KR20250122027A - Monoclonal antibody against Feline Serum amyloid A, hybridoma cell line producing same, and uses thereof - Google Patents

Abstract

The present invention relates to a monoclonal antibody (mAb) against a protein of feline serum amyloid A (fSAA), a fusion cell line producing the same, and uses thereof.
When using the hybridoma cell line of the present invention and the monoclonal antibody generated therefrom, since it does not bind (or react) with other proteins in the cat's body and selectively (specifically) binds only to Serum Amyloid A, Serum Amyloid A can be distinguished and detected or diagnosed without cross-reactivity.

Description

Monoclonal antibody against feline serum amyloid A, hybridoma cell line producing same, and uses thereof

The present invention relates to a monoclonal antibody (mAb) against a protein of feline serum amyloid A (fSAA), a fusion cell line producing the same, and uses thereof.

Inflammation tests are performed to assess health status and detect clinically undetected abnormalities, thereby reducing complications and mortality from infections, trauma, tumors, and surgery. CRP (C-reactive protein) is a fundamental test for inflammation, and elevated blood levels indicate acute infection or inflammation. CRP is insensitive in the early stages of the acute phase, leading to false negatives. Furthermore, its levels increase without significant differences during bacterial and viral infections, the primary causes of inflammation, making it difficult to prescribe antibiotics. Serum Amyloid A (SAA) is a protein rapidly produced in the liver during early inflammatory conditions. SAA increases more rapidly than CRP during acute inflammatory conditions, making it an important inflammatory marker for human inflammation testing.

The acute phase response (APR) is the initial response to inflammatory stimuli such as infection/trauma/tumor/surgery, and one of the characteristics of this response is that a heterogeneous group of plasma proteins called acute phase proteins (APPs), including C-reactive protein (CRP), serum amyloid A (SAA), haptoglobin (Hp), and α1-acid glycoprotein, are produced in the liver by inflammatory cytokines, resulting in an increase in the blood concentration of acute phase proteins.

Major APPs such as CRP, SAA, and Hp are usually present at concentrations of less than 1 ug/ml in healthy animals, and increase up to 1,000-fold after inflammatory stimulation, reaching a peak within 24–48 hours and then rapidly decreasing upon recovery.

Among APPs, SAA is a biomarker of inflammation and infection in various species, including dogs, cats, horses, and ruminants. In cats, SAA concentrations increase significantly in the early stages of disease compared to CRP. Accordingly, SAA is considered to have a greater rate of change, and it is clinically applied as a biomarker for determining the course of disease diagnosis and treatment. SAA concentrations are known to increase in cats with infection-related diseases, tissue damage, trauma, surgery, tumors, and immune-mediated diseases (Takashi Tamamoto et al., J Vet Diagn Invest. 2013 May;25(3):428-32).

This APP diagnosis can be seen as an important tool for continuously monitoring post-surgical reinfection and recovery as well as a diagnostic aid for inflammation and disease in companion animals.

Typically, SAA is detected by collecting serum or plasma in heparin or serum tubes and placing a small drop onto a large device. However, this existing method has the disadvantage of taking a long time to determine the result and being somewhat expensive.

Feline Serum Amyloid A (fSAA) is one of the major acute-phase proteins in cats. It is produced in the liver by cytokines in response to disease, inflammatory responses, and tissue damage. The blood concentration of fSAA can increase by more than 1000 times during an acute-phase response, so it can be used as a major marker for disease or inflammatory responses.

Like CRP, fSAA is normally present at very low concentrations, but rapidly increases and then decreases during illness, infection, or surgery, allowing for monitoring recovery or selecting appropriate treatment.

In cats with an inflammatory response, levels of SAA, alpha 1-acid glycoprotein (alpha 1-AG), and haptoglobin (HP) increased, while levels of feline C-Reactivity Protein (fCRP) remained unchanged. Because fSAA increases early after the acute phase reaction, followed by increases in alpha 1-AG and HP, fSAA can be used as a marker for early detection of the acute phase reaction.

Against this backdrop, the inventors of the present invention have made extensive efforts to develop a monoclonal antibody against fSAA, and have completed the present invention by producing a monoclonal antibody having excellent specificity and sensitivity, with high binding affinity specifically for fSAA.

