Peptide antigens for formylmethionine antibody production

Abstract

The present invention relates to a novel mixed peptide antigen for producing a formylmethionine antibody. A formylmethionine-specific antibody produced using the antigen peptide of the present invention can detect formylmethionine by universally binding to a peptide or protein containing formylmethionine, and has the effect of having higher sensitivity compared to a conventional antibody using a single antigen peptide.

Description

Technology Field

[0001] The present invention relates to a novel mixed peptide antigen for the production of formylmethionine antibodies. Background Technology

[0002] Methionine is the common starting amino acid residue for growing peptide chains (e.g., protein synthesis) in most biological systems. The two most prominent modifications of methionine, the initiating amino acid, involve the addition of an N-formyl group to the methionine molecule prior to the start of messenger RNA (mRNA) translation, and the subsequent removal of the N-formyl group from the amino(N-)-terminal methionine of the subsequent immature peptide. N-terminal formylation is a type of protein modification observed primarily in bacteria, mitochondria, and chloroplasts, and is known to be observed in the cytoplasm of eukaryotic cells under specific stress conditions. The enzyme that plays a major role in the formylation of intracellular proteins is formylmethionyl-transferase, which produces formylmethionyl-tRNA by attaching a formyl group to methionyl-tRNA. Subsequently, when formylmethionyl-tRNA is used to initiate protein synthesis, a protein containing formylmethionine at the N-terminus is synthesized. The N-formyl group is removed by peptide diformylase. Peptide diformylase (PDF) detaches the formyl group from most immature formylmethionine-peptides in substrate-specific reactions. However, there are exceptions to the general action of PDF. For example, some E. coli proteins exist partially in a formylated state (Housechild-Logat, P., 1968; Marasco, WA et al., 1984; and Milligan, DL and Koshland, Jr., DE, 1990).

[0003] Meanwhile, methods for constructing antibodies to specifically detect N-terminal formylation of proteins have long been proposed. For example, to produce an antibody that recognizes the peptide known as formyl-MLF, a method has been used in which a formyl-MLF-like peptide is attached to a carrier protein and used as the antigen to construct the antibody. However, constructing antibodies using a single peptide antigen in this manner has the disadvantage that the antibody strongly recognizes only peptides / proteins containing structures or sequences similar to the peptide antigen, while weakly failing to recognize peptides / proteins with structures or sequences different from the antigen. For instance, in the case of a polyclonal antibody recently produced using fMGSGC as an antigen, it strongly recognizes peptides starting with fMG but relatively weakly recognizes other peptides starting with fM, which also contributes to lowering the sensitivity of the antibody itself. Therefore, when using existing methods, antibodies that recognize each formyl peptide must be produced separately, and inefficiency becomes pronounced when considering the diversity of protein sequences.

[0004] Therefore, it is necessary to develop antibodies that can overcome the limitations of conventional technology and universally recognize peptides / proteins. The problem to be solved

[0005] The object of the present invention is to provide an antigen peptide for producing an antibody specific to formylmethionine (fMet, fM).

[0006] In addition, the objective of the present invention is to provide a composition for producing an antibody specific to formylmethionine.

[0007] In addition, the objective of the present invention is to provide a kit for producing an antibody specific to formylmethionine.

[0008] In addition, the objective of the present invention is to provide a method for producing an antibody specific to formylmethionine.

[0009] In addition, the objective of the present invention is to provide an antibody specific to formylmethionine.

[0010] In addition, the objective of the present invention is to provide a composition for detecting formylmethionine.

[0011] In addition, the objective of the present invention is to provide a kit for detecting formylmethionine.

[0012] In addition, the objective of the present invention is to provide a method for detecting a peptide or protein containing formylmethionine. means of solving the problem

[0013] To solve the above problem, the present invention provides an antigen peptide for producing an antibody specific to formylmethionine.

[0014] In addition, the present invention provides a composition for producing an antibody specific to formylmethionine comprising the antigen peptide.

[0015] In addition, the present invention provides a kit for producing an antibody specific to formylmethionine comprising the antigen peptide.

[0016] In addition, the present invention provides a method for producing an antibody specific to formylmethionine.

[0017] In addition, the present invention provides an antibody specific to formylmethionine.

[0018] In addition, the present invention provides a composition for detecting formylmethionine comprising the above antibody.

[0019] In addition, the present invention provides a kit for detecting formylmethionine comprising the above antibody.

