JP7751834B2 - Methods for detecting cholera toxin - Google Patents

Description

The present invention relates to a method for detecting cholera toxin, an antibody used in the method, and a kit for use in the method.

Vibrio cholerae is a gram-negative bacillus of the genus Vibrio in the family Vibrionaceae. It is classified into many serotypes based on its surface antigen (O antigen), but only bacteria that exhibit either the O1 or O139 serotype and produce cholera toxin are considered to be the cause of cholera. Cholera is transmitted by oral ingestion of food or water contaminated with V. cholerae in the feces of patients or carriers. Serotype O1 cholera has caused seven major global epidemics since 1817. Many of these outbreaks were imported infections among travelers from endemic countries in India, Southeast Asia, and other regions. However, outbreaks have decreased in areas with improved sanitation. The majority of reported cases in developed countries are due to infection by travelers to developing countries where cholera is still endemic, or through food or drink brought in from developing countries. The number of reported cases of cholera in Japan has been decreasing year by year, and while there were around 50 to 100 cases per year in the early 2000s, in recent years the number has often fallen to less than 10 cases per year.

Cholera toxin (also known as cholera toxin or CT) is an oligomeric protein with a molecular weight of approximately 84 kDa, consisting of one A subunit molecule with a molecular weight of approximately 27 kDa and five B subunit molecules with a molecular weight of approximately 11.6 kDa. Details of the amino acid sequence and mechanism of action of cholera toxin have already been studied (Patent Document 1, Non-Patent Document 1).

To make a definitive diagnosis of cholera, it is necessary to isolate Vibrio cholerae by stool culture, confirm that the serotype is O1 or O139, and further verify the presence of toxin production or the toxin gene. Commercially available kits for detecting Vibrio cholerae include Denka's Vibrio cholerae AD "Seiken." Methods for detecting Vibrio cholerae include common bacterial detection methods such as isolation and culture testing and PCR testing, as well as methods using anti-Vibrio cholerae monoclonal antibodies (Patent Documents 2 to 4) and oligonucleotides for detecting Vibrio cholerae (Patent Documents 5 and 6). However, isolation and culture testing requires specialized laboratories, is cumbersome, and requires time for culturing and outsourcing. Furthermore, PCR requires specialized equipment, is cumbersome, and requires time for outsourcing.

A kit for detecting cholera toxin is commercially available, such as the VET-RPLA "Seiken" manufactured by Denka Corporation. The most common method for detecting cholera toxin is the Reversed Passive Latex Agglutination Test (RPLA), which is also used by the VET-RPLA "Seiken." However, because it detects cholera toxin from cultured bacteria, this method has problems, such as requiring a day of testing time and a minimum detection sensitivity of 1-2 ng/mL. Furthermore, as stated in the package insert for the VET-RPLA "Seiken," it also has problems in that it detects Escherichia coli heat-labile enterotoxin (LT), a cholera toxin homolog, with the same sensitivity.

Japanese Unexamined Patent Publication No. 58-222033 Japanese Patent Application Laid-open No. 62-265993 Japanese Unexamined Patent Publication No. 62-265994 Japanese Unexamined Patent Publication No. 62-265998 Japanese Patent Application Publication No. 5-276996 Japanese Patent Application Publication No. 7-8279

R.-G. Zhang, D. L. Scott, M. L. Westbrook, S. Nance, B. D. Spangler, G. G. Shipley and E. M. Westbrook. 1995 The three-dimensional crystal structure of cholera toxin. Journal of Molecular Biology 251563-573

The problem that this invention aims to solve is to provide a simple, rapid, and highly sensitive method for detecting cholera toxin (CT), which is required in a variety of situations, including medical settings, food contamination sites, and laboratories.

The inventors conducted extensive research to solve the above-mentioned problems. First, an antigen solution containing the B subunit of cholera toxin (CTxB) and an adjuvant was inoculated into BALB/c mice. After inoculation, the mice's splenocytes or lymph node cells were fused with P3U1 to produce hybridomas. From these, hybridomas producing monoclonal antibodies highly reactive to CTxB were selected, and four hybridomas producing antibodies highly reactive to CTxB were successfully isolated.

These monoclonal antibodies were then used to detect CTxB and full-length cholera toxin (CTx) using immunochromatography, and the results showed that CTxB and CTx could be detected with high sensitivity.

In a first aspect, the present invention provides a method for detecting cholera toxin in a sample by immunochromatography (hereinafter abbreviated as the detection method of the present invention), which is characterized in that the method uses the same or two different antibodies selected from the following group (hereinafter abbreviated as the antibody group of the present invention) as a labeled antibody and a capture antibody, respectively:
1) 2G6 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) the 3F7 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) 4E6 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) 8A5 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; and 5) an antibody having one or two amino acid mutations in each of the VH CDR3 region and/or VL CDR3 region of the antibodies of 1) to 4) above.

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

More preferably, an antibody belonging to the group of antibodies of the present invention has an equilibrium dissociation constant KD for cholera toxin of 5.0×10 ⁻⁹ M or less, more preferably 1.0×10 ⁻⁹ M or less, and even more preferably 5.0 ^{×10 −10} M or less.

In the antibody described in 5) 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.

In further preferred embodiments, the antibodies of the present invention have the following characteristics:
1) 2G6 antibody having at least the amino acid sequence of SEQ ID NO: 7 as the VH CDR1 region, the amino acid sequence of SEQ ID NO: 8 as the VH CDR2 region, the amino acid sequence of SEQ ID NO: 9 as the VH CDR3 region, the amino acid sequence of SEQ ID NO: 10 as the VL CDR1 region, the amino acid sequence of SEQ ID NO: 11 as the VL CDR2 region, and the amino acid sequence of SEQ ID NO: 12 as the VL CDR3 region;
2) a 3F7 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) 4E6 antibody having at least the amino acid sequence of SEQ ID NO: 27 as the VH CDR1 region, the amino acid sequence of SEQ ID NO: 28 as the VH CDR2 region, the amino acid sequence of SEQ ID NO: 29 as the VH CDR3 region, the amino acid sequence of SEQ ID NO: 30 as the VL CDR1 region, the amino acid sequence of SEQ ID NO: 31 as the VL CDR2 region, and the amino acid sequence of SEQ ID NO: 32 as the VL CDR3 region;
4) 8A5 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 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 antibodies described above in 1) to 4).

In a more preferred embodiment, the antibodies of the present invention have the following characteristics:
1) 2G6 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) 3F7 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) 4E6 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) 8A5 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; and 5) an antibody having 80% or more sequence identity with the antibodies of 1) to 4) above.

In a second aspect, the present invention provides an antibody for detecting cholera toxin, which is any one antibody selected from the above-mentioned group of antibodies of the present invention, or a combination of two or more identical or different antibodies. The antibody combination is, for example, a combination of two identical or different antibodies used for detecting cholera toxin by immunochromatography. More preferably, it is a combination of the same antibody, which is the 8A5 antibody in 4) above or an antibody derived from the 8A5 antibody in 4) among the antibodies in 5), and most preferably a combination of the same antibody, which is the 8A5 antibody in 4) above.

In a third aspect, the present invention provides an immunochromatography kit for detecting cholera toxin, characterized by comprising any one antibody or combination of antibodies selected from the above-described group of antibodies of the present invention. The kit, for example, comprises the 8A5 antibody of 4), which is sensitized with gold colloid to form a labeled antibody, and the 8A5 antibody of 4) is immobilized linearly on a solid phase to form a capture antibody.

By using the present invention, cholera toxin, the cause of food poisoning, can be detected easily (e.g., by preparing a kit, a special laboratory is not required), quickly (test time: less than 1 hour, preferably less than 30 minutes, e.g., 15 minutes), and with high sensitivity (detection sensitivity: up to 1 ng/mL, preferably up to 0.25 ng/mL) even in medical settings. Furthermore, antibodies belonging to the antibody group of the present invention can only detect the B subunit (LTB) of Escherichia coli heat-labile enterotoxin, a homologue of cholera toxin, at a high concentration of 100 ng/mL, and are therefore considered to have high specificity for cholera toxin detection.

1 shows a scheme for producing hybridomas to obtain the antibody group of the present invention. CTxB reactivity of 10 candidate antibodies is shown. The left bar shows the OD 450 in ELISA using the antigen CTxB, and the right bar shows the OD ₄₅₀ in ELISA using killed Enterobacterium cells as a negative control. The reactivity of six purified candidate antibodies to CTxB is shown. The left bar shows the OD _{450 in ELISA when the antigen CTxB concentration was 1.0 μg/mL, and the right bar shows the OD 450} when the antigen concentration was 0.1 μg/mL. The reactivity of six purified candidate antibodies to CTx is shown. The left bar shows the OD _{450 in ELISA when the antigen CTx concentration was 1.0 μg/mL, and the right bar shows the OD 450} when the antigen concentration was 0.1 μg/mL. 1 shows the affinity of six purified candidate antibodies to CTxB as determined by surface plasmon resonance. Immunochromatographs are shown for each combination of gold colloid-sensitized candidate antibody and candidate antibody used for the test line, using water, PBS alone, or CTxB at 0.1, 1, 10, and 100 ng/mL. Immunochromatographs are shown for each combination of gold colloid-sensitized candidate antibody and candidate antibody used for the test line, in the presence of 1% PBS alone, CTxB 10 ng/mL, and LTB 10 ng/mL. CTx (0, 0.25, 0.5, 1.0, or 10 ng/mL), CTxB (0, 0.25, 0.5, 1.0, or 10 ng/mL), or LTB (0, 0.25, 0.5, 1.0, 10, or 100 ng/mL) was prepared and suspended in a developing solution of 1% NP40-PBS, and the immunochromatography was developed using a gold colloid-sensitized: 8A5, test line: 8A5 immunochromatography reagent. CTx (0, 1.0, or 10 ng/mL), CTxB (0, 1.0, or 10 ng/mL), or LTB (0, 1.0, 10, or 100 ng/mL) was suspended in healthy human feces, and the suspension was prepared in a developing solution of 1% NP40-PBS. The immunochromatograph was developed using an immunochromatographic reagent with gold colloid sensitization: 8A5 and test line: 8A5.

The present invention is described in detail below. Terms used in this specification are understood in accordance with 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.

In a first aspect, as described above, the present invention provides a method for detecting cholera toxin in a sample using immunochromatography. Immunochromatography is understood to be an immunoassay method that detects an antigen using a labeled antibody and a capture antibody. Typically, the steps of immunochromatography involve 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 using 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.

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

In the detection method of the present invention, the labeled antibody is any antibody selected from the group of antibodies of the present invention that has been labeled. The labeling method may be any method commonly used in the art, and typically includes, but is not limited to, methods of binding metal colloids, fluorescent dyes, magnetic latex beads, etc. to the antibody, such as sensitizing the antibody to gold colloid particles as a metal colloid.

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., cholera toxin. 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 cholera toxin, 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 detection area.

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, solid layers made of microporous materials such as glass fiber, nylon, nitrocellulose, polyvinylidene difluoride, and cellulose acetate. Furthermore, functionally, the solid layer includes a sample application area, an electrophoresis area, and a determination area.

In the detection method of the present invention, the method for developing the sample is selected appropriately depending on the solid layer. For example, development may be due to capillary action in the solid layer, or electrophoresis using an external force such as electricity. Electrophoresis using capillary action in the solid layer is preferred because it requires less equipment and is simpler.

In the detection method of the present invention, the method for determining or confirming the determination portion is selected appropriately depending on the labeled antibody. For example, such determination or confirmation of the determination portion can be performed by visual inspection or measurement using a fluorescence measurement device. Visual inspection is preferred due to its simplicity, as it requires less equipment, but fluorescence measurement has the advantage of enabling quantitative measurement based on fluorescence intensity.

Cholera toxin, which is the target of detection in the detection method of the present invention, is composed of an A subunit consisting of the amino acid represented by SEQ ID NO: 1 and a B subunit consisting of five amino acids represented by SEQ ID NO: 2. The detailed structure of cholera toxin is registered in public databases (such as GenBank). The amino acid sequences of the A and B subunits of cholera toxin are specifically shown below. Cholera toxin is abbreviated as CT, and when simply referred to as cholera toxin, both the full length and fragments, such as subunits, are implied. In particular, the full length of cholera toxin is referred to as CTx, the A subunit as CTxA, and the B subunit as CTxB. It is to be understood that for all amino acid sequences, the one-letter and three-letter abbreviations are unambiguously interchangeable.

Amino acid sequence of CTxA (SEQ ID NO: 1):
MVKIIFVFFIFLSSFSYANDDKLYRADSRPDEIKQSGGLMPRGQSEYFDRGTQMNINLYDHAR GTQTGFVRHDDGYVSTSISLRSAHLVGQTILSGHSTYYIYVIATAPNMFNVNDVLGAYSPHPDEQ EVSALGGIPYSQIYGWYRVHFGVLDEQLHRNRGYRDRYYSNLDIAPAADGYGLAGFPPEHRAWR EEPWIHHAPPGCGNAPRSSMSNTCDEKTQSLGVKFLDEYQSKVKRQIFSGYQSDIDTHNRIKDEL

Amino acid sequence of CTxB (SEQ ID NO:2):
MIKLKFGVFFTVLLSSAYAHGTPQNITDLCAEYHNTQIYTLNDKIFSYTESLAGKREMAIIT FKNGAIFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMAN

In the present invention, antibodies may be so-called complete antibodies, as well as various other 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.

Antibodies are typically tetramers consisting of two pairs of light and heavy chains. The amino-terminal portion of each chain contains 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 (CDR), and this is where amino acid sequence variation is most pronounced. "Variable" refers to the fact that certain segments of the variable region vary extensively 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 15-30 amino acids separated by short regions of extreme variability called "hypervariable regions," each 9-15 amino acids or longer. Each VH and VL consists of three hypervariable regions (also known as "complementarity-determining regions" or "CDRs") and four FRs, arranged from the amino terminus to the carboxy terminus in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Among the CDRs, CDR3 is the segment that is particularly important for determining complementarity, followed by the other CDRs. The FRs contribute little or nothing to determining complementarity. Therefore, when specifying an antibody in terms of its antigen-binding specificity, it is possible to sufficiently identify the antibody by specifying only the CDR sequence, for example, the amino acid sequence of CDR2 or the amino acid sequences of CDRs 1 to 3, rather than specifying the entire amino acid sequence of the antibody.

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, a bivalent fragment comprising 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., 1989 ... al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:5879-5883, incorporated by reference in their entireties); (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. U.S.A. 90:6444-6448, all incorporated by reference in their entireties).

In some embodiments, the antibody may be a mixture of antibodies derived from different species, such as a chimeric antibody and/or a humanized antibody. That is, in the present invention, CDR sets may be used with framework and constant regions other than those specifically described by sequence herein.

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 one or more variable regions from a mouse (or in some cases, a rat) and one or more constant regions 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. Some or all of the CDRs are encoded by nucleic acid from a non-human organism and 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 WO 92/11018; Jones, 1986, Nature 321:522-525; Verhoeyen et al. , 1988, Science 239:1534-1536, all of which are incorporated herein by reference in their entirety.

In one embodiment, the antibody is a minibody. A minibody is a minimized antibody-like protein comprising 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 all of the hinge region.

Antibodies of the present invention are typically isolated or recombinant. "Isolated," as used to describe the various polypeptides disclosed herein, refers to a polypeptide that has been identified and separated and/or recovered from the cell or cell culture in which it 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 having different antigenic specificities. For example, an isolated antibody that specifically binds to cholera toxin is substantially free of antibodies that specifically bind to antigens other than cholera toxin.

Multiple isolated monoclonal antibodies can be used in combination in the detection method of the present invention. For example, in the case of immunochromatography, one can be labeled to form a labeled antibody, and the other can be immobilized on a solid phase to form a capture antibody.

An antibody selected from the group of antibodies of the present invention specifically binds to cholera toxin. "Specific binding" or "specifically binds," or "specific" for a particular antigen or antigen fragment, means binding that is measurably different from non-specific interactions. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which is typically a molecule of similar structure that has no binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target.

Specific binding to a particular antigen or antigen fragment is exhibited by an antibody having a Kd (1/s) for the antigen or antigen fragment of, for example, at least about ^1.0x10-1 , at least about ^5.0x10-2 , or preferably at least about ^1.0x10-2. Here, Kd refers to the dissociation rate constant of a particular antibody-antigen interaction. Typically, an antibody that specifically binds to an antigen has a Kd that is 20-, 50-, 100-, 500-, 1000-, or 5,000-fold or more higher than that of a control molecule for the antigen or antigen fragment.

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, or 5,000 times or more higher than that for a control, where Ka refers to the association rate of a specific antibody-antigen interaction.

Furthermore, the binding characteristics between a specific antibody and an antigen or antigen fragment that specifically binds to the antibody are determined as the equilibrium dissociation constant KD (M) 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 KD for cholera toxin of ^5.0x10-9 M or less, more preferably ^1.0x10-9 M or less, and even more preferably ^5.0x10-10 M or less at 25°C, and a KD of ^1.0x10-9 M or less, more preferably ^5.0x10-10 M or less, and even more preferably ^1.0x10-10 M or less at 37°C.

The binding rate constant, dissociation rate constant, and equilibrium dissociation constant between an antibody and an antigen can all be easily determined by methods known in the art. Such methods often include, but are not limited to, surface plasmon resonance methods, including antigen cross-linking methods, antibody cross-linking methods, and capture methods.

In a second aspect, the present invention provides a group of antibodies that specifically bind to cholera toxin. The antibodies included in the group of antibodies of the present invention can be used alone or in combination of two or more types. It is also possible to use any one antibody selected from the group of antibodies of the present invention in combination with another antibody from the group of antibodies of the present invention, for example, another antibody that binds to cholera toxin.

In the most preferred embodiment, antibody 1) 2G6 of the present invention has a VH having the amino acid sequence represented by SEQ ID NO: 3, and a VL having the amino acid sequence represented by SEQ ID NO: 5. Furthermore, the gene encoding the VH of antibody 1) has a nucleotide sequence represented by SEQ ID NO: 4, and the gene encoding the VL of antibody 1) is represented by SEQ ID NO: 6. The amino acid sequence of antibody 1) and the nucleotide sequence of the gene encoding it are specifically shown below.

Amino acid sequence of VH of antibody 1) (SEQ ID NO: 3):
EGQLQESGAELAKPGASVKMSCKASGYTFTSYWMHWVKQRPGQGLEWIGFINPYTGFTEY SQKFKDKATLTADKSSSTAYIQLNSLTSEDSAVYYCSRRFNYGSAYFDFWGQGTTLTVSS

Nucleotide sequence of the gene encoding VH of antibody 1) (SEQ ID NO: 4):
gagggcagc ttcaggagtc tgggctgaa ctggcaaaac ctgggccctc agtgaagatg tcctgcaagg cttctggcta caccttact agctactgga tgcactgggt aaaacagagg cctggacagg gtctggaatg gattggattc attaatccctt acactggttt tactgagtac agtcagaagt tcaaggacaa ggccacattg actgcagaca aatccctccag tacagcctac atacaactga acagcctgac atctgaggac tctgcagtct attackgttc aagaaggttt aactacggta gtgcctactt tgacttctgg ggccaaggca ccactctcac agtctcctca

Amino acid sequence of VL of antibody 1) (SEQ ID NO: 5):
DILLTQSPALMAASPGEKVTITCSVSSSISSSNLHWYQQKSETSPKPWIYGTSNLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSNYPLTFGGGTKLEVK

Nucleotide sequence of the gene encoding VL of antibody 1) (SEQ ID NO: 6):
gacattttgc tcactcagtc tccagcactc atggctgcat ctccagggga gaaggtcacc atcacctgta gtgtcagctc aagtataagt tccagcaact tgcactggta ccagcagaag tcagaaacct cccccaaacc ctggatttat ggcacatcca acctggcttc tggagtccct gttcgcttca gtggcagtgg atctgggac tcttattctc tcacaatcag cagcatggag gctgaagatg ctgccactta ttactgtcaa cagtggagta attacccact cacgttcgga ggggggacca agctggaggt aaaa

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

VH CDR1 amino acid sequence of Antibody 1) (SEQ ID NO:7):
GYTFTSYWMH
VH CDR2 amino acid sequence of Antibody 1) (SEQ ID NO:8):
FINPYTGFTEYSQKFKD
VH CDR3 amino acid sequence of Antibody 1) (SEQ ID NO:9):
RFNYGSAYFDF
VL CDR1 amino acid sequence of Antibody 1) (SEQ ID NO: 10):
SVSSSISSSNLH
VL CDR2 amino acid sequence of Antibody 1) (SEQ ID NO:11):
GTSNLAS
VL CDR3 amino acid sequence of Antibody 1) (SEQ ID NO: 12):
QQWSNYPLT

In the most preferred embodiment, antibody 2) 3F7 of the present invention has a VH having the amino acid sequence represented by SEQ ID NO: 13, and a VL having the amino acid sequence represented by SEQ ID NO: 15. Furthermore, the gene encoding the VH of antibody 2) has the nucleotide sequence represented by SEQ ID NO: 14, and the gene encoding the VL of antibody 2) is represented by SEQ ID NO: 16. The amino acid sequence of antibody 2) and the nucleotide sequence of the gene encoding it are specifically shown below.

Amino acid sequence of VH of antibody 2) (SEQ ID NO: 13):
QAYLQQSGPELMKPGASVKISCKASGYSFTSYYMHWVRQRHGKSLEWIGYIDPFNGDTRY NQKFKGKATLTVDKSSSTAYMHLSSLTSEDSAVHYCARWAYYDSYYFDYWGQGTTLTVSS

Nucleotide sequence of the gene encoding VH of antibody 2) (SEQ ID NO: 14):
caggcttatc tgcagcagtc tggacctgag ctgatgaagc ctgggcttc agtgaagata tcctgcaagg cttccggtta ctctttcact agctactaca tgcactgggt gaggcagaga catggaaaga gccttgagtg gattggatat attgatcctt tcaatggtga tactaggtac aaccagaaat tcaaggcaa ggccacattg actgtagaca aatcttccag cacagcctac atgcatctca gcagcctgac atctgaggac tctgcagtcc attackgtgc aagatgggct tactacgata gttattattt tgactactgg ggccaaggca ccactctcac agtctcctca

Amino acid sequence of VL of antibody 2) (SEQ ID NO: 15):
DVVVTQTPLSLPVSLGEQASISCRSSQGFVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCLQGSHVPLTFGAGTKLELK

Nucleotide sequence of the gene encoding VL of antibody 2) (SEQ ID NO: 16):
gatgttgtgg tgacccaaac tccactctcc ctgcctgtca gtcttggaga gcaagcctcc atctcttgca gatctagtca gggctttgta catagtaatg gaaacaccta tttagaatgg tacctgcaga aaccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt tctgggtcc cagacaggtt cagtggcagt ggatcaggga cagtttcac actcaagatc agcagagtgg aggctgagga tctgggagtt tattactgtc ttcaaggttc acatgttccg ctcacgttcg gtgctgggac caagctggag ctgaaa

Furthermore, antibody 2) was 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 its VH CDR1 is represented by SEQ ID NO: 17, its VH CDR2 is represented by SEQ ID NO: 18, and its VH CDR3 is represented by SEQ ID NO: 19, and its VL CDR1 is represented by SEQ ID NO: 20, its VL CDR2 is represented by SEQ ID NO: 21, and its 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.

VH CDR1 amino acid sequence of antibody 2) (SEQ ID NO: 17):
GYSFTSYYMH
VH CDR2 amino acid sequence of antibody 2) (SEQ ID NO: 18):
YIDPFNGDTRYNQKFKG
VH CDR3 amino acid sequence of antibody 2) (SEQ ID NO: 19):
WAYYDSYYFDY
Antibody 2) VL CDR1 amino acid sequence (SEQ ID NO:20):
RSSQGFVHSNGNTYLE
VL CDR2 amino acid sequence of antibody 2) (SEQ ID NO:21):
KVSNRFS
VL CDR3 amino acid sequence of antibody 2) (SEQ ID NO:22):
LQGSHVPLT

In the most preferred embodiment, antibody 3) 4E6 of the present invention has a VH having the amino acid sequence represented by SEQ ID NO: 23, and a VL having the amino acid sequence represented by SEQ ID NO: 25. Furthermore, the gene encoding the VH of antibody 3) has the nucleotide sequence represented by SEQ ID NO: 24, and the gene encoding the VL of antibody 3) is represented by SEQ ID NO: 26. The amino acid sequence of antibody 3) and the nucleotide sequence of the gene encoding it are specifically shown below.

Amino acid sequence of VH of antibody 3) (SEQ ID NO:23):
EVRLQQSGPELVRPGASMKISCKASGYSFTGYTMNWVKQSHGKNLDWIGLIHPYNGRTTYNQKFKGKATLTVDKSSSTAYMELLSLTSEDSAVYYCAKEYDWYFDVWGAGTTVTVSS

Nucleotide sequence of the gene encoding VH of antibody 3) (SEQ ID NO: 24):
gaggttcggc tgcaacagtc tggacctgag ctggtgaggc ctggagcttc aatgaagata tcctgcaagg cttctggtta ctcattcact ggctacacca tgaactgggt gaaacagagc catggaaaga accttgactg gattggactt attcatccctt acaatggtcg tactacctac aaccagaagt tcaagggcaa ggccacatta actgtagaca agtcatccag cacagcctac atggagctcc tcagtctgac atctgaggac tctgcagtct attackgtgc aaaagagtac gactggtact tcgatgtctg gggcgcagg accacggtca ccgtctcctca a

Amino acid sequence of VL of antibody 3) (SEQ ID NO:25):
DIQMTQSPSSLSASLGDTITITCHASQNINIWLSWYQQKPGNIPKLLIYQASNLHTGVPSRFSGSGSGTGFKLTIRSLQPEDIATYYCQQGQSYPRTFGGGTKLEIK

Nucleotide sequence of the gene encoding VL of antibody 3) (SEQ ID NO: 26):
gacatccaga tgacacagtc tccatccagt ctgtctgcat cccttggaga cacaattacc atcacttgcc atgccagtca gaacattaat atttggttaa gctggtacca gcagaaacca ggaaattc ctaaactt gatctatcag gcttccaact tgcacacag cgtcccatca aggtttagtg gcagtggatc tggaacaggt ttcaaattaa ccatccgcag cctgcagcct gaagacattg ccacttacta ctgtcaacag ggtcaaagtt atccctcggac gttcggtgga ggcaccaagc tggaaatcaa a

Furthermore, antibody 3) was 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 its VH CDR1 is represented by SEQ ID NO: 27, its VH CDR2 is represented by SEQ ID NO: 28, and its VH CDR3 is represented by SEQ ID NO: 29, and its VL CDR1 is represented by SEQ ID NO: 30, its VL CDR2 is represented by SEQ ID NO: 31, and its 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.

VH CDR1 amino acid sequence of antibody 3) (SEQ ID NO:27):
GYSFT GYTMN
VH CDR2 amino acid sequence of antibody 3) (SEQ ID NO:28):
LIHPYNGRTTYNQKF
VH CDR3 amino acid sequence of antibody 3 (SEQ ID NO:29):
EYDWYFDV
Antibody 3) VL CDR1 amino acid sequence (SEQ ID NO:30):
HASQNINIWLS
Antibody 3) VL CDR2 amino acid sequence (SEQ ID NO:31):
QASNLHT
VL CDR3 amino acid sequence of antibody 3 (SEQ ID NO:32):
QQGQSYPRT

In the most preferred embodiment, antibody 4) 8A5 of the present invention has a VH having the amino acid sequence represented by SEQ ID NO: 33, and a VL having the amino acid sequence represented by SEQ ID NO: 35. Furthermore, the gene encoding the VH of antibody 4) has the nucleotide sequence represented by SEQ ID NO: 34, and the gene encoding the VL of antibody 4) is represented by SEQ ID NO: 36. The amino acid sequence of antibody 4) and the nucleotide sequence of the gene encoding it are specifically shown below.

Amino acid sequence of VH of antibody 4) (SEQ ID NO: 33):
QVQLQQSAAELARPGASVKMSCKASGFTFTSYTLHWVKQRPGQGLEWIGYINPRGYTEY HQKFKDKTTLTADKSSSTAYIQLNSLTSEDSAVYYCARYDGFFLYALDYWGQGTSVTVSS

Nucleotide sequence of the gene encoding VH of antibody 4) (SEQ ID NO: 34):
caggtccaac tgcagcagtc tgcagctgaa ctggcaagac ctgggcctc agtgaagatg tcctgcaagg cttctggctt caccttact agctacacgt tacactgggt aaaacagagg cctggacag gtctggaatg gattggatac attaatcccc gtggatatac tgagtaccat cagaagttca aggacaagac cacattgact gcagacaaat cctccagcac agcctacata caactgaaca gcctgacatc tgaggactct gcggtctatt actgtgcaag atatgatggt ttcttcctct atgctttgga ctattggggt caaggaacct cagtcaccgt ctcctca

Amino acid sequence of VL of antibody 4) (SEQ ID NO:35):
DIQMTQSPSSMYASLGERVTITCKASQDINSNLSWFQQKPGKSPKTLIYRASRLVHGVPSRFSGSGSGQDYSLISSLEYEDMGIYSCLQYDEFPYTFGGGTKLEIK

Nucleotide sequence of the gene encoding VL of antibody 4) (SEQ ID NO: 36):
gacatccaga tgacacagtc tccatcttcc atgtatgcat ctctagga gagagtcact atcacttgca aggcgagtca ggacattaat agcaatttaa gctggttcca gcagaaacca gggaaatctc ctaagaccct gatctatcgt gcaagcagat tggtacatgg ggtcccatca aggttcagtg gcagtggatc tgggcaagat tattctctca ccatcagcag cctggagtat gaagatatgg gaatttattc ttgtctacag tatgatgagt ttccgtacac gttcggagg gggaccaagc tggaaataaa a

Furthermore, antibody 4) was 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.

VH CDR1 amino acid sequence of antibody 4) (SEQ ID NO:37):
GFTFTSYTLH
VH CDR2 amino acid sequence of antibody 4) (SEQ ID NO:38):
YINPRGYTEYHQKFKD
VH CDR3 amino acid sequence of antibody 4) (SEQ ID NO:39):
YDGFFLYALDY
VL CDR1 amino acid sequence of antibody 4) (SEQ ID NO:40):
KASQDINSNLS
VