Method for detecting Clostridium perfringens enterotoxin

Abstract

To detect Clostridium perfringens enterotoxin (CPE) simply, quickly and with high sensitivity.SOLUTION: An antigen liquid containing fusion protein (VT2B-C-CPE) at a verotoxin receptor binding site and a Clostridium perfringens enterotoxin C terminal, and adjuvant is inoculated into a BALB / c mouse. After inoculation, a spleen cell or lymph node cell of the mouse are fused with P3U1 to create hybridoma, from which hybridoma producing a monoclonal antibody indicating high reactivity to CPE is selected, and acquisition of 5 hybridomas producing antibody showing excellent reactivity to CPE succeeds. Next, when the monoclonal antibodies are used to detect CPE with immunochromatography, a result that CPE can be detected with high sensitivity is obtained.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for detecting Clostridium perfringens enterotoxin, an antibody used in the method, and a kit for use in the method. [Background technology]

[0002] Clostridium perfringens is a gram-positive anaerobic bacillus belonging to the genus Clostridium. Clostridium perfringens is classified into five types, A to E, depending on the type of primary toxin it produces, and among these, the type that produces the enterotoxin that causes food poisoning is classified as type A. In Japan, food poisoning caused by enterotoxin-producing Clostridium perfringens is not very common, occurring in the range of 20 to 40 cases per year, but it often becomes a large-scale incident, with an average of more than 80 patients per case.

[0003] Enterotoxin is a general term for enterotoxin proteins, which are produced by various bacteria such as Staphylococcus aureus and Salmonella. The enterotoxin produced by Clostridium perfringens (Clostridium perfringens enterotoxin, hereafter abbreviated as CPE) is a protein consisting of 319 amino acid residues with a molecular weight of approximately 35 kDa, and details of its amino acid sequence, mechanism of action, etc. have already been studied (Patent Document 1, Non-Patent Document 1).

[0004] Current known methods for testing Clostridium perfringens or CPE include isolation and culture testing, enterotoxin production testing (RPLA method), and genetic testing (PCR method) (Patent Documents 2 to 4). However, isolation and culture testing requires a specialized laboratory, is cumbersome, and requires time for culturing and outsourcing. Furthermore, the RPLA method detects enterotoxins from cultured bacteria, which requires one day of testing time and has a minimum detection sensitivity of 1 to 2 ng / mL. Furthermore, the PCR method requires specialized equipment, which makes the method cumbersome and requires time for outsourcing. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Re-tabled publication 2018 / 159476 [Patent Document 2] Japanese Patent Application Laid-Open No. 2013-48611 [Patent Document 3] Japanese Patent Application Laid-Open No. 2004-069504 [Patent Document 4] Japanese Patent Application Publication No. 09-133684 [Non-patent literature]

[0006] [Non-Patent Document 1] Kengo Kitadokoro et al., Journal of the Crystallographic Society of Japan, Vol. 55, No. 3, 2013 Summary of the Invention [Problem to be solved by the invention]

[0007] The problem to be solved by the present invention is to detect Clostridium perfringens enterotoxin (CPE) simply, quickly and with high sensitivity. [Means for solving the problem]

[0008] The present inventors conducted extensive research to solve the above-mentioned problems. First, BALB / c mice were inoculated with an antigen solution containing a fusion protein (VT2B-C-CPE) of the verotoxin receptor binding site and the C-terminus of Clostridium perfringens enterotoxin and an adjuvant. After inoculation, splenocytes or lymph node cells from the mice were fused with P3U1 to produce hybridomas. From these, hybridomas producing monoclonal antibodies highly reactive to CPE were selected, and five hybridomas producing antibodies highly reactive to CPE were successfully isolated.

[0009] Next, we used these monoclonal antibodies to detect CPE by immunochromatography, and found that CPE could be detected with high sensitivity.

[0010] In a first aspect, the present invention provides a method for detecting Clostridium perfringens enterotoxin in a sample by immunochromatography (hereinafter abbreviated as the detection method of the present invention), which is characterized in that two different antibodies selected from the following group (hereinafter abbreviated as the antibody group of the present invention) are used as a labeled antibody and a capture antibody, respectively: 1) L4C8D6 antibody having at least the amino acid sequence of SEQ ID NO: 9 as the VH CDR3 region and the amino acid sequence of SEQ ID NO: 12 as the VL CDR3 region; 2) an L5C2E3 antibody having at least the amino acid sequence of SEQ ID NO: 19 as the VH CDR3 region and the amino acid sequence of SEQ ID NO: 22 as the VL CDR3 region; 3) S8D3F2 antibody having at least the amino acid sequence of SEQ ID NO: 29 as the VH CDR3 region and the amino acid sequence of SEQ ID NO: 32 as the VL CDR3 region; 4) L9E5E1 antibody having at least the amino acid sequence of SEQ ID NO: 39 as the VH CDR3 region and the amino acid sequence of SEQ ID NO: 42 as the VL CDR3 region; 5) L10E1G2 antibody having at least the amino acid sequence of SEQ ID NO: 49 as the VH CDR3 region and the amino acid sequence of SEQ ID NO: 52 as the VL CDR3 region; and 6) An antibody having one or two amino acid mutations in each of the VH CDR3 region and / or VL CDR3 region of the antibody of 1) to 5) above.

[0011] The antibodies belonging to the antibody group of the present invention are preferably isolated antibodies. The antibodies belonging to the antibody group of the present invention may be either polyclonal or monoclonal, but are preferably monoclonal in order to prevent erroneous detection due to non-specific reactions.

[0012] More preferably, the antibody belonging to the group of antibodies of the present invention has a 5.0x10 antibody against Clostridium perfringens enterotoxin. -10 M or less, preferably 3.0x10 -10 It has an equilibrium dissociation constant KD less than or equal to M.

[0013] In the antibody of 6) above, the amino acid mutation may be any of substitution, deletion, or insertion of an amino acid residue, but substitution is preferred, and conservative amino acid substitution is particularly preferred from the viewpoint of maintaining immunological specificity.

[0014] In further preferred embodiments, the antibodies of the present invention have the following characteristics: 1) an L4C8D6 antibody having at least the amino acid sequence of SEQ ID NO: 7 as a VH CDR1 region, the amino acid sequence of SEQ ID NO: 8 as a VH CDR2 region, the amino acid sequence of SEQ ID NO: 9 as a VH CDR3 region, the amino acid sequence of SEQ ID NO: 10 as a VL CDR1 region, the amino acid sequence of SEQ ID NO: 11 as a VL CDR2 region, and the amino acid sequence of SEQ ID NO: 12 as a VL CDR3 region; 2) an L5C2E3 antibody having at least the amino acid sequence of SEQ ID NO: 17 as a VH CDR1 region, the amino acid sequence of SEQ ID NO: 18 as a VH CDR2 region, the amino acid sequence of SEQ ID NO: 19 as a VH CDR3 region, the amino acid sequence of SEQ ID NO: 20 as a VL CDR1 region, the amino acid sequence of SEQ ID NO: 21 as a VL CDR2 region, and the amino acid sequence of SEQ ID NO: 22 as a VL CDR3 region; 3) an S8D3F2 antibody having at least the amino acid sequence of SEQ ID NO: 27 as a VH CDR1 region, the amino acid sequence of SEQ ID NO: 28 as a VH CDR2 region, the amino acid sequence of SEQ ID NO: 29 as a VH CDR3 region, the amino acid sequence of SEQ ID NO: 30 as a VL CDR1 region, the amino acid sequence of SEQ ID NO: 31 as a VL CDR2 region, and the amino acid sequence of SEQ ID NO: 32 as a VL CDR3 region; 4) an L9E5E1 antibody having at least the amino acid sequence of SEQ ID NO: 37 as a VH CDR1 region, the amino acid sequence of SEQ ID NO: 38 as a VH CDR2 region, the amino acid sequence of SEQ ID NO: 39 as a VH CDR3 region, the amino acid sequence of SEQ ID NO: 40 as a VL CDR1 region, the amino acid sequence of SEQ ID NO: 41 as a VL CDR2 region, and the amino acid sequence of SEQ ID NO: 42 as a VL CDR3 region; 5) an L10E1G2 antibody having at least the amino acid sequence of SEQ ID NO: 47 as a VH CDR1 region, the amino acid sequence of SEQ ID NO: 48 as a VH CDR2 region, the amino acid sequence of SEQ ID NO: 49 as a VH CDR3 region, the amino acid sequence of SEQ ID NO: 50 as a VL CDR1 region, the amino acid sequence of SEQ ID NO: 51 as a VL CDR2 region, and the amino acid sequence of SEQ ID NO: 52 as a VL CDR3 region; and 6) An antibody having one or two amino acid mutations in each of the VH CDR1 region, VH CDR2 region, VH CDR3 region, VL CDR1 region, VL CDR2 region and / or VL CDR3 region of the antibody of 1) to 5) above.

[0015] In a more preferred embodiment, the antibodies of the present invention have the following characteristics: 1) L4C8D6 antibody having the amino acid sequence of SEQ ID NO: 3 as the VH region and the amino acid sequence of SEQ ID NO: 5 as the VL region; 2) L5C2E3 antibody having the amino acid sequence of SEQ ID NO: 13 as the VH region and the amino acid sequence of SEQ ID NO: 15 as the VL region; 3) S8D3F2 antibody having the amino acid sequence of SEQ ID NO: 23 as the VH region and the amino acid sequence of SEQ ID NO: 25 as the VL region; 4) L9E5E1 antibody having the amino acid sequence of SEQ ID NO: 33 as the VH region and the amino acid sequence of SEQ ID NO: 35 as the VL region; 5) L10E1G2 antibody having the amino acid sequence of SEQ ID NO: 43 as the VH region and the amino acid sequence of SEQ ID NO: 45 as the VL region; and 6) An antibody having 80% or more sequence identity with the antibodies of 1) to 5) above.

[0016] In a second aspect, the present invention provides an antibody for detecting Clostridium perfringens enterotoxin, which is any one antibody or a combination of two or more antibodies selected from the above-mentioned group of antibodies of the present invention. The antibody combination is, for example, a combination of two antibodies for detecting Clostridium perfringens enterotoxin by immunochromatography.

