclonal antibodies against human soluble SR-A

[0041] Using the human soluble scavenger receptor A protein prepared in Example 2 as the immunogen, 6-8 week old female BALB / c mice were immunized by subcutaneous injection of 100 µg / mouse antigen with an equal volume of Freund's complete adjuvant after thorough emulsification, followed by intraperitoneal injection in the back. A second immunization was performed 4 weeks later, specifically by subcutaneous injection of 50 µg / mouse antigen with an equal volume of incomplete Freund's adjuvant after thorough emulsification in the back and intraperitoneal injection. A third immunization was performed 8 weeks later, with the same dosage as the second immunization. One week after the third immunization, blood was collected from the tail vein of the mice to detect the immune serum titer. Mice with the highest titer were selected for intraperitoneal booster immunization, and spleen cells were harvested 3 days later for fusion. Spleen cells were prepared from the spleens of the immunized mice, and splenocytes were fused with SP20 myeloma cells at a ratio of 9:1 using standard procedures. When the fused cells covered approximately 60% of the bottom of the well, the cell culture supernatant was collected, and a rapid screening method was used to screen for high-affinity positive clones. The earlier a significant color change occurred during the reaction, the higher the affinity of the monoclonal antibody secreted by that positive clone for the antigen protein. The specific method was as follows: The human soluble SR-A protein prepared in Example 2 was diluted with carbonate coating buffer to a concentration of 2.0 μg / mL, and 150 μL was coated per well, incubated overnight at 4°C; the plate was washed twice with washing buffer; 200 μL / well blocking buffer was added and the plate was blocked at room temperature for 6 hours; the plate was washed 5 times with washing buffer. After adding 100 μL of sample dilution buffer to each well, 10 μL of cell culture supernatant was added, and the plate was incubated with shaking at room temperature for 15 min, then the supernatant was discarded. Wash the plate 5 times, invert it onto absorbent paper to dry, add 100 μl / well of HRP-labeled goat anti-mouse IgG antibody, and incubate with shaking at room temperature. Continuously observe the plate, selecting the first positive clone to show a significant color change as the positive clone secreting the highest affinity monoclonal antibody. Name this cell line secreting the high affinity monoclonal antibody mouse hybridoma cell line 5B41 or 5B41. Continue to expand the culture, adding 1×10⁻⁶ cells... 6 5B41 mouse hybridoma cells were injected into the peritoneal cavity of mice. Ascites fluid was collected 2 weeks later, and antibodies were purified using the Montage Antibody Purification Kit with PROSEP-G (Millipore, catalog number LSK2 ABG 20). The purified antibodies were aliquoted into 1 mg vials and stored at -20°C.

[0042] Example 4: Preparation of rabbit anti-human soluble SR-A protein polyclonal antibody

[0043] Polyclonal antibodies against SR-A protein were prepared using the human soluble SR-A protein prepared in Example 2 as an immunogen. The specific preparation process is as follows: One healthy New Zealand white rabbit was selected, and 1.0 mg of the human soluble SR-A protein prepared in Example 2 was mixed with 1.0 mL of Freund's complete adjuvant. After thorough emulsification with a stirrer, 0.2 mL was injected subcutaneously at both sides of the rabbit's spine. Four weeks later, 1.0 mg of the human soluble SR-A protein was mixed with 1.0 mL of Freund's incomplete adjuvant. After thorough emulsification with a stirrer, a second immunization was performed at different points on the above site, with 0.1 mL injected at each point. A third booster immunization was performed four weeks later, with 0.1 mL injected at each point. This was used to prepare polyclonal antibody serum. One week later, blood was collected from the heart, centrifuged at 5000 rpm for 15 minutes, and the serum was aliquoted and stored at -20°C for later use. The indirect ELISA method for determining the titer of polyclonal antibodies is as follows: The ELISA plate is coated with human soluble SR-A protein at a concentration of 2.0 μg / mL, 150 μL per well, and incubated overnight at 4°C; the plate is washed twice with washing buffer; 200 μL / well blocking buffer is added and the plate is blocked at room temperature for 6 hours; the plate is washed 5 times with washing buffer. Rabbit serum was diluted with PBS at dilution ratios of 1:2000, 1:8000, 1:32000, 1:128000, 1:512000, 1:1024000, and 1:2048000, with 100 μL added to each well. The plate was incubated at 37°C for 45 min. The plate was washed 5 times with washing buffer (200 μL per well). HRP-labeled goat anti-rabbit secondary antibody was added and incubated at 37°C for 45 min. The plate was washed 5 times with washing buffer (200 μL per well). Freshly prepared substrate solution was added (100 μL per well) and incubated at 37°C for 10 min. The reaction was terminated by adding 50 μL of 2 M H₂SO₄ to each well. The absorbance of each well was measured using a microplate reader at 450 nm, and the readings were taken within 10 minutes after termination. Results are as follows: Figure 3 As shown, using pre-immunization rabbit serum as a negative control, the titer of the prepared rabbit anti-human soluble SR-A protein polyclonal antibody reached 1:1024000.

[0044] Example 5: Identification of Recognition Specificity of Monoclonal and Polyclonal Antibodies Against Human Soluble SR-A Protein

[0045] In this embodiment, the specific recognition of human SR-A protein by the monoclonal antibody 5B41 and polyclonal antibody against human soluble SR-A protein prepared in this invention was identified by Western blotting. Commercially available SR-A protein (catalog number: 10427-H07H) from Sino Biological was used for eukaryotic expression in HEK293 cells. 10 μg of sample was loaded per well. After SDS-PAGE separation, the protein was transferred to a PVDF membrane and blocked with 5% skim milk at room temperature for 1 h. Then, 5.0 μg / ml of monoclonal antibody 5B41, rabbit anti-human soluble SR-A protein polyclonal antibody, and commercially available anti-SR-A monoclonal antibody MM03 (catalog number 10427-MM03) from Sino Biological were added, and the membrane was incubated overnight at 4°C. After washing three times with 0.1% TBST, goat anti-mouse HRP antibody and goat anti-rabbit HRP antibody were added, and the membrane was incubated at room temperature for 2 h. After washing three times with 0.1% TBST, the membrane was developed using ECL chemiluminescence buffer. The results are shown below. Figure 4 As shown, similar to the commercial antibody MM03 (lane 3), the monoclonal antibody 5B41 (lane 1) against human soluble SR-A protein and the rabbit polyclonal antibody against SR-A protein (lane 2) prepared in this invention can specifically recognize human SR-A protein, showing a specific band at a molecular weight of ~70kDa. At the same time, all three antibodies show bands at a molecular weight of ~180kDa, which are presumably SR-A protein trimer bands. In addition, there are no other non-specific bands.

[0046] Example 6: Establishment and process optimization of a specific detection method for human soluble SR-A protein

[0047] The purpose of this embodiment is to establish a double-antibody sandwich method for the specific detection of human soluble SR-A protein by using the high-affinity monoclonal antibody 5B41, prepared according to the present invention, as the capture antibody to coat an ELISA plate, and using a horseradish peroxidase (HRP)-labeled rabbit anti-human soluble SR-A protein polyclonal antibody as the detection antibody. The specific method is as follows: Monoclonal antibody 5B41 was used as the capture antibody to coat the ELISA plate at a concentration of 2.0 μg / mL, with 120 μL added to each well. The plate was incubated overnight at 4°C, and washed twice with washing buffer. 150 μL / well blocking buffer was added and incubated overnight at 4°C, then the buffer was discarded and the plate was air-dried. Three positive serum samples (P1, P2, and P3) and three negative serum samples (N1, N2, and N3) were added to different wells, each 100 μL. All samples were confirmed using a commercial SR-A protein assay kit (catalog number: SEK10427) purchased from Sino Biologicals, and the procedure was performed according to the kit instructions. Three different reaction conditions were set up: incubation at 37°C for 1 hour, 37°C for 45 minutes, and 37°C for 30 minutes, respectively. After discarding the buffer, the plate was washed 5 times. 100 μL of HRP-labeled rabbit anti-human soluble SR-A protein polyclonal antibody was added to each well, and the plate was incubated at 37°C for 30 minutes. The plate was washed 5 times, patted dry, and 50 μL each of TMB chromogenic solutions A and B were added to each well. The plate was incubated at room temperature in the dark for 10 minutes. 50 μL of 2 M H₂SO₄ stop solution was added to each well to terminate the reaction. The OD450nm value was measured using a microplate reader within 10 minutes. Each concentration of sample was tested three times, and the average value was calculated. Then, the P / N ratio was calculated. The results are shown in Table 1. The P / N values ​​of the three double-antibody sandwich detection methods established in this invention are all significantly greater than those of the control reagent. Among them, method three has the largest P / N value and the shortest reaction time, with a sample incubation time of 30 minutes, an antibody incubation time of 30 minutes, and a color development time of 10 minutes. Under these reaction conditions, it can accurately distinguish between positive and negative samples. In contrast, the sample incubation time of the control reagent is 2 hours, the antibody incubation time is 1 hour, and the color development time is 20 minutes. The total reaction time of double-antibody sandwich detection method three established in this invention is 1.5 hours, which is significantly shorter than the 4 hours of the control reagent.

[0048] Table 1. Optimization of the process for detecting human soluble SR-A protein using a double-antibody sandwich assay.

[0049]

[0050] Example 7: Limit of Detection for Specific Detection of Human Soluble SR-A Protein

[0051] The linear range and limit of detection of the double-antibody sandwich method for the specific detection of human soluble SR-A protein, established in Example 6, were evaluated. The specific method was as follows: Commercially available SR-A protein (catalog number: 10427-H07H) purchased from Sino Biological was serially diluted to concentrations of 7680, 3840, 1920, 960, 480, 240, 120, 60, 30, 15, and 0 pg / mL. For detection, 100 μL of the serially diluted SR-A protein was added to each well, incubated at 37°C for 30 min, and the solution was discarded followed by washing the plate 5 times. 100 μL of HRP-labeled rabbit anti-human soluble SR-A protein polyclonal antibody was added to each well, and the plate was incubated at 37°C for 30 min. After washing the plate 5 times and drying it, 50 μL each of TMB chromogenic solutions A and B were added to each well, and the plate was incubated at room temperature in the dark for 15 min. Add 50 μL of 2 M H₂SO₄ stop solution per well to terminate the reaction. Measure the OD450nm value using a microplate reader within 10 minutes. Repeat the test three times for each concentration of sample. The results are shown in Table 2. The present invention's method for the specific detection of soluble SR-A protein detects human soluble SR-A protein. Using three times the detection value of the blank well as the cutoff value, the detectable range is 15–7680 pg / mL, and the limit of detection (LOD) reaches 15 pg / mL. In contrast, the detectable range of the comparative reagent is 78.125–5000 pg / mL, and the LOD is 78.13 pg / mL (the comparative reagent's detection data are from the instructions for the commercial SR-A protein assay kit (catalog number: SEK10427) from Sino Biologicals). The sensitivity of the present invention's method for the specific detection of soluble SR-A protein is significantly higher than that of the comparative reagent.

[0052] Table 2. Limit of Detection for Double Antibody Sandwich Detection Method of Human Soluble SR-A Protein

[0053]

[0054] Example 8: Specific determination of human soluble SR-A protein in in vitro biological samples

[0055] The method for specifically detecting human soluble SR-A protein, as established above, was used to simultaneously detect 36 in vitro serum samples from RA patients and 20 in vitro serum samples from healthy individuals. The specific detection steps were the same as those described in Method 3 of Example 6. The results are as follows... Figure 5As shown, the average level of human soluble SR-A protein in 36 ex vivo serum samples from RA patients was 543.50 pg / mL, while the average level in 20 ex vivo serum samples from healthy individuals was 17.49 pg / mL, with a significant difference between the two groups (p<0.0001). Using the average level of human soluble SR-A protein in ex vivo serum samples from healthy individuals + 3SD as the cutoff value (Cutoff=23.40), the positive detection rate of human soluble SR-A protein in ex vivo serum samples from RA patients was 61.11%, with a specificity of 100%, indicating that the method of this invention can be used for the specific detection of human soluble SR-A protein.

[0056] Example 9: Identification of the 5B41 subtype of the anti-human soluble SR-A high-affinity monoclonal antibody

[0057] The heavy and light chain isotypes of the mouse monoclonal antibody 5B41 of this invention were identified using the mouse antibody subtype rapid detection card (catalog number THJ-ISO-M8a, batch number 052725) from Antaiji (Beijing) Biotechnology Co., Ltd. 100 μL of the supernatant from the mouse hybridoma cell line 5B41 was added to the sample wells of the mouse antibody subtype rapid detection card, and the results were observed and recorded after standing for 5-10 min. The results are as follows: Figure 6 As shown, the anti-human soluble SR-A high-affinity monoclonal antibody 5B41 is a mouse IgG1 subtype, and the antibody light chain is an Igκ subtype.

[0058] Example 10: Determination of the amino acid sequence of the variable region of anti-human soluble SR-A high-affinity monoclonal antibody 5B41

[0059] Mouse hybridoma cell line 5B41, secreting a high-affinity monoclonal antibody against human soluble SR-A, was cultured. Total RNA was extracted from the hybridoma cells using the Trizol method, and cDNA was reverse transcribed. PCR amplification was then performed using primers for the Fab fragment of the mouse monoclonal antibody synthesized by Beijing Qingke Biotechnology Co., Ltd. The primer sequences are available in *Recombinant Antibodies* (Science Press, 2005), edited by Shen Beifen. Amplification conditions were as follows: preheating at 95℃ for 2 min, followed by 30 cycles of 95℃ for 30 seconds, 58℃ for 30 seconds, and 72℃ for 30 seconds, with a final extension at 72℃ for 5 min. The PCR product was ligated into the pMD18-T vector and transformed into *E. coli* JM109. Positive clones were selected for sequencing. The sequenced data was compared with the mouse-derived monoclonal antibody CDR region sequence using IgBLAST (https: / / www.ncbi.nlm.nih.gov / igblast / ) in the NCBI website.

[0060] Sequence analysis revealed that the heavy chain variable region contains 125 amino acids, with the following sequence: QVQLEESGADLVRPGTSVKVSCKAS GYAFTNYL IEWVKQRPGQGLEWIGV INPGSGIT HYNEKFKGKATLTADKSSSTAYMQLSSLTSDDSAVYFC ARWLYYGNSRDDYYAMDY WGQGTSVTVSS (SEQ ID NO.1) has three CDR regions indicated by underscores: CDR1 is located at 26-33 aa with the amino acid sequence GYAFTNYL (SEQ ID NO.2); CDR2 is located at 51-58 aa with the amino acid sequence INPGSGIT (SEQ ID NO.3); and CDR3 is located at 97-114 aa with the amino acid sequence ARWLYYGNSRDDYYAMDY (SEQ ID NO.4). The light chain variable region has 109 amino acids, and its sequence is as follows: DIVLTQSPASLAVSLGQRATISYRAS KSVSTSGYSY MHWNQQKPGQPPRLLIY LVS NLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYC QHIRELTR SEGAPSWKS (SEQ ID NO.5) has three CDR regions indicated by underscores. CDR1 is located at 27-36 aa and has the amino acid sequence KSVSTSGYSY (SEQ ID NO.6); CDR2 is located at 54-56 aa and has the amino acid sequence LVS; and CDR3 is located at 93-100 aa and has the amino acid sequence QHIRELTR (SEQ ID NO.7).

Claims

1. A monoclonal antibody or antigen-binding fragment thereof that recognizes human soluble scavenger receptor A, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, and the light chain variable region comprises light chain CDR1, light chain CDR2, and light chain CDR3, characterized in that, The amino acid sequence of the heavy chain CDR1 is the sequence shown in SEQ ID NO.2; The amino acid sequence of the heavy chain CDR2 is the sequence shown in SEQ ID NO.3; The amino acid sequence of the heavy chain CDR3 is the sequence shown in SEQ ID NO.4; The amino acid sequence of the light chain CDR1 is the sequence shown in SEQ ID NO.6; The amino acid sequence of the light chain CDR2 is LVS; The amino acid sequence of the light chain CDR3 is the sequence shown in SEQ ID NO.

7.

2. The monoclonal antibody or its antigen-binding fragment according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO.

5.

3. The monoclonal antibody according to claim 2, characterized in that, It is secreted by mouse hybridoma cell line 5B41 with accession number CGMCC No.46796.

4. The monoclonal antibody or its antigen-binding fragment according to claim 1 or 2, characterized in that, The monoclonal antibody or antigen-binding fragment is a Fab fragment, Fab' fragment, F(ab')2 fragment, single-chain antibody, or humanized antibody.

5. A nucleic acid molecule, characterized in that, It comprises a nucleic acid encoding the monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 4.

6. An expression carrier, characterized in that, It comprises the nucleic acid molecule as described in claim 5.

7. A recombinant, characterized in that, It comprises the nucleic acid molecule of claim 5 or the expression vector of claim 6.

8. The recombinant according to claim 7, characterized in that, It can be a mammalian cell recombinant, an insect cell recombinant, a yeast cell recombinant, or a bacterial cell recombinant.

9. A mouse hybridoma cell line that secretes a monoclonal antibody recognizing human soluble scavenger receptor A, characterized in that, It is the mouse hybridoma cell line 5B41 with accession number CGMCC No.46796.

10. The use of the monoclonal antibody or its antigen-binding fragment according to any one of claims 1 to 4 in the preparation of a kit for detecting human soluble scavenger receptor A.

11. A method for detecting human soluble scavenger receptor A, characterized in that, This includes the use of the monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 4.

12. The method of claim 11, characterized in that, The method is a double antibody sandwich method, wherein the monoclonal antibody or its antigen-binding fragment described in any one of claims 1 to 4 is used as a capture antibody.

13. A kit for detecting human soluble scavenger receptor A, characterized in that, It comprises the monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 4.

14. The kit according to claim 13, characterized in that, The kit is a double-antibody sandwich kit, wherein the monoclonal antibody or its antigen-binding fragment described in any one of claims 1 to 4 is used as a capture antibody.

Images