Detection kit for organisms S100A8 and S100A9 or protein complexes thereof

Abstract

The invention relates to an antibody specifically bound with S100A8 protein, an antigen binding fragment or a variant thereof, an antibody specifically bound with S100A9 protein, an antigen binding fragment or a variant thereof and application of the antibody and the antigen binding fragment or the variant thereof in preparation of a detection kit for bacterial infectious diseases. The detection kit has high detection sensitivity and specificity on organism bacterial infection, and can be used for detecting bacterial infectious diseases of clinical samples.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a detection kit for S100A8, S100A9 or their protein complexes in organisms. Background Technology

[0002] Pathogen infection is a common health challenge in clinical medicine, affecting almost all organs and systems of the human body and causing a variety of diseases. Infectious diseases are a significant health problem with persistently high morbidity and mortality rates worldwide. With the increasing aging population, the growing number of immunocompromised individuals, the overuse of antibiotics, and the increasing drug resistance of pathogens, the clinical management of infectious diseases faces severe challenges.

[0003] Despite significant advancements in etiological diagnosis and anti-infective treatment in modern medicine, early identification, severity assessment, and prognosis of infectious diseases remain challenging. Depending on the site of infection and the causative bacteria, bacterial infections can lead to diseases such as sepsis, pneumonia, tonsillitis, bacterial dysentery, bacterial liver abscess, cystitis, and osteomyelitis.

[0004] The severity of infectious diseases is closely related to the type of pathogen, the site of infection, and the host's immune status. When local infections are not effectively controlled, pathogens and their toxins may enter the bloodstream, triggering a systemic inflammatory response and even developing into life-threatening sepsis. This pathological process highlights the dynamic nature of infectious diseases: from local infection to a systemic inflammatory response, which may ultimately lead to multiple organ dysfunction syndrome (MODS).

[0005] Due to the high heterogeneity of clinical manifestations of infectious diseases and the different immune responses triggered by different pathogens, their diagnosis and treatment face enormous challenges. In 2020, the World Health Organization listed sepsis as a major global health problem, further highlighting the urgency of infectious disease management.

[0006] Early diagnosis and timely intervention are crucial in the clinical management of infectious diseases, especially for high-risk patients. Currently, clinical diagnosis mainly relies on traditional biomarkers such as C-reactive protein (CRP), procalcitonin (PCT), and white blood cell count (WBC), combined with microbial culture. However, these indicators have significant limitations. In immunosuppressed or early-stage infected patients, CRP and PCT may not be significantly elevated, leading to an increased risk of missed diagnoses; CRP and PCT can also be nonspecifically elevated in non-infectious inflammation (such as trauma, surgery, and autoimmune diseases), making it difficult to accurately distinguish infection from other inflammatory states; these markers typically rise significantly only hours to days after infection, exhibiting a time lag that hinders very early diagnosis; and existing indicators are insufficient for accurately assessing the severity of infection and the risk of progression to sepsis or organ failure.

[0007] Therefore, exploring more sensitive and specific biomarkers is a key direction for improving the accuracy of diagnosis and treatment of infectious diseases. Summary of the Invention

[0008] To address the technical problems existing in the prior art, this invention proposes an antibody that specifically binds to the S100A8 protein, its antigen-binding fragment, or a variant thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: 8H-CDR1, the sequence of which is shown in SEQ ID NO.1; 8H-CDR2, the sequence of which is shown in SEQ ID NO.2; and 8H-CDR3, the sequence of which is shown in SEQ ID NO.3; the light chain variable region comprises: 8L-CDR1, the sequence of which is shown in SEQ ID NO.4; 8L-CDR2, the sequence of which is shown in SEQ ID NO.5; and 8L-CDR3, the sequence of which is shown in SEQ ID NO.6.

[0009] The antibody, its antigen-binding fragment, or a variant thereof as described above, wherein: the sequence of the heavy chain variable region is as shown in SEQ ID NO. 7; and the sequence of the light chain variable region is as shown in SEQ ID NO. 8.

[0010] An antibody that specifically binds to the S100A9 protein, its antigen-binding fragment, or a variant thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: 9H-CDR1, the sequence of which is shown in SEQ ID NO. 9; 9H-CDR2, the sequence of which is shown in SEQ ID NO. 10; and 9H-CDR3, the sequence of which is shown in SEQ ID NO. 11; and the light chain variable region comprises: 9L-CDR1, the sequence of which is shown in SEQ ID NO. 12; 9L-CDR2, the sequence of which is shown in SEQ ID NO. 13; and 9L-CDR3, the sequence of which is shown in SEQ ID NO. 14.

[0011] The antibody, its antigen-binding fragment, or a variant thereof as described above, wherein: the sequence of the heavy chain variable region is as shown in SEQ ID NO. 15; and the sequence of the light chain variable region is as shown in SEQ ID NO. 16.

[0012] The antibody, its antigen-binding fragment, or a variant thereof, as described above, further comprises a heavy chain constant region and a light chain constant region, wherein: the antibody heavy chain constant region is selected from one or more of IgG, IgM, IgA, IgE, or IgD; and the light chain constant region is selected from the κ or λ chain.

[0013] The antibodies, their antigen-binding fragments, or variants thereof as described above, wherein the IgG series antibodies are selected from one or more of IgG1, IgG2, and IgG4.

[0014] The antibody, its antigen-binding fragment, or a variant thereof as described above, wherein the antibody is selected from the group consisting of: whole antibodies, bispecific antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.

[0015] The antibody, its antigen-binding fragment, or a variant thereof as described above, wherein the antigen-binding fragment is selected from the group consisting of: Fab fragment, Fab' fragment, F(ab)2 fragment, Fv fragment, and ScFv.

[0016] A fusion protein comprising an antibody, an antigen-binding fragment thereof, or a variant thereof as described above; preferably, the fusion protein further comprises biotin.

[0017] One or more isolated nucleic acid molecules that encode an antibody, its antigen-binding fragment or a variant thereof as described above, or a fusion protein as described above.

[0018] One or more vectors containing one or more isolated nucleic acid molecules as described above.

[0019] A cell comprising one or more isolated nucleic acid molecules as described above or one or more carriers as described above.

[0020] A method for producing an antibody, its antigen-binding fragment or a variant thereof, or a fusion protein as described above, comprising culturing cells as described above under conditions that enable the expression of the antibody, its antigen-binding fragment or a variant thereof, or a fusion protein as described above.

[0021] The use of antibodies, antigen-binding fragments thereof, or variants thereof that specifically bind to the S100A8 protein as described above, or fusion proteins as described above, in the preparation of diagnostic kits for bacterial infection-related diseases in subjects.

[0022] The use of antibodies, antigen-binding fragments thereof, or variants thereof that specifically bind to the S100A9 protein as described above, or fusion proteins as described above, in the preparation of diagnostic kits for bacterial infection-related diseases in subjects.

[0023] This invention relates to the use of antibodies, antigen-binding fragments thereof, or variants thereof that specifically bind to S100A8 and S100A9 proteins, respectively, in the preparation of diagnostic kits for bacterial infection-related diseases in subjects; wherein the antibody, antigen-binding fragment thereof, or variant thereof that specifically binds to S100A8 protein includes a heavy chain variable region and a light chain variable region; preferably, the heavy chain variable region includes: 8H-CDR1, the sequence of which is shown in SEQ ID NO.1; 8H-CDR2, the sequence of which is shown in SEQ ID NO.2; 8H-CDR3, the sequence of which is shown in SEQ ID NO.3; the light chain variable region includes: 8L-CDR1, the sequence of which is shown in SEQ ID NO.4; 8L-CDR2, the sequence of which is shown in SEQ ID NO.5; 8L-CDR3, the sequence of which is shown in SEQ ID NO.6; the antibody, antigen-binding fragment thereof, or variant thereof that specifically binds to S100A9 protein includes a heavy chain variable region and a light chain variable region; preferably, the heavy chain variable region includes: 9H-CDR1, the sequence of which is shown in SEQ ID NO. As shown in NO.9; 9H-CDR2, the sequence of which is shown in SEQ ID NO.10; 9H-CDR3, the sequence of which is shown in SEQ ID NO.11; the light chain variable region includes: 9L-CDR1, the sequence of which is shown in SEQ ID NO.12; 9L-CDR2, the sequence of which is shown in SEQ ID NO.13; 9L-CDR3, the sequence of which is shown in SEQ ID NO.14.

[0024] According to any of the above-described applications, the bacteria are Enterobacter aerogenes, Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Candida krusei, Staphylococcus aureus, Streptococcus pneumoniae, Candida tropicalis, Ornithine-lysin-Rauvolfia, Klebsiella pneumoniae, Salmonella typhimurium serotype, Enterococcus faecalis, Acinetobacter baumannii, Candida albicans, Haemophilus influenzae, or Moraxella catarrhalis.

[0025] According to any of the applications described above, the bacterial infection-related diseases include the common cold, influenza, asthma, pneumonia, atelectasis, bronchitis, pharyngitis, otitis media, sinusitis, meningitis, sepsis, septicemia, catheter-related infection, artificial joint or heart valve infection, postoperative wound infection, blood infection, periodontitis, endocarditis, abscess, cellulitis, food poisoning, toxic shock syndrome, osteomyelitis, urinary tract infection, gastroenteritis, and bladder infection. Inflammation, otitis externa, retinitis, mononucleosis, pertussis, thrush, vaginitis, vulvitis, cervicitis, endometritis, salpingitis, pelvic inflammatory disease, invasive candidiasis, esophagitis, conjunctivitis, trachoma candidiasis, viral hepatitis, poliomyelitis, measles, chickenpox, herpes zoster, AIDS, plague, brucellosis, cholera, scarlet fever, tuberculosis, rickettsial disease, syphilis, relapsing fever, Lyme disease, Legionnaires' disease, or tinea.