[0017] In a third aspect, the present invention provides an immunochromatography kit for detecting Clostridium perfringens enterotoxin, comprising any one of antibodies or a combination of antibodies selected from the above-described group of antibodies of the present invention. The kit comprises, for example, a combination of the L5C2E3 antibody (2) and the L9E5E1 antibody (4), in which the L9E5E1 antibody (4) is sensitized with gold colloid to form a labeled antibody, and the L5C2E3 antibody (2) is immobilized linearly on a solid phase to form a capture antibody. [Effects of the Invention]

[0018] The antibodies of the present invention all have sufficiently low KD values ​​for Clostridium perfringens enterotoxin and can withstand the labeling required for immunochromatography. Therefore, by using the antibodies of the present invention, the detection method of the present invention enjoys the advantages of immunochromatography, such as simplicity, speed, and high sensitivity. [Brief explanation of the drawings]

[0019] [Figure 1] 1 shows a scheme for producing hybridomas to obtain the antibody group of the present invention. [Figure 2] This shows the C-CPE reactivity of 10 candidate antibodies. The left bar shows the OD450 measured by ELISA using C-CPE as the antigen, and the right bar shows the OD450 measured by ELISA using killed Enterobacterium bacteria as a negative control. [Figure 3] The reactivity of 10 purified candidate antibodies to C-CPE is shown. The left bar shows the OD450 in ELISA when the antigen C-CPE was used at a concentration of 1.0 μg / mL, and the right bar shows the OD450 when the antigen C-CPE was used at a concentration of 0.1 μg / mL. [Figure 4] The reactivity of 10 purified candidate antibodies to CPE is shown. The left bar shows the OD450 in ELISA when the antigen CPE concentration was 1.0 μg / mL, and the right bar shows the OD450 when the antigen CPE concentration was 0.1 μg / mL. [Figure 5] The affinity of eight purified candidate antibodies to C-CPE determined by surface plasmon resonance is shown. [Figure 6]Immunochromatographs are shown for each combination of gold colloid-sensitized candidate antibody and candidate antibody used for the test line, using water alone or C-CPE at 1, 10, 100, and 1000 ng / mL. [Figure 7] The figure shows an immunochromatograph in which a developer solution of 2% NP40-PBS in which CPE or stool + CPE was suspended was developed on immunochromatographic reagents of gold colloid sensitization: L9E5E1 and test line: L5C2E3. DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention is described in detail below. The terms used in this specification are understood according to their general definitions in the fields of microbiology, biochemistry, molecular biology, medicine, and pharmacology. However, for terms specifically explained or defined in this specification, the explanations or definitions in this specification take precedence. Furthermore, the literature cited in this specification is incorporated herein by reference.

[0021] In a first aspect, the present invention provides a method for detecting Clostridium perfringens enterotoxin in a sample by immunochromatography, as described above. Immunochromatography is understood to be an immunoassay method that detects an antigen using a labeled antibody and a capture antibody. Typically, the immunochromatographic process involves contacting a sample with a labeled antibody, applying the sample to a sample application portion of a solid phase, developing the labeled antibody-antigen conjugate in the sample, and capturing the labeled antibody-antigen conjugate with a capture antibody immobilized on a determination portion to form a labeled antibody-antigen-capture antibody conjugate, which is then determined visually or by an appropriate measurement device. Therefore, the detection method of the present invention typically includes the steps of: 1) contacting the sample with a labeled antibody; 2) developing the sample contacted with the labeled antibody on a solid phase having a determination portion to which a capture antibody is immobilized; and 3) confirming the determination portion.

[0022] In the detection method of the present invention, the specimen may be any specimen used in food poisoning tests, such as feces, beverages, or foods, and may be a specimen diluted or suspended in water, a buffer solution, etc. The term "specimen" also encompasses not only specimens or specimen dilutions or suspensions, but also mixtures of these with labeled antibodies.

[0023] In the detection method of the present invention, the labeled antibody is a labeled antibody selected from the group of antibodies of the present invention. The labeling method may be any method commonly used in the art, and typically includes, but is not limited to, binding a metal colloid, a fluorescent dye, magnetic latex beads, or the like to the antibody, such as sensitizing the antibody to gold colloid particles as a metal colloid.

[0024] In the detection method of the present invention, the capture antibody is an antibody that binds to the same antigen as the labeled antibody, i.e., CPE. Typically, the capture antibody is immobilized on a solid layer on which the sample is developed. The antigen bound to the labeled antibody, i.e., the labeled antibody-bound CPE, is captured on the solid layer by the capture antibody. Typically, the capture antibody is immobilized on a portion of the solid layer, for example, in a line perpendicular to the direction of electrophoresis, to form a test area.

[0025] In the detection method of the present invention, the solid layer may be any solid layer commonly used in immunochromatographic assays, including, but not limited to, a solid layer made of a microporous material such as glass fiber, nylon, nitrocellulose, polyvinylidene difluoride, cellulose acetate, etc. Furthermore, functionally, the solid layer includes a sample application portion, an electrophoresis portion, and a determination portion.

[0026] In the detection method of the present invention, the method of developing the sample is appropriately selected depending on the solid layer. For example, the development may be based on capillary action in the solid layer or on electrophoresis with the application of an external force such as electricity. Electrophoresis utilizing capillary action in the solid layer is preferred because it requires less equipment and is simpler.

[0027] In the detection method of the present invention, the determination or confirmation of the determination portion is appropriately selected depending on the labeled antibody. For example, such determination or confirmation of the determination portion includes visual inspection as well as measurement using a fluorescence measurement device. Visual inspection is preferred because it requires less equipment, but fluorescence measurement has the advantage of enabling quantitative measurement based on fluorescence intensity.

[0028] Clostridium perfringens enterotoxin (also known as CPE), which is the target of detection in the detection method of the present invention, is a protein consisting of 319 amino acids and represented by SEQ ID NO: 1, and the gene encoding it has the nucleotide sequence represented by SEQ ID NO: 2. The gene encoding CPE (SEQ ID NO: 2) is registered in a known database (GenBank, etc.) under the accession number M98037. The amino acid sequence of CPE and the nucleotide sequence of the gene encoding it are specifically shown below. It should be understood that for all amino acid sequences, the one-letter and three-letter codes can be unambiguously converted into each other.

[0029] Amino acid sequence of CPE (SEQ ID NO: 1): MLSNNLNPMVFENAKEVFLISEDLKTPINITNSNSNLSDGLYVIDKGDGWILGEPSVVSSQILNPNETGTFSQSLTKSKEVSINVNFSVGFTSEFIQASVEYGFGITIGEQNTIERSVST TAGPNEYVYYKVYATYRKYQAIRISHGNISDDGSIYKLTGIWLSKTSADSLGNIDQGSLIETGERCVLTVPSTDIEKEILDLAAATERLNLTDALNSNPAGNLYDWRSSNSYPWTQKLNL HLTITATGQKYRILASKIVDFNIYSNNFNNLVKLEQSLGDGVKDHYVDISLDAGQYVLVMKANSSYSGNYPYSILFQKF

[0030] Nucleotide sequence of the gene encoding CPE (SEQ ID NO: 2): gtttataata tataatatta tgtttagtga aattatgtta attactact tatttcttct tattattaa ttacatttc aacttgatct ctttaacgta tatctcttt attacccaag ctttaattcc ttcagcatta attacataa atgtccatgt agaaatata tcaatattattattattatatagataga tccaaaataa aaacttttaa attatatatt ataaaaaa aattagaat aaggagatgt taatataat atgcttagta acaatttaaa tccaatggtg ttcgaaatg ctaagaagt atttcttatt tctgaggatt taaaaacacc aattaatatt acaacttata attagatcaggattagt actataggattagta atattagggg aaccctcagt agtttcaagt siaattctta atcctaatga aacaggtacc tttagccaat cattaactta atctaagaa gtatctaa atgtaattt ttcagttgga tttactctg aatttataca agcatctgta gatatggat ttggataac taggaatac haaatattacaat tagactatacatagtactactac tcatactac tcatactac aaggtttag caacttatag aaagtatcaa gctattagaa tttctcatgg taatatctct gatgatggat caatttaata attacagga atatggctta gtaaaacatc tgcagatagc ttaggaata ttgatcagg ttcattaatt gaaactggtg aagatgtgt ttcattagatt gatatagaaccatag attagaatcatactactgctacaga aagattaaat ttaactgatg cattaaactc aaatccagct ggtaatttat atgattggcg ttcttctaac tcataccctt ggactcaaaa gcttaattta cacttaacaa ttacagctac tggacaaaaa tatagaatct tagctagcaa aattgttgat tttaatattt attcaaataa ttttaataat ctagtgaaat tagaacagtc cttaggtgat ggagtaaaag atcattatgt tgatataagc ttagatgctg gacaatatgt tcttgtaatg aaagctaatt catcatatag tggaaattac cctattcaa tattatttca aaaattttaa tattttaaaa taatataatc aaattaattt acaaaagaca gtatgtaata ttaaattatt acatactgtc taatttttt attataattt aatttttcat

[0031] In the present invention, antibodies may be so-called complete antibodies as well as various structures, such as, but not limited to, antibody fragments, monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies, chimeric antibodies, humanized antibodies, antibody fusions, and fragments of each of these.

[0032] Antibodies are typically tetramers consisting of two pairs of light and heavy chains, with the amino-terminal portion of each chain containing a variable region primarily responsible for antigen recognition. Three loops in the variable region assemble in each V domain of the heavy and light chains (also called VH for the heavy chain and VL for the light chain) to form the antigen-binding site. Each loop is also called a complementarity-determining region (hereinafter also referred to as "CDR"), and this region exhibits the most significant amino acid sequence variation. "Variable" refers to the fact that certain segments of the variable region vary widely among antibody sequences. The variability within the variable region is not uniformly distributed. Instead, V domains consist of relatively invariant structures called framework regions (FRs) of 15 to 30 amino acids separated by short, highly variable regions called "hypervariable regions," each 9 to 15 amino acids long or longer. Each VH and VL consists of three hypervariable regions (also known as "complementarity-determining regions" or "CDRs") and four FRs, which are arranged from the amino terminus to the carboxy terminus in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Among the CDRs, CDR3 is particularly important for determining complementarity, followed by the other CDRs. FRs contribute little or nothing to determining complementarity. Therefore, when specifying an antibody based on its antigen-binding specificity, it is possible to adequately identify the antibody by specifying only the CDR sequence, for example, the amino acid sequence of CDR2 or the amino acid sequences of CDR1 to CDR3, rather than specifying the entire amino acid sequence of the antibody.

[0033] In one embodiment, the antibody is an antibody fragment. Specific antibody fragments include, but are not limited to, (i) a Fab fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a Fd fragment consisting of the VH and CH1 domains; (iii) a Fv fragment consisting of the VL and VH domains of a single antibody; (iv) a dAb fragment consisting of a single variable region (Ward et al., 1989, Nature 341:544-546, incorporated by reference in its entirety); (v) an isolated CDR region; (vi) a F(ab')2 fragment, which is a bivalent fragment containing two linked Fab fragments; and (vii) a single-chain Fv molecule (scFv), in which the VH and VL domains are linked by a peptide linker that allows the two domains to associate to form an antigen-binding site (Bird et al., 1988, Science 242:423-426, Huston et al., 2002). al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883, incorporated by reference in its entirety); (viii) bispecific single-chain Fvs (WO 03 / 11161, incorporated by reference herein); and (ix) "diabodies" or "triabodies," which are multivalent or multispecific fragments constructed by gene fusion (Tomlinson et al., 2000, Methods Enzymol. 326:461-479; WO 94 / 13804; Holliger et al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-6448, all incorporated by reference in their entirety).

[0034] In some embodiments, the antibodies may be a mixture of different species, e.g., chimeric and / or humanized antibodies, etc. That is, in the present invention, CDR sets may be used with framework and constant regions other than those specifically set forth by sequence herein.

[0035] In general, both "chimeric antibody" and "humanized antibody" refer to antibodies that combine regions from two or more species. For example, a "chimeric antibody" traditionally contains variable region(s) from a mouse (or in some cases, a rat) and constant region(s) from a human. A "humanized antibody" typically refers to a non-human antibody that has variable domain framework regions swapped for sequences found in a human antibody. Generally, in a humanized antibody, the entire antibody, except for the CDRs, is encoded by a polynucleotide of human origin or is identical to such an antibody except within its CDRs. The CDRs, some or all of which are encoded by nucleic acid from a non-human organism, are grafted onto the beta-sheet framework of a human antibody variable region to create an antibody whose specificity is determined by the grafted CDRs. The production of such antibodies is described, for example, in WO92 / 11018, Jones, 1986, Nature 321:522-525, Verhoeyen et al., 1988, Science 239:1534-1536, all of which are incorporated herein by reference in their entireties.

[0036] In one embodiment, the antibody is a minibody. A minibody is a minimized antibody-like protein that contains an scFv linked to a CH3 domain (Hu et al., 1996, Cancer Res. 56:3055-3061, incorporated by reference in its entirety). In some cases, the scFv may be linked to an Fc region and may include some or the entire hinge region.

[0037] The antibodies of the present invention are typically isolated or recombinant. "Isolated," when used to describe various polypeptides disclosed herein, refers to a polypeptide that has been identified and separated and / or recovered from a cell or cell culture in which the polypeptide is expressed. Typically, an isolated polypeptide is prepared by at least one purification step. An "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigen specificities. For example, an isolated antibody that specifically binds to CPE is substantially free of antibodies that specifically bind to antigens other than CPE.

[0038] Isolated monoclonal antibodies with different specificities can be used in combination in the detection method of the present invention, for example, in the case of immunochromatography, by labeling one to form a labeled antibody and immobilizing the other on a solid phase to form a capture antibody.

[0039] An antibody selected from the antibody group of the present invention specifically binds to a CPE. "Specific binding" or "specifically binds," or "specific" for a particular antigen or antigen fragment, refers to binding that is measurably different from non-specific interactions. Specific binding can be measured, for example, by determining the binding of a molecule compared to the binding of a control molecule, which is usually a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target.

[0040] Specific binding to a particular antigen or antigen fragment is, for example, at least about 10 -1 , at least about 10 -2 , at least about 10 -3 , at least about 2.0x10 -3 , at least about 5.0x10 -3 , at least about 2.0x10 -4This is demonstrated by an antibody having a Kd (1 / s) for an antigen or antigen fragment of 20, 50, 100, 500, 1000, 5,000, or 10,000 times or more higher than that of a control molecule for the antigen or antigen fragment.

[0041] Furthermore, specific binding to a particular antigen or antigen fragment is exhibited by an antibody having an association rate constant Ka (1 / Ms) for the antigen or antigen fragment that is at least 20-, 50-, 100-, 500-, 1000-, 5,000-, or 10,000-fold higher than that for the antigen or antigen fragment relative to a control, where Ka refers to the association rate of a particular antibody-antigen interaction.

[0042] Furthermore, the binding characteristics of a specific antibody to an antigen or antigen fragment that specifically binds to the antibody are expressed as the equilibrium dissociation constant KD (M), calculated by Kd / Ks. The equilibrium dissociation constant is also called affinity. High binding characteristics are expressed as a low KD value. For example, the antibody used in the detection method of the present invention has a binding affinity of 5.0 x 10 for CPE. -10 M or less, preferably 3.0x10 -10 Has a KD of M or less.

[0043] The binding rate constant, dissociation rate constant, and equilibrium dissociation constant between an antibody and an antigen can all be easily determined by techniques known in the art, including, but not limited to, the so-called surface plasmon resonance method, which includes an antigen cross-linking method, an antibody cross-linking method, and a capture method.

[0044] In a second aspect, the present invention provides a group of antibodies that specifically bind to CPE. The antibodies included in the group of antibodies of the present invention can be used alone or in combination of two or more. Furthermore, any one antibody selected from the group of antibodies of the present invention can be used in combination with another antibody from the group of antibodies of the present invention, for example, another antibody that binds to CPE.

[0045] In the most preferred embodiment, antibody 1) L4C8D6 of the present invention has a VH having the amino acid sequence shown in SEQ ID NO: 3 and a VL having the amino acid sequence shown in SEQ ID NO: 5. Furthermore, the gene encoding the VH of antibody 1) has the nucleotide sequence shown in SEQ ID NO: 4, and the gene encoding the VC of antibody 1) is shown in SEQ ID NO: 6. The amino acid sequence of antibody 1) and the nucleotide sequence of the gene encoding it are specifically shown below.

[0046] Amino acid sequence of VH of antibody 1) (SEQ ID NO: 3): EVQAQESGPGLVKPSQTVSLTCTVTGISITTGNYRWSWIRQFPGNKLEWIGYIYYGGTVTYNPSLTSRTTITRDTSKNQLFLEMNSLTAEDTATYYCARVFGNFGYWYFDVWGAGTTVTVSS

[0047] Nucleotide sequence of the gene encoding VH of antibody 1) (SEQ ID NO: 4): gaggtgcaag ctcaggagtc aggacctggt ctggtgaaac cttctcagac agtgtccctc acctgcactg tcactggcat ctccatcacc actggaaatt acagatggag ctggatccgg cagtttccag gaaacaaact ggagtggata gggtacatat actacggtgg taccgttacc tataatccat ctctcacaag tcgaaccacc atcactagag acacttccaa gaaccaactc ttcctggaaa tgaactcttt gactgctgaa gacacagcca catactactg tgcacgcgtg tttggcaact tcggatactg gtacttcgat gtctggggcg cagggaccac ggtcaccgtc tcctca

[0048] Amino acid sequence of VL of antibody 1) (SEQ ID NO: 5): DNVLTQSPTTMAASPGEKITIACSASSSISSNYLHWYQQKPGFSPKLLIYRTSNLASGVPVRFSGSGSGTSYSLTIGTMEAEDVATYYCQQGSSVPLTFGAGTKLELK

[0049] Nucleotide sequence of the gene encoding VL of antibody 1) (SEQ ID NO: 6): gacaatgttc tcacccagtc tccaaccacc atggctgcat ctcccgggga gaagatcact atcgcctgca gtgccagctc aagtataagt tccaattact tgcattggta tcagcagaag ccaggattct cccctaaact cttgatttat aggacatcca atctggcttc tggagtccca gttcgcttca gtggcagtgg gtctgggacc tcttactctc tcacaattgg caccatggag gcagaagatg ttgccactta ctactgccag cagggtagta gtgtaccgct cacgttcggt gctgggacca agctggagct gaaa

[0050] Antibody 1) was further analyzed in detail, and CDRs 1 to 3 of VH and VL were identified. Antibody 1) of the present invention has an amino acid sequence in which VH CDR1 is represented by SEQ ID NO: 7, VH CDR2 is represented by SEQ ID NO: 8, and VH CDR3 is represented by SEQ ID NO: 9, and an amino acid sequence in which VL CDR1 is represented by SEQ ID NO: 10, VL CDR2 is represented by SEQ ID NO: 11, and VL CDR3 is represented by SEQ ID NO: 12. The amino acid sequences of CDRs 1 to 3 of VH and VL are specifically shown below.

[0051] VH CDR1 amino acid sequence of Antibody 1) (SEQ ID NO:7): GISITTGNYRWS VH CDR2 amino acid sequence of Antibody 1) (SEQ ID NO: 8): YIYYGGTVTYNPSLTS VH CDR3 amino acid sequence of Antibody 1) (SEQ ID NO: 9): VFGNFGYWYFDV VL CDR1 amino acid sequence of antibody 1) (SEQ ID NO: 10): SASSSISSNYLH VL CDR2 amino acid sequence of antibody 1) (SEQ ID NO: 11): RTSNLAS VL CDR3 amino acid sequence of antibody 1) (SEQ ID NO: 12): QQGSSVPLT

[0052] In the most preferred embodiment, antibody 2) L5C2E3 of the present invention has a VH having the amino acid sequence shown in SEQ ID NO: 13 and a VL having the amino acid sequence shown in SEQ ID NO: 15. The gene encoding the VH of antibody 2) has the nucleotide sequence shown in SEQ ID NO: 14, and the gene encoding the VC of antibody 2) is shown in SEQ ID NO: 16. The amino acid sequence of antibody 2) and the nucleotide sequence of the gene encoding it are specifically shown below.

[0053] Amino acid sequence of VH of antibody 2) (SEQ ID NO: 13): QVQLQQSGADLVRPGTSLRVSCKASGYAFTNYLIEWIKQRPGQGLEWIGVINPGSGGTNYNENFKGKATLTADKSSSTAYMQLSSLTSDDSAVYFCARHGNYGNYDMDYWGQGTSVTVSS

[0054] Nucleotide sequence of the gene encoding VH of antibody 2) (SEQ ID NO: 14): caggtccaac tgcagcagtc tggagctgac ctggtcaggc ctgggacttc attgaggtg tcctgcaagg cttctggata cgccttcact aattacttga tagagtggat aaagcagagg cctggacagg gccttgagtg gattggagtg attaatcctg gaagtggtgg tactaattac aatgagaact tcaagggcaa ggcaacactg actgcagaca aatcctccag cactgcctac atgcagctca gcagcctgac atctgatgac tctgcggtct atttctgtgc aagacatggt aactacggga actatgatat ggactactgg ggtcaaggaa cctcagtcac cgtctcctca

[0055] Amino acid sequence of VL of antibody 2) (SEQ ID NO: 15): DVVMTQSPVTLSVTPGDRVSLSCRASQSISDYLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGYGFPLTFGAGTKLELK

[0056] Nucleotide sequence of the gene encoding VL of antibody 2) (SEQ ID NO: 16): gatgttgtga tgacccagtc tccagtcacc ctgtctgtga ctccaggaga tagagtctct ctttcctgca gggccagcca gagtattagc gactacttac actggtatca acaaaaatca catgagtctc caaggcttct catcaaatat gcttcccaat ccatctctgg gatcccctcc aggttcagtg gcagtggatc agggtcagat ttcactctca gtatcaacag tgtggaacct gaagatgttg gagtgtatta ctgtcaaaat ggttacggct ttccgctcac gttcggtgct gggaccaagc tggagctgaa a

[0057] Antibody 2) was further analyzed in detail, and CDR1 to CDR3 of VH and VL were identified. Antibody 2) of the present invention has an amino acid sequence in which VH CDR1 is represented by SEQ ID NO: 17, VH CDR2 is represented by SEQ ID NO: 18, and VH CDR3 is represented by SEQ ID NO: 19, and an amino acid sequence in which VL CDR1 is represented by SEQ ID NO: 20, VL CDR2 is represented by SEQ ID NO: 21, and VL CDR3 is represented by SEQ ID NO: 22. The amino acid sequences of CDR1 to CDR3 of VH and VL are specifically shown below.

[0058] VH CDR1 amino acid sequence of antibody 2) (SEQ ID NO: 17): GYAFTNYLIE VH CDR2 amino acid sequence of antibody 2) (SEQ ID NO: 18): VINPGSGGTNYNENF VH CDR3 amino acid sequence of antibody 2) (SEQ ID NO: 19): HGNYGNYDMDY Antibody 2) VL CDR1 amino acid sequence (SEQ ID NO: 20): RASQSISDYLH VL CDR2 amino acid sequence of antibody 2) (SEQ ID NO: 21): YASQSIS VL CDR3 amino acid sequence of antibody 2) (SEQ ID NO: 22): QNGYGFPLT

[0059] In the most preferred embodiment, antibody 3) S8D3F2 of the present invention has a VH having the amino acid sequence shown in SEQ ID NO: 23 and a VL having the amino acid sequence shown in SEQ ID NO: 25. The gene encoding the VH of antibody 3) has the nucleotide sequence shown in SEQ ID NO: 24, and the gene encoding the VC of antibody 3) is shown in SEQ ID NO: 26. The amino acid sequence of antibody 3) and the nucleotide sequence of the gene encoding it are specifically shown below.

[0060] Amino acid sequence of VH of antibody 3) (SEQ ID NO: 23): QESGPGLVKPSQTMSLTCTVTGISITSGNYRWSWIRQFPGNKLEWIGYIYYSGTVTYNPSLTSRTTITRDTSKNQFFLEMNSLTAEDTATYYCARVYGSFGYWFFDVWGAGTTVTVSS

[0061] Nucleotide sequence of the gene encoding VH of antibody 3) (SEQ ID NO: 24): caggagtcag gacctggtct ggtgaaacct tctcagacaa tgtccctcac ctgcactgtc actggcatct ccatcacctc tggaaattac agatggagct ggatccggca atttccagga aacaaactgg agtggatagg gtacatatac tacagtggta ccgttaccta caatccatct ctcacaagtc gaaccaccat cactcgagac acttccaaga accaattctt cctggaaatg aactctttga ctgctgaaga cacagccaca tattactgtg cccgcgtgta tggtagcttc ggatactggt tcttcgatgt ctggggcgca gggaccacgg tcaccgtctc ctca

[0062] Amino acid sequence of VL of antibody 3) (SEQ ID NO: 25): DNVLTQSPTTMTASPGEKITITCSASSNISSNYLHWYQQKPGFSPKLLIYRTSNLASGVPGRFSGSGSGTSYTLTIGSMEAEDVASYYCQQGNTIPFTFGAGTELELK

[0063] Nucleotide sequence of the gene encoding VL of antibody 3) (SEQ ID NO: 26): gacaatgttc tcacccagtc tccaaccacc atgactgcat ctcccgggga gaagatcact atcacctgca gtgccagctc aaatataagt tccaattact tgcattggta tcagcagaag ccaggattct cccctaaact cttgatttat aggacatcca atctggcatc tggagtccca ggtcgcttca gtggcagtgg gtctgggacc tcttacactc tcacaattgg ctccatggag gctgaagatg ttgcctctta ctactgccag cagggtaata ctattccgtt cacgttcggt gctgggaccg agctggagct gaaa

[0064] Antibody 3) was further analyzed in detail, and CDR1 to 3 of VH and VL were identified. Antibody 3) of the present invention has an amino acid sequence in which VH CDR1 is represented by SEQ ID NO: 27, VH CDR2 is represented by SEQ ID NO: 28, and VH CDR3 is represented by SEQ ID NO: 29, and an amino acid sequence in which VL CDR1 is represented by SEQ ID NO: 30, VL CDR2 is represented by SEQ ID NO: 31, and VL CDR3 is represented by SEQ ID NO: 32. The amino acid sequences of CDR1 to 3 of VH and VL are specifically shown below.

[0065] VH CDR1 amino acid sequence of antibody 3) (SEQ ID NO: 27): GISITSGNYRWS VH CDR2 amino acid sequence of antibody 3) (SEQ ID NO: 28): YIYYSGTVTYNPSLTS VH CDR3 amino acid sequence of antibody 3) (SEQ ID NO: 29): VYGSFGYWFFDV Antibody 3) VL CDR1 amino acid sequence (SEQ ID NO: 30): SASSNISSNYLH Antibody 3) VL CDR2 amino acid sequence (SEQ ID NO: 31): RTSNLAS VL CDR3 amino acid sequence of antibody 3) (SEQ ID NO: 32): QQGNTIPFT

[0066] In the most preferred embodiment, antibody 4) L9E5E1 of the present invention has a VH having the amino acid sequence shown in SEQ ID NO: 33 and a VL having the amino acid sequence shown in SEQ ID NO: 35. The gene encoding the VH of antibody 4) has the nucleotide sequence shown in SEQ ID NO: 34, and the gene encoding the VC of antibody 4) is shown in SEQ ID NO: 36. The amino acid sequence of antibody 4) and the nucleotide sequence of the gene encoding it are specifically shown below.

[0067] Amino acid sequence of VH of antibody 4) (SEQ ID NO: 33): EVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMGYISYSGNTSYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCAYFGNPFYHAIDYWGQGTSVTVSS

[0068] Nucleotide sequence of the gene encoding VH of antibody 4) (SEQ ID NO: 34): gaggtgcagc ttcaggagtc gggacctggc ctggtgaaac cttctcagtc tctgtccctc acctgcactg tcactggcta ctcaatcacc agtgattatg cctggaactg gatccggcag tttccaggaa acaaactgga gtggatgggc tacataagct acagtggtaa cactagctac aacccatctc tcaaaagtcg aatctctatc actcgagaca catccaagaa ccagttcttc ctgcagttga attctgtgac tactgaggac acagccacat attactgtgc ctactttggt aacccatttt accatgctat tgactactgg ggtcaaggaa cctcagtcac cgtctcctca

[0069] Amino acid sequence of VL of antibody 4) (SEQ ID NO: 35): HPETQSSSSFSVSLGDRVTITCKASEDIYNRFAWYQQKPGNVPRLLISGATSLETGVPSRFSGSGSGKDCTLSISSLQTEDVATYYCQQYWSMPITFGAGTKLELK

[0070] Nucleotide sequence of the gene encoding VL of antibody 4) (SEQ ID NO: 36): gatgttgtga tgacccagtc tccagtcacc ctgtctgtga ctccaggaga tagagtctct ctttcctgca gggccagcca gagtattagc gactacttac actggtatca acaaaaatca catgagtctc caaggcttct catcaaatat gcttcccaat ccatctctgg gatcccctcc aggttcagtg gcagtggatc agggtcagat ttcactctca gtatcaacag tgtggaacct gaagatgttg gagtgtatta ctgtcaaaat ggttacggct ttccgctcac gttcggtgct gggaccaagc tggagctgaa a

[0071] Antibody 4) was further analyzed in detail, and CDR1 to CDR3 of VH and VL were identified. Antibody 4) of the present invention has an amino acid sequence in which its VH CDR1 is represented by SEQ ID NO: 37, its VH CDR2 is represented by SEQ ID NO: 38, and its VH CDR3 is represented by SEQ ID NO: 39, and its VL CDR1 is represented by SEQ ID NO: 40, its VL CDR2 is represented by SEQ ID NO: 41, and its VL CDR3 is represented by SEQ ID NO: 42. The amino acid sequences of CDR1 to CDR3 of VH and VL are specifically shown below.

[0072] VH CDR1 amino acid sequence of antibody 4) (SEQ ID NO: 37): GYSITSDYAWN VH CDR2 amino acid sequence of antibody 4) (SEQ ID NO: 38): YISYSGNTSYNPSL VH CDR3 amino acid sequence of antibody 4) (SEQ ID NO: 39): FGNPFYHAIDY VL CDR1 amino acid sequence of antibody 4) (SEQ ID NO: 40): KASEDIYNRFA VL CDR2 amino acid sequence of antibody 4) (SEQ ID NO: 41): GATSLET VL CDR3 amino acid sequence of antibody 4) (SEQ ID NO: 42): QQYWSMPIT

[0073] In the most preferred embodiment, antibody 5) L10E1G2 of the present invention has a VH having the amino acid sequence shown in SEQ ID NO: 43 and a VL having the amino acid sequence shown in SEQ ID NO: 45. The gene encoding the VH of antibody 5) has the nucleotide sequence shown in SEQ ID NO: 44, and the gene encoding the VC of antibody 5) is shown in SEQ ID NO: 46. The amino acid sequence of antibody 5) and the nucleotide sequence of the gene encoding it are specifically shown below.

[0074] Amino acid sequence of VH of antibody 5) (SEQ ID NO: 43): QVQLQQSGAELVRPGTSVKVSCKASGYAFTNYLIEWIKQRPGQGLEWIGVINPGSGGTNYNEKFKGKATLTGDKSSSTVYMQLSSLTSDDSAVYYCARHGNYGNYDMDYWGQGTSVTVSS

[0075] Nucleotide sequence of the gene encoding VH of antibody 5) (SEQ ID NO: 44): caggtccaac tgcagcagtc tggagctgag ctggtaaggc ctgggacttc agtgaaggtg tcctgcaagg cttctggata cgccttcact aattacttga tagagtggat aaagcagagg cctggacagg gccttgagtg gattggagtg attaatcctg gaagtggtgg tactaactac aatgagaagt tcaagggcaa ggcaacactg actggagaca aatcttccag cactgtctac atgcaactca gcagcctgac atctgatgac tctgcggtct attactgtgc aaggcatggt aactacggga actatgatat ggactactgg ggtcaaggaa cctcagtcac cgtctcctca

[0076] Amino acid sequence of VL of antibody 5) (SEQ ID NO: 45): DVVMTQSPATLSVTPGDRVSLSCRASQSIYDYLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLTINSVEPEDVGVYYCQNGHGFPLTFGAGTKLELK

[0077] Nucleotide sequence of the gene encoding VL of antibody 5) (SEQ ID NO: 46): gatgttgtga tgacccagtc tccagccacc ctgtctgtga ctccaggaga tagagtctct ctttcctgca gggccagcca gagtatttac gactacttac actggtatca acaaaaatca catgagtctc caaggcttct catcaagtat gcttcccaat ccatctctgg gatcccctcc aggttcagtg gcagtggatc agggtccgat ttcactctca ctatcaacag tgtggaacct gaagatgttg gagtgtatta ctgtcaaaat ggtcacggct ttccgctcac gttcggtgct gggaccaagc tggagctgaa a

[0078] Antibody 5) was further analyzed in detail, and CDR1 to 3 of VH and VL were identified. Antibody 5) of the present invention has an amino acid sequence in which its VH CDR1 is represented by SEQ ID NO: 47, its VH CDR2 is represented by SEQ ID NO: 48, and its VH CDR3 is represented by SEQ ID NO: 49, and its VL CDR1 is represented by SEQ ID NO: 50, its VL CDR2 is represented by SEQ ID NO: 51, and its VL CDR3 is represented by SEQ ID NO: 52. The amino acid sequences of CDR1 to 3 of VH and VL are specifically shown below.

[0079] VH CDR1 amino acid sequence of antibody 5) (SEQ ID NO: 47): GYAFTNYLIE VH CDR2 amino acid sequence of antibody 5) (SEQ ID NO: 48): VINPGSGGTNYNEKFKG VH CDR3 amino acid sequence of antibody 5) (SEQ ID NO: 49): HGNYGNYDMDY VL CDR1 amino acid sequence of antibody 5) (SEQ ID NO: 50): RASQSIYDYLH VL CDR2 amino acid sequence of antibody 5) (SEQ ID NO: 51): YASQSIS VL CDR3 amino acid sequence of antibody 5) (SEQ ID NO: 52): QNGHGFPLT

[0080] The present invention also provides mutant antibodies, i.e., the antibodies of the present invention may have a number of modifications, including, but not limited to, amino acid modifications in the CDRs (affinity maturation), amino acid modifications in the Fc region, glycosylation variants, and other types of covalent modifications.

[0081] As used herein, a "variant" refers to a polypeptide sequence that differs from that of a parent polypeptide by at least one amino acid modification, which may include substitutions, insertions, and deletions, with substitutions, especially conservative substitutions, being preferred in many cases.

[0082] Generally, as described herein, variants may include any number of modifications as long as the functionality of the antibody is still present, i.e., in the case of amino acid variants made in the CDRs of any of the antibodies of the invention, for example, the antibody should still specifically bind to the CPE.

[0083] In general, however, the goal in most cases is to alter function with the minimum number of modifications, so that 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions are typically utilized. In some cases, 1-5 modifications are present, and many embodiments also recognize the use of 1-2, 1-3, and 1-4 modifications.

[0084] It should be noted that the number of amino acid modifications may be within the range of a functional domain: for example, it may be desirable to have 1 to 5 modifications in the Fc region of a wild-type or variant protein, e.g., 1 to 5 modifications in the Fv region. Preferably, the variant polypeptide sequence has at least about 80%, 85%, 90%, 95%, or up to 98 or 99% identity with the parent sequence (e.g., the variable region, constant region, and / or heavy and light chain sequences of any antibody selected from the group of antibodies of the present invention). It should be noted that the percentage of identity is determined by the number of amino acids, although this depends on the sequence size.

[0085] As used herein, "amino acid substitution" or "substitution" refers to the replacement of an amino acid at a specific position in a parent polypeptide sequence with another amino acid. For example, the substitution S100A refers to a mutant polypeptide in which the serine at position 100 is replaced with an alanine. As used herein, "amino acid insertion" or "insertion" refers to the addition of an amino acid at a specific position in a parent polypeptide sequence. As used herein, "amino acid deletion" or "deletion" refers to the removal of an amino acid at a specific position in a parent polypeptide sequence.

[0086] As used herein, "parent polypeptide," "parent protein," "precursor polypeptide," or "precursor protein" refers to an unmodified polypeptide that is subsequently modified to generate a variant. Typically, the parent polypeptide herein is any antibody selected from the group of antibodies of the present invention. The parent polypeptide may refer to the polypeptide itself, a composition comprising the parent polypeptide, or the amino acid sequence encoding it. Thus, as used herein, "parent Fc polypeptide" refers to an Fc polypeptide that is modified to generate a variant, and as used herein, "parent antibody" refers to an antibody that is modified to generate a variant antibody.

[0087] As used herein, "wild-type" or "WT" or "native" refers to an amino acid sequence or nucleotide sequence found in nature, including allelic variations. A WT protein, polypeptide, antibody, immunoglobulin, IgG, etc., has an amino acid sequence or nucleotide sequence that has not been intentionally modified.

[0088] As used herein, a "variant Fc region" refers to an Fc sequence that differs from that of a wild-type Fc sequence by virtue of at least one amino acid modification. An Fc variant may refer to the Fc polypeptide itself, a composition comprising the Fc variant polypeptide, or the amino acid sequence.

[0089] In some embodiments, one or more amino acid modifications are made in one or more CDRs of antibody.Generally, only one or two amino acids are substituted in any single CDR, and usually 3, 4, 5, 6, 7, 8, 9 or 10 or more changes are not made in a set of CDRs.However, in any CDR, any combination of no substitution, one or two, or three or more substitutions can be made, and any other substitutions can be independently and optionally combined.

[0090] In some cases, amino acid modifications in the CDRs are referred to as "affinity maturation." An "affinity matured" antibody has one or more changes in one or more CDRs that result in improved affinity of the antibody for the antigen compared to a parent antibody that does not have those changes. In some cases, although rare, it may be desirable to reduce the affinity of an antibody for its antigen, but this is generally not preferred.

[0091] Affinity maturation can be performed to increase the binding affinity of an antibody for an antigen by at least about 10%, 50%, 100%, 150%, or 1-5 times compared to the "parent" antibody. Preferred affinity-matured antibodies have nanomolar or even picomolar affinities for the target antigen. Affinity-matured antibodies are produced by known procedures. See, for example, Marks et al., 1992, Biotechnology 10:779-783, which describes affinity maturation by heavy chain variable (VH) and light chain variable (VL) domain shuffling. Methods for random mutagenesis of CDR and / or framework residues are described, for example, in Barbas, et al. 1994, Proc. Nat. Acad. Sci. USA 91:3809-3813; Shier et al., 1995, Gene 169:147-155; Yelton et al., 1995, J. Immunol. 155:1994-2004; Jackson et al., 1995, J. Immunol. 154(7):3310-9; and Hawkins et al., 1992, J. Mol. Biol. 226:889-896.

[0092] Alternatively, amino acid modifications can be made in one or more CDRs of an antibody of the invention that are "silent" or "conservative," e.g., that do not significantly alter the affinity of the antibody for antigen. These can be made for a variety of reasons, including to optimize expression (which can be made to a nucleic acid encoding an antibody of the invention).

[0093] It is well known in the art that a certain amino acid can be substituted with another amino acid having a similar hydrophobicity index and still produce a protein having a similar biological function (e.g., a protein equivalent in enzymatic activity; hereinafter, "bioisequence"). In such amino acid substitutions, the hydrophobicity index is preferably within ±2, more preferably within ±1, and even more preferably within ±0.5. It is understood in the art that such amino acid substitutions based on hydrophobicity are efficient. The hydrophilicity index is also taken into consideration in creating bioisosteres. As described in U.S. Patent No. 4,554,101, the following hydrophilicity indices are assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0±1); glutamic acid (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5±1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). In such amino acid substitutions, the hydrophilicity index is preferably within ±2, more preferably within ±1, and even more preferably within ±0.5.

[0094] Therefore, in the present invention, polypeptides may be subjected to mutations or conservative substitutions. Such mutations may be substitutions, insertions, or deletions. Methods for such mutations are well known in the art and are not limited to any particular method. A conservative substitution refers to a substitution in which the hydrophilicity index and / or hydrophobicity index of the original amino acid and the substituted amino acid are similar as described above, and is a typical example of a silent modification. Examples of such conservative substitutions are well known to those skilled in the art, and include, but are not limited to, substitutions within the following groups: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; valine, leucine, and isoleucine.

[0095] Thus, variant CDRs and antibodies are included within the definition of the CDRs and antibodies of the invention; i.e., the antibodies of the invention may comprise amino acid modifications in one or more CDRs. Furthermore, as outlined below, amino acid modifications may optionally be made independently in any region outside the CDRs (including framework and constant regions).

[0096] In some embodiments, mutant antibodies specific for CPE are described. The antibodies consist of six CDRs, each of which independently differs from each of the above amino acid sequences by 0, 1, or 2 amino acid substitutions. In contrast, the FR region can be mutated more extensively, for example, to achieve about 80%, 85%, or 90% identity with the parent FR region sequence, because the amino acid sequence does not significantly affect the binding specificity of the antibody.

[0097] In addition to the modifications outlined above, other modifications can be made. For example, the molecule can be stabilized by the incorporation of disulfide bridges linking the VH and VL domains (Reiter et al., 1996, Nature Biotech. 14:1239-1245, incorporated by reference in its entirety). Furthermore, there are a variety of covalent modifications of antibodies that can be made, as outlined below.

[0098] Covalent modifications of antibodies are included within the scope of this invention and are usually, but not always, done post-translationally. For example, several types of covalent antibody modifications are introduced into the molecule by reacting specific amino acid residues of the antibody with organic derivatizing agents capable of reacting with selected side chains or with the N- or C-terminal residues.

[0099] Cysteinyl residues most commonly are reacted with α-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues may also be derivatized by reaction with bromotrifluoroacetone, α-bromo-β-(5-imidozoyl)propionic acid, chloroacetylphosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole, and the like.

[0100] Histidyl residues are derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain. Para-bromophenacyl bromide is also useful; the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0.

[0101] Lysinyl and amino-terminal residues are reacted with succinic or other carboxylic acid anhydrides. Derivatization with this agent has the effect of reversing the charge of the lysinyl residues. Other suitable agents for derivatizing α-amino-containing residues include imidoesters such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydrides, trinitrobenzenesulfonic acid, O-methylisourea, 2,4-pentanedione, and aminotransferase-catalyzed reactions with glyoxylic acid.

[0102] Arginyl residues are modified by reaction with one or more conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be performed under alkaline conditions because of the high pKa of the guanidine functional group. Furthermore, these reagents may react with lysine groups and the arginine epsilon-amino group.

[0103] The specific modification of tyrosyl residues, where there is particular interest in introducing spectral labels into tyrosyl residues, may be made by reaction with aromatic diozonium compounds or tetranitromethane. Most commonly, N-acetylimidazole and tetranitromethane are used to form O-acetyltyrosyl species and 3-nitro derivatives, respectively. Tyrosyl residues are iodinated with 125I or 131I to prepare labeled proteins for use in radioimmunoassay, the chloramine T method (described above as suitable).

[0104] Carboxyl side groups (aspartyl or glutamyl) are selectively modified by reaction with carbodiimides (R'-N=C=N-R'), where R and R' are optionally different alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide and 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Further, aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

[0105] Derivatization with bifunctional agents is useful for crosslinking antibodies to water-insoluble support matrices or surfaces, for use in a variety of methods in addition to those described below. Commonly used crosslinking agents include, for example, 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, such as esters with 4-azidosalicylic acid, homobifunctional imidoesters (including disuccinimidyl esters such as 3,3'-dithiobis(succinimidyl propionate)), and bifunctional maleimides (such as bis-N-maleimido-1,8-octane). Derivatization agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate produce photoactivatable intermediates that can form crosslinks in the presence of light. Alternatively, reactive water-insoluble matrices and reactive substrates such as cynomolgusogen bromide-activated carbohydrates (as described in U.S. Patent Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440, all of which are incorporated by reference) are used for protein immobilization.

[0106] Glutaminyl and asparaginyl residues are frequently deamidated to the corresponding glutamyl and aspartyl residues, respectively. Alternatively, these residues are deamidated under mildly acidic conditions. Both forms of these residues are within the scope of this invention.

[0107] Other modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl group of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine, and histidine side chains (Tecreighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86

[1983] , incorporated by reference in its entirety), acetylation of N-terminal amines, and amidation of C-terminal carboxyl groups.

[0108] Additionally, as will be appreciated by those skilled in the art, labels (including fluorescent, enzymatic, magnetic, radioactive, etc.) can all be added to antibodies (and other compositions of the invention).

[0109] The present invention provides numerous antibodies, each with a specific set of CDRs (including some amino acid substitutions, as outlined above). As outlined above, antibodies can be defined by a set of six CDRs, a variable region, or full-length heavy and light chains (including constant regions). Furthermore, amino acid substitutions can be made, as outlined above. Generally, in the context of changes within a CDR, amino acid modifications are typically described in terms of the number of amino acid modifications that can be made due to the relatively short length of the CDRs. While this also applies to discussing the number of amino acid modifications that can be introduced into a variable, constant, or full-length sequence, it is also appropriate to define these changes in terms of "% identity" in addition to the number of changes. Thus, as described herein, antibodies encompassed within the present invention are 80, 85, 90, 95, 98, or 99% identical to the sequences set forth in the SEQ ID NOs listed herein.

[0110] The present invention further provides methods for producing any antibody selected from the group of antibodies of the present invention. These methods include culturing host cells containing one or more isolated nucleic acids encoding an antibody of the present invention. As will be understood by those skilled in the art, this can be performed in a variety of ways depending on the characteristics of the antibody. In some embodiments, when the antibody of the present invention is a full-length conventional antibody, e.g., a heavy chain variable region and a light chain variable region, the method is performed under conditions that allow the antibody to be produced and isolated.

[0111] Generally, nucleic acids encoding the antibodies of the present invention are provided. Such polynucleotides encode both the variable and constant regions of the heavy and light chains, respectively, although other combinations are contemplated by the present invention in accordance with the compositions described herein. The present invention also contemplates oligonucleotide fragments derived from the disclosed polynucleotides and nucleic acid sequences complementary to these polynucleotides.

[0112] Polynucleotides may be in the form of RNA or DNA. Polynucleotides in the form of DNA, cDNA, genomic DNA, nucleic acid analogs, and synthetic DNA are within the scope of the present invention. The DNA may be double-stranded or single-stranded, and if single-stranded, may be the coding (sense) strand or non-coding (antisense) strand. The coding sequence encoding a polypeptide may be identical to the coding sequence provided herein, or it may be a different coding sequence that, as a result of redundancy or degeneracy in the genetic code, encodes the same polypeptide as the DNA provided herein.

[0113] In some embodiments, one or more nucleic acids encoding an antibody of the present invention are incorporated into an expression vector, which can be designed to be extrachromosomal or integrated into the genome of the host cell into which it is introduced. Expression vectors can contain any number of appropriate regulatory sequences (including, but not limited to, transcriptional and translational regulatory sequences, promoters, ribosomal binding sites, enhancers, origins of replication, etc.) or other elements (such as selection genes), all operably linked as is well known in the art. In some cases, two nucleic acids can be used, each in a different expression vector (e.g., the heavy chain in a first expression vector and the light chain in a second expression vector) or in the same expression vector. It will be understood by those skilled in the art that the design of one or more expression vectors, including the selection of regulatory sequences, can depend on factors such as the choice of host cell and the desired level of protein expression.

[0114] Generally, nucleic acids and / or expression vectors are introduced into a suitable host cell such that one or more nucleic acids are operably linked to one or more expression control elements (e.g., in a vector, in a construct produced by a cellular process, integrated into the genome of the host cell) using any method appropriate for the host cell selected (e.g., transformation, transfection, electroporation, infection, etc.) to produce a recombinant host cell. The resulting recombinant host cell can be maintained under conditions appropriate for expression (e.g., in the presence of an inducer, in a suitable non-human animal, in media supplemented with appropriate salts, growth factors, antibiotics, nutritional supplements, etc.), thereby producing one or more encoded polypeptides. In some cases, the heavy chain is produced in one cell and the light chain is produced in another cell.

[0115] Mammalian cell lines available as hosts for expression are known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC), Manassas, VA, including, but not limited to, Chinese hamster ovary (CHO) cells, HEK 293 cells, NSO cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and numerous other cell lines. Non-mammalian cells, including, but not limited to, bacteria, yeast, insects, and plants, can also be used to express recombinant antibodies. In some embodiments, antibodies can be produced in transgenic animals, such as cows and chickens.

[0116] General methods of antibody molecular biology, expression, purification and screening are described, for example, in Antibody Engineering, edited by Kontermann & Dubel, Springer, Heidelberg, 2001 and 2010; Hayhurst & Georgiou, 2001, Curr Opin Chem Biol 5:683-689; Maynard & Georgiou, 2000, Annu Rev Biomed Eng 2:339-76; and Morrison, S. (1985) Science 229:1202.

[0117] The present invention also provides an immunochromatography kit for detecting Clostridium perfringens enterotoxin, comprising any one antibody or combination of antibodies selected from the antibody group of the present invention. One embodiment of such a kit comprises a detection section on a matrix made of a porous material such as a nitrocellulose membrane, in which a capture antibody is immobilized linearly, and a labeled reagent zone upstream of the detection section, in which a labeled detection antibody (labeled antibody) is carried. Typically, the labeled reagent zone is composed of a porous pad carrying the labeled detection antibody. A developer tank containing a developer solution is provided at the upstream end of the matrix. Furthermore, typically, downstream of the detection zone, a development confirmation section in which an anti-labeled antibody is immobilized linearly to confirm the development of the labeled antibody is provided. Further downstream of the development confirmation section, a developer solution absorption section equipped with a porous absorbent pad for absorbing the developer solution is provided. Furthermore, if the label is an enzyme label, a substrate zone carrying a substrate for the labeling enzyme is provided upstream of the labeled reagent zone.

[0118] During use, a sample is added to the labeled reagent zone, and the developer pad is inserted into the developer tank by applying pressure to the pusher and moving the protrusion, and developer is supplied to the matrix through the developer pad. As the developer passes through the substrate zone, the substrate is dissolved into the developer, causing the developer containing the substrate to flow. As the developer passes through the labeled reagent zone, the labeled antibody and sample are dissolved into the developer, causing the developer containing the substrate, labeled antibody, and sample to flow. If the sample contains CPE, the CPE and labeled antibody bind via an antigen-antibody reaction. When they reach the detection zone, the immobilized antibody and CPE bind via an antigen-antibody reaction in the detection zone. As a result, the labeled antibody is immobilized in the detection zone via the CPE. CPE is detected by measuring the label in the detection zone. If the sample does not contain CPE, the labeled antibody does not immobilize in the detection zone and moves further downstream, resulting in no label being detected in the detection zone. In addition, since the anti-labeled antibody is immobilized in the developer confirmation section downstream of the detection zone, the labeled antibody is fixed to the developer confirmation section. Therefore, if the label is detected in the developer confirmation section, it means that the developer has been properly developed. The developer is finally absorbed by the absorbent pad downstream.

[0119] The present invention also provides a kit for use in the method of the present invention, which comprises at least the monoclonal antibody of the present invention or a combination thereof.

[0120] When using a method that uses the sandwich method as the detection principle, at least one of the immobilized antigen capture antibody and the detection antibody, preferably both, is the monoclonal antibody of the present invention. Furthermore, standard specimen reagents (various concentrations), control reagents, sample dilutions, dilution cartridges, washing solutions, etc. can be combined. When an enzyme label is used, a substrate or reaction stop solution necessary for detecting the label can be included. When the detection antibody is not labeled, for example, a labeled substance that binds to the detection antibody can be included in the kit.

[0121] When immunochromatography is used as the sandwich method, the kit can include a device equipped with a membrane carrier on which an antigen-capturing antibody is immobilized in a detection zone and a pad carrying a labeled detection antibody. The device can also include other components suitable for immunochromatography, such as a developer pad and an absorbent pad.

[0122] The kit of the present invention may further include instructions for use of the kit. [Example]

[0123] The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to the following examples. It is not something that can be determined.

[0124] 1. Antibody Production The following media were used: FBS(-) medium: DMEM (high glucose, Nakarai) 500 mL, 100 mM sodium pyruvate 5 mL, MEM non-essential amino acid solution (NEAA) (Nacalai) 5 mL, 2-mercaptoethanol 0.5 mL, penicillin-streptomycin mixed solution (Nacalai) 5 mL; FBS(+) medium: FBS(-) medium supplemented with 10% FBS (Invitrogen); HAT medium: 250 mL of FBS(+) medium, 5 mL of HAT (×50 conc. MP Bio), 10 ng / mL of IL-6 (PeproTech recombinant mouse IL-6); HT medium: DMEM (high glucose, Nakarai) 500 mL, 100 mM sodium pyruvate 5 mL, MEM non-essential amino acid solution (NEAA) (Nacalai) 5 mL, 2-mercaptoethanol 0.5 mL, FBS (Invitrogen) 50 mL, IL-6 or BM Condimed H 5 mL, HT (×50 conc. MP Bio) 5 mL, penicillin-streptomycin mixed solution (Nacalai) 5 mL; HyColne LM, ADCF Mab: HyColne LM ADCF MAb 1000 mL, penicillin-streptomycin mixed solution (Nacalai) 10.1 mL (100x)

[0125] 1-1.Cell fusion The fusion protein VT2B-C-CPE was mixed with Sigma Adjuvant system at a 1:1 ratio and injected twice into the footpad of BALB / c mice at two-week intervals (Figure 1). Lymph nodes and spleens were harvested from the mice and added to FBS(+) medium. The tissues were placed in a 5-cm-diameter dish containing 5 mL of FBS(+) medium. After removing any attached fat, splenocytes or lymph node cells were strained using a 100 μm cell strainer and a disposable syringe plunger. Splenocytes or lymph node cells were mixed with mouse myeloma cells P3U1, and the fused cells were diluted in HAT medium and seeded at 200 μL per well in a 96-well plate. The cells were cultured at 37°C with 10% CO2, with 100 μL of HAT medium replaced every 2–3 days, until colonies appeared and proliferated. Ten hybridomas were obtained. The 10 hybridomas were designated as L2F11H7, L4C8D6, L5A4C5, L5C2E3, L6C12D8, L9E5E1, L10E1G2, S7E10G11, S7F10G2, and S8D3F2, and the antibodies obtained from each hybridoma were designated using the same names.

[0126] 1-2. Primary screening (ELISA) The antigen, C-CPE, was diluted with PBS, and lyophilized mouse intestinal bacteria (negative control) was dissolved in carbonate-bicarbonate buffer (killed mouse intestinal bacteria: 1.0 mg / mL, C-CPE: 5.0 μg / mL). The antigen and killed mouse intestinal bacteria were added to a 96-well plate at 100 μL per well and allowed to stand overnight at 4°C for immobilization. The solution was discarded, and 170 μL of 1% BSA-PBS was added to each well. The plate was then left to stand at room temperature for 2 hours for blocking, followed by washing three times with 250 μL of PBS-T. Each hybridoma culture supernatant was added at 50 μL per well, allowed to stand at room temperature for 2 hours, and then washed three times with 250 μL of PBS-T. Next, 100 μL / well of anti-mouse IgG (whole molecule)-HRP (1031-05, Southern Biotech) diluted in 1% BSA-PBS-T was added, and the plate was incubated at 37°C for 1 hour. Then, the plate was washed three times with 250 μL of PBS-T. Then, 100 μL / well of TMB (KPL 50-76-03 TMB Microwell Peroxidase Substrate System) was added. After exactly 2 minutes at room temperature, 50 μL / well of 0.5N HCl was added. The absorbance at 450 nm was measured using a spectrophotometer. The results are shown in Figure 2. For each hybridoma, the left bar indicates the absorbance of C-CPE, and the right bar indicates the absorbance of killed enterobacterial cells. Antibodies obtained from all hybridomas showed significantly higher reactivity with C-CPE than killed enterobacterial cells.

[0127] 1-3. Cloning (limiting dilution method) Ten hybridoma lines were cloned by limiting dilution, diluted with HAT medium to 0.5, 1, or 2 cells / 200 μL / well, and seeded onto 96-well plates. The supernatant (100 μL / well) was used for screening (ELISA). If a single colony was found in a positive well, the results were scaled up in HT medium to prepare a frozen stock. Large-scale antibody preparations were made using the culture supernatant or mouse ascites fluid, as needed. If a single colony was not found, the cloning was repeated.

[0128] 1-4. Monoclonal antibody purification HT medium was warmed to 37°C in an incubator, and the frozen hybridomas were thawed in the incubator at 37°C. The thawed hybridomas were added to 10 mL of HT medium. After centrifugation (1,200 rpm, 3 minutes), the supernatant was removed, and the pellet was loosened and suspended in 10 mL of HT medium. The cell suspension was added to a 10 cm cell dish and cultured at 37°C. After confirming hybridoma growth, the cells were suspended and added to a flask containing 10 mL of HT medium and cultured at 37°C. After confirming hybridoma growth, the cells were suspended and added to a flask containing 40 mL of HT medium and cultured at 37°C. After confirming hybridoma growth, the supernatant was removed, the cells were washed twice with 15 mL of PBS, and then 50 mL of ADCF MAb was added and cultured at 37°C. After 2 days of culture, the supernatant was collected, 50 mL of ADCF MAb was added, and the cells were cultured at 37°C. The collected supernatant was stored at 4°C.

[0129] The culture medium was centrifuged (1,200 rpm, 5 minutes) to collect the supernatant, which was then filtered through a 0.45 μm filter. The antibody was purified and isolated by affinity chromatography using Protein G Sepharose. The absorbance (A280) of the purified antibody elution fraction was measured using an ultra-microspectrophotometer (NanoDrop™ Lite, Thermo Fisher Scientific). The pooled collected fractions were added to an Amicon Ultra-15 30K (UFC903024, Merck), and 4–5 mL of PBS was added. After concentration and centrifugation, the absorbance (A280) was measured in the same manner, and the concentration was calculated.

[0130] 1-5. Purified antibody reactivity confirmation test (ELISA) C-CPE and CPE were dissolved in carbonate-bicarbonate buffer and adjusted to the desired concentrations (C-CPE: 0.1, 1.0 μg / mL, CPE: 0.1, 1.0 μg / mL). Each antigen was added to a 96-well plate at 100 μL per well and allowed to stand overnight at 4°C for immobilization. The solution was discarded, and 170 μL of 1% BSA-PBS was added to the 96-well plate at 170 μL per well. The plate was then allowed to stand at room temperature for 2 hours for blocking. After washing with PBS-T, 50 μL of hybridoma culture supernatant was added per well and allowed to stand at room temperature for 2 hours, followed by washing with PBS-T. Anti-mouse IgG (whole molecule)-HRP (1031-05, Southern Biotech) diluted in 1% BSA-PBS-T was added at 100 μL per well and allowed to stand at 37°C for 1 hour, followed by washing with PBS-T. TMB (KPL 50-76-03 TMB Microwell Peroxidase Substrate System) was added at 100 μL per well. After exactly 2 minutes at room temperature, 50 μL per well of 0.5 N HCl was added, and the absorbance at 450 nm was measured using a spectrophotometer. The results are shown in Figure 3 (C-CPE) and Figure 4 (CPE). For each antibody, the left bar represents the absorbance at 1.0 μg / mL, and the right bar represents the absorbance at 0.1 μg / mL. All antibodies showed high reactivity with C-CPE at high concentrations, but their reactivity generally decreased at lower concentrations. In particular, L6C12D8 showed almost no reactivity with C-CPE at low concentrations. Furthermore, the low reactivity of L6C12D8 against CPE was particularly notable.

[0131] 1-6. Cross-reactivity test (ELISA) The following heat-killed bacterial cells were prepared (60°C, 60 minutes) and freeze-dried. C.jejuni, P.aeruginosa, S.liquefaciens, E.coli DH5α, S.Enteritidis, B.vulgatus, L.gasseri, P.mirabilis, C.freundii, E.freundii, K.pneumoniae, K.aerogenes, E.cloacae, E.faecalis Antigens and freeze-dried heat-killed bacterial cells were dissolved and adjusted to a concentration in carbonate-bicarbonate buffer (C-CPE: 1.0 μg / mL, CPE: 1.0 μg / mL, various killed bacterial cells: 1.0 mg / mL). 100 μL of each solution was added to a 96-well plate and allowed to stand overnight at 4°C for immobilization. The solution was discarded, and 170 μL of 1% BSA-PBS was added to the 96-well plate at 170 μL / well. The plate was then allowed to stand at room temperature for 2 hours for blocking. After washing with PBS-T, 50 μL of hybridoma culture supernatant was added to the plate and allowed to stand at room temperature for 2 hours, followed by washing with PBS-T. 100 μL of anti-mouse IgG (whole molecule)-HRP (1031-05, Southern Biotech) diluted in 1% BSA-PBS-T was added to the plate and allowed to stand at 37°C for 1 hour, followed by washing with PBS-T. TMB (KPL 50-76-03 TMB Microwell Peroxidase Substrate System) was added at 100 μL per well, and after exactly 2 minutes at room temperature, 0.5 N HCl was added at 50 μL per well. The absorbance at 450 nm was measured using a spectrophotometer. Reactivity was observed with the antigens C-CPE and CPE, but not with any of the above bacterial cells.

[0132] Sandwich ELISA We performed a sandwich ELISA test to detect CPE using various combinations of the 10 antibodies obtained in sections 1-4 above (L2F11H7, L4C8D6, L5A4C5, L5C2E3, L6C12D8, L9E5E1, L10E1G2, S7E10G11, S7F10G2, and S8D3F2). First, the antibodies were labeled. The modifier solution from the HRP Conjugation Kit (Abcam AB102890-300) was added to a monoclonal antibody (>1 mg / mL F) solution and mixed (1 μL modifier / 10 μL antibody). The monoclonal antibody with the modifier solution added was then added to the HRP mix and mixed. The antibody-HRP mixed solution was left to react in the dark at 20-25°C for 3 hours to overnight. A quencher solution was added, and the mixture was reacted in the dark at 20 to 25°C for 30 minutes (1 µL quencher / 10 µL antibody).

[0133] Next, the capture antibody was immobilized, and the reaction of the labeled antibody was examined. The monoclonal antibody was prepared at a concentration of 2.0 μg / mL in Carbonate-Bicarbonate Buffer. 100 μL / well of the solution was added to a 96-well plate and incubated overnight at 4°C. The plate was washed three times with 300 μL of PBS-T, after which the capture antibody solution was removed. 200 μL / well of 1% BSA-PBS was added to the 96-well plate and incubated for 2 hours at room temperature or overnight at 4°C. 100 μL / well of an antigen CPE (0.1 μg / mL) solution diluted with 1% BSA-PBST was added, and the plate was incubated for 90 minutes at 37°C. After removing the solution, the plate was washed three times with 300 μL of PBS-T. 100 μL / well of a 0.5 μg / mL HRP-labeled labeled antibody solution diluted with 1% BSA-PBST was added, and the plate was incubated for 2 hours at room temperature. After removing the solution, the plate was washed three times with 300 μL of PBS-T. 100 μL of TMB was added to each well, and after exactly 2 minutes at room temperature, 50 μL of 0.5 N HCl was added to each well. The absorbance at 450 nm was measured using a spectrophotometer.

[0134] First, as expected from the principle of sandwich ELISA, when the labeled and capture antibodies were the same, none of the antibodies detected the antigen. When S7E10G11 or S7F10G2 was used as the labeled antibody, none of the antibodies used as the capture antibody detected the antigen. Furthermore, when L5A4C5, L6C12D8, S7E10G11, and S7F10G2 were immobilized as capture antibodies, none of the antibodies used as the labeled antibody detected the antigen. Therefore, S7E10G11 and S7F10G2 were completely eliminated as candidates, and L5A4C5 and L6C12D8 were also concluded to be of limited utility. The other six antibodies were often able to detect the antigen sufficiently when different antibodies were used as the labeled and capture antibodies. In particular, L4C8D6, S8D3F2, and L9E5E1 were able to detect the antigen in almost all combinations.

[0135] 2. Antibody affinity analysis Antibody affinity was measured by surface plasmon resonance using a Biacore T200 (Cytiva). Anti-mouse IgG antibodies were immobilized on a CM5 chip using a Mouse Antibody Capture Kit (Cytiva), and various anti-CPE monoclonal antibodies were captured. Running buffer (0.1 M HEPES, 1.5 M NaCl, 30 mM EDTA, 0.5% v / v Surfactant P 20) containing C-CPE was added at a flow rate of 30 μL / min (association phase), and the running buffer was then passed over the sensor chip (dissociation phase). The association rate constant (Ka), dissociation rate constant (Kd), and equilibrium dissociation constant (KD) were calculated using BIAEvaluation 3.1. The results are shown in Figure 5. L5C2E3 exhibited an extremely low KD, followed by L4C8D6, S8D3F2, L9E5E1, and L10E1G2, which also exhibited sufficiently low KD.

[0136] 3. Immunochromatography 3-1. Preparation of immunochromatography kit Three antibodies, L5C2E3, S8D3F2, and L9E5E1, were prepared to the determined concentrations for the coating protein amounts. KH2PO4 at the optimal pH was dispensed, and a 40 nm diameter colloidal gold solution (BB International) was added, stirred, and allowed to stand. 1% PEG20,000 and 10% BSA were added, followed by centrifugation, removal of the supernatant, and ultrasonic dispersion of the precipitate. Gold colloid storage buffer was added, and the centrifugation process was repeated to obtain the antibody-sensitized colloidal gold solution.

[0137] S8D3F2, L5C2E3, and L9E5E1 antibodies were prepared for use in the test lines and applied to nitrocellulose membranes (150CNPH-N-SS40). After application, the membranes were heat-dried at 50°C for 30 minutes. After drying, blocking and stabilization treatments were performed, followed by further drying to obtain antibody-immobilized membranes. Next, the antibody-sensitized gold colloid solution, DW, and gold colloid application buffer were mixed in a 1:1:2 ratio. The mixture was evenly applied to a Glass Fiber Diagnostic Pad (MILLIPORE) and dried under reduced pressure in a desiccator to obtain a conjugate pad.

[0138] 100 μL each of water alone or C-CPE 1, 10, 100, and 1000 ng / mL aqueous solutions was dropped onto the prototype (full strip), and the color development of the line was confirmed after approximately 20 minutes. The results are shown in Figure 6. For all full strips, clear color development was confirmed at concentrations of 10 to 1000 ng / mL, and color development was also confirmed at 1 ng / mL. In particular, sufficient color development was confirmed even at 1 ng / mL for the full strips with gold colloid (L9E5E1) and test line (L5C2E3).

[0139] 3-2. Verification of the developing solution Using immunochromatographic reagents (gold colloid: L9E5E1, test line: L5C2E3), 100 μL of CPE suspended in 2% NP40-PBS was developed and analyzed after 20 minutes. A pseudo-sample was prepared by suspending healthy human stool in 2% NP40-PBS and adjusting the concentration to 10 ng / mL CPE. 100 μL of this sample was developed on the immunochromatographic reagents (gold colloid: L9E5E1, test line: L5C2E3) and analyzed after 20 minutes. The results are shown in Figure 7. Since bands were observed on both the test line and the control, it was determined that the development was successful.

[0140] 3-3. Immunochromatographic reagent cross-reactivity test The following bacteria were prepared in 2% NP40-PBS to a McFarland concentration of 0.5 (approximately 1.0x108 cfu / mL), and 100µL was applied to the immunochromatography reagent (colloidal gold: L9E5E1, test line: L5C2E3). After 20 minutes, the results were analyzed to verify cross-reactivity. The immunochromatography reagent (colloidal gold: L9E5E1, test line: L5C2E3) showed no cross-reactivity with any of the bacteria. C. jejuni, C. coli, S. typhimurium, C. difficile, P. mirabilis, P. aeruginosa B. cereus, C. freundii, S. aureus, K. pneumonia, K. aerogenes, S. liquefaciens, E. cloacae, E. faecalis, E hermannii, S. epidermidis, E coli O114, C tetani

[0141] 4. Amino acid and gene sequence analysis Various hybridomas (10 6 After RNA was extracted from the cells, cDNA was synthesized. Using the cDNA as a template, VH and VL were amplified and cloned using degenerate primers, and the VH and VL gene sequences were analyzed. The results are shown in the sequence tables.

Claims

1. A method for detecting Clostridium perfringens enterotoxin in a sample by immunochromatography, characterized in that two different antibodies selected from the following group are used as a labeled antibody and a capture antibody, respectively: 1) an antibody having at least the amino acid sequence of SEQ ID NO: 7 as a VH CDR1 region, the amino acid sequence of SEQ ID NO: 8 as a VH CDR2 region, the amino acid sequence of SEQ ID NO: 9 as a VH CDR3 region, the amino acid sequence of SEQ ID NO: 10 as a VL CDR1 region, the amino acid sequence of SEQ ID NO: 11 as a VL CDR2 region, and the amino acid sequence of SEQ ID NO: 12 as a VL CDR3 region; 2) an antibody having at least the amino acid sequence of SEQ ID NO: 17 as a VH CDR1 region, the amino acid sequence of SEQ ID NO: 18 as a VH CDR2 region, the amino acid sequence of SEQ ID NO: 19 as a VH CDR3 region, the amino acid sequence of SEQ ID NO: 20 as a VL CDR1 region, the amino acid sequence of SEQ ID NO: 21 as a VL CDR2 region, and the amino acid sequence of SEQ ID NO: 22 as a VL CDR3 region; 3) an antibody having at least the amino acid sequence of SEQ ID NO: 27 as a VH CDR1 region, the amino acid sequence of SEQ ID NO: 28 as a VH CDR2 region, the amino acid sequence of SEQ ID NO: 29 as a VH CDR3 region, the amino acid sequence of SEQ ID NO: 30 as a VL CDR1 region, the amino acid sequence of SEQ ID NO: 31 as a VL CDR2 region, and the amino acid sequence of SEQ ID NO: 32 as a VL CDR3 region; 4) an antibody having at least the amino acid sequence of SEQ ID NO: 37 as the VH CDR1 region, the amino acid sequence of SEQ ID NO: 38 as the VH CDR2 region, the amino acid sequence of SEQ ID NO: 39 as the VH CDR3 region, the amino acid sequence of SEQ ID NO: 40 as the VL CDR1 region, the amino acid sequence of SEQ ID NO: 41 as the VL CDR2 region, and the amino acid sequence of SEQ ID NO: 42 as the VL CDR3 region; and 5) An antibody having at least the amino acid sequence of SEQ ID NO: 47 as the VH CDR1 region, the amino acid sequence of SEQ ID NO: 48 as the VH CDR2 region, the amino acid sequence of SEQ ID NO: 49 as the VH CDR3 region, the amino acid sequence of SEQ ID NO: 50 as the VL CDR1 region, the amino acid sequence of SEQ ID NO: 51 as the VL CDR2 region, and the amino acid sequence of SEQ ID NO: 52 as the VL CDR3 region.

2. The method of claim 1, wherein the antibody is an isolated antibody.

3. The method of claim 1 or 2, wherein the antibody is a monoclonal antibody.

4. The antibody reacted with Clostridium perfringens entrotoxin with a 5.0 x 10 －１０ The method according to any one of claims 1 to 3, characterized in that the compound has an equilibrium dissociation constant KD of M or less.

5. The antibody reacted with Clostridium perfringens entrotoxin with a 3.0 x 10 －１０ 5. The method of claim 4, wherein the compound has an equilibrium dissociation constant KD of M or less.

6. The method according to any one of claims 1 to 5, wherein the antibody further has the following characteristics: 1) an antibody having the amino acid sequence of SEQ ID NO: 3 as the VH region and the amino acid sequence of SEQ ID NO: 5 as the VL region; 2) an antibody having the amino acid sequence of SEQ ID NO: 13 as the VH region and the amino acid sequence of SEQ ID NO: 15 as the VL region; 3) an antibody having the amino acid sequence of SEQ ID NO: 23 as the VH region and the amino acid sequence of SEQ ID NO: 25 as the VL region; 4) an antibody having the amino acid sequence of SEQ ID NO: 33 as the VH region and the amino acid sequence of SEQ ID NO: 35 as the VL region; 5) an antibody having the amino acid sequence of SEQ ID NO: 43 as the VH region and the amino acid sequence of SEQ ID NO: 45 as the VL region; and 6) An antibody having the VH CDR1-3 and VL CDR1-3 regions of claim 1 in each of the amino acid sequences of 1) to 5) above, and having 80% or more sequence identity with each of the amino acid sequences of 1) to 5) above in other regions.

7. The method according to any one of claims 1 to 6, wherein the antibody is a combination of the antibody of 2) and the antibody of 4).

8. An antibody for detecting Clostridium perfringens enterotoxin, 1) an antibody having at least the amino acid sequence of SEQ ID NO: 7 as a VH CDR1 region, the amino acid sequence of SEQ ID NO: 8 as a VH CDR2 region, the amino acid sequence of SEQ ID NO: 9 as a VH CDR3 region, the amino acid sequence of SEQ ID NO: 10 as a VL CDR1 region, the amino acid sequence of SEQ ID NO: 11 as a VL CDR2 region, and the amino acid sequence of SEQ ID NO: 12 as a VL CDR3 region; 2) an antibody having at least the amino acid sequence of SEQ ID NO: 17 as a VH CDR1 region, the amino acid sequence of SEQ ID NO: 18 as a VH CDR2 region, the amino acid sequence of SEQ ID NO: 19 as a VH CDR3 region, the amino acid sequence of SEQ ID NO: 20 as a VL CDR1 region, the amino acid sequence of SEQ ID NO: 21 as a VL CDR2 region, and the amino acid sequence of SEQ ID NO: 22 as a VL CDR3 region; 3) an antibody having at least the amino acid sequence of SEQ ID NO: 27 as a VH CDR1 region, the amino acid sequence of SEQ ID NO: 28 as a VH CDR2 region, the amino acid sequence of SEQ ID NO: 29 as a VH CDR3 region, the amino acid sequence of SEQ ID NO: 30 as a VL CDR1 region, the amino acid sequence of SEQ ID NO: 31 as a VL CDR2 region, and the amino acid sequence of SEQ ID NO: 32 as a VL CDR3 region; 4) an antibody having at least the amino acid sequence of SEQ ID NO: 37 as the VH CDR1 region, the amino acid sequence of SEQ ID NO: 38 as the VH CDR2 region, the amino acid sequence of SEQ ID NO: 39 as the VH CDR3 region, the amino acid sequence of SEQ ID NO: 40 as the VL CDR1 region, the amino acid sequence of SEQ ID NO: 41 as the VL CDR2 region, and the amino acid sequence of SEQ ID NO: 42 as the VL CDR3 region; and 5) An antibody having at least the amino acid sequence of SEQ ID NO: 47 as the VH CDR1 region, the amino acid sequence of SEQ ID NO: 48 as the VH CDR2 region, the amino acid sequence of SEQ ID NO: 49 as the VH CDR3 region, the amino acid sequence of SEQ ID NO: 50 as the VL CDR1 region, the amino acid sequence of SEQ ID NO: 51 as the VL CDR2 region, and the amino acid sequence of SEQ ID NO: 52 as the VL CDR3 region. A single antibody or a combination of two or more antibodies selected from the group consisting of:

9. The antibody or antibody combination of claim 8, wherein said antibody is an isolated antibody.

10. 10. The antibody or antibody combination of claim 8 or 9, wherein said antibody is a monoclonal antibody.

11. The antibody reacted with Clostridium perfringens entrotoxin with a 5.0 x 10 －１０ The antibody or antibody combination according to any one of claims 8 to 10, characterized in that it has an equilibrium dissociation constant KD of less than or equal to M.

12. The antibody reacted with Clostridium perfringens entrotoxin with a 3.0 x 10 －１０ 12. The antibody or antibody combination of claim 11, characterized in that it has an equilibrium dissociation constant KD of less than or equal to M.

13. An antibody or antibody combination according to any one of claims 8 to 12, characterized in that said antibody further has the following characteristics: 1) an antibody having the amino acid sequence of SEQ ID NO: 3 as the VH region and the amino acid sequence of SEQ ID NO: 5 as the VL region; 2) an antibody having the amino acid sequence of SEQ ID NO: 13 as the VH region and the amino acid sequence of SEQ ID NO: 15 as the VL region; 3) an antibody having the amino acid sequence of SEQ ID NO: 23 as the VH region and the amino acid sequence of SEQ ID NO: 25 as the VL region; 4) an antibody having the amino acid sequence of SEQ ID NO: 33 as the VH region and the amino acid sequence of SEQ ID NO: 35 as the VL region; 5) an antibody having the amino acid sequence of SEQ ID NO: 43 as the VH region and the amino acid sequence of SEQ ID NO: 45 as the VL region; and 6) An antibody having the VH CDR1-3 and VL CDR1-3 regions according to claim 8 in each of the amino acid sequences of 1) to 5) above, and having 80% or more sequence identity with each of the amino acid sequences of 1) to 5) above in other regions.

14. An immunochromatographic kit for detecting Clostridium perfringens enterotoxin, comprising the antibody or combination of antibodies according to any one of claims 8 to 13.

15. The kit of claim 14, characterized in that the antibody combination is a combination of antibody 2) and antibody 4), in which antibody 4) is sensitized with gold colloid to form a labeled antibody, and antibody 2) is immobilized in a linear form on a solid phase to form a capture antibody.

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