Specific detection method for human s100ββ homodimer

Abstract

The application discloses a method for specifically detecting human S100beta beta homodimer by using a single monoclonal antibody. The monoclonal antibody can specifically and highly recognize the human S100beta subunit, and the amino acid sequences of the heavy chain and light chain variable regions are shown as SEQ ID NO. 1 and SEQ ID NO. 5. The monoclonal antibody or an antigen-binding fragment thereof is used as a capture antibody and a detection antibody to simultaneously combine with the same epitope sites on two subunits of the S100beta beta homodimer, so that a specific S100beta beta homodimer double-antibody sandwich detection method is established. The minimum detection limit of the method for the S100beta beta homodimer reaches 0.005 ng / mL, and the linear detection range is 0.005-10 ng / mL. The method is suitable for specific detection of the S100beta beta homodimer.

Description

Technical Field

[0001] This invention relates to the field of medical immunoassay technology, specifically to a specific detection method for human S100ββ homodimer. Background Technology

[0002] S100 proteins are a group of low-molecular-weight acidic calcium-binding proteins widely distributed in various tissues. They were first discovered in bovine brain tissue in 1965. The "S" in their name stands for "soluble," and "100" refers to their 100% solubility in a saturated ammonium sulfate solution at neutral pH. To date, the literature reports at least 25 members in the S100 protein family, including S100A1-S100A18, S100B, S100C, S100P, S100Z, CALB3, Profilaggrin, and Trichohyalin, making it the largest family of EF-handed calcium-binding proteins. S100A1 and S100B were the first members of the S100 protein family to be discovered. S100A1, initially called S100α, is mainly found in skeletal muscle, cardiac muscle, and kidneys; S100B, initially called S100β, is mainly distributed in glial cells and Schwann cells of the central and peripheral nervous systems. Most members of the S100 protein family exist in dimer form, and S100A1 and S100B also exist in three different dimer forms: S100αα (S100A1A1), S100αβ (S100A1B), and S100ββ (S100BB).

[0003] Because S100β is specifically distributed in glial cells and Schwann cells of the central and peripheral nervous systems, it is the most abundant, active, and closely watched member of the S100 family in the brain. In normal individuals, due to the integrity of brain cells and the blood-brain barrier, the levels of S100β in the blood and cerebrospinal fluid are extremely low. However, certain diseases or causes, such as cerebral hemorrhage, viral encephalitis, childhood concussion, neonatal hypoxia, brain tumors, neuroinflammation, and neurodegeneration, can impair the integrity of the blood-brain barrier. This leads to the release of intracellular S100β into the extracellular space, into the cerebrospinal fluid, and across the damaged blood-brain barrier into the bloodstream, resulting in a significant increase in S100β levels in both the blood and cerebrospinal fluid. Therefore, S100β is also known as a central nervous system-specific biological protein, serving as a specific biomarker for brain injury. Monitoring changes in its levels can help determine the progression and prognosis of certain diseases.

[0004] However, a literature review revealed that in the vast majority of studies, S100αβ heterodimer and S100ββ homodimer are collectively referred to as S100B, and it is believed that the overall levels of S100αβ heterodimer and S100ββ homodimer are associated with brain injury-related diseases. Only a very few studies have clearly distinguished between these different forms of dimer. However, the research of Nancy Pham et al. has shown that only S100ββ homodimer is the true biomarker closely related to brain injury (Pham N, Fazio V, Cucullo L, Teng Q, Biberthaler P, et al. (2010) Extracranial Sources of S100B Do Not Affect Serum Levels. PLoSONE5(9): e12691. doi:10.1371 / journal.pone.0012691). However, due to the long-standing ambiguity in the concept, and the fact that both S100αβ heterodimer and S100ββ homodimer contain the S100β subunit, all existing detection reagents target the S100β subunit. Moreover, they all employ a double-antibody sandwich detection method, which uses two different monoclonal antibodies paired together or one monoclonal antibody paired with a polyclonal antibody. This results in the inability to distinguish between S100αβ heterodimer and S100ββ homodimer, as well as between S100β monomer and S100ββ homodimer, leading to test results that do not accurately correspond to clinical symptoms.

[0005] To address the above shortcomings and achieve specific detection of human S100ββ homodimer, this invention uses bioinformatics analysis to screen for dominant antigenic epitopes specific to the human S100β subunit protein. Multiple monoclonal antibodies were prepared by immunizing mice with these epitopes. The monoclonal antibody with the highest affinity and specific recognition of the S100β subunit was selected. This monoclonal antibody is used both as a capture antibody to bind to a specific epitope on one S100β subunit and as a detection antibody to bind to the same epitope on another S100β subunit, thus achieving specific detection of S100ββ homodimer. For S100αβ heterodimer and free S100β subunit monomers, since they contain only one S100β subunit (i.e., only one specific epitope), the detection antibody cannot bind to them and therefore will not interfere with the detection results of S100ββ homodimer. The specific detection method for S100ββ homodimer of the present invention can specifically and accurately detect S100ββ homodimer in human body fluids without interference from S100αβ heterodimer and free S100β subunit monomers. Summary of the Invention

[0006] Therefore, the purpose of this invention is to provide a method for the specific detection of human S100ββ homodimer using a single monoclonal antibody. This method for the specific detection of human S100ββ homodimer is specifically and accurately detected only in human ex vivo body fluids, and is not affected by S100αβ heterodimers and free S100β subunit monomers.

[0007] The present invention also provides a monoclonal antibody prepared using mouse hybridoma technology that can recognize the S100β subunit with high affinity and high specificity. The monoclonal antibody serves as both a capture antibody and a detection antibody, and achieves specific detection of human S100ββ homodimer through a double antibody sandwich method.

[0008] Therefore, a first aspect of the present invention relates to a monoclonal antibody or antigen-binding fragment thereof that recognizes the human S100β subunit with high affinity and high specificity, 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.

[0009] 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.

[0010] 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 can specifically recognize the human S100β subunit because it retains the variable regions of the light chain and the heavy chain, or only retains the variable region of the heavy chain.

[0011] 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.

[0012] 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.

[0013] 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 bacterial recombinant or a yeast recombinant.

[0014] The fifth aspect of this invention relates to a monoclonal antibody mouse hybridoma cell line that secretes the aforementioned monoclonal antibody capable of specifically recognizing the human S100β subunit. Further, the monoclonal antibody mouse hybridoma cell line is the 6F16 monoclonal antibody mouse hybridoma cell line with accession number CGMCC No. 46784.

[0015] 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 S100ββ homodimer.

[0016] The seventh aspect of this invention relates to a method for detecting S100ββ homodimer in human ex vivo biological samples. The method utilizes the aforementioned monoclonal antibody or its antigen-binding fragment as both a capture antibody and a detection antibody to achieve specific detection of S100ββ homodimer. Further, the method is a double-antibody sandwich method, wherein the monoclonal antibody or its antigen-binding fragment is used simultaneously as both a capture antibody and a detection antibody. More further, the method includes: step (1), labeling fluorescent microspheres with the aforementioned monoclonal antibody or its antigen-binding fragment as a detection antibody, and coating a nitrocellulose membrane with the aforementioned monoclonal antibody or its antigen-binding fragment as a capture antibody, thus assembling a conventional double-antibody sandwich fluorescent immunochromatographic assay card; step (2), dropping the human ex vivo biological sample into the sample well of the double-antibody sandwich fluorescent immunochromatographic assay card and allowing it to stand for several minutes; step (3), reading the results using a fluorescent immunoassay analyzer. Further, the human ex vivo biological sample is human serum, plasma, or whole blood.

[0017] An eighth aspect of the present invention relates to a kit for detecting human S100ββ homodimer, the kit comprising the aforementioned monoclonal antibody or its antigen-binding fragment for capturing and detecting human S100ββ homodimer. Further, the kit is a double-antibody sandwich type kit, wherein the monoclonal antibody or its antigen-binding fragment serves simultaneously as both a capture antibody and a detection antibody. Further still, the kit is an enzyme-linked immunosorbent assay (ELISA) kit or a fluorescence immunochromatographic assay kit.

[0018] Instructions for the Preservation of Biological Materials

[0019] The monoclonal antibody mouse hybridoma cell line 6F16 of this invention has been deposited at the China General Microbiological Culture Collection Center (CGMCC), with registration number CGMCC No. 46784, deposit date March 4, 2026, and classification name: Monoclonal Antibody 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

[0020] Figure 1 This is an SDS-PAGE electrophoresis image showing the expression of human S100β and human S100α subunit proteins in prokaryotes, where M is the molecular weight standard; 1 represents the dimer (band a) and monomer (band b) of human S100β subunit protein under non-reducing conditions; 2 represents the monomer (band b) of human S100β subunit protein under reducing conditions; 3 represents the dimer (band c) and monomer (band d) of human S100α subunit protein under non-reducing conditions; and 4 represents the monomer (band d) of human S100α subunit protein under reducing conditions.

[0021] Figure 2 This is an SDS-PAGE electrophoresis image showing S100ββ homodimer and S100αβ heterodimer under non-reducing conditions, where M is the molecular weight standard.

[0022] Figure 3 This is the detection result of the enzyme-linked immunosorbent assay (ELISA) method for the specific detection of human S100ββ homodimer, specifically for three proteins: S100β monomer, S100αβ heterodimer, and S100ββ homodimer.

[0023] Figure 4 This is the linear range of the fluorescence immunochromatographic method for the specific detection of human S100ββ homodimer.

[0024] Figure 5 This is a comparative test result of clinical samples from a healthy physical examination group and a traumatic brain injury group using a fluorescence immunochromatographic method for the specific detection of human S100ββ homodimer.

[0025] Figure 6 This is a diagram showing the identification results of the 6F16 subtype of the anti-human S100β subunit specific high-affinity monoclonal antibody. Detailed Implementation

[0026] The purpose of this invention is to provide a method for the specific detection of human S100ββ homodimer using a single monoclonal antibody. This method for the specific detection of human S100ββ homodimer in human body fluids is specifically and accurately detected only, without interference from S100αβ heterodimer and free S100β subunit monomers.

[0027] This invention also provides a monoclonal antibody prepared using mouse hybridoma technology that can recognize the S100β subunit with high affinity and high specificity. High affinity refers to its strong binding ability to a specific antigenic epitope of the S100β subunit, and high specificity means that it recognizes only the S100β subunit and not the S100α subunit. This monoclonal antibody is used as both a capture antibody and a detection antibody, achieving specific detection of human S100ββ homodimer through a double-antibody sandwich method, unaffected by interference from S100αβ heterodimer and free S100β subunit monomers.

[0028] The specific preparation process is as follows: First, a dominant antigenic epitope specific to the human S100β subunit protein was screened using bioinformatics analysis. Multiple monoclonal antibodies were prepared by immunizing mice with this epitope. The monoclonal antibody with the highest affinity and specific recognition of the human S100β subunit protein was selected. The mouse hybridoma cell line secreting this monoclonal antibody is named 6F16 or monoclonal antibody mouse hybridoma cell line 6F16 in this patent application. The inventors deposited this monoclonal antibody mouse hybridoma cell line at the China General Microbiological Culture Collection Center (CGMCC) on March 4, 2026, with accession number CGMCC No. 46784, and classified it as a monoclonal antibody mouse hybridoma cell line.

[0029] Simultaneously, the human S100β and human S100α subunit proteins were routinely expressed in prokaryotes in *E. coli*. Using the prokaryotically expressed human S100β and human S100α subunit proteins, and purified by conventional molecular sieves, S100ββ homodimer and S100αβ heterodimer were obtained.

[0030] Next, the inventors utilized the prepared monoclonal antibody 6F16, which exhibits high specificity and high affinity for recognizing the human S100β subunit protein, as both a capture antibody and a detection antibody, establishing a specific detection method for human S100ββ homodimer based on the double-antibody sandwich principle. This method specifically detects only the S100ββ homodimer, and the detection results are unaffected by interference from S100αβ heterodimers and free S100β subunit monomers.

[0031] The human S100ββ homodimer-specific fluorescence immunochromatographic detection method established in this invention achieves a detection limit of 0.005 ng / mL and a linear range of 0.005–10 ng / mL. The detection time is only 15–20 minutes, making it suitable for rapid testing in outpatient and emergency departments.

[0032] The inventors amplified and sequenced the gene sequence of the monoclonal antibody 6F16 secreted by the mouse hybridoma cell line CGMCC No. 46784. 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 122 amino acids, specifically: VQLQQSGPELVKPGASVRISCKAS GYTFTSYY IHWVKQRPGQGLEWIGW IYPGNVNT KYNEKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYFC ARGGYDGYFVPSYFDV WGAGTSVTVSS (SEQ ID NO.1) has three CDR regions indicated by underscores: CDR1 is located at 25-32 aa with the amino acid sequence GYTFTSYY (SEQ ID NO.2); CDR2 is located at 50-57 aa with the amino acid sequence IYPGNVNT (SEQ ID NO.3); and CDR3 is located at 96-111 aa with the amino acid sequence ARGGYDGYFVPSYFDV (SEQ ID NO.4). The light chain variable region has 109 amino acids, and its sequence is as follows: DIVLTQSPASLAVSLGQRATISYRAS KSVSTSGYSY MHWNQQKPGQPPRLLIY LVS NLESGVPARFSGSGPGTDFTLNIHPVEEEDAATYYC QHIREL TR SEGGPSWKS (SEQ ID NO.5) has three CDR regions indicated by underscores, where CDR1 is located at 27-36 aa with the amino acid sequence KSVSTSGYSY (SEQ ID NO.6); CDR2 is located at 54-56 aa with the amino acid sequence LVS; and CDR3 is located at 93-100 aa with the amino acid sequence QHIRELTR (SEQ ID NO.7).

[0033] 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 conservative 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 S100β subunit protein with high specificity and high affinity, and are therefore also within the scope of protection of this invention.

[0034] Those skilled in the art can also use existing techniques in the art to prepare various antibody fragments, i.e., antigen-binding fragments, capable of recognizing human S100β subunit protein with high specificity and 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 a region in the antibody structure that can bind to the antigen, consisting 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 fragment 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, and the F(ab')2 fragment is further reduced to form two Fab' fragments. Because the above antigen-binding fragments can still bind to the S100β subunit protein, they can be used to detect human S100ββ homodimer and to prepare kits for detecting human S100ββ homodimer. Therefore, the aforementioned antigen-binding fragments are also within the scope of protection of this invention.

[0035] 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 characteristic of recognizing the human S100β subunit protein with high specificity and high affinity, and therefore can be used to detect human S100ββ homodimer, and in the preparation of kits for detecting human S100ββ homodimer. Therefore, the above-mentioned single-chain antibody is also within the scope of protection of the present invention.

[0036] Those skilled in the art can design and synthesize nucleic acid molecules encoding the variable region of the monoclonal antibody that recognizes the human S100β subunit protein with high specificity and high affinity, based on the aforementioned amino acid sequence. They can also insert the synthesized nucleic acid molecules into a nucleic acid vector to construct an expression vector capable of expressing a monoclonal antibody or its antigen-binding fragment that recognizes the human S100β subunit protein with high specificity and 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, bacterial cells, or yeast cells to obtain mammalian cell recombinants, insect cell recombinants, bacterial recombinants, or yeast 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 the human S100β subunit protein with high specificity and high affinity. Therefore, the aforementioned nucleic acid molecules, expression vectors, and mammalian cell recombinants, insect 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.

[0037] As described above, the antibody or its antigen-binding fragment of the present invention can recognize the human S100β subunit protein with high specificity and high affinity, and can be used in methods for detecting S100ββ homodimer in human ex vivo biological samples. The method can be a double antibody sandwich method, wherein the monoclonal antibody or its antigen-binding fragment is used as both a capture antibody and a detection antibody. Further, the method includes: step (1), labeling the monoclonal antibody or its antigen-binding fragment as a detection antibody onto fluorescent microspheres, and coating the monoclonal antibody or its antigen-binding fragment as a capture antibody onto a nitrocellulose membrane, assembling a conventional double antibody sandwich fluorescent immunochromatographic assay card; step (2), dropping the human ex vivo biological sample into the sample well of the double antibody sandwich fluorescent immunochromatographic assay card and allowing it to stand for several minutes; step (3), reading the results using a fluorescence immunoassay analyzer. In the above steps, labeling the monoclonal antibody or its antigen-binding fragment as a detection antibody onto fluorescent microspheres, coating the monoclonal antibody or its antigen-binding fragment as a capture antibody onto a nitrocellulose membrane, assembling them into a conventional double-antibody sandwich fluorescent immunochromatographic assay card, preparing human biological samples, adding human biological samples to the sample wells of the double-antibody sandwich fluorescent immunochromatographic assay card, and reading the results using a fluorescent immunoassay analyzer are all conventional techniques in the field and will not be elaborated further here.

[0038] S100B has been found to increase in various pathological conditions of the nervous system, including acute traumatic brain injury, neurodegenerative diseases, multiple sclerosis, congenital / perinatal diseases, and mental illnesses. Furthermore, research on S100B as a biomarker involves various body fluids, such as serum, plasma, whole blood, cerebrospinal fluid, cord blood, amniotic fluid, urine, and saliva, all of which contain detectable S100B (Lin Meijun, Qu Mingqian, Sun Furong, et al. Clinical significance and research progress of S100B protein in central nervous system diseases. Hainan Medical Journal, 2020, 31(6): 767-771). Therefore, in conjunction with the following embodiments of the present invention, the antibody or its antigen-binding fragment of the present invention can recognize the human S100β subunit protein in human ex vivo biological samples such as serum, plasma, or whole blood with high specificity and high affinity.

[0039] Similarly, since the antibodies or antigen-binding fragments of the present invention can recognize human S100β subunit protein with high specificity and high affinity, they can be used to prepare kits for detecting human S100β β-homodimeric protein. These kits can be any kit utilizing the binding reaction between the antibodies or antigen-binding fragments of the present invention and human S100β subunit 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. To illustrate the technical content, objectives, and effects of the technical solution in detail, specific embodiments are described below.

[0040] Example 1: Analysis of human S100β subunit-specific antigenic epitopes and preparation of synthetic peptides

[0041] The protein reference sequences for the human S100β and human S100α subunits were found in the GenBank database of the National Center for Biotechnology Information (NCBI) in the United States (https: / / www.ncbi.nlm.nih.gov / ). They are NP_006263.1 (also known as S100-B / S100beta) and NP_006262.1 (also known as S100A1 / S100 / S100A / S100-alpha), respectively. The human S100β subunit is 92 amino acids long, with the sequence mselekamvalidvfhqysgregdkhklkkselkelinnelshfleeikeqevvdkvmetldndgdgecdfqefmafvamvttacheffehe (SEQ ID NO. 8), containing two calcium-binding regions, 19-32 amino acids and 62-73 amino acids, and two cysteine ​​residues C69 and C85 involved in dimer formation; the human S100α subunit is 94 amino acids long, with the sequence mgseletametlinvfhahsgkegdkyklskkelkellqtelsgfldaqkdvdavdkvmkeldengdgevdfqeyvvlvaaltvacnnffwens (SEQ ID NO. 8). NO.9), which also contains two calcium-binding regions, 20-33 aa and 63-74 aa, and one cysteine ​​residue C86 involved in dimer formation. First, the amino acid sequences of the S100β and S100α subunits were aligned using the bioinformatics software DNAMAN 6.0. The results showed a high homology (57.45%) between the S100β and S100α subunits. Subsequently, the distribution of B-cell epitopes in the S100β and S100α subunits was analyzed using the bioinformatics software BIOSUN. The predicted epitopes and their scores are shown in Table 1. Epitopes B1, B5, A1, and A5 had low scores and were not dominant antigenic epitopes. Furthermore, epitopes B5 and A5 contain cysteine ​​residues, which participate in dimer formation. Epitopes B2, B4, A2, and A4 are all located in calcium-binding regions; conformational changes after binding calcium ions in vivo may lead to epitope alterations. The sequence of S100β subunit epitope B3 is completely different from S100α subunit epitope A3 and other sequences, exhibiting S100β subunit specificity and a high score. Therefore, epitope B3 of the S100β subunit was selected as the immunogen for preparing specific monoclonal antibodies. Shanghai Dechi Biotechnology Co., Ltd. was commissioned to prepare the synthetic peptide of the S100β subunit-specific antigenic epitope and perform KLH conjugation. To ensure epitope integrity, the sequence was extended by two amino acids before and after epitope B3. Additionally, to facilitate KLH conjugation, a cysteine ​​residue (C) was added to the carboxyl terminus. The final synthetic peptide sequence of the S100β subunit-specific antigenic epitope was CEE. IKEQEVV DK (SEQ ID NO.10).

[0042] Table 1. B-cell epitopes of human S100β and human S100α subunits

[0043]

[0044] Example 2: Prokaryotic expression of the human S100β subunit

[0045] The purpose of this embodiment is to prepare the human S100β subunit protein. First, based on the genetic code preferences of *E. coli*, the optimized nucleotide sequence suitable for expression in the *E. coli* expression system was deduced as ATGAGCGAACTGGAGAAAGCGATGGTGGCCTTGATTGATGTTTTTCATCAGTATTCTGGCCGTGAAGGTGACAAGCACAAACTGAAAAAGTCCGAATTAAAAGAGCTGATCAACAATGAACTTTCGCATTTCCTGGAAGAGATTAAAGAACAAGAAGTGGTCGATAAGGTTATGGAGACCCTCGATAACGACGGCGATGGTGAATGCGACTTTCAGGAGTTCATGGCGTTTGTAGCAATGGTGACGACTGCTTGTCACGAGTTCTTTGAGCATGAG (SEQ ID NO. 11). The optimized nucleotide sequence of the S100β subunit was synthesized by Beijing Qingke Biotechnology Co., Ltd., and conventional molecular biology techniques were used to ligate it into the pCold-I vector to construct the recombinant expression vector pC-S100β. pC-S100β was then transformed into… E. coliSingle colonies of BL21(DE3) competent cells were picked and cultured overnight at 37°C with shaking in 2 mL of LB broth containing ampicillin sodium. The next day, the cells 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 cells were collected by centrifugation at 4°C, 6000 rpm for 10 min. The cells were resuspended in 25 mM Tris-HCl (pH 8.5) and sonicated on ice. The supernatant was collected by centrifugation at 4°C, 12000 rpm for 10 min. SDS-PAGE gel electrophoresis showed that the S100β subunit was mainly expressed in soluble form in the supernatant. Ni column affinity chromatography was used for purification. Elution was performed with 25 mM Tris-HCl (pH 8.5) solution containing 25 mM imidazole and 250 mM imidazole. The S100β subunit protein was mainly present in the 250 mM imidazole elution buffer. Simultaneously, SDS-PAGE electrophoresis under both reducing and non-reducing conditions was performed to analyze the purified product. The results are as follows: Figure 1 As shown, under non-reducing conditions (lane 1), the S100β subunit protein exists in two forms: a dimer protein and a monomeric protein, with molecular weights of approximately 30 kDa (band a) and 15 kDa (band b), respectively; under reducing conditions (lane 2), the S100β subunit protein exists in monomeric form, with a molecular weight of approximately 15 kDa (band b).

[0046] Example 3: Prokaryotic expression of the human S100α subunit

[0047] The purpose of this embodiment is to prepare the human S100α subunit protein. First, based on the genetic code preferences of *E. coli*, an optimized nucleotide sequence suitable for expression in the *E. coli* expression system was derived: ATGGGTAGCGAACTGGAGACCGCGATGGAAACGTTGATTAACGTGTTTCATGCCCACTCTGGTAAAGAAGGCGATAAGTATAAACTGTCCAAAAAGGAGTTAAAAGAACTGCTTCAGACTGAACTGTCGGGTTTCCTCGACGCGCAAAAAGATGTTGATGCAGTGGACAAGGTCATGAAAGAGCTGGATGAAAATGGCGACGGTGAAGTTGATTTTCAGGAGTACGTAGTGCTGGTTGCTGCGTTGACCGTCGCCTGCAACAATTTCTTCTGGGAAAACAGC (SEQ ID NO. 12). The optimized nucleotide sequence of the S100α subunit was synthesized by Beijing Qingke Biotechnology Co., Ltd., and then ligated into the pGEX-4T-2 vector using conventional molecular biology techniques to construct the recombinant expression vector pGEX-S100α. pGEX-S100α conversion E. coli BL21(DE3) competent cells were induced to express the protein using the same procedure as in Example 2. SDS-PAGE gel electrophoresis showed that the S100α subunit was expressed in a soluble form in the supernatant. The recombinant vector-expressed antigen was purified using a GST column. The processed sample was slowly loaded, and the translucent solution was collected when the absorbance began to rise. After equilibration, the protein was eluted with elution buffer (50 mM Tris-HCl, 1% L-GST, pH 8.3), and the elution peak was collected. The purified protein was analyzed by SDS-PAGE electrophoresis under reducing and non-reducing conditions. The results are shown below. Figure 1 As shown, under non-reducing conditions (lane 3), the S100α subunit protein is mostly present as a monomeric protein with a molecular weight of approximately 37 kDa (d band); a small amount exists as a homodimer with a molecular weight of approximately 75 kDa (c band); under reducing conditions (lane 4), the S100α subunit protein is entirely present as a monomeric protein with a molecular weight of approximately 37 kDa (d band).

[0048] Example 4: Preparation of S100ββ homodimer and S100αβ heterodimer

[0049] The purpose of this embodiment is to prepare S100ββ homodimer and S100αβ heterodimer using the S100β and S100α subunit proteins prepared in Example 2 or 3. Based on the molecular weight of their monomeric proteins, the molecular weight of the S100ββ homodimer can be estimated to be approximately 30 kDa, and the molecular weight of the S100αβ heterodimer can be estimated to be approximately 52 kDa.

[0050] For the preparation of S100ββ homodimer, the protein product obtained in Example 2 was purified using a standard Sepdadex G-75 gel column. 2.0 mL of sample was slowly injected into the column at a flow rate of 1 mL / min to ensure sufficient interaction between the sample and the gel. When a protein peak appeared, elution was performed with 50 mM Tris-HCl (pH 8.5) buffer at a constant flow rate of 0.5 mL / tube. 10-20 tubes of protein peaks were collected, with larger molecular weight proteins eluted first. The collected proteins were then analyzed by SDS-PAGE under non-reducing conditions to select the purified S100ββ homodimer protein with a molecular weight of 30 kDa.

[0051] For the preparation of S100αβ heterodimers, equal volumes of purified S100α and β subunits were first mixed and incubated overnight in a shaker at 2-5°C to obtain different dimer mixtures. Then, molecular sieve purification was performed using a standard Sepdadex G-100 gel column, following the same procedure. The proteins were collected and analyzed by SDS-PAGE under non-reducing conditions to select the purified S100αβ heterodimer protein with a molecular weight of 52 kDa.

[0052] The SDS-PAGE electrophoresis results of the prepared S100ββ homodimer and S100αβ heterodimer proteins under non-reducing conditions are as follows: Figure 2 As shown.

[0053] Example 5: Preparation and screening of high-affinity monoclonal antibodies against human S100β subunit

[0054] Using the S100β subunit-specific epitope synthetic peptide KLH conjugate prepared in Example 1 as the immunogen, 6-8 week old female BALB / c mice were immunized with 100 µg of antigen per mouse plus an equal volume of Freund's complete adjuvant, thoroughly emulsified, and injected subcutaneously and intraperitoneally into the back. A second immunization was performed 4 weeks later, with 50 µg of antigen per mouse plus incomplete Freund's adjuvant, thoroughly emulsified, and injected subcutaneously and intraperitoneally into the back. A third immunization was performed 8 weeks later. One week after the third immunization, blood was collected from the tail vein of the mice to detect the titer of the immune serum. Mice with the highest titer were selected for a booster immunization via intraperitoneal injection. Three days later, spleen cells were harvested for fusion. Spleen cells were prepared from the spleens of the immunized mice to form a spleen cell suspension. Spleen cells and SP20 myeloma cells were fused 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 positive clones with high specificity and affinity for the S100β subunit were screened using an enzyme-linked immunosorbent assay (ELISA). The specific method is as follows: Dilute the S100β and S100α subunit proteins prepared in Examples 2 and 3 respectively with 0.05 M carbonate coating buffer (pH 9.6) to a concentration of 2.5 μg / ml, and coat each well with 150 μl, incubating overnight at 4°C; wash the plate twice with washing buffer; add 200 μl / well blocking buffer and block at room temperature for 6 hours, washing the plate 5 times; add 90 μl of sample diluent to each well, then add 10 μl of cell culture supernatant, incubate at 37°C for 30 min, discard the supernatant, wash the plate 5 times, and blot dry; add 100 μl / well of HRP-labeled goat anti-mouse IgG antibody, incubate at 37°C for 30 min, wash the plate 5 times, and blot dry; add 50 μl each of TMB chromogenic solution A and B to each well, and develop the color at room temperature in the dark for 10 min; add 50 μl of 2 M H2SO4 to each well to stop the reaction, and measure the absorbance of each well at a wavelength of 450 nm using a microplate reader within 10 minutes. The results are shown in Table 2. Through experimental screening, a monoclonal antibody hybridoma cell line was identified that recognizes only the S100β subunit, shows no cross-reactivity with the S100α subunit, and has the highest detection value. This line was named 6F16, and the monoclonal antibody it secretes can recognize the human S100β subunit with high affinity and high specificity. 1×10 6 6F16 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 LSK2ABG 20). The purified antibodies were aliquoted into 1 mg vials and stored at -20°C.

[0055] Table 2. Affinity and specificity identification results of monoclonal antibodies secreted by mouse hybridoma cell lines (OD450nm)

[0056] Example 6: Establishment of a specific detection method for human S100ββ homodimer

[0057] The purpose of this embodiment is to establish a specific detection method for human S100ββ homodimer using a highly specific and high-affinity monoclonal antibody against the human S100β subunit prepared from the mouse hybridoma cell line 6F16 obtained in Example 5. Since the S100ββ homodimer has two S100β subunits, each containing one S100β subunit-specific antigenic epitope IKEQEVV (i.e., epitope B3 in Table 1 above), the same monoclonal antibody can be used both as a capture antibody binding to the IKEQEVV epitope on one S100β subunit and as a detection antibody binding to the IKEQEVV epitope on the other S100β subunit. This allows for the specific detection of the S100ββ homodimer. Since both the S100αβ heterodimer and the free S100β subunit monomer contain only one S100β subunit, i.e. one IKEQEVV epitope, the detection antibody of this invention cannot bind to them and therefore will not interfere with the detection results of the S100ββ homodimer.

[0058] The specific detection method is as follows: Coat the ELISA plate with 6F16 monoclonal antibody at a concentration of 2.0 μg / mL, adding 100 μL to each well. Coat overnight at 4°C, and wash twice with washing buffer. Add 110 μL / well blocking buffer and incubate overnight at 4°C, then discard the buffer and air dry. Add 100 μL of the S100ββ homodimer, S100αβ heterodimer, and S100β subunit monomer protein solutions prepared in Example 4 to different wells, each at a concentration of 1.0 ng / mL, and incubate at 37°C for 45 min, then discard the buffer. Wash the plate 5 times, add 100 μL of the corresponding HRP-labeled 6F16 monoclonal antibody to each well, and incubate at 37°C for 30 min. Wash the plate 5 times, blot dry, and add 50 μL each of TMB chromogenic solutions A and B to each well, incubating at room temperature in the dark for 15 min. The reaction was stopped by adding 50 μL / well of 2 M H₂SO₄ stop solution, and the OD value at 450 nm was measured using a microplate reader within 10 minutes. Each sample was tested three times, and the average value was calculated. Results are as follows: Figure 3 As shown, the double-antibody sandwich detection method established by using 6F16 monoclonal antibody as both the capture antibody and the detection antibody can only specifically detect S100ββ homodimer, and the detection results are not affected by S100αβ heterodimer and free S100β subunit monomers.

[0059] Example 7: Specific Fluorescent Immunochromatographic Assay for Human S100ββ Homodimer

[0060] Since S100ββ homodimer is a true marker of brain injury, its concentration in serum is extremely low under normal conditions, approximately 0.01~0.05 ng / mL. However, after brain injury, the blood-brain barrier is disrupted, leading to a sharp increase in serum S100ββ homodimer levels, which can even reach 7.0~8.0 ng / mL. Therefore, the linear range of the detection reagent needs to cover both the lower limit of normal and the upper limit of abnormal levels. On the other hand, since brain injury is an acute and critical condition, rapid and immediate detection is required. To meet these needs, this embodiment establishes a simple, rapid, and wide-linear-range fluorescence immunochromatographic assay method. Specifically, the method uses the high-specificity, high-affinity monoclonal antibody 6F16 against the human S100β subunit of this invention as the detection antibody-labeled fluorescent microspheres, and also uses it as the capture antibody to coat a nitrocellulose membrane. A double-antibody sandwich fluorescence immunochromatographic assay method is established according to conventional techniques and configured into a double-antibody sandwich fluorescence immunochromatographic assay card. The S100ββ homodimer prepared according to Example 4 was serially diluted with 0.01M phosphate buffer (pH 7.2–7.4) to concentrations of 10, 5, 1.0, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, and 0 ng / mL to establish a linear equation. For detection, 100 µL of the serially diluted S100ββ homodimer was vertically added to the sample well of the detection card and allowed to react at room temperature for 15 min. The AFS-1000 dry fluorescence immunoassay analyzer was used for testing. After the sample was added to each well of the reagent card, it migrated to the chromatography zone via capillary action. At the test and control lines, fluorescent microsphere-labeled particles accumulated due to antigen-antibody reactions, forming complexes and either forming or not forming fluorescent microsphere reaction bands. Under the excitation light source, the fluorescent substances in the microspheres emitted fluorescence signals of specific wavelengths. The fluorescence immunoassay analyzer captured these signals and calculated the ratio of the T-line fluorescence value to the C-line fluorescence value (T / C) through signal conversion. The amount of S100ββ protein in the sample was positively correlated with the signal intensity of the fluorescent antibody. Each concentration of sample was tested three times. Results are as follows: Figure 4 The results show that the linear equation for the detection of S100ββ homodimer using a dual-antibody sandwich fluorescence immunochromatographic assay, in which monoclonal antibody 6F16 serves as both the capture and detection antibody, is y = 1.022x - 0.465 (R²). 2 The linear range is 0.005–10 ng / mL, and the limit of detection can reach 0.005 ng / mL. The detection time of this method is only 15–20 minutes, making it suitable for rapid testing in outpatient and emergency departments.

[0061] Example 8: Specific determination of S100ββ homodimer in in vitro biological samples

[0062] The method for specific fluorescence immunochromatographic assay of S100ββ homodimer in human ex vivo biological samples, established as described above, was used to simultaneously detect 10 ex vivo serum samples from healthy individuals and 10 ex vivo serum samples from patients with traumatic brain injury. The specific detection procedure was the same as above: 50 μL of serum sample was taken, and 50 μL of sample diluent 0.01 M phosphate buffer (pH 7.2–7.4) was added, mixed well, and then added to the sample well.

[0063] The results are as follows Figure 5 As shown, the average value of human S100ββ homodimer in 10 in vitro serum samples from healthy individuals was 0.325±0.322 ng / mL, while the average value of human S100ββ homodimer in 10 in vitro serum samples from patients with traumatic brain injury was 12.980±5.831 ng / mL. The difference between the two groups was significant (p<0.001), indicating that the method of the present invention can be used for the specific detection of S100ββ homodimer in human in vitro biological samples.

[0064] Example 9: Identification of the 6F16 subtype of the anti-human S100β subunit-specific high-affinity monoclonal antibody

[0065] The heavy and light chain subtypes of mouse antibodies were identified using the rapid mouse antibody subtype detection card (catalog number THJ-ISO-M8a, batch number 052725) from Antaiji (Beijing) Biotechnology Co., Ltd. 100 μL of supernatant from the culture of mouse hybridoma cell line 6F16 was added to the sample wells of the rapid mouse antibody subtype 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 S100β subunit specific high-affinity monoclonal antibody 6F16 is mouse IgG1 subtype, and the antibody light chain is Igκ subtype.

[0066] Example 10: Determination of the amino acid sequence of the variable region of the anti-human S100β subunit specific high-affinity monoclonal antibody 6F16

[0067] Mouse hybridoma cell line 6F16, secreting a high-affinity monoclonal antibody specific to the human S100β subunit, 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. (primer sequences are available in *Recombinant Antibodies*, edited by Shen Beifen, published by Science Press, 2005). 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, transformed into *E. coli* JM109, and 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.

[0068] Sequence analysis revealed that the heavy chain variable region consists of 122 amino acids, with the following sequence: VQLQQSGPELVKPGASVRISCKAS GYTFTSYY IHWVKQRPGQGLEWIGW IYPGNVNT KYNEKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYFC ARGGYDGYFVPSYFDV WGAGTSVTVSS (SEQ ID NO.1) has three CDR regions indicated by underscores: CDR1 is located at 25-32 aa with the amino acid sequence GYTFTSYY (SEQ ID NO.2); CDR2 is located at 50-57 aa with the amino acid sequence IYPGNVNT (SEQ ID NO.3); and CDR3 is located at 96-111 aa with the amino acid sequence ARGGYDGYFVPSYFDV (SEQ ID NO.4). The light chain variable region has 109 amino acids, and its sequence is as follows: DIVLTQSPASLAVSLGQRATISYRAS KSVSTSGYSY MHWNQQKPGQPPRLLIY LVS NLESGVPARFSGSGPGTDFTLNIHPVEEEDAATYYC Q HIRELTR SEGGPSWKS (SEQ ID NO.5) has three CDR regions indicated by underscores, where CDR1 is located at 27-36 aa with the amino acid sequence KSVSTSGYSY (SEQ ID NO.6); CDR2 is located at 54-56 aa with the amino acid sequence LVS; and CDR3 is located at 93-100 aa with the amino acid sequence QHIRELTR (SEQ ID NO.7).

Claims

1. A monoclonal antibody or antigen-binding fragment thereof that recognizes the human S100β subunit with high specificity and high affinity, 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 the monoclonal antibody mouse hybridoma cell line 6F16 with accession number CGMCC No.46784.

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 bacterial recombinant, or a yeast recombinant.

9. A mouse hybridoma cell line that secretes a monoclonal antibody with high specificity and high affinity for recognizing the human S100β subunit, characterized in that, It is the monoclonal antibody mouse hybridoma cell line 6F16 with accession number CGMCC No.46784.

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 S100ββ homodimer.

11. A kit for detecting human S100ββ homodimer, characterized in that, It comprises the monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 4.

12. The kit according to claim 11, 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 simultaneously as a capture antibody and a detection antibody.

13. The kit according to claim 12, characterized in that, The kit is an enzyme-linked immunosorbent assay kit or a fluorescence immunochromatographic assay kit.

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