[0026] A diagnostic kit for bacterial infection-related diseases, comprising an antibody that specifically binds to the S100A8 protein as described above, its antigen-binding fragment or a variant thereof, or a fusion protein as described above.

[0027] A diagnostic kit for bacterial infection-related diseases, comprising an antibody that specifically binds to the S100A9 protein as described above, its antigen-binding fragment or a variant thereof, or a fusion protein as described above.

[0028] A diagnostic kit for bacterial infection-related diseases includes antibodies that specifically bind to S100A8 and S100A9 proteins, their antigen-binding fragments, or variants thereof; wherein the antibody specifically binding to the S100A8 protein, its antigen-binding fragment, or variants thereof includes a heavy chain variable region and a light chain variable region; preferably, the heavy chain variable region comprises: 8H-CDR1, the sequence of which is shown in SEQ ID NO.1; 8H-CDR2, the sequence of which is shown in SEQ ID NO.2; 8H-CDR3, the sequence of which is shown in SEQ ID NO.3; the light chain variable region comprises: 8L-CDR1, the sequence of which is shown in SEQ ID NO.4; 8L-CDR2, the sequence of which is shown in SEQ ID NO.5; 8L-CDR3, the sequence of which is shown in SEQ ID NO.6; the antibody specifically binding to the S100A9 protein, its antigen-binding fragment, or variants thereof includes a heavy chain variable region and a light chain variable region; preferably, the heavy chain variable region comprises: 9H-CDR1, the sequence of which is shown in SEQ ID NO. As shown in NO.9; 9H-CDR2, the sequence of which is shown in SEQ ID NO.10; 9H-CDR3, the sequence of which is shown in SEQ ID NO.11; the light chain variable region includes: 9L-CDR1, the sequence of which is shown in SEQ ID NO.12; 9L-CDR2, the sequence of which is shown in SEQ ID NO.13; 9L-CDR3, the sequence of which is shown in SEQ ID NO.14.

[0029] In the kit described above, the antibody that specifically binds to the S100A9 protein, its antigen-binding fragment, or a variant thereof is biotin-labeled.

[0030] The kit described above further includes streptavidin labeled with horseradish peroxide.

[0031] In the kit described above, the concentration of the antibody, its antigen-binding fragment, or a variant thereof that specifically binds to the S100A8 protein is 1-4 μg / mL; preferably, the concentration is about 1-3 μg / mL; more preferably, the concentration is about 2 μg / mL.

[0032] In the kit described above, the concentration of the antibody, its antigen-binding fragment, or a variant thereof that specifically binds to the S100A9 protein is 1-4 μg / mL; preferably, the concentration is 1-3 μg / mL; more preferably, the concentration is about 1 μg / mL.

[0033] The kit described above further includes a coating solution; preferably, the coating solution is selected from PBS buffer, Tris-HCl buffer, or CBS buffer.

[0034] The kit described above further includes a blocking solution, preferably a BSA solution; more preferably, the BSA solution concentration is 1%-5%; even more preferably, the BSA solution concentration is about 1%-2%.

[0035] A method for detecting bacterial infection in an organism using the above-described kit includes: diluting the antibody, its antigen-binding fragment, or a variant thereof as described above to approximately 2 μg / mL with CBS buffer; incubating at 25-37°C for 50-70 min followed by overnight incubation at 4°C; adding 1%-2% BSA solution; incubating at 25-37°C for 30-90 min; preferably, incubating for 60-90 min; adding the sample to be tested or a standard; incubating at 25-37°C for 30-120 min; preferably, incubating for 60-90 min. Add biotin-labeled antibodies, antigen-binding fragments thereof, or variants thereof as described above; incubate at 25-37°C for 30-120 minutes; preferably, incubate for 60-90 minutes; add horseradish peroxide-labeled streptavidin; incubate at 25-37°C for 30-60 minutes; preferably, about 45 minutes; add chromogenic substrate; preferably, the chromogenic substrate is a TMB substrate; incubate at 25-37°C for 5-15 minutes; preferably, incubate at 25°C for 10 minutes; add stop solution to terminate the reaction, and read the absorbance using a specified instrument.

[0036] This test kit exhibits high sensitivity and specificity for detecting bacterial infections in organisms and can be used to detect bacterial infectious diseases in clinical samples. Attached Figure Description

[0037] The preferred embodiments of the present invention will now be described in further detail with reference to the accompanying drawings, wherein:

[0038] Figure 1 This is a titer analysis of a monoclonal antibody according to an embodiment of the present invention;

[0039] Figure 2 The results are the sensitivity analysis results of a double-antibody sandwich ELISA method according to an embodiment of the present invention; and

[0040] Figure 3 shows the clinical sample detection results of the S100A8 / A9 protein double antibody sandwich reagent kit according to an embodiment of the present invention; wherein, Figure 3A ROC curves for detecting bacterial infectious diseases using the antibodies of this application; Figure 3B ROC curve for CRP method in detecting bacterial infectious diseases; Figure 3C ROC curve for PCT method in detecting bacterial infections. Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0042] In the following detailed description, reference can be made to the accompanying drawings, which form part of this application and illustrate specific embodiments of the present application. In the drawings, similar reference numerals describe substantially similar components in different figures. Specific embodiments of the present application are described in sufficient detail below to enable those skilled in the art to implement the technical solutions of the present application. It should be understood that other embodiments or modifications to the embodiments of the present application may also be utilized.

[0043] Bacterial infection is a common systemic or local inflammatory response caused by bacterial pathogens, and it is one of the leading causes of hospital-acquired and community-acquired infections. Following a bacterial infection, the host's innate immune system is rapidly activated to fight the invading pathogen; this activation may trigger a cytokine storm, characterized by a significant increase in the levels of pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and interferon-γ (IFN-γ). Excessive production of these cytokines can lead to cellular damage and organ dysfunction.

[0044] Timely and effective antibacterial treatment is a key measure to reduce mortality in patients with bacterial infections. Early identification and appropriate treatment of bacterial infections can significantly improve patients' chances of survival and recovery speed.

[0045] Bacterial infections involve various immune cells, among which neutrophils play a crucial role. As the main effector cells of the innate immune system, neutrophils play a central role in the early defense against bacterial invasion. They clear pathogens through mechanisms such as phagocytosis, degranulation, and the release of neutrophil extracellular traps (NETs), thus playing a key role in the initiation and regulation of the inflammatory response. In the pathological process of bacterial infection, neutrophils migrate from the bloodstream to the site of infection and enhance the killing effect on pathogens by forming NETs. However, excessive activation of neutrophils can also lead to the formation of immune thrombosis, which in turn can trigger disseminated intravascular coagulation (DIC), potentially exacerbating microcirculatory dysfunction and tissue damage. Therefore, screening strategies based on neutrophil-based biomarkers for bacterial infection may reveal specific biomarkers for the early diagnosis of bacterial infections, which is of great significance for improving treatment efficacy and patient prognosis.

[0046] The proper nouns used in this article have the following meanings:

[0047] The pathogens referred to in this article are pathogenic bacteria, viruses, fungi, parasites, etc. Pathogens invade and multiply in an organism, potentially triggering local or systemic pathological reactions. Among these, bacterial infections are systemic inflammatory response syndromes caused by pathogen infection, commonly seen in severe trauma or infectious diseases. According to one embodiment of this application, pathogens include, but are not limited to: bacteria such as Escherichia coli, Staphylococcus aureus, Streptococcus, Mycobacterium tuberculosis, and Klebsiella pneumoniae; viruses such as influenza virus and novel coronavirus; and fungi such as Candida and Aspergillus.

[0048] The public datasets mentioned in this article include databases containing information on samples with clinically confirmed pathogen infections, such as the MIMIC database. This database contains a large amount of information on bacterial infection samples caused by different reasons. By analyzing these samples, we can obtain information on the sensitivity and specificity of bacterial infection detection under different detection methods.

[0049] The antibodies discussed in this article generally refer to immunoglobulin molecules composed of two pairs of identical polypeptide chains, each pair having a "light" (L) chain and a "heavy" (H) chain. The light chain of an antibody can be classified as κ and λ light chains. The heavy chain can be classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both the light and heavy chains, variable and constant regions are linked by "J" regions of approximately 12 or more amino acids, and the heavy chain also includes "D" regions of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (V...H ) and heavy chain constant region (C H It consists of three structural domains (C). The heavy chain constant region consists of three structural domains (C). H 1. C H 2 and C H 3) Composition. Each light chain consists of a light chain variable region (V L ) and light chain constant region (C L It consists of a light chain constant region composed of a structural domain C. L Composition. The constant region of an antibody mediates the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (such as effector cells) and the first component (Clq) of the classical complement system. V H and V L The region can be further subdivided into highly variable regions called complementary determination regions (CDRs), which are interspersed among more conservative regions called framing regions (FRs). Each V H and V L Arranged from N-terminus to C-terminus in the following order, consisting of 3 CDRs and 4 FRs: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable region (V) of each heavy / light chain pair... H and V L These amino acids form antibody binding sites. The distribution of amino acids to regions or domains follows the definitions of Kabat sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)) or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917, Chothia et al. (1989) Nature 342:878-883. The amino acid positions described in this invention are based on online comparisons using the abysis tool (http: / / www.bioinf.org.uk / abysis / index.html) and do not represent actual positions in the amino acid sequence. The term "antibody" is not limited to any antibody production method. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.