One object of the present invention is to provide a monoclonal antibody (mAb) that specifically binds to feline serum amyloid A (SAA) protein.

Another object of the present invention is to provide a fusion cell line producing the monoclonal antibody of the present invention.

Another object of the present invention is to provide a composition for detecting feline serum amyloid A, comprising a monoclonal antibody that specifically binds to feline serum amyloid A protein.

Another object of the present invention is to provide a method for detecting feline serum amyloid A in vitro using the composition for detecting feline serum amyloid A of the present invention.

Another object of the present invention is to provide a kit for detecting feline serum amyloid A comprising the monoclonal antibody of the present invention.

Another object of the present invention is to provide an information-providing method for determining the presence or absence of antibodies to feline serum amyloid A using the kit of the present invention.

Another object of the present invention is to provide a method for detecting antibodies to feline serum amyloid A using the kit of the present invention.

Another object of the present invention is to provide a method for providing information necessary for detecting feline serum amyloid A, which comprises a step of contacting a monoclonal antibody of the present invention with a biological sample isolated from a feline body fluid to confirm the formation of an antigen-antibody complex.

This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention can also be applied to each other description and embodiment. In other words, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Furthermore, the scope of the present invention should not be considered limited by the specific descriptions described below.

One aspect of the present invention provides a monoclonal antibody (mAb) that specifically binds to feline serum amyloid A (SAA) protein.

The term "Serum Amyloid A (SAA) of the present invention" refers to an amino acid sequence of amyloid A (APP) found in many species, including cats, dogs, and horses. SAA is secreted into plasma from the liver and is also produced by various extrahepatic tissues at sites of inflammation. Most circulating SAA is bound to high-density lipoproteins (HDL), and similar to other AAPPs, SAA concentrations increase in response to inflammatory stimuli. Cats and dogs have 111 a.a.r. SAA, whereas horses have 110 a.a.r. SAA. Cats, dogs, and horses have eight amino acids inserted into the central portion of the molecule compared to human or mouse SAA. The primary amino acid sequences of SAA from various mammalian species are highly conserved, sharing 61-80% sequence identity, so that all SAA families share a common structural fold. SAAs are found in diseases such as urinary tract disorders or after surgery, pancreatitis, injury, infection-related diseases, tumors, kidney diseases, mammary tumors, lymphomas, It is a useful diagnostic aid for peritonitis and sepsis, and is independent of breed, sex, age, neutering status, and SAA concentration. Cats with high SAA levels had a significantly shorter average survival time of 72 days compared to 571 days in cats with low SAA levels, suggesting its potential use as a prognostic biomarker.

The term "Feline Serum Amyloid A (SAA)" of the present invention refers to Feline Serum Amyloid A (fSAA), which is one of the major acute-phase proteins in cats and is produced in the liver by cytokines due to diseases, inflammatory reactions, tissue damage, etc., and fSAA is a lipoprotein trimer structural substance of 11.4 to 12.5 kDa and is composed of 104 to 112 amino acids, and the concentration of fSAA in the blood can increase by more than 1000 times during an acute-phase reaction, so it can be used as a major marker for diseases or inflammatory reactions. When an inflammatory reaction occurs in the body, macrophages are activated and inflammatory cytokines such as Interleukin (IL)-1, IL-6, and Tumor necrosis factor-α (TNF-α) are secreted. These inflammatory cytokines cause SAA to be produced in hepatocytes, starting 6 hours after immune stimulation, reaching peak levels 21 to 24 hours later, and returning to normal levels within 1 to 2 weeks.

The blood SAA concentration of a healthy cat is 0.6 to 5 mg/L, and a level of 5 mg/L or less is generally considered normal. In addition, it has been reported that the blood SAA concentration increases to over 300 mg/L in cats with symptoms such as injury, renal failure, or infection, and the level is also greatly increased in feline infectious peritonitis (FIP, feline infectious peritonitis), so it can be used as an auxiliary means of diagnosis in the current situation where a specific diagnostic method for FIP does not exist.

The term "monoclonal antibody (mAb)" of the present invention refers to an antibody that has the specificity to recognize and react with only one antigenic determinant (epitope), and is formed by a single antibody-forming cell and consists of a single molecular structure. A complete antibody has an overall Y shape and is composed of two long heavy chains (H) and two short light chains (L). Each heavy chain and light chain are connected to each other by a sulfur bond, and are divided into a variable region (V), which is a site that reacts with an antigen, and a constant region (C), which is a site that expresses an effector function. The variable region contains a complementarity-determining region (CDR), which determines the structure of the variable region so that it can form a specific binding with an antigen and controls the antibody binding strength.

The term "heavy chain" of the present invention may be interpreted to mean a full-length heavy chain and fragments thereof, including a variable region domain VH comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen and three constant region domains CH1, CH2 and CH3.

The term "light chain" of the present invention may be interpreted to mean a full-length light chain and fragments thereof, including a variable region domain VL and a constant region domain CL, which include an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen.

The term "CDR (complementarity determining region)" of the present invention refers to the amino acid sequence of the hypervariable region of the heavy and light chains of an immunoglobulin. The heavy and light chains may each include three CDRs (CDRH1, CDRH2, CDRH3 and CDRL1, CDRL2, CDRL3). The CDRs may provide key contact residues for antibody binding to an antigen or epitope.

The term "antigen-binding fragment" of the present invention refers to a fragment of the entire immunoglobulin structure, and a portion of a polypeptide that includes a portion capable of binding an antigen. For example, it may be F(ab')2, Fab', Fab, Fv, or scFv, but is not limited thereto. Among the antigen-binding fragments, Fab has a structure having variable regions of the light and heavy chains, a constant region of the light chain, and the first constant region (CH1) of the heavy chain, and has one antigen-binding site. Fab' differs from Fab in that it has a hinge region containing one or more cysteine residues at the C-terminus of the heavy chain CH1 domain. F(ab')2 antibodies are produced when the cysteine residues in the hinge region of Fab' form a disulfide bond. Fv is the minimum antibody fragment that has only the heavy chain variable region and the light chain variable region, and recombinant techniques for producing Fv fragments are widely known in the art. A two-chain Fv has a heavy chain variable region and a light chain variable region linked non-covalently, and a single-chain Fv has a heavy chain variable region and a single chain variable region covalently linked via a peptide linker or directly linked at the C-terminus, so that they can form a dimer-like structure like a two-chain Fv. The antigen-binding fragment can be obtained using a proteolytic enzyme (for example, a Fab can be obtained by restriction digestion of a whole antibody with papain, and an F(ab')2 fragment can be obtained by digestion with pepsin) or can be produced through genetic recombination technology.

In the present invention, the monoclonal antibody of the present invention may specifically bind to feline serum amyloid A protein.

The amino acid sequence of the above cat serum amyloid A protein can be obtained from a known database, but is not limited thereto.

As another example, the cat serum amyloid A protein may have, comprise, consist of, or consist essentially of the amino acid sequence set forth in SEQ ID NO: 1.

The cat serum amyloid A protein may comprise an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 80%, 80.3%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% homology or identity thereto. In addition, it is obvious that a protein having an amino acid sequence in which a part of the sequence is deleted, modified, substituted, conservatively substituted, or added is also included within the scope of the present application, as long as it has such homology or identity and exhibits an effect corresponding to the protein comprising the amino acid sequence of SEQ ID NO: 1.

For example, if the amino acid sequence has sequence additions or deletions, naturally occurring mutations, silent mutations or conservative substitutions that do not alter the function of the protein of the present application at the N-terminus, C-terminus and/or within the amino acid sequence.

The term "conservative substitution" refers to the replacement of one amino acid with another amino acid having similar structural and/or chemical properties. Such amino acid substitutions may generally be based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or amphipathic nature of the residues. Typically, conservative substitutions may have little or no effect on the activity of a protein or polypeptide.

In this application, the terms "homology" or "identity" refer to the degree of similarity between two given amino acid sequences or base sequences, which may be expressed as a percentage. The terms "homology" and "identity" are often used interchangeably.

Sequence homology or identity of conserved polynucleotides or polypeptides is determined by standard alignment algorithms, and may be combined with default gap penalties established by the program being used. In practice, homologous or identical sequences can generally hybridize with all or part of the sequence under moderate or high stringency conditions. It should be appreciated that hybridization also includes hybridization with polynucleotides containing common codons or codons that take codon degeneracy into account.

Whether any two polynucleotide or polypeptide sequences are homologous, similar or identical can be determined using known computer algorithms such as the "FASTA" program using default parameters, for example, as in Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444. Alternatively, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later) can be determined using the GCG program package (Devereux, J., et al, Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego, 1994, and [CARILLO et al.](1988) SIAM J Applied Math 48: 1073). For example, homology, similarity, or identity can be determined using BLAST or ClustalW from the National Center for Biotechnology Information database.

Homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information, for example, using a GAP computer program such as that of Needleman et al. (1970), J Mol Biol. 48:443, as disclosed, for example, in Smith and Waterman, Adv. Appl. Math (1981) 2:482. In brief, the GAP program can be defined as the total number of symbols in the shorter of the two sequences divided by the number of similarly arranged symbols (i.e., nucleotides or amino acids). Default parameters for the GAP program include (1) a binary comparison matrix (containing values of 1 for identity and 0 for non-identity) and (2) a comparison matrix as disclosed by Gribskov et al. (1986) Nucl. Acids Res. 48:443, as disclosed by Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979). 14: 6745 weighted comparison matrix (or EDNAFULL (EMBOSS version of NCBI NUC4.4) permutation matrix); (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or a gap opening penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for terminal gaps.

In the present invention, a polynucleotide encoding feline serum amyloid A protein may be included, but is not limited thereto.

The polynucleotide encoding the above feline serum amyloid A protein can have various modifications in the coding region within a range that does not change the amino acid sequence of the feline serum amyloid A protein, taking into account the degeneracy of the codon or the preferred codon in an organism that is to express the feline serum amyloid A protein. Specifically, the polynucleotide encoding the feline serum amyloid A protein can have, include, consist of, or consist essentially of a base sequence having a homology or identity of 70% or more, 75% or more, 76% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, or 98% or more with the base sequence of SEQ ID NO: 2.

Additionally, probes that can be prepared from known genetic sequences, for example, sequences encoding proteins that hybridize under stringent conditions with a complementary sequence to all or part of the polynucleotide sequence, and have the activity of a protein consisting of the amino acid sequence of SEQ ID NO: 1, may be included without limitation. The "stringent conditions" above refer to conditions that enable specific hybridization between polynucleotides. The appropriate stringency for hybridizing polynucleotides depends on the length and degree of complementarity of the polynucleotides, and the variables are well known in the art (see Sambrook et al., supra, 9.50-9.51, 11.7-11.8).

The method for producing the monoclonal antibody of the present invention is not particularly limited, and may be manufactured by a general method known in the art. For example, it may be manufactured by a chemical peptide synthesis method, it may be manufactured by amplifying a gene encoding the monoclonal antibody by PCR (polymerase chain reaction) or synthesizing it by a known method, cloning it into an expression vector, and expressing it, it may be manufactured using a hybridoma cell line derived from lymphocytes obtained by injecting an immunogen of the monoclonal antibody into an animal, and it may be manufactured using a hybridoma cell line produced by inoculating an animal with a protein derived from undifferentiated dental pulp stem cells to which the monoclonal antibody specifically binds as an immunogen, and using lymphocytes obtained from the animal.

The monoclonal antibody of the present invention, like conventional monoclonal antibodies, may be composed of two full-length heavy chains and two full-length light chains. In this case, the heavy chains and light chains are not particularly limited.

In addition, some amino acid sequences constituting the monoclonal antibody may be mutated. In proteins and polypeptides, amino acid exchanges that do not overall change the activity of the molecule are well known in the art. The most common exchanges are exchanges between amino acid residues Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly, and a monoclonal antibody having a sequence mutated from the amino acid sequence of the monoclonal antibody provided by the present invention through such exchanges may be included in the scope of the present invention. In addition, a mutated monoclonal antibody having increased structural stability against heat, pH, etc. or increased antibody activity of the monoclonal antibody due to a mutation or modification in the amino acid sequence may also be included in the scope of the present invention.

The above monoclonal antibody may be produced from a fusion cell line deposited under the deposit number KCTC19152P.

The term "fusion cell line" of the present invention refers to a cell created by artificially fusing two different types of cells, and refers to a cell or cell line in which two or more homologous or heterologous cells are fused using a substance that causes cell fusion, such as polyethylene glycol, or a type of virus, thereby integrating different functions of each cell into a single cell. In particular, hybrid cells created by fusing myeloma cells with B cells, which are precursors of antibody-producing cells among lymphocytes contained in the spleen or lymph nodes, produce monoclonal antibodies and are therefore widely applied in research and clinical practice. In addition, T cells that produce lymphokines, which are physiologically active substances, and hybridomas, which are tumor cells thereof, are also being commercialized.

In one embodiment, the fusion cell line of the present invention may be prepared by culturing a mixture of spleen cells and myeloma cells from a mouse immunized with feline serum amyloid A protein. Among the fusion cell lines, the fusion cell line Anti-fSAA mAb 4A11, which produces a monoclonal antibody specific for feline serum amyloid A protein, was deposited with the Biological Resource Center of the Korea Research Institute of Bioscience and Biotechnology on December 20, 2023, and was assigned the accession number KCTC19152P.

Another aspect provides a fusion cell line producing the monoclonal antibody.

At this time, the description of the “monoclonal antibody” and “fusion cell line” is as described above.

Specifically, the fusion cell line may be deposited under the deposit number KCTC19152P. The fusion cell line may be cultured in large quantities in vitro or in vivo.

Additionally, the monoclonal antibody produced by the above-mentioned fusion cell line can be used without purification, or can be purified to a high purity (e.g., 95% or higher) using methods well known in the art. Such purification techniques include, for example, gel electrophoresis, dialysis, salt precipitation, and chromatography, allowing separation from culture medium or ascites fluid.

In the present invention, the monoclonal antibody of the present invention may be IgG or an isotype thereof.

For example, the monoclonal antibody of the present invention may be an immunoglobulin subtype of IgG2a. In another example, the subtype of the mAb produced from the fusion cell line of the present invention, Anti-fSAA mAb 4A11, may be IgG2a, but is not limited thereto.

Another aspect of the present invention provides a composition for detecting feline serum amyloid A, comprising the monoclonal antibody of the present invention.

The terms used herein are as described above.

The composition for detecting antibodies to feline serum amyloid A of the present invention comprises the monoclonal antibody that can be used to specifically bind to feline serum amyloid A protein and thus ultimately detect antibodies to feline serum amyloid A, and therefore can be usefully used for detecting antibodies to feline serum amyloid A.

Another aspect of the present invention provides a method for detecting cat serum amyloid A in vitro using the composition of the present invention.

Another aspect of the present invention provides a kit for detecting feline serum amyloid A, comprising the monoclonal antibody of the present invention.

The terms used herein are as described above.

In the present invention, the assay system for use in the kit is not limited thereto, but may include an ELISA plate, a dipstick device, an immunochromatographic assay device, a radiation fractionation immunoassay device, and a flow-through device. For example, a diagnostic kit in the form of a strip or device using an immunochromatographic assay (ICA) may be used.

In the present invention, the kit may additionally include the monoclonal antibody conjugated to a label; or a secondary antibody and/or chromogenic substrate capable of binding to the monoclonal antibody.

Examples of the above markers include horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase, luciferase, β-D-galactosidase, malate dehydrogenase (MDH), acetylcholinesterase, colloidal gold, metal nanoparticles, silica nanoparticles, organic fluorescent materials, fluorescent proteins, quantum dots, radioactive materials, isotopes, dyes, carbon nanotubes, graphene, and fullerene. May include, but is not limited to, the following:

Examples of the above chromogenic substrates may include, but are not limited to, DAB (Diaminobenzidine), AEC (3-amino-9-ethylcarbazole), BCIP/NBT (5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium), BCIP/INT (5-bromo-4-chloro-3-indolyl phosphate/iodonitrotetrazolium), NF (New fuchsin), FRT (Fast Red TR Salt), TMB (3,3',5,5'-tetramethyl bezidine), ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)), and OPD (o-phenylenediamine).

Another aspect of the present invention provides a composition for detecting antibodies to feline serum amyloid A, comprising a monoclonal antibody that specifically binds to feline serum amyloid A protein.

Another aspect of the present invention provides a kit for detecting antibodies to feline serum amyloid A, comprising a monoclonal antibody that specifically binds to feline serum amyloid A protein.

The terms used herein are as described above.

In the present invention, the antibody against feline serum amyloid A may be feline-derived IgG or a subtype thereof.

For example, the kit may detect antibodies to feline serum amyloid A as an immunoglobulin subtype of IgG2a.

In the present invention, the assay system for use in the kit may include, but is not limited to, an ELISA plate, a dipstick device, an immunochromatographic assay device, a radiation fractionation immunoassay device, and a flow-through device. For example, a diagnostic kit in the form of a strip or device using an immunochromatographic assay (ICA) may be used. As another example, reference may be made to the antibody detection kit disclosed in Korean Patent No. 10-2128453.

The monoclonal antibody of the present invention can be used to objectively analyze the presence or absence of feline Serum Amyloid A (fSAA) and proteins contained in clinical specimens such as cat tissue, blood, body fluid, etc. or samples for inflammation-related research.

Another aspect of the present invention provides a method for detecting antibodies to feline serum amyloid A, comprising the step of contacting a biological sample isolated from an individual with a monoclonal antibody of the present invention.

Another aspect of the present invention provides a method for providing information necessary for detecting feline serum amyloid A, comprising the step of contacting a monoclonal antibody of the present invention with a biological sample isolated from a feline body fluid to confirm the formation of an antigen-antibody complex.

The terms used herein are as described above.

The term "biological sample" of the present invention includes, but is not limited to, whole blood, plasma, serum, tissue, tissue fragments, cells, cell fragments, body fluids, saliva, synovial fluid, cerebrospinal fluid, lymph, feces, and urine.

Additionally, in the present invention, the biological sample may be used interchangeably with “specimen”, “test solution” or “sample”.

The method of the present invention may further include, but is not limited to, a step of confirming the formation of an antigen-antibody complex between the cat serum amyloid A protein and the monoclonal antibody in the sample.

The term "antigen-antibody complex" of the present invention means a complex formed by combining feline serum amyloid A protein and a monoclonal antibody of the present invention that binds to feline serum amyloid A protein.

At this time, the monoclonal antibody of the present invention may be conjugated to a label. Alternatively, the monoclonal antibody of the present invention may be conjugated to a secondary antibody and/or a chromogenic substrate capable of binding to the monoclonal antibody of the present invention. The label, secondary antibody, and chromogenic substrate are as described above in the previous embodiment.

The formation of the antigen-antibody complex may be detected by a method such as, but not limited to, a colorimetric method, an electrochemical method, a fluorometric method, a luminometry method, a particle counting method, an absorbance measurement method, a spectroscopic method, a Raman spectroscopic method, a surface plasmon resonance method, an interferometric method, a visual assessment method, and a scintillation counting method.

The monoclonal antibody of the present invention has a high binding affinity specifically for only the protein of feline serum amyloid A (SAA) and does not react with other proteins, and thus can be usefully used for the detection of feline serum amyloid A or the diagnosis of diseases caused by feline serum amyloid A infection. In addition, by utilizing the above characteristics, it can be usefully used in related research and development such as prevention, treatment, prognosis observation, and titer measurement of feline serum amyloid A.

Figure 1 shows the results of electrophoresis of feline serum amyloid A (fSAA) protein using polyacrylamide gel (abbreviation: SM, protein size marker).
Figure 2 shows the results of electrophoresis using a polyacrylamide gel of a monoclonal antibody (mAb) against feline serum amyloid A protein produced from the fusion cell line of the present invention (Anti-fSAA mAb 4A11) (Abbreviation: SM, protein size marker).
Figure 3 shows the results of confirming the isotype of mAb against feline serum amyloid A protein produced from the fusion cell line of the present invention (Anti-fSAA mAb 4A11).
Figure 4 is a diagram showing the dissociation constant (K _d ) of a mAb produced from the fusion cell line of the present invention (Anti-fSAA mAb 4A11) against feline serum amyloid A protein.
Figure 5 shows the results confirming that the monoclonal antibody produced from the fusion cell line (Anti-fSAA mAb 4A11) of the present invention exhibits specificity only for feline serum amyloid A protein.

Hereinafter, to aid understanding of the present invention, examples and other embodiments will be described in detail. However, the embodiments according to the present invention may be modified in various ways, and the scope of the present invention should not be construed as being limited to the following examples. The embodiments of the present invention are provided to more fully explain the present invention to those of average skill in the art.

Example 1. Production of monoclonal antibody (mAb)

Example 1-1. Immunization and cell fusion

To produce monoclonal antibodies specific to the feline SAA protein, mice were immunized to produce a fusion cell line that produces monoclonal antibodies specific to the feline SAA protein.

A suspension containing 100 μg of feline SAA protein and an equal volume of complete Freund's adjuvant was prepared and injected intraperitoneally into 6-week-old female mice (BALB/C, Daehan Biolink Co., Ltd., Korea). Three weeks later, incomplete Freund's adjuvant was injected once more using the same method, and then three weeks later, the mice were immunized once more using the same method. Two days after the last injection, tail blood was collected to obtain serum, which was diluted 1/1,000 in phosphate buffer and the antibody titer was determined by enzyme-linked immunosorbent assay (ELISA). If the titer was low, the mice were immunized again three weeks later using the same method.

After that, the spleen was removed from the immunized mice, mashed with a tissue grinder, and the cell suspension was placed in a cell culture flask and centrifuged to recover myeloma cells and spleen cells. Myeloma cells and spleen cells were recovered, suspended in DMEM medium, and the number of cells was counted. ^1Х107 myeloma cells and ^1Х108 spleen cells were transferred to a 50 ml container, an appropriate amount of DMEM medium was added, and centrifuged at 200Хg for 5 minutes to recover the cells. The recovered cells were suspended in DMEM medium, and 1 ml of PEG (50% polyethylene glycol) solution was added for 1 minute while gently shaking. After 5 minutes, 1 ml of DMEM medium was slowly added over 30 seconds, and then 3 ml of DMEM culture medium was added over 30 seconds. Again, 17 ml of DMEM culture medium was added over 1 minute, then 20 ml of DMEM culture medium was added, and the reaction was continued for 5 minutes. After centrifugation at 200 Хg for 5 minutes, the medium was removed. After carefully resuspending in 50 ml of DMEM culture medium containing 1% HAT (hypoxathine-aminopterin-thymidine), 100 ㎕ of the cells were added per well of a 96-well cell culture plate containing feeder cells, and cultured in a 37°C, 5% _CO2 incubator.

Example 1-2. Cloning of a fusion cell line and production of monoclonal antibodies using ascites

After culturing the cells cultured as in Example 1-1 for 10 more days, it was confirmed that the fused cells formed colonies and grew, then about 100 ㎕ of the medium was taken from a 96-well cell culture plate, and the wells in which cells producing specific antibodies were growing were screened using a plate coated with cat SAA protein. The cells in the wells were harvested and transferred to a 24-well cell culture vessel, cultured, and cloning was continued until a stable fused cell line producing a monoclonal antibody specific to the cat SAA protein was obtained. Upon completion of cloning, the cells were transferred to a T flask, cultured in bulk, frozen, and then stored in liquid nitrogen.

The fusion cell line Anti-fSAA mAb 4A11, which produces a monoclonal antibody specific for the feline SAA protein obtained above, was deposited at the Biological Resource Center of the Korea Research Institute of Bioscience and Biotechnology on December 20, 2023 and assigned the accession number KCTC19152P.

In addition, for mass production of antibodies, 0.5 ml of incomplete Freund's adjuvant was injected into the peritoneal cavity of 6-8 week-old mice (BALB/C). On the 1st day, the fused cell line (Anti-fSAA mAb 4A11) was suspended in phosphate buffer and counted. 1.5 Х 10 ⁶ cells per mouse were suspended in 0.5 ml of phosphate buffer and injected into the peritoneal cavity. After 1-2 weeks, when ascitic fluid had accumulated appropriately, the ascitic fluid was collected using a syringe and then frozen.

Example 1-3. Purification of monoclonal antibodies

Ammonium sulfate was added to the ascites collected in the same manner as in Example 1-2, mixed for 30 minutes, centrifuged at 8,000 rpm for 30 minutes, and the supernatant was collected. Ammonium sulfate was added to the supernatant again at a concentration of 50%, and left to stand at 4°C for 30 minutes. Centrifuged again at 8,000 rpm for 30 minutes, the supernatant was discarded, and the precipitate was suspended in 20 mM PBS. This suspension was dialyzed against 20 mM PBS for more than 18 hours, and the dialysate was injected into a Protein G-coupled column equilibrated with 20 mM PBS (pH 7.4). After all the injections were completed, unadsorbed substances were removed using PBS. The antibody bound to the column was eluted with a 10 mM glycine solution (pH 2.9). At this time, 1 M Tris (pH 9.0) solution was added in a volume of 1/10 of the eluate to adjust the pH to 7.0–7.5. The eluted antibody solution was concentrated and dialyzed against PBS, and after dialysis was completed, electrophoresis was performed on a polyacrylamide gel.

As a result, bands corresponding to the heavy chain and light chain of the monoclonal antibody were formed at the 50 kDa and 25 kDa regions, respectively, confirming that the mAb produced from the fusion cell line (Anti-fSAA mAb 4A11) was separated and purified (Fig. 2).

Example 1-4. Monoclonal antibody isotype and dissociation constant (K) of monoclonal antibody _d ) check

As a result of confirming the subtype of mAb specific for feline SSA protein produced from the fusion cell line (Anti-fSAA mAb 4A11) in Examples 1-1 to 1-3 above, it was confirmed that the subtype of mAb produced from the fusion cell line, Anti-fSAA mAb 4A11, was IgG2a (Fig. 3).

In addition, as a result of confirming the dissociation constant (K _d ) of the mAb, it was confirmed that the K _d value of the mAb produced from the fusion cell line Anti-fSAA mAb 4A11 was 0.610 ± 0.019 nM (Fig. 4).

Example 3. Monoclonal antibody specificity test

To confirm whether the fusion cell line of the present invention (Anti-fSAA mAb 4A11) exhibits specificity only for the feline SSA protein produced, it was compared with feline infectious peritonitis virus (FIPV), feline coronavirus (FCoV), and feline herpesvirus (FHV).

As a result, it was confirmed that the monoclonal antibody produced from the fusion cell line (Anti-fSAA mAb 4A11) did not show specificity for FIPV, FCoV, and FHV antigens, but only for the feline SAA protein (Fig. 5).

As can be seen from the results of the above examples, the fusion cell line (Anti-fSAA mAb 4A11) of the present invention has a high binding affinity only for the feline SSA protein and does not react with other proteins, so it can be usefully used for the detection of the feline SSA protein or the diagnosis of diseases caused by infection in cats. In addition, by utilizing the above characteristics, it can be usefully used in related research and development such as prevention, treatment, prognosis observation, and titer measurement.

From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without altering its technical spirit or essential characteristics. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as encompassing all changes or modified forms derived from the meaning and scope of the following claims and their equivalent concepts, rather than the detailed description above.

Korea Research Institute of Bioscience and Biotechnology, Biological Resource Center (KCTC) KCTC19152P 20231220

Attach an electronic file of the sequence list

Claims (13)

A monoclonal antibody that specifically binds to feline serum amyloid A protein.
A monoclonal antibody according to claim 1, wherein the cat serum amyloid A protein comprises an amino acid sequence of sequence number 1.
A fusion cell line producing the monoclonal antibody of claim 1.
In the third paragraph, the fusion cell line is a fusion cell line deposited under the deposit number KCTC19152P.
A composition for detecting feline serum amyloid A, comprising the monoclonal antibody of claim 1.
A method for detecting cat serum amyloid A in vitro using the composition of claim 5.
A kit for detecting feline serum amyloid A, comprising the monoclonal antibody of claim 1.
In claim 7, the kit is characterized in that it detects antibodies to feline serum amyloid A by distinguishing them as IgG2a.
A method for providing information for determining the presence of antibodies to feline serum amyloid A using the kit of Article 7.
A method for detecting antibodies to feline serum amyloid A using the kit of Article 7.
A method for providing information necessary for detecting feline serum amyloid A, comprising a step of contacting the monoclonal antibody of claim 1 with a biological sample isolated from a cat's body fluid to confirm the formation of an antigen-antibody complex.
A method according to claim 11, wherein the biological sample separated from the cat's body fluid is at least one selected from the group consisting of whole blood, plasma, serum, tissue, tissue lysate, cells, cell lysate, body fluid, saliva, synovial fluid, cerebrospinal fluid, lymph, feces, and urine.
In claim 11, the antigen-antibody complex formation is detected by at least one method selected from the group consisting of a colorimetric method, an electrochemical method, a fluorometric method, a luminometry method, a particle counting method, an absorbance measurement method, a spectroscopic method, a Raman spectroscopic method, a surface plasmon resonance method, an interferometric method, a visual assessment method, and a scintillation counting method.