[0020] In addition, the present invention provides a method for detecting a peptide or protein containing formylmethionine. Effects of the invention

[0021] The formylmethionine-specific antibody produced using the antigen peptide of the present invention can detect formylmethionine by universally binding to peptides or proteins containing formylmethionine, and has the effect of having higher sensitivity compared to conventional antibodies using a single antigen peptide. Brief explanation of the drawing

[0022] Figure 1 is a figure confirming the antigen-binding ability of the formylmethionine antibody of the present invention to formylmethionine in Escherichia coli: Act: Actinonin; CBB staining: Coomassie staining image; anti-fMGSGC serum: Serum containing antibodies produced using conventional fMGSGC antigens; anti-fMdPEG4C serum: serum containing an antibody produced using the fMdPEG4C antigen; and anti-fMXC serum: Serum containing antibodies produced using the mixed antigen fMXC. FIG. 2 is a figure confirming the antigen-binding ability of the formylmethionine antibody of the present invention to formylmethionine in mammalian cells: Act: Actinonin; anti-fMGSGC serum: Serum containing antibodies produced using conventional fMGSGC antigens; anti-fMdPEG4C serum: serum containing an antibody produced using the fMdPEG4C antigen; and anti-fMXC serum: Serum containing antibodies produced using the mixed antigen fMXC. Specific details for implementing the invention

[0023] Hereinafter, the present invention will be described in detail with respect to embodiments of the present invention. However, the following embodiments are presented as examples of the present invention and are not intended to limit the present invention, and various modifications and applications are possible within the scope of the claims set forth below and the equivalents interpreted therefrom.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. Any method and material similar or equivalent to those described herein may be used in practice to test the invention, but preferred materials and methods are described herein.

[0025] Throughout this specification, not only are conventional one- and three-character codes for naturally occurring amino acids used, but generally accepted three-character codes for other amino acids, such as Aib (α-aminoisobutyric acid), Sar (N-methylglycine), etc., are also used. Additionally, amino acids referred to by abbreviations in this invention are described according to the IUPAC-IUB nomenclature as follows:

[0026] Alanine: Ala, A; Arginine: Arg, R; Asparagine: Asn, N; Aspartic acid: Asp, D; Cysteine: Cys, C; Glutamic acid: Glu, E; Glutamine: Gln, Q; Glycine: Gly, G; Histidine: His, H; Isoleucine: IIe, I; Leucine: Leu, L; Lysine: Lys, K; Methionine: Met, M; Phenylalanine: Phe, F; Proline: Pro, P; Serine: Ser, S; Threonine: Thr, T; Tryptophan: Trp, W; Tyrosine: Tyr, Y; and Valine: Val, V.

[0028] In one aspect, the present invention relates to an antigen peptide for producing an antibody specific to formylmethionine (fMet, fM), comprising the amino acid sequence of the following general formula 1:

[0029] fM-XC (General Formula 1, Sequence No. 1)

[0030] In the above formula,

[0031] fM is formylmethionine; and

[0032] X is G(Gly), A(Ala), V(Val), L(Leu), I(Ile), T(Thr), S(Ser), C(Cys), M(Met), D(Asp), N(Asn), E(Glu), Q(Gln), K(Lys), R(Arg), H(His), F(Phe), Y(Tyr), W(Trp) or P(Pro).

[0033] In one embodiment, X may be one or more selected from the group consisting of G(Gly), A(Ala), V(Val), L(Leu), I(Ile), T(Thr), S(Ser), C(Cys), M(Met), D(Asp), N(Asn), E(Glu), Q(Gln), K(Lys), R(Arg), H(His), F(Phe), Y(Tyr), W(Trp), and P(Pro).

[0034] In one embodiment, the antigen peptide of the present invention comprises a peptide comprising an amino acid sequence of fMGC, a peptide comprising an amino acid sequence of fMAC, a peptide comprising an amino acid sequence of fMVC, a peptide comprising an amino acid sequence of fMLC, a peptide comprising an amino acid sequence of fMIC, a peptide comprising an amino acid sequence of fMTC, a peptide comprising an amino acid sequence of fMSC, a peptide comprising an amino acid sequence of fMCC, a peptide comprising an amino acid sequence of fMMC, a peptide comprising an amino acid sequence of fMDC, a peptide comprising an amino acid sequence of fMNC, a peptide comprising an amino acid sequence of fMEC, a peptide comprising an amino acid sequence of fMQC, a peptide comprising an amino acid sequence of fMKC, a peptide comprising an amino acid sequence of fMRC, a peptide comprising an amino acid sequence of fMHC, a peptide comprising an amino acid sequence of fMFC, and a peptide comprising an amino acid sequence of fMYC. It may include a peptide, a peptide containing the amino acid sequence of fMWC, or a peptide containing the amino acid sequence of fMPC.

[0035] In one embodiment, the antigen peptide of the present invention may be a mixed peptide comprising fMGC (SEQ No. 2), fMAC (SEQ No. 3), fMVC (SEQ No. 4), fMLC (SEQ No. 5), fMIC (SEQ No. 6), fMTC (SEQ No. 7), fMSC (SEQ No. 8), fMCC (SEQ No. 9), fMMC (SEQ No. 10), fMDC (SEQ No. 11), fMNC (SEQ No. 12), fMEC (SEQ No. 13), fMQC (SEQ No. 14), fMKC (SEQ No. 15), fMRC (SEQ No. 16), fMHC (SEQ No. 17), fMFC (SEQ No. 18), fMYC (SEQ No. 19), fMWC (SEQ No. 20), and fMPC (SEQ No. 21).

[0036] In one embodiment, the antigen peptide may further comprise a carrier protein, and the carrier protein may be keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), or ovalbumin (OVA).

[0037] In one embodiment, a carrier protein may be attached to the antigen peptide through a linker, and the linker may be a chemical linker and may be sulfo-SMCC (sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate).

[0038] In one embodiment, the carrier protein may be bound / attached to the cysteine ​​of the antigen peptide.

[0039] In one embodiment, the carrier protein may be bound to / attached to the C-terminus of the antigen peptide and may be bound to / attached to the carboxyl group of the C-terminus.

[0040] In one embodiment, the cysteine ​​(Cys) group of the antigen peptide and the lysine (Lys) group of the carrier protein keyhole-limpet hemocyanin can be combined / linked using Sulfo-SMCC.

[0041] In the present invention, formylation refers to the action of introducing a formyl group (CHO-) into a compound. In the present invention, "formylation" refers to the formylation of the α-amino group of an amino acid.

[0042] In the present invention, "peptide" refers to a polymer of amino acids; typically, a form in which a small number of amino acids are linked is called a peptide, and a form in which many amino acids are linked is called a protein. In the structure of a peptide or protein, the connection between amino acids consists of amide bonds or peptide bonds. A peptide bond refers to a bond in which water (H2O) is removed between a carboxyl group (-COOH) and an amino group (-NH2) to form a -CO-NH- structure. The peptide of the present invention can be prepared according to chemical synthesis methods known in the art, particularly solid-phase synthesis techniques (Merrifield, J. Amer. Chem. Soc. 85:2149-54 (1963); Stewart, et al., Solid Phase Peptide Synthesis, 2nd. ed., Pierce Chem. Co.: Rockford, 111 (1984)), and can also be produced by genetic engineering techniques.

[0043] Even if the present invention is described as "a peptide composed of a specific sequence number," if it has the same or corresponding activity as a peptide composed of the amino acid sequence of the said sequence number, it does not exclude meaningless sequence additions before or after the amino acid sequence of the said sequence number, naturally occurring mutations, or silent mutations thereof, and it is obvious that even in cases having such sequence additions or mutations, it falls within the scope of the present invention.

[0044] In the present invention, modifications to the peptide sequence may involve some amino acids being modified through any one of substitution, addition, deletion, and modification, or a combination of these methods. Such modifications include modifications using L-type or D-type amino acids and / or non-natural amino acids; and / or modifications by modifying the natural sequence, e.g., modification of side chain functional groups, intramolecular covalent bonds, e.g., inter-side chain ring formation, methylation, acylation, ubiquitination, phosphorylation, aminohexalation, biotinylation, etc. The substituted or added amino acids may include the 20 amino acids typically observed in human proteins as well as atypical or non-naturally occurring amino acids. Commercial sources of atypical amino acids include Sigma-Aldrich, ChemPep, and Genzyme Pharmaceuticals. Peptides containing these amino acids and typical peptide sequences can be synthesized and purchased through commercial peptide synthesis companies, for example, American Peptide Company or Bachem in the United States, or Anygen in Korea.

[0045] In addition, the range of antigen peptides of the present invention includes functional equivalents of a peptide comprising the amino acid sequence of SEQ ID NO. 1, more preferably functional equivalents of a peptide comprising the amino acid sequence of SEQ ID NO. 1, and salts thereof.

[0046] The above-mentioned functional equivalent refers to a peptide having at least 75%, preferably 90%, and more preferably 95% sequence homology (i.e., identity) with the peptide of SEQ ID NO. 1 as a result of the addition, substitution, or deletion of amino acids, such as having sequence homology of 75, 76, 77, 78, 79, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%, and having substantially the same physiological activity as the peptide of SEQ ID NO. 1. In this specification, sequence homology and homology are defined as the percentage of amino acid residues of the candidate sequence relative to the amino acid sequence of SEQ ID NO. 1 after aligning the candidate sequence with the amino acid sequence of SEQ ID NO. 1 and introducing gaps. If necessary, conservative substitutions are not considered as part of the sequence homology in order to obtain the maximum percentage sequence homology. The N-terminus, C-terminus, or internal extension, deletion, or insertion of the amino acid sequence of SEQ ID NO. 1 is not interpreted as a sequence affecting the sequence homology or homology.

[0047] In addition, the sequence homogeneity can be determined by general standard methods used to compare similar portions of the amino acid sequences of two polypeptides. Computer programs such as BLAST or FASTA align two polypeptides so that their respective amino acids are optimally matched (along the full length of one or two sequences or along a predicted portion of one or two sequences). The program provides a default opening penalty and a default gap penalty, and provides a scoring matrix such as PAM250 (standard scoring matrix) that can be used in conjunction with the computer program. For example, percentage homogeneity can be calculated as follows: multiply the total number of identical matches by 100 and then divide by the sum of the length of the longer sequence within the corresponding matched span and the number of gaps introduced into the longer sequence to align the two sequences.

[0048] The scope of functional equivalents of the present invention includes derivatives and mimetics (peptidomimetics), and "derivative" is a general term for similar peptides in which a portion of the chemical structure of the peptide is modified while maintaining the basic framework of the peptide of SEQ ID NO. 1, and preferably, one or more amino acids are substituted with other amino acids, one or more amino acids are added, one or more amino acids are deleted, or a compound that increases the half-life of the peptide (e.g., polyethylene glycol, etc.) is fused. In the present invention, the stability, storage stability, volatility, or solubility of the peptide according to the present invention may be maintained, increased, or decreased by the derivative.

[0049] In the present invention, "antibody" refers to a functional component of serum and is often referred to as a collection of molecules (antibodies or immunoglobulins) or a single molecule (antibody molecule or immunoglobulin molecule). An antibody molecule may bind to or react with specific antigenic determinants (antigens or antigenic epitopes) to induce, in turn, immunological effector mechanisms. Individual antibody molecules are generally considered to be unispecific, and the composition of antibody molecules may be monoclonal (i.e., composed of identical antibody molecules) or polyclonal (i.e., composed of different antibody molecules reacting with identical or different epitopes on the same antigen or distinct antigens). Each antibody molecule possesses a unique structure that enables it to specifically bind to its corresponding antigen, and all natural antibody molecules have the same overall basic structure of two identical light chains and two identical heavy chains. Antibodies are also collectively known as immunoglobulins. The term antibody or antibodies used in the present invention is used in the broadest sense and includes original antibodies, chimeric antibodies, humanized antibodies, complete human and short-chain antibodies, as well as binding fragments of antibodies, such as Fab, Fv fragments or scFv fragments, and multimeric forms, such as dimer IgA molecules or pentavalent IgM.

[0050] In the present invention, "epitope" is generally used to describe a portion of a larger molecule or a part of a larger molecule (e.g., an antigen or antigenic site) that possesses antigenic or immunogenic activity in animals, preferably mammals, and most preferably humans. An epitope possessing immunogenic activity is a portion of a larger molecule that elicits an antibody response in animals. An epitope possessing antigenic activity is a portion of a larger molecule to which an antibody immunospecifically binds, determined by any method widely known in the art, for example, the immunoassay described herein. An antigenic epitope does not necessarily have to be immunogenic. An antigen is a substance to which an antibody or antibody fragment immunospecifically binds, such as a toxin, virus, bacterium, protein, or DNA. An antigen or antigenic site often possesses more than one epitope, unless they are very small, and can often stimulate an immune response. Antibodies that bind to different epitopes on the same antigen can have varying effects on the activity of the antigen to which they bind, depending on the location of the epitope. While antibodies that bind to an epitope at the active site of the antigen completely block the function of the antigen, other antibodies that bind to different epitopes may have no or almost no effect on the activity of the antigen.

[0051] In the present invention, "polyclonal (polyclonal) antibody" refers to a composition of different (various) antibody molecules capable of binding to or reacting with several different specific antigenic determinants / epitopes on the same or different antigens, wherein each individual antibody in the composition can react with a specific epitope. Generally, the variability of polyclonal antibodies lies in the so-called variability regions of polyclonal antibodies, particularly the CDR1, CDR2, and CDR3 regions. In the present invention, polyclonal antibodies may be produced in a single pot or may be a mixture of different polyclonal antibodies. A mixture of monoclonal antibodies is produced in individual batches and does not necessarily have to come from the same individual or cell line, and thus is not considered as a polyclonal antibody itself, for example, because it would result in differences in post-translational modification. However, if a mixture of monoclonal antibodies provides the same antigen / epitope coverage as the polyclonal antibody of the present invention, it will be considered equivalent to the polyclonal antibody.

[0052] In one aspect, the present invention relates to a peptide comprising an amino acid sequence of fMGC, a peptide comprising an amino acid sequence of fMAC, a peptide comprising an amino acid sequence of fMVC, a peptide comprising an amino acid sequence of fMLC, a peptide comprising an amino acid sequence of fMIC, a peptide comprising an amino acid sequence of fMTC, a peptide comprising an amino acid sequence of fMSC, a peptide comprising an amino acid sequence of fMCC, a peptide comprising an amino acid sequence of fMMC, a peptide comprising an amino acid sequence of fMDC, a peptide comprising an amino acid sequence of fMNC, a peptide comprising an amino acid sequence of fMEC, a peptide comprising an amino acid sequence of fMQC, a peptide comprising an amino acid sequence of fMKC, a peptide comprising an amino acid sequence of fMRC, a peptide comprising an amino acid sequence of fMHC, a peptide comprising an amino acid sequence of fMFC, a peptide comprising an amino acid sequence of fMYC, and an amino acid sequence of fMWC. The present invention relates to an antigen peptide mixture comprising a peptide containing a sequence and a peptide containing the amino acid sequence of fMPC.

[0053] In one embodiment, each peptide in the antigen peptide mixture may be an isolated (e.g., synthetic and / or purified) peptide.

[0054] In the present invention, the peptide of the "peptide mixture" refers to a peptide that functions as an antigen and has a length of three amino acids. Accordingly, the "peptide mixture" refers to a mixture of the said peptides.

[0055] In one aspect, the present invention relates to a composition for producing an antibody specific to formylmethionine comprising the antigen peptide of the present invention or a mixture of said antigen peptides.

[0056] In one embodiment, the composition comprises a peptide comprising an amino acid sequence of fMGC, a peptide comprising an amino acid sequence of fMAC, a peptide comprising an amino acid sequence of fMVC, a peptide comprising an amino acid sequence of fMLC, a peptide comprising an amino acid sequence of fMIC, a peptide comprising an amino acid sequence of fMTC, a peptide comprising an amino acid sequence of fMSC, a peptide comprising an amino acid sequence of fMCC, a peptide comprising an amino acid sequence of fMMC, a peptide comprising an amino acid sequence of fMDC, a peptide comprising an amino acid sequence of fMNC, a peptide comprising an amino acid sequence of fMEC, a peptide comprising an amino acid sequence of fMQC, a peptide comprising an amino acid sequence of fMKC, a peptide comprising an amino acid sequence of fMRC, a peptide comprising an amino acid sequence of fMHC, a peptide comprising an amino acid sequence of fMFC, and a peptide comprising an amino acid sequence of fMYC. It may include a mixture of antigen peptides, comprising a peptide containing the amino acid sequence of fMWC and a peptide containing the amino acid sequence of fMPC.

[0057] In one embodiment, the composition may be a reagent composition for producing an antibody specific to formylmethionine.

[0058] In one embodiment, the antibody specific to formylmethionine can detect the first formylmethionine regardless of the second amino acid type of the peptide or protein.

[0059] In one aspect, the present invention relates to a kit for producing an antibody specific to formylmethionine comprising the antigen peptide of the present invention, the antigen peptide mixture, or the composition.

[0060] The antibody manufacturing composition and kit according to the present invention may further include known components for maintaining and preserving the antigen peptide.

[0061] In one embodiment, the antibody manufacturing composition and kit may further include a known agent for inducing and promoting an immune response within a host.

[0062] In one aspect, the present invention relates to a method for producing an antibody specific to formylmethionine, comprising the steps of: inoculating a host other than a human with the antigen peptide of the present invention or a mixture of the antigen peptides multiple times to induce an immunization reaction; and obtaining serum from the blood of the host.

[0063] In one embodiment, the host may be a mammal other than a human, and the mammal other than a human may be a mouse, rabbit, rat, guinea pig, horse, dog, sheep, goat, cat, chicken, duck, monkey, or primate, and is more preferably a rabbit, but is not limited thereto.

[0064] In one embodiment, the serum may contain a formylmethionine antibody.

[0065] In one embodiment, the step of purifying the formylmethionine antibody from the serum may be further included.

[0066] In one aspect, the present invention relates to an antibody specific to formylmethionine produced by the method of the present invention.

[0067] In one embodiment, the antibody may be a polyclonal antibody.

[0068] In one embodiment, the antibody may specifically bind to a peptide or protein containing formylmethionine, and the formylmethionine may be the first amino acid of the peptide or protein, and the antibody specific to formylmethionine of the present invention may detect the first formylmethionine regardless of the type of the second amino acid of the peptide or protein.

[0069] In one aspect, the present invention relates to a composition for detecting formylmethionine comprising the antibody of the present invention.

[0070] In one embodiment, the composition may be a reagent composition.

[0071] In one aspect, the present invention relates to a kit for detecting formylmethionine comprising the antibody of the present invention.

[0072] In one aspect, the present invention relates to a method for detecting a peptide or protein containing formylmethionine, comprising the step of treating a sample with the antibody of the present invention to induce an antigen-antibody reaction.

[0073] In one embodiment, the peptide or protein may be a peptide or protein in which the first amino acid is formylmethionine.

[0075] The present invention will be explained in more detail through the following examples. However, the following examples are intended only to illustrate the content of the present invention and do not limit the present invention.

[0077] Example 1. Preparation of fMXC mixed peptide antigen

[0078] To produce an antibody specific to formylmethionine (fMet), fMXC peptides were synthesized to produce a mixed antigen. Specifically, the first amino acid fM of the peptide antigen is formylmethionine, the second amino acid X is one of glycine (Gly, G), alanine (Ala, A), valine (Val, V), leucine (Leu, L), isoleucine (Ile, I), threonine (Thr, T), serine (Ser, S), cysteine ​​(Cys, C), methionine (Met, M), aspartic acid (Asp, D), asparagine (Asn, N), glutamic acid (Glu, E), glutamine (Gln, Q), lysine (Lys, K), arginine (Arg, R), histidine (His, H), phenylalanine (Phe, F), tyrosine (Tyr, Y), tryptophan (Trp, W), and proline (Pro, P), and the third amino acid, which is a crosslinkable residue, is cysteine ​​(C). Mixed antigen fMXC was produced by synthesizing a mixture of 20 antigen peptides (fMGC, fMAC, fMVC, fMLC, fMIC, fMTC, fMSC, fMCC, fMMC, fMDC, fMNC, fMEC, fMQC, fMKC, fMRC, fMHC, fMFC, fMYC, fMWC, and fMPC). In addition, a keyhole limpet hemocyanin (KLH) transport protein was attached to the synthesized peptides using the chemical linker sulfo-SMCC (sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) to finally produce fMXC-(sulfo-SMCC)-KLH.

[0080] Example 2. Production of formylmethionine-specific antibody

[0081] The mixed antigen fMXC-(sulfo-SMCC)-KLH prepared in Example 1 above, and its control antigens fMGSGC-(sulfo-SMCC)-KLH and fMdPEG4-(sulfo-SMCC)-KLH, were each mixed with complete Freund's adjuvant (CFA) and injected subcutaneously into rabbits (New Zealand white). Immunoboosting was performed in the same manner at weeks 4, 6, and 8 of the initial immunization time, and at week 9, blood was collected from the heart to obtain final serum containing antibodies (anti-fMGSGC serum, anti-fMdPEG4C serum, and anti-fMXC serum).

[0083] Example 3. Confirmation of the antigen-binding ability of formylmethionine-specific antibodies

[0084] 3-1. Antigen-binding ability of antibodies in E. coli

[0085] To confirm the antigen-binding ability of the three formylmethionine-specific antibodies (anti-fMGSGC serum, anti-fMdPEG4C serum, and anti-fMXC serum) prepared in Example 2 above in E. coli, Coomassie blue staining and Western blot analysis were performed. Specifically, E. coli DH5α was inoculated into LB medium (LB broth 25 g / L) and cultured overnight in a 37°C incubator. The next day, the antibodies were diluted 100-fold in LB medium and cultured again; when the OD600 reached 0.6, the antibodies were treated with ethanol or the peptide deformylase inhibitor actinonin (Act) for 30 minutes. At this time, actinonin was prepared by dissolving it in ethanol at a concentration of 20 mg / ml and treated with E. coli to achieve a final concentration of 2 μg / ml. Afterwards, the culture medium was centrifuged at 12,000g for 2 minutes at 4°C to obtain an E. coli pellet, which was then dissolved in a 2% SDS solution containing a protease inhibitor cocktail. The pellet was sonicated for several seconds, then centrifuged at 12,000g for 10 minutes at 4°C to obtain the supernatant, and its protein concentration was measured using a BCA protein assay. The supernatant diluted to a concentration of 2 μg / μl was mixed with 4X SDS sample buffer at a ratio of 3:1 (v / v), heated at 70°C for 10 minutes, and the membrane obtained by electrophoresis was blocked for 1 hour with 5% skim milk (in PBS-T [(137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4 and 0.1% Tween 20 {w / v}]), and the formylmethionine-specific antibody anti-fMXC serum (unpurified serum) prepared in Example 2 and two control antibodies (anti-fMGSGC serum and anti-fMdPEG4C serum) were each reacted overnight at 4°C with solutions diluted at a ratio of 1:1000 in 5% skim milk.Afterward, the membrane was washed three times with PBS-T for 10 minutes each, reacted with a secondary antibody (Goat anti-rabbit IgG HRP conjugate, diluted 1:5000 in 5% skim milk) for 1 hour, and washed three times with PBS-T for 10 minutes each. Formylmethionine was detected on the washed membrane in chemiluminescence mode using a GE Amersham Imager AI680 instrument and an HRP substrate.

[0086] As a result, the formylmethionine antibody produced using the mixed antigen fMXC was found to specifically bind to a protein containing formylmethionine produced in E. coli (Fig. 1). Furthermore, even when the production of a protein containing formylmethionine in E. coli was increased by actinonin treatment, both the antibody produced using the conventional fMGSGC antigen and the antibody produced using the mixed antigen fMXC of the present invention exhibited high antigen binding affinity (Fig. 1). On the other hand, the antibody produced using the fMdPEG4C antigen showed relatively lower antigen binding affinity compared to the antibody produced using the mixed antigen fMXC (Fig. 1).

[0088] 3-2. Antigen-binding ability of antibodies in mammalian cells

[0089] Western blot analysis was performed to confirm the antigen-binding ability of the three formylmethionine-specific antibodies (anti-fMGSGC serum, anti-fMdPEG4C serum, and anti-fMXC serum) prepared in Example 2 above in mammalian cells. Specifically, HK2 cell lines, which are human kidney-derived cells, were cultured in DMEM / F-12 + 10% FBS medium, and when the cell density reached 90%, they were treated with ethanol or actinonin, a peptide diformylase inhibitor, for 10 hours. At this time, actinonin was prepared by dissolving it in ethanol at a concentration of 20 mg / ml and treated to E. coli to a final concentration of 10 μg / ml. Subsequently, cells were harvested using trypsin enzyme and lysed in RIPA buffer supplemented with a protease inhibitor cocktail. After sonicating the sample for a few seconds, the supernatant was obtained by centrifuging at 12,000 g for 10 minutes at 4°C. The supernatant, diluted to a protein concentration of 2 μg / μl, was mixed with 4X SDS sample buffer at a ratio of 3:1 (v / v) and heated at 70°C for 10 minutes. The membrane obtained by electrophoresis was blocked with 5% skim milk for 1 hour, and the formylmethionine-specific antibody anti-fMXC serum (unpurified serum) prepared in Example 2 and two control antibodies (anti-fMGSGC serum and anti-fMdPEG4C serum), each diluted in 5% skim milk at a ratio of 1:1000, were reacted overnight at 4°C with the solution. Afterward, the membrane was washed three times with PBS-T for 10 minutes each, reacted with a secondary antibody (Goat anti-rabbit IgG HRP conjugate, diluted 1:5000 in 5% skim milk) for 1 hour, and washed three times with PBS-T for 10 minutes each. Formylmethionine was detected on the washed membrane in chemiluminescence mode using a GE Amersham Imager AI680 instrument and an HRP substrate.

[0090] As a result, the formylmethionine antibody produced using the mixed antigen fMXC was found to specifically bind to proteins containing formylmethionine produced within the cell (Fig. 2). Furthermore, since the synthesis of proteins containing formylmethionine mainly occurs only in mitochondria in human-derived cells, antibodies produced using the conventional fMGSGC antigen and the antibody produced using the fMdPEG4C antigen showed low antigen binding affinity for proteins containing formylmethionine that were slightly increased by actinonin treatment, whereas the antibody produced using the mixed antigen fMXC showed significantly higher antigen binding affinity compared to them (Fig. 2).

[0092] Through this, it was confirmed that the antibody produced using the mixed antigen fMXC of the present invention has superior sensitivity to formylmethionine compared to conventional antibodies.

[0095] Sequence information for SEQ ID NOs 1 to 21, which have fewer than 4 defined amino acids as described in this specification, is provided below. *fM: formylmethionine (fMet, fM)

Claims

Claim 1 Peptide composed of the amino acid sequence of fMGC, peptide composed of the amino acid sequence of fMAC, peptide composed of the amino acid sequence of fMVC, peptide composed of the amino acid sequence of fMLC, peptide composed of the amino acid sequence of fMIC, peptide composed of the amino acid sequence of fMTC, peptide composed of the amino acid sequence of fMSC, peptide composed of the amino acid sequence of fMCC, peptide composed of the amino acid sequence of fMMC, peptide composed of the amino acid sequence of fMDC, peptide composed of the amino acid sequence of fMNC, peptide composed of the amino acid sequence of fMEC, peptide composed of the amino acid sequence of fMQC, peptide composed of the amino acid sequence of fMKC, peptide composed of the amino acid sequence of fMRC, peptide composed of the amino acid sequence of fMHC, peptide composed of the amino acid sequence of fMFC, peptide composed of the amino acid sequence of fMYC, peptide composed of the amino acid sequence of fMWC An antigen peptide for producing an antibody specific to formylmethionine (fMet, fM), comprising a peptide and a peptide composed of the amino acid sequences of fMPC. Claim 2 delete Claim 3 delete Claim 4 An antigen peptide for producing an antibody specific to formylmethionine, further comprising a carrier protein in claim 1. Claim 5 In claim 4, an antigen peptide for producing an antibody specific to formylmethionine, wherein a carrier protein is bound through a linker. Claim 6 In claim 5, the linker is sulfo-SMCC (sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate), an antigen peptide for producing an antibody specific to formylmethionine. Claim 7 A composition for producing an antibody specific to formylmethionine, comprising the antigen peptide of claim 1. Claim 8 delete Claim 9 A kit for producing an antibody specific to formylmethionine, comprising the antigen peptide of claim 1. Claim 10 a) a step of inducing an immunization reaction by inoculating a host other than a human multiple times with the antigen peptide of claim 1 or the composition of claim 7; and b) a step of obtaining serum from the blood of the host, comprising a method for producing an antibody specific to formylmethionine. Claim 11 A method for producing an antibody specific to formylmethionine, wherein, in claim 10, the step of purifying the formylmethionine antibody from serum is further included. Claim 12 An antibody specific to formylmethionine prepared by the method of claim 10. Claim 13 In claim 12, the antibody is a polyclonal antibody, an antibody specific to formylmethionine. Claim 14 In claim 12, the antibody is a formylmethionine-specific antibody that specifically binds to a peptide or protein containing formylmethionine. Claim 15 A composition for detecting formylmethionine comprising the antibody of claim 12. Claim 16 A kit for detecting formylmethionine containing the antibody of claim 12. Claim 17 A method for detecting a peptide or protein containing formylmethionine, comprising the step of treating a sample with the antibody of claim 12 to induce an antigen-antibody reaction. Claim 18 A method for detecting a peptide or protein containing formylmethionine, wherein, in claim 17, the first amino acid is formylmethionine, the peptide or protein.

Images