[0050] The antigen-binding fragments referred to herein generally refer to one or more fragments of a full-length antibody that retain the ability to bind to the same antigen (e.g., S100A8 or S100A9 protein) to which the antibody is bound, and compete with the intact antibody for antigen-specific binding. Antigen-binding fragments can be generated using recombinant DNA technology or by enzymatic or chemical cleavage of the intact antibody. In some cases, antigen-binding sites include Fab, Fab', F(ab')2, F(ab)2, Fd, Fv, dAb, and complementarity-determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, biantibodies, and peptides, which contain at least a portion of an antibody sufficient to confer specific antigen-binding ability to the peptide.

[0051] The variants referred to herein generally refer to proteins that differ from their parent molecule (e.g., a polypeptide) by at least one amino acid. A variant can refer to the molecule itself, or a composition containing that molecule. When such a molecule is a polypeptide or a protein, it can also refer to the amino acid sequence of the molecule. In some cases, a variant differs from its parent molecule (e.g., a protein) by the addition, deletion, or substitution of one or more amino acids, such as 1-50, 1-40, 1-30, 1-20, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 amino acids. In some cases, the variant may have at least about 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher) sequence homology with the amino acid sequence of its parent molecule.

[0052] The S100A8 and S100A9 proteins mentioned in this paper are specific proteins produced by the immune system after an organism is invaded by pathogens. S100A8 / A9 refers to the heterodimeric complex (main functional form) formed by S100A8 and S100A9 monomers through non-covalent bonds. This application prepares specific antibodies against S100A8 and S100A9 proteins and uses these specific antibodies to detect whether an organism is infected with pathogens, achieving higher sensitivity and specificity than CRP detection.

[0053] The anti-S100A8 antibodies described herein are antibodies that specifically bind to the S100A8 protein in animals and further serve as detection and / or capture antibodies against diseases caused by related pathogens. Examples include antibodies numbered 24E3, 24E2, 6B10, and 27C1 mentioned in this application.

[0054] The anti-S100A9 antibodies described herein are antibodies that specifically bind to the S100A9 protein in animals and further serve as detection and / or capture antibodies against diseases caused by related pathogens. Examples include antibodies numbered 7C4, 1F1, and 7G2 mentioned in this application.

[0055] The binding specificity referred to herein generally means the ability of one substance to specifically bind to another substance and not readily bind to any other substance randomly. This includes the ability to specifically bind (e.g., to an immune response) to a given target (while not binding to or substantially not binding to non-targets). For example, a protein may bind specifically to another protein due to its specific structure. Targeting portions may exhibit binding specificity to the corresponding antigen. The antibody (or its antigen-binding fragment or variant) of this application may be monospecific and contain one or more binding sites that specifically bind to a target, or it may be multispecific (e.g., bispecific or trispecific) and contain two or more binding sites that specifically bind to the same or different targets.

[0056] Biotin, also known as vitamin B7, vitamin H, or coenzyme R, is a water-soluble vitamin with a molecular weight of only 244.31 Da. In cells, it acts as an important coenzyme, participating in multiple metabolic pathways, such as carbohydrate metabolism, fatty acid synthesis, amino acid metabolism, and gluconeogenesis. Biotin exists in the body in both bound and free forms; approximately 12% is covalently bound, 7% is reversibly bound, and the remaining 80% is free. The human body needs to obtain biotin from external sources to meet its requirements, usually through dietary supplementation. Biotin is naturally found in many foods (such as eggs, meat, grains, and green vegetables) and can also be synthesized by intestinal bacteria. Higher doses of biotin are also found in multivitamins, prenatal vitamins, and supplements that promote hair, skin, and nail growth. In addition, high doses of biotin can be used over-the-counter for medical purposes in diabetes, lipid metabolism disorders, biotin synthase deficiency, carboxylase deficiency, and peripheral neuropathy.

[0057] The avidin discussed in this article is a glycoprotein with a molecular weight of 67–68 kDa. Each molecule consists of four subunits and can bind tightly to four biotin molecules. Streptavidin, isolated from Streptomyces avidinii, is widely used due to its lower nonspecificity.

[0058] The double-antibody sandwich ELISA method discussed in this article refers to the Biotin-(Strept) Avidin System (BAS), a novel bioreaction amplification system developed in the late 1970s. Biotin can covalently bind to the amino groups of most proteins, enzymes, and other molecules to form conjugates. Avidin can also covalently link to modified magnetic beads, solid-phase supports, and other media. One molecule of avidin can bind to four molecules of biotin in a non-covalent manner with high affinity, and it is resistant to pH changes, detergents, multiple elution steps, chelating agents, and other extreme reaction conditions (such as temperature). The high affinity and cascade amplification effect between biotin and avidin make BAS immunolabeling and related tracer analyses more sensitive, leading to its widespread application in medical testing across various detection systems. Approximately 85% of common chemiluminescence analyzers employ BAS-based detection methods.

[0059] The term "basically not" as used in this article typically means very little or almost no binding of a particular substance. For example, very little or almost none (e.g., less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.1%, or less than 0.01%).

[0060] The term "monoclonal antibody" as used in this article generally refers to a group of substantially homologous antibodies, meaning that the individual antibodies comprising this group are identical except for the possibility of naturally occurring mutations present in trace amounts. Monoclonal antibodies are highly specific, targeting a single antigenic site directly. Furthermore, unlike polyclonal antibody preparations which include different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on an antigen. The modifier "monoclonal" is not interpreted as requiring any special method to produce the antibody. For example, monoclonal antibodies can be prepared using hybridoma technology or generated in bacterial, eukaryotic, or plant cells using recombinant DNA methods. Monoclonal antibodies can also be obtained from phage antibody libraries using techniques described, for example, those described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., Mol. Biol., 222:581-597 (1991).

[0061] The chimeric antibodies discussed in this article generally refer to antibodies where a portion of the amino acid sequence of each heavy or light chain is homologous to, or belongs to, a corresponding amino acid sequence from a specific species, while the remaining segments of that chain are homologous to a corresponding sequence from another species. For example, the variable regions of both the light and heavy chains may originate from the variable region of an antibody from one animal species (e.g., mouse, rat), while the constant region is homologous to an antibody sequence from another species (e.g., human). For instance, to obtain chimeric antibodies, the variable region can be generated using non-human B cells or hybridoma cells, while the combined constant region is derived from humans. The variable region has the advantage of being easy to prepare, and its specificity is not affected by the source of the combined constant region. Furthermore, because the constant region of a chimeric antibody can be derived from humans, the likelihood of the chimeric antibody eliciting an immune response upon injection is lower than with antibodies using a non-human source of the constant region.

[0062] The humanized antibodies discussed in this article typically refer to chimeric antibodies that contain fewer sequences derived from non-human immunoglobulins, thereby reducing the immunogenicity of xenobiotic antibodies when introduced into humans, while maintaining the antibody's complete antigen-binding affinity and specificity. For example, CDR transplantation (Jones et al., Nature 321:522(1986)) and its variants can be used; including “reshaping” (Verhoeyen et al., 1988 Science 239:1534-1536; Riechmann et al., 1988 Nature 332:323-337; Tempest et al., Bio / Technol 19919:266-271), and “hyperchimerization” (Queen et al., 1989 Proc Natl Acad Sci USA 86:10029-10033; Co et al., 1991 Proc Natl Acad Sci USA 88:2869-2873; Co et al., 1992 J Immunol 148:1149-1154) and techniques such as veneering (Mark, et al., "Derivation of therapeutically active humanized and veneered anti-CD18 antibodies." In: Metcalf B W, Dalton BJ, eds. Cellular adhesion: molecular definition to therapeutic potential. New York: Plenum Press, 1994:291-312), and surface reconstruction (US Patent US5639641) can humanize non-human-derived binding domains. If other regions, such as hinge regions and constant region structural domains, are also derived from non-human sources, these regions can also be humanized.

[0063] The fully human antibodies discussed in this article generally refer to therapeutic antibodies containing antibody regions derived from fully human amino acid sequences, wherein antigen specificity has been selected in vivo using genetically modified mice or through antibody engineering methods involving binding screening. Compared to mouse or chimeric antibodies, fully human antibodies and humanized antibodies have a lower risk of inducing an immune response in humans.

[0064] The bispecific antibodies discussed in this article generally refer to artificial proteins capable of simultaneously binding to two different types of antigens. The main types of manufacturing methods include quadromas, chemical conjugation, and genetic recombination. IgG-like forms retain the structure of a conventional monoclonal antibody (mAb) with two Fab arms and an Fc region, in addition to binding to two different antigens at two Fab sites. Each heavy chain and light chain pair comes from a unique mAb. The Fc region, formed by the two heavy chains, forms a third binding site. Non-IgG-like forms include chemically linked Fabs, consisting only of Fab regions, and various types of bivalent and trivalent single-chain variable fragments (scFvs). There are also fusion proteins that mimic the variable domains of two antibodies. Bispecific antibodies have high cytotoxic potential and bind to antigens expressed relatively weakly at lower effective doses. Furthermore, targeting more than one molecule can be used to circumvent parallel pathway regulation and avoid resistance to treatment.

[0065] The Fab fragments discussed in this article generally refer to a portion of an immunoglobulin molecule (such as an antigen-binding fragment). A Fab fragment may contain a portion of a light chain and a heavy chain, and has a single antigen-binding site. Fab fragments can be obtained by digesting immunoglobulin molecules with papain. For example, a Fab fragment may consist of a constant domain and a variable domain for each heavy and light chain. The variable domain may contain a complementary site (antigen-binding site) containing a set of complementarity-determining regions at the amino terminus of the immunoglobulin molecule. Papain can be used to cleave immunoglobulin molecules into two Fab fragments and one Fc fragment. Pepsin cleaves below the hinge region, resulting in an F(ab')2 fragment and a pFc' fragment. The divalent F(ab)2 or F(ab')2 fragment has two antigen-binding regions linked by disulfide bonds. Reduction of the F(ab)2 or F(ab')2 fragment produces two monovalent Fab or Fab' fragments, which have free thiol groups that can be used for conjugation with other molecules.

[0066] The Fv fragments discussed in this article generally refer to the smallest fragments produced by the enzymatic cleavage of IgG and IgM antibodies. Fv fragments possess characteristics derived from V... H and V L Antigen binding sites are formed in the region, but they lack C. H 1 and C L Region. Through non-covalent interactions, V H and V L The chains are joined together in the Fv fragment.

[0067] The ScFv mentioned in this article generally refers to single-chain antibody fragments. ScFv can also refer to recombinant single-chain polypeptide molecules in which the variable regions of the light and heavy chains of the antibody are linked by peptide linkers. Single-chain antibodies (ScFvs) typically do not include the portion of the antibody's Fc region involved in effector functions, and are therefore naked antibodies, although methods are known to add such regions to known ScFv molecules (if desired). See Helfrich et al., Arapid and versatile method for harnessing ScFvantibody fragments with various biological functions. J Immunol Methods 237:131-145 (2000) and de Haard et al., Creating and engineering human antibodies for immunotherapy. Advanced Drug Delivery Reviews 31:5-31 (1998).

[0068] The term "one or more isolated nucleic acid molecules" as used in this article generally refers to a polymer of any length of nucleotide (whether deoxyribonucleotide, ribonucleotide, or analogue) isolated from its natural environment or synthesized artificially.

[0069] The sequence homology mentioned in this article usually refers to the obvious similarity of the amino acid sequences of homologous proteins.

[0070] As used herein, the term "about" generally refers to an approximation of a given value that can be reasonably inferred based on ordinary techniques in the art, including equivalent and approximate values ​​resulting from the experimental and / or measurement conditions of that given value. For example, it can refer to a value that is no more than 10% higher or lower than the value modified by the term. For example, the term "about 5 μg / kg" refers to a range of 4.5 μg / kg to 5.5 μg / kg. As another example, "about 1 hour" means a range of 48 minutes to 72 minutes.

[0071] This application has screened antibodies that specifically bind to the S100A8 protein, their antigen-binding fragments or variants thereof, which can be used for the detection of the S100A8 protein or the S100A8 / A9 protein complex in organisms.

[0072] This application has screened antibodies, antigen-binding fragments thereof, or variants thereof that specifically bind to the S100A9 protein, which can be used for the detection of the S100A9 protein or the S100A8 / A9 protein complex in organisms.

[0073] In some embodiments, the antibody that specifically binds to the S100A8 protein, its antigen-binding fragment, or a variant thereof includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: 8H-CDR1, the sequence of which is shown in SEQ ID NO.1; 8H-CDR2, the sequence of which is shown in SEQ ID NO.2; and 8H-CDR3, the sequence of which is shown in SEQ ID NO.3; and the light chain variable region comprises: 8L-CDR1, the sequence of which is shown in SEQ ID NO.4; 8L-CDR2, the sequence of which is shown in SEQ ID NO.5; and 8L-CDR3, the sequence of which is shown in SEQ ID NO.6.

[0074] In some embodiments, an antibody that specifically binds to the S100A8 protein, its antigen-binding fragment or a variant thereof, wherein: the sequence of the heavy chain variable region is as shown in SEQ ID NO.7; and the sequence of the light chain variable region is as shown in SEQ ID NO.8.

[0075] In some embodiments, the antibody that specifically binds to the S100A9 protein, its antigen-binding fragment, or a variant thereof includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: 9H-CDR1, the sequence of which is shown in SEQ ID NO. 9; 9H-CDR2, the sequence of which is shown in SEQ ID NO. 10; and 9H-CDR3, the sequence of which is shown in SEQ ID NO. 11; and the light chain variable region comprises: 9L-CDR1, the sequence of which is shown in SEQ ID NO. 12; 9L-CDR2, the sequence of which is shown in SEQ ID NO. 13; and 9L-CDR3, the sequence of which is shown in SEQ ID NO. 14.

[0076] In some embodiments, the antibody specifically binds to the S100A9 protein, its antigen-binding fragment, or a variant thereof, wherein: the sequence of the heavy chain variable region is as shown in SEQ ID NO.15; and the sequence of the light chain variable region is as shown in SEQ ID NO.16.

[0077] In some embodiments, the antibody, its antigen-binding fragment, or a variant thereof described herein further includes a heavy chain constant region and a light chain constant region, wherein: the antibody heavy chain constant region is selected from one or more of IgG, IgM, IgA, IgE, or IgD; and the light chain constant region is selected from the κ or λ chain. In some embodiments, the IgG series antibody is selected from one or more of IgG1, IgG2, and IgG4.

[0078] In some embodiments, the antibody is selected from the group consisting of: whole antibodies, bispecific antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.

[0079] In some embodiments, the antigen-binding fragments referred to in this application are selected from the group consisting of: Fab fragments, Fab' fragments, F(ab)2 fragments, Fv fragments, and ScFv.

[0080] In some embodiments, this application relates to a fusion protein comprising the antibody described above, its antigen-binding fragment, or a variant thereof; preferably, the fusion protein further comprises biotin. In some embodiments, the fusion protein may be a fusion protein of the antibody, its antigen-binding fragment, or a variant thereof that specifically binds to the S100A8 protein or the S100A9 protein, specifically bound to the S100A9 protein, and biotin.

[0081] This application further relates to one or more isolated nucleic acid molecules that encode antibodies, antigen-binding fragments thereof, or variants thereof that specifically bind to the S100A8 protein or the S100A9 protein as described in this application, or the fusion proteins mentioned above.

[0082] In some embodiments, the heavy chain variable region nucleic acid molecule sequence of the antibody that specifically binds to the S100A8 protein, its antigen-binding fragment, or a variant thereof is shown in SEQ ID NO.17; the heavy chain variable region nucleic acid molecule sequence is shown in SEQ ID NO.18.

[0083] In some embodiments, the heavy chain variable region nucleic acid molecule sequence of the antibody that specifically binds to the S100A9 protein, its antigen-binding fragment, or a variant thereof is shown in SEQ ID NO.19; the heavy chain variable region nucleic acid molecule sequence is shown in SEQ ID NO.20.

[0084] This application further relates to one or more vectors containing the one or more isolated nucleic acid molecules.

[0085] This application further relates to a cell that contains one or more isolated nucleic acid molecules or carriers.

[0086] A method for generating the antibody, its antigen-binding fragment, or a variant thereof described in this application comprises culturing the cell under conditions that enable the antibody, its antigen-binding fragment, or a variant thereof or fusion protein described herein to be expressed.

[0087] This application relates to a detection kit for bacterial infection-related diseases, comprising an antibody that specifically binds to the S100A8 protein, its antigen-binding fragment or a variant thereof, or a fusion protein.

[0088] This application relates to a detection kit for bacterial infection-related diseases, comprising an antibody that specifically binds to the S100A9 protein, its antigen-binding fragment or a variant thereof, or a fusion protein.

[0089] This application relates to a detection kit for bacterial infection-related diseases, comprising antibodies that specifically bind to S100A8 and S100A9 proteins, their antigen-binding fragments, or variants thereof.

[0090] The antibody, its antigen-binding fragment, or a variant thereof that specifically binds to the S100A8 protein includes a heavy chain variable region and a light chain variable region; preferably, the heavy chain variable region comprises: 8H-CDR1, the sequence of which is shown in SEQ ID NO.1; 8H-CDR2, the sequence of which is shown in SEQ ID NO.2; 8H-CDR3, the sequence of which is shown in SEQ ID NO.3; and the light chain variable region comprises: 8L-CDR1, the sequence of which is shown in SEQ ID NO.4; 8L-CDR2, the sequence of which is shown in SEQ ID NO.5; and 8L-CDR3, the sequence of which is shown in SEQ ID NO.6.

[0091] The antibody, its antigen-binding fragment, or a variant thereof that specifically binds to the S100A9 protein includes a heavy chain variable region and a light chain variable region; preferably, the heavy chain variable region comprises: 9H-CDR1, the sequence of which is shown in SEQ ID NO. 9; 9H-CDR2, the sequence of which is shown in SEQ ID NO. 10; 9H-CDR3, the sequence of which is shown in SEQ ID NO. 11; the light chain variable region comprises: 9L-CDR1, the sequence of which is shown in SEQ ID NO. 12; 9L-CDR2, the sequence of which is shown in SEQ ID NO. 13; 9L-CDR3, the sequence of which is shown in SEQ ID NO. 14.

[0092] In some embodiments, antibodies that specifically bind to the S100A9 protein, their antigen-binding fragments, or variants thereof are labeled with biotin.

[0093] In some embodiments, the kit also includes streptavidin labeled with horseradish peroxide.

[0094] In some embodiments, the concentration of the antibody, its antigen-binding fragment, or a variant thereof that specifically binds to the S100A8 protein is 1-4 μg / mL; preferably, the concentration is about 1-3 μg / mL; more preferably, the concentration is about 2 μg / mL.

[0095] In some embodiments, the concentration of the antibody, its antigen-binding fragment, or a variant thereof that specifically binds to the S100A9 protein is 1-4 μg / mL; preferably, the concentration is 1-3 μg / mL; more preferably, the concentration is about 1 μg / mL.

[0096] In some embodiments, the kit further includes a coating solution; preferably, the coating solution is selected from PBS buffer, Tris-HCl buffer, or CBS buffer.

[0097] In some embodiments, the kit further includes a blocking solution, preferably a BSA solution; more preferably, the BSA solution concentration is 1%-5%; even more preferably, the BSA solution concentration is about 1%-2%.

[0098] In some embodiments, the bacteria are pathogenic bacteria such as Enterobacter aerogenes, Salmonella, Enterococcus faecalis, Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Enterobacteriaceae, Candida krusei, Staphylococcus aureus, Streptococcus pneumoniae, Candida tropicalis, Acinetobacter baumannii, Salmonella, Ornithine-lysine-lactamase Raouli, Klebsiella pneumoniae, Salmonella typhimurium serotype, Enterococcus faecalis, Acinetobacter baumannii, Candida albicans, Haemophilus influenzae, or Moraxella catarrhalis.

[0099] In some embodiments, bacterial infection-related diseases include the common cold, influenza, asthma, pneumonia, atelectasis, bronchitis, pharyngitis, otitis media, sinusitis, meningitis, sepsis, septicemia, catheter-related infection, artificial joint or heart valve infection, postoperative wound infection, blood infection, periodontitis, endocarditis, abscess, cellulitis, food poisoning, toxic shock syndrome, osteomyelitis, urinary tract infection, gastroenteritis, cystitis, otitis externa, retinitis, mononucleosis, pertussis, thrush, vaginitis, vulvitis, cervicitis, endometritis, salpingitis, pelvic inflammatory disease, invasive candidiasis, esophagitis, conjunctivitis, trachoma candidiasis, viral hepatitis, poliomyelitis, measles, chickenpox, herpes zoster, AIDS, plague, brucellosis, cholera, scarlet fever, tuberculosis, rickettsial disease, syphilis, relapsing fever, Lyme disease, Legionnaires' disease, or tinea, etc.

[0100] Therefore, this application relates to a method for detecting bacterial infection in organisms using the kit of this application, comprising: diluting the antibody, its antigen-binding fragment, or a variant thereof as described in any one of claims 1-2 or 5-8 with CBS buffer to about 2 μg / mL; incubating at 25-37°C for 50-70 min followed by overnight incubation at 4°C; adding 1%-2% BSA solution; incubating at 25-37°C for 30-90 min; preferably, incubating for 60-90 min; adding the sample to be tested or a standard; incubating at 25-37°C for 30-120 min; preferably, incubating for 60-90 min. minutes; add biotin-labeled antibody as described in any one of claims 3-4 or 5-8, its antigen-binding fragment or a variant thereof; incubate at 25-37°C for 30-120 minutes; preferably, incubate for 60-90 minutes; add horseradish peroxide-labeled streptavidin; incubate at 25-37°C for 30-60 minutes; preferably, about 45 minutes; add chromogenic substrate; preferably, the chromogenic substrate is a TMB substrate; incubate at 25°C-37°C for 5-15 minutes; preferably, incubate at 25°C for 10 minutes; add stop solution to terminate the reaction, and read the absorbance using a specified instrument.

[0101] The technical solution of this application will be described below through specific embodiments. Those skilled in the art should understand that the following embodiments are merely one implementation of the technical solution of this application, and are not intended to limit the technical solution of this application.

[0102] Example 1: Preparation of anti-S100A8 and anti-S100A9 monoclonal antibodies

[0103] (1) Obtain S100A8 and S100A9 proteins and use them as antigens to immunize animals in order to prepare corresponding monoclonal antibodies. The S100A8 and S100A9 proteins can be isolated by methods known in the art or purchased as commercial products, which will not be described in detail in this application.

[0104] (2) Animal Immunization: Systemic immunization was performed on 6-8 week old female Balb / C mice. Pre-blood collection was conducted by collecting blood through the orbital cavity and retaining only serum to avoid hemolysis. For the initial immunization, multiple subcutaneous injections were administered on the back: some mice were injected with a mixture of 100 μL S100A8 / S100A9 antigen and Freund's complete adjuvant (FCA), while other mice were injected with a mixture of 50 μL antigen and Freund's incomplete adjuvant (FIA). Second and third immunizations were performed by multiple subcutaneous injections of 50 μL antigen and FIA on the back. After the third immunization, serum samples were collected and ELISA titers were measured to assess the immunization effect. Mice with titers below the target were given supplementary immunizations until the titers met the experimental requirements. Subsequently, a pulse immunization was performed. After the pulse immunization, blood was collected again through the orbital cavity, serum was separated, and stored under appropriate conditions. Throughout the process, hemolysis was strictly avoided, and the temperature was maintained to ensure serum quality.

[0105] (3) ELISA detection of serum titer: Dilute the antigen to 1 μg / mL with PBS, add 100 μL to the ELISA plate, and coat overnight at 2–8℃. The next day, gently pat the ELISA plate dry and wash three times with 180 μL PBST buffer, 5 min each time. Then, add 100 μL 1% BSA blocking buffer and block at 37℃ for 60 min. Dilute the serum after the third immunization with blocking buffer 1000 times and perform 3-fold serial dilutions to prepare a total of 7 gradients; at the same time, dilute the blank serum 1000 times as a negative control. After patting the blocked ELISA plate dry, add 100 μL of diluted serum and incubate at 37℃ for 60 min. After incubation, wash three times again with 180 μL PBST buffer, 5 min each time. Then, dilute the HRP-labeled secondary antibody to the appropriate concentration with blocking buffer, add 50 μL to the ELISA plate, and incubate at 37℃ for 60 min. After the secondary antibody incubation, wash three times with 180 μL PBST buffer, 5 min each time. Add 50 μL TMB chromogenic solution and incubate in the dark for about 10 min. Adjust the reaction time as needed based on the color development. Then add stop solution to terminate the reaction. Finally, read the absorbance of each well at 450 nm using a microplate reader. Serum titer is defined as the maximum dilution where the OD value reaches at least 2.5 times that of the negative control serum.

[0106] The experimental results showed that the serum antibody titer of mouse No. 4 in the S100A8 immunization group was the highest (81,000), the titer of mouse No. 2 in the S100A9 immunization group reached 243,000, and the others were 81,000. Therefore, mice No. 4 in S100A8 and No. 2 and No. 5 in S100A9 were selected for cell fusion to prepare monoclonal antibodies.

[0107] (4) Preparation of anti-S100A8 and anti-S100A9 monoclonal antibodies:

[0108] ①SP2 / 0 Cell Preparation: 7 to 10 days before cell fusion, remove SP2 / 0 myeloma cells from liquid nitrogen storage and quickly thaw them in a preheated 37°C water bath. Then, resuscitate and culture them using culture medium, passing them 2 to 3 times. Select cells with uniform morphology, clear outlines, and no impurities in the culture medium, and expand the culture to two T75 culture flasks. Before cell fusion, aspirate the cells from the culture flasks with sterile PBS and collect them into 50mL centrifuge tubes for subsequent use.

[0109] ② Preparation of feeder cells: Healthy female Balb / c mice were used as the source of feeder cells. After euthanasia via cervical dislocation, the experimental animals were immediately immersed in 75% ethanol solution for surface sterilization for 10 minutes, and then transferred to a biosafety cabinet for aseptic processing. The experimental animals were fixed on a sterile dissection table, and the epidermis and muscle layer were dissected layer by layer along the abdominal midline using sterilized scissors to fully expose the abdominal cavity. After euthanasia of blank mice, the abdominal epidermis was dissected to expose the peritoneum. 15–20 mL of sterile PBS was carefully injected, gently rubbed, and then aspirated and transferred to a 50 mL centrifuge tube. This process was repeated 2–3 times, and the extracted macrophages were temporarily stored in a 37°C incubator.

[0110] ③ Preparation of immune spleen cells: Balb / c mice that had undergone immunization and whose serum antibody titers met the target were selected and euthanized after whole blood collection via ocular venous plexus puncture. The experimental animals were then sterilized by immersing their bodies in 75% ethanol solution for 5 minutes, and spleen tissue was harvested according to aseptic procedures. Observation showed that the spleen volume significantly increased and became congested after immunization. The spleen was placed on a sterile cell sieve and gently ground with a sterile grinding rod. The mixture was washed and filtered with 40 mL of pre-cooled serum-free culture medium, and the filtrate was collected into a 50 mL centrifuge tube. After centrifugation (3000 rpm, 10 min), the supernatant was discarded, and the cell suspension volume was adjusted to 20 mL with complete culture medium. Cell concentration was measured using a hemocytometer. Whole blood samples were centrifuged (3000 rpm, 10 min), and the supernatant was collected to obtain high-titer positive serum.

[0111] ④ Cell Fusion: Rinse the cell sieve with pre-warmed complete culture medium to collect residual cells. Determine the required number of SP2 / 0 myeloma cells based on the total number of spleen lymphocytes, typically mixing spleen lymphocytes to myeloma cells at a ratio of 1:10. After thoroughly mixing the two cell suspensions, centrifuge (1000 rpm, 10 min) to remove the supernatant, and gently tap the bottom of the tube to loosen the cell clumps. Then, under continuous gentle shaking, add 1 mL of pre-warmed 50% PEG2000 solution (37℃) dropwise using a sterile dropper, strictly controlling the addition process to be completed within 60 seconds. Immediately transfer the mixture to a 37℃ constant temperature water bath and incubate for 60 seconds to induce fusion. Subsequently, following the principle of serial dilution, slowly add 1 mL, 2 mL, 3 mL, 4 mL, and 5 mL of serum-free culture medium sequentially within the first to 5 minutes, adjusting the final volume to 30 mL to terminate the fusion reaction. Centrifuge again (parameters as above) to remove the supernatant. The cell pellet was gently resuspended in HAT-selective medium and transferred to a serum-free culture system pre-coated with feeder cells (macrophages). After thorough mixing, the cells were aliquoted into 96-well cell culture plates at 200 μL / well using a sterile dispensing tank (6-15 plates can typically be prepared from a single mouse spleen cell). The culture plates were incubated at 37°C in a 5% CO2 incubator for 5-7 days, and the growth status of the fusion cells was observed daily. During the first medium change, 100 μL of the original medium was removed and an equal volume of fresh HAT medium was added. After 36-48 hours, the medium was completely replaced with HT medium. When the fusion cells grew to approximately 20% confluence, the HT medium was replaced again, and the cell supernatant was collected after 24-36 hours for antibody detection.

[0112] ⑤ Hybridoma polyclonal / monoclonal screening: Dilute the antigen to 1 μg / mL with PBS, add 50 μL to the ELISA plate, and coat overnight at 2–8℃. The next day, gently pat the coated ELISA plate dry and wash three times with 200 μL PBST buffer, 5 min each time. Then, prepare 1% BSA blocking buffer, add 100 μL to the ELISA plate, and block at 37℃ for 60 min. After blocking, gently pat the ELISA plate dry, add 50 μL of hybridoma cell supernatant, and incubate at 37℃ for 60 min. After primary antibody incubation, gently pat the ELISA plate dry and wash three times with 200 μL PBST buffer, 5 min each time. Next, dilute the HRP-labeled secondary antibody to 0.01% with blocking buffer, add 50 μL to the ELISA plate, and incubate at 37℃ for 60 min. After the secondary antibody incubation, gently pat the ELISA plate dry and wash it three times with 200 μL PBST buffer, 5 min each time. Then add 50 μL TMB chromogenic solution and incubate in the dark for approximately 10 min (adjust the reaction time as needed based on visual observation). Finally, add stop solution to terminate the reaction. Use a microplate reader to read the absorbance of each well at 450 nm, selecting the wells with the highest OD values ​​as positive wells. Transfer the positive clones to a 24-well plate for further culture.

[0113] ⑥ Subcloning of hybridoma cells: Select positive hybridoma cells with stable cell state and uniform clonal morphology. Gently suspend the cells in 1 mL of serum-free medium containing HT, and measure cell density using a hemocytometer. Based on the test results, perform serial dilutions, seeding approximately 200 cells into 10 mL of HT selective medium to prepare an initial suspension. Use a stepwise dilution method for cell plating: aliquot the initial suspension at 200 μL per well into two rows (A and B) of a 96-well plate (approximately 4 cells / well); then add 5 mL of medium to the remaining suspension and mix well, aliquoting into rows (C and D) (approximately 2 cells / well); finally, add another 5 mL of medium and dilute again, aliquoting into four rows (E and H) (approximately 1 cell / well). All culture plates are labeled with batch and clonal gradient information and incubated at 37°C with 5% CO2 for 5-7 days, monitoring clonal formation daily. After the monoclonal cells have stabilized, the supernatant is collected for specific ELISA detection. Positive wells are screened, and the subcloning process is repeated 3-4 times. Finally, hybridoma cell lines with 100% monoclonal positivity are selected for amplification culture. After establishing stable cell lines, they are aliquoted into cryovials and stored in liquid nitrogen for long-term use.

[0114] On day 7 after cell fusion, microscopic observation revealed cell clusters in each well, indicating a high fusion rate. After 14 days of culturing the fused cells, the supernatant was used to detect antibody secretion. Preliminary screening results are as follows: Monoclonal antibody screening using S100A8 protein as the immunogen: Nine hybridoma cell lines with good growth and stable monoclonal antibody secretion were initially screened. After four rounds of subclonal screening, four positive hybridoma cell lines that could stably secrete specific antibodies were finally identified and named 24E3, 24F2, 6B10, and 27C1, respectively. Monoclonal antibody screening using S100A9 protein as the immunogen: Thirteen hybridoma cell lines with good growth and stable monoclonal antibody secretion were initially screened. After four rounds of subclonal screening, three positive hybridoma cell lines that could stably secrete specific antibodies were finally identified and named 7C4, 1F1, and 7G2, respectively.

[0115] ⑦ Antibody Purification: After equilibrating the Protein G column with equilibration buffer, add the dialyzed antibody and wait for it to completely flow through the column. Wash the column with equilibration buffer until no protein flows out. After equilibration, elute the antibody with Tri-Gly elution buffer until no protein flows out. Neutralize the eluted antibody with neutralization buffer and perform SDS-PAGE analysis on a small amount. Mix the eluent containing the antibody and dialyze overnight at 2–8°C. Filter the antibody through a 0.22 μm filter and store at -20°C.

[0116] In this embodiment, after culture, the monoclonal antibody was collected and purified from the cell supernatant. The concentration of the purified monoclonal antibody was determined using an ultra-micro spectrophotometer, and the results are shown in Table 1 below:

[0117] Table 1. Concentration of purified monoclonal antibodies

[0118]

[0119] To evaluate the titers of different monoclonal antibodies, this example involved serial dilutions of seven monoclonal antibodies, and their titers were detected using ELISA. The experimental results are as follows: Figure 1 As shown, with OD 450 The dilution value of the enzyme-labeled antibody was determined by using a dilution of 2.5 times that of the blank well as the standard for dilution titer. The titers of each monoclonal antibody were as follows: monoclonal antibody 7C4: 1:81000; monoclonal antibody 1F1: 1:81000; monoclonal antibody 7G2: 1:81000; monoclonal antibody 24E3: 1:9000; monoclonal antibody 24F2: 1:9000; monoclonal antibody 6B10: 1:243000.

[0120] Example 2 Monoclonal Antibody Pairing Analysis

[0121] (1) Biotinylated antibody labeling: Anti-S100A8 and anti-S100A9 antibodies, prepared and purified according to the method in Example 1, were dissolved in an amine-free buffer (such as PBS) with a pH of 7.2-8.0. An appropriate amount of EZ-LinkSulfo-NHS-Biotin reagent was used, and the required volume was calculated based on a 20-fold molar excess to prepare a 10 mM biotin solution. The biotin solution was added to the antibody solution, gently mixed, and incubated at room temperature for 30 min or at 4°C for 60 min. After the reaction was complete, unreacted biotin and byproducts were removed using a desalting column or dialysis apparatus, and the labeled antibody was transferred to a suitable buffer. Finally, the biotinylation effect was verified by ELISA. The labeled antibody can be stored at -20°C for later use.

[0122] (2) Establishment of the double-antibody sandwich ELISA detection system: The capture antibody was diluted to 1 μg / mL with PBS and added to a 96-well plate, 50 μL / well, and incubated overnight at 4°C. The capture antibodies were anti-S100A8 and anti-S100A9 antibodies prepared and purified according to the method in Example 1. The next day, the plate was washed three times with PBST, and 100 μL / well of 1% BSA was added, and the plate was blocked at 37°C for 60 min. After washing, 50 μL / well of the sample or standard was added, and the plate was incubated at 37°C for 60 min. After washing, 50 μL / well of biotin-labeled detection antibody was added, wherein the detection antibody was anti-S100A9 and anti-S100A8 antibodies prepared and purified according to the method in Example 1; the plate was incubated at 37°C for 60 min. After washing, 50 μL / well of streptavidin-HRP was added, and the plate was incubated at 37°C for 30 min. After washing, add 50 μL of TMB substrate per well and incubate at room temperature in the dark for 10-30 min. Then, add 50 μL of stop solution per well to terminate the reaction. Read the absorbance at 450 nm using a microplate reader.

[0123] (3) Screening of the best antibody pairing combination: Since the S100A8 / A9 protein is composed of S100A8 and S100A9 proteins in the form of heterodimer proteins, in order to detect the content of S100A8 / A9 in serum, the antibodies of S100A8 and S100A9 are paired to detect the S100A8 / A9 protein, that is, 24E3, 24F2, 6B10 and 27C1 are paired with 7C4, 1F1 and 7G2. The antibody coating concentration is 2μg / mL and the concentration of recombinant S100A8 / A9 protein standard is 1μg / mL.

[0124] The specific procedure was as follows: Seven monoclonal antibodies (four anti-S100A8 and three anti-S100A9) were used as capture antibodies and coated into 96-well ELISA plates at a concentration of 2 μg / mL, 50 μL / well, and incubated overnight at 4°C. The next day, the plates were washed three times with PBST, and 100 μL / well of 1% BSA blocking buffer was added, and the plates were blocked at 37°C for 60 min. After washing, 50 μL / well of standard S100A8 / A9 protein (1 μg / mL) was added, and the plates were incubated at 37°C for 60 min. After washing, 50 μL / well of biotin-labeled detection antibodies (four anti-S100A8 and three anti-S100A9) were added, and the plates were incubated at 37°C for 60 min. After washing, 50 μL / well of TMB substrate was added, and the plates were incubated for 10 min at room temperature in the dark. Finally, 50 μL of stop solution was added to each well to terminate the colorimetric reaction.

[0125] The absorbance of each well was measured using a full-wavelength microplate reader (detection wavelength 450 nm), and the results showed that all tested antibody combinations were successfully paired. Through further clinical serum reactivity testing and antibody yield optimization evaluation, 24F2 and 7C4-bio were ultimately determined to be the optimal antibody pairing combination.

[0126] Table 2 Screening for optimal antibody pairing combinations

[0127]

[0128]

[0129] This application further sequenced the 24F2 and 7C4-bio monoclonal antibodies with the optimal antibody pairing combination. The sequencing results are shown in Tables 3-1 and 3-2, where H represents the heavy chain of the antibody, L represents the light chain of the antibody, and the numbers 8 and 9 indicate that the sequence belongs to the anti-S100A8 antibody or the anti-S100A9 antibody, respectively.

[0130] Table 3-1 Nucleotide sequences of monoclonal antibodies

[0131]

[0132]

[0133] Table 3-2 Monoclonal Antibody DNA Sequences

[0134]

[0135]

[0136]

[0137]

[0138] (4) Determining the optimal working concentration of the antibody: Optimization was performed using checkerboard titration. Monoclonal antibody 24F2 was used as the capture antibody, and biotin-labeled 7C4 was used as the detection antibody. Checkerboard titration was performed using four different concentrations of capture antibody (1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL) and four different concentrations of detection antibody (1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL). The optimal working concentration was determined by the P / N ratio. The results are shown in Table 4. The optimal capture antibody concentration was approximately 2 μg / mL, and the optimal detection antibody concentration was approximately 1 μg / mL.

[0139] Table 4. Determination of antibody concentration using the checkerboard titration method.

[0140]

[0141] (5) Determination of the working concentration of HRP-labeled streptavidin: After determining the working concentrations of the capture antibody and the detection antibody, three different dilutions of HRP-labeled streptavidin were used (1:2000, 1:3000, 1:4000, and 1:5000). The optimal working concentration was determined by the P / N ratio, and the results are shown in Table 5. The biotin-HRP enzyme-labeled antibody dilution factor was approximately 1:3000.

[0142] Table 5. Determination of the optimal biotin-HRP enzyme-labeled antibody dilution factor using the checkerboard titration method.

[0143]

[0144] Example 3: Optimization of the S100A8 / A9 double-antibody sandwich ELISA detection system

[0145] In this embodiment, different optimized systems were constructed using the experimental system with the optimal working concentration of the antibody and other optimal reaction parameters. Specifically, monoclonal antibody 24F2 was used as the capture antibody at a concentration of approximately 2 μg / mL; biotin-labeled 7C4 was used as the detection antibody at a concentration of approximately 1 μg / mL; and the biotin-HRP enzyme-labeled antibody was diluted approximately 1:3000.

[0146] Furthermore, the detection system and specific experimental methods of the double-antibody sandwich ELISA method in this embodiment can be referred to the description in Example 1, and will not be repeated here.

[0147] (1) Determination of the optimal coating solution: In this embodiment, three different buffer systems were constructed for comparative analysis. Further, in this embodiment, the capture antibody was diluted to 2 μg / mL with PBS (pH=7.4), Tris-HCl (pH=8.0), and CBS (pH=9.4), respectively. The optimal coating solution was determined by the checkerboard method. The results are shown in Table 6. The optimal coating solution was CBS (pH=9.4), with a P / N value of 13.691.

[0148] Table 6. Determination of Optimal Capture Antibody Coating Solution Using the Checkerboard Titration Method

[0149]

[0150]

[0151] (2) Optimization of solid-phase coating conditions: In this embodiment, the capture antibody was diluted to 2 μg / mL using CBS (pH=9.4) and then coated. The optimal coating conditions were explored using the checkerboard method. The results are shown in Table 7. The optimal coating conditions were 37℃ for 60 min + 4℃ overnight, with a P / N value of 9.084.

[0152] Table 7. Determination of Optimal Capture Antibody Coating Solution Using the Checkerboard Titration Method

[0153]

[0154] (3) Optimal blocking reagent: In this example, the capture antibody was diluted to 2 μg / mL with CBS (pH=9.4) and coated overnight at 37℃ for 60 min + 4℃. The optimal blocking solution was explored by checkerboard method. The results are shown in Table 8. The optimal blocking solution is 2% BSA with a P / N value of 13.854.

[0155] Table 8. Determination of the Optimal Blocking Solution Using the Checkerboard Titration Method

[0156]

[0157]

[0158] (4) Determination of the optimal blocking time for the capture antibody: In this embodiment, three blocking times were set: 30 min, 60 min, and 90 min. The optimal blocking time was determined by the P / N ratio. The detection results are shown in Table 9. Approximately 60-90 minutes is the optimal blocking time.

[0159] Table 9. Determination of Optimal Closure Time Using the Checkerboard Titration Method

[0160]

[0161] (5) Determination of the optimal incubation time for antigen: As shown in Table 10, this embodiment can determine that the optimal incubation time is about 60 minutes.

[0162] Table 10. Determination of Optimal Antigen Incubation Time Using the Checkerboard Titration Method

[0163]

[0164] (6) Determination of the optimal incubation time for antibody detection: As shown in Table 11, the optimal incubation time is about 60 minutes.

[0165] Table 11. Determination of Optimal Incubation Time for Antibody Detection Using the Checkerboard Titration Method

[0166]

[0167] (7) Determination of the optimal incubation time for HRP-labeled streptavidin: As shown in Table 12, the optimal incubation time for HRP-labeled streptavidin was determined to be approximately 45 min, with a P / N value of 20.185.

[0168] Table 12. Determination of Optimal Incubation Time for HRP-Labeled Streptavidin Using the Checkerboard Titration Method

[0169]

[0170] (8) Determination of optimal color development time: As shown in Table 12, this embodiment designs six combinations of color development conditions, including two temperature gradients (37℃ and 25℃) and three time gradients (5, 10, and 15 min), and determines the optimal color development conditions by the P / N ratio. As shown in Table 13, when the color development condition is 25℃ for 10 min, the P / N value is 17.750.

[0171] Table 13 Determining the Optimal Color Development Time Using the Checkerboard Titration Method

[0172]

[0173] Example 4: Evaluation of the S100A8 / A9 double-antibody sandwich ELISA detection system

[0174] (1) Sensitivity detection of S100A8 / A9 protein double antibody sandwich ELISA detection method: Based on the optimized reaction parameter system of Example 1 and Example 2, the recombinant S100A8 / A9 standard was serially diluted 8 times with a starting concentration of 4000 ng / mL to generate a series of concentration gradients covering 5.49-4000 ng / mL for detection.

[0175] As shown in Table 14, the S100A8 / A9 protein concentration exhibits a good linear relationship between 49.383 ng / mL and 4000 ng / mL. Within this detection range, a linear regression equation can be obtained: y = 0.0008x + 0.2256, R0. 2 =0.991 (e.g.) Figure 2 As shown in the figure, the horizontal axis represents protein concentration and the vertical axis represents absorbance value; the minimum detectable concentration of this detection method is 49.383 ng / mL.

[0176] Table 14 Results of ELISA Method Sensitivity Analysis

[0177] Antigen concentration (ng / mL) 4000.000 1333.333 444.444 148.148 49.383 16.461 5.487 0 OD value (A450) 3.479 1.468 0.754 0.37 0.22 0.123 0.098 0.08

[0178] (2) Accuracy test of the S100A8 / A9 protein double-antibody sandwich ELISA detection method: Accuracy is the degree of closeness between the detection results of an analytical method and the true or reference value, generally expressed as recovery rate (%). The S100A8 / A9 protein standard was diluted to three concentration gradients: high, medium, and low. Three parallel wells were set up for each concentration in the same experiment, and the OD450 absorbance value was measured. The corresponding recovery rates were then calculated, and the results are shown in Table 15. According to the data, the recovery rate ranged from 94.934% to 103.414%.

[0179] Table 15 Accuracy Test of S100A8 / A9 Protein Double Antibody Sandwich ELISA Detection Method

[0180] Added concentration (ng / mL) Measured concentration (ng / mL) Average recovery rate (%) 4000 4136.544 103.414 2000 1898.691 94.934 100 102.762 102.762

[0181] (3) Precision detection of the S100A8 / A9 protein double antibody sandwich ELISA method: The accuracy of the double antibody sandwich ELISA was assessed by measuring intra-assay variability. Intra-assay variability assessment: The S100A8 / A9 protein standard was diluted to three concentration gradients: 4000 ng / mL, 2000 ng / mL, and 1000 ng / mL. Six parallel wells were set up for each concentration in the same experiment, and the OD450 absorbance value was measured. The coefficient of variation (CV = (standard deviation SD / mean MN) × 100%) was calculated. The results are shown in Table 16. The intra-assay CV of the S100A8 / A9 protein sandwich ELISA method established in this experiment for the detection of the standard was between 1.500% and 9.114%, and the intra-assay variability CV values ​​were all less than 10%.

[0182] Table 16 Precision determination of the S100A8 / A9 protein double antibody sandwich ELISA detection method

[0183] S100A8 / A9 concentration (ng / mL) Intra-batch variation CV±SD (%) 4000.000 1.500％±0.046 1333.333 2.596％±0.051 444.444 4.834％±0.050 148.148 7.837％±0.034 49.383 5.099％±0.012 16.461 4.956％±0.007 5.487 5.221％±0.006 0 9.114％±0.007

[0184] Example 5: Application of the S100A8 / A9 double-antibody sandwich ELISA detection system

[0185] (1) Inclusion and exclusion criteria for patients with bacterial infections: Patients must have typical symptoms of bacterial infection (e.g., fever >38.3℃, local redness, swelling, heat, pain, or purulent discharge) or supporting imaging evidence (e.g., pulmonary infiltrates, abscesses), and must have a positive microbial culture. Patients with serious concurrent diseases (e.g., malignant tumors, autoimmune diseases), long-term use of immunosuppressants, pregnant or lactating women, recent history of major surgery or trauma, concurrent active infections (e.g., tuberculosis, HIV), or serum sample quality that does not meet the requirements will be excluded.

[0186] (2) Inclusion and Exclusion Criteria for the Healthy Control Group: The inclusion criteria for the healthy control group included no age or gender restrictions, no history of major illnesses, no chronic infectious diseases, and no history of mental illness. Furthermore, participants' physical examinations should show normal vital signs such as blood pressure, heart rate, and body temperature, and laboratory test results such as complete blood counts should be normal. Exclusion criteria included a recent (e.g., within 6 months) history of major surgery or trauma, long-term use of immunosuppressants or hormone medications, a history of drug or alcohol abuse, pregnancy or lactation, and any health condition or treatment history that might affect the study results.

[0187] In this embodiment, a double-antibody sandwich ELISA method was used to detect 174 clinical samples, including 133 samples from patients with bacterial infections and 41 samples from healthy controls. The expression of S100A8 / A9 under different detection methods showed a significant difference between patients with bacterial infections and healthy controls (P < 0.01, as shown in Figure 3). The 133 bacterial infections were caused by bacteria such as *Enterobacter aerogenes*, *Pseudomonas aeruginosa*, *Staphylococcus hemolyticus*, *Escherichia coli*, *Candida krusei*, *Staphylococcus aureus*, *Streptococcus pneumoniae*, *Candida tropicalis*, *Ornithine-Rauvolfia*, *Klebsiella pneumoniae*, *Salmonella typhimurium* serotype, *Enterococcus faecalis*, *Acinetobacter baumannii*, *Candida albicans*, *Haemophilus influenzae*, or *Moraxella catarrhalis*. Figures 3A-3B As shown, the area under the detection curve (AUC) using the antibody of this application was 0.9799, the cut-off value was 363.58 ng / mL, the sensitivity was 90.24%, and the specificity was 95.49%, which are far better than the detection results of the commonly used CRP and PCT methods in clinical practice.

[0188] The above embodiments are for illustrative purposes only and are not intended to limit the invention. Those skilled in the art can make various changes and modifications without departing from the scope of the invention. Therefore, all equivalent technical solutions should also fall within the scope of the invention.

Claims

1. An antibody, antigen-binding fragment thereof or variant thereof specifically binding to S100A8 protein, comprising a heavy chain variable region and a light chain variable region, wherein, the heavy chain variable region comprises: 8H-CDR1, the sequence of which is shown in SEQ ID NO. 1; 8H-CDR2, the sequence of which is shown in SEQ ID NO. 2; 8H-CDR3, the sequence of which is shown in SEQ ID NO. 3; the light chain variable region comprises: 8L-CDR1, the sequence of which is shown in SEQ ID NO. 4; 8L-CDR2, the sequence of which is shown in SEQ ID NO. 5; 8L-CDR3, the sequence of which is shown in SEQ ID NO.

6. Preferably, the sequence of the heavy chain variable region is shown in SEQ ID NO. 7; and the sequence of the light chain variable region is shown in SEQ ID NO.

8. 2.An antibody, antigen-binding fragment thereof or variant thereof specifically binding to S100A9 protein, comprising a heavy chain variable region and a light chain variable region, wherein, the heavy chain variable region comprises: 9H-CDR1, the sequence of which is shown in SEQ ID NO. 9; 9H-CDR2, the sequence of which is shown in SEQ ID NO. 10; 9H-CDR3, the sequence of which is shown in SEQ ID NO. 11; the light chain variable region comprises: 9L-CDR1, the sequence of which is shown in SEQ ID NO. 12; 9L-CDR2, the sequence of which is shown in SEQ ID NO. 13; 9L-CDR3, the sequence of which is shown in SEQ ID NO.

14. Preferably, the sequence of the heavy chain variable region is shown in SEQ ID NO. 15; and the sequence of the light chain variable region is shown in SEQ ID NO.

16. 3.Use of an antibody, antigen-binding fragment thereof or variant thereof specifically binding to S100A8 protein and S100A9 protein respectively in the preparation of a diagnostic kit for a bacterial infection related disease in a subject; the antibody, antigen-binding fragment thereof or variant thereof specifically binding to S100A8 protein comprises a heavy chain variable region and a light chain variable region; Preferably, the heavy chain variable region comprises: 8H-CDR1, the sequence of which is shown in SEQ ID NO. 1; 8H-CDR2, the sequence of which is shown in SEQ ID NO. 2; 8H-CDR3, the sequence of which is shown in SEQ ID NO. 3; the light chain variable region comprises: 8L-CDR1, the sequence of which is shown in SEQ ID NO. 4; 8L-CDR2, the sequence of which is shown in SEQ ID NO. 5; 8L-CDR3, the sequence of which is shown in SEQ ID NO. 6; the antibody, antigen-binding fragment thereof or variant thereof specifically binding to S100A9 protein comprises a heavy chain variable region and a light chain variable region; Preferably, the heavy chain variable region comprises: 9H-CDR1, the sequence of which is shown in SEQ ID NO. 9; 9H-CDR2, the sequence of which is shown in SEQ ID NO. 10; 9H-CDR3, the sequence of which is shown in SEQ ID NO. 11; wherein ​ ​ ​ ​ The light chain variable region comprises: 9L-CDR1, the sequence of which is shown in SEQ ID NO. 12; 9L-CDR2, the sequence of which is shown in SEQ ID NO. 13; and 9L-CDR3, the sequence of which is shown in SEQ ID NO.

14.

4. The use according to claim 3, wherein the bacteria is Enterobacter aerogenes, Enterococcus faecalis, Pseudomonas aeruginosa, Staphylococcus haemolyticus, Escherichia coli, Candida krusei, Staphylococcus aureus, Streptococcus pneumoniae, Candida tropicalis, Raoultella ornithinolytica, Klebsiella pneumoniae, Salmonella typhimurium, Acinetobacter baumannii, Candida albicans, Haemophilus influenzae, or Moraxella catarrhalis.

5. The use according to claim 3, wherein the bacterial infection related disease is common cold, influenza, asthma, pneumonia, atelectasis, bronchitis, pharyngitis, otitis media, sinusitis, meningitis, sepsis, sepsis, catheter-related infection, artificial joint or heart valve infection, postoperative wound infection, blood infection, periodontitis, endocarditis, abscess, cellulitis, food poisoning, toxic shock syndrome, osteomyelitis, urinary tract infection, gastroenteritis, cystitis, otitis externa, retinitis, mononucleosis, whooping cough, thrush, vaginitis, vulvitis, cervicitis, endometritis, salpingitis, pelvic inflammation, invasive candidiasis, esophagitis, conjunctivitis, trachoma candidiasis, viral hepatitis, poliomyelitis, measles, varicella, herpes zoster, AIDS, plague, brucellosis, cholera, scarlet fever, tuberculosis, rickettsial disease, syphilis, relapsing fever, Lyme disease, Legionnaires' disease, or tinea.

6. A detection kit for a bacterial infection related disease, comprising an antibody, an antigen-binding fragment thereof, or a variant thereof that specifically binds to S100A8 protein and S100A9 protein, respectively; wherein The antibody, the antigen-binding fragment thereof, or the variant thereof that specifically binds to S100A8 protein comprises a heavy chain variable region and a light chain variable region; Preferably, the heavy chain variable region comprises: 8H-CDR1, the sequence of which is shown in SEQ ID NO. 1; 8H-CDR2, the sequence of which is shown in SEQ ID NO. 2; and 8H-CDR3, the sequence of which is shown in SEQ ID NO. 3; and the light chain variable region comprises: 8L-CDR1, the sequence of which is shown in SEQ ID NO. 4; 8L-CDR2, the sequence of which is shown in SEQ ID NO. 5; and 8L-CDR3, the sequence of which is shown in SEQ ID NO. 6; The antibody, the antigen-binding fragment thereof, or the variant thereof that specifically binds to S100A9 protein comprises a heavy chain variable region and a light chain variable region; Preferably, the heavy chain variable region comprises: 9H-CDR1, the sequence of which is shown in SEQ ID NO. 9; 9H-CDR2, the sequence of which is shown in SEQ ID NO. 10; and 9H-CDR3, the sequence of which is shown in SEQ ID NO.

11. The light chain variable region comprises: 9L-CDR1, the sequence of which is shown in SEQ ID NO. 12; 9L-CDR2, the sequence of which is shown in SEQ ID NO. 13; and 9L-CDR3, the sequence of which is shown in SEQ ID NO.

14.

7. The kit of claim 6, wherein the antibody, antigen-binding fragment thereof, or variant thereof that specifically binds to S100A9 protein or the antibody, antigen-binding fragment thereof, or variant thereof that specifically binds to S100A9 protein is biotin-labeled.

8. The kit of claim 6, wherein, the concentration of the antibody, antigen-binding fragment thereof, or variant thereof that specifically binds to S100A8 protein is 1-4 μg / mL; preferably, the concentration is about 1-3 μg / mL; more preferably, the concentration is about 2 μg / mL; the concentration of the antibody, antigen-binding fragment thereof, or variant thereof that specifically binds to S100A9 protein is 1-4 μg / mL; preferably, the concentration is 1-3 μg / mL; more preferably, the concentration is about 1 μg / mL.

9. The kit of claim 6, further comprising: a coating solution; preferably, the coating solution is selected from a PBS buffer, a Tris-HCl buffer, or a CBS buffer; a blocking solution, preferably, the blocking solution comprises a BSA solution; more preferably, the concentration of the BSA solution is 1%-5%; more preferably, the concentration of the BSA solution is about 1%-2%.

10. A method for detecting bacterial infection of an organism using the kit of any one of claims 6-9, comprising: diluting the antibody, antigen-binding fragment thereof, or variant thereof of any one of claims 1-2 or 5-8 to about 2 μg / mL with a CBS buffer, and standing for 50-70 min in an environment of 25-37°C and then treating overnight at 4°C; adding a BSA solution at 1%-2%, and standing for 30-90 min in an environment of 25-37°C; preferably, standing for 60-90 min; adding a sample to be tested or a standard sample, and standing for 30-120 min in an environment of 25-37°C; preferably, standing for 60-90 min; adding the biotin-labeled antibody, antigen-binding fragment thereof, or variant thereof of any one of claims 3-4 or 5-8, and standing for 30-120 min in an environment of 25-37°C; preferably, standing for 60-90 min; adding horseradish peroxide-labeled streptavidin, and standing for 30-60 min in an environment of 25-37°C; preferably, about 45 min; adding a color developing substrate; preferably, the color developing substrate is a TMB substrate; and standing for 5-15 min in an environment of 25-37°C; preferably, standing for 10 min at 25°C; adding a stop solution to terminate the reaction, and reading the absorbance by a designated instrument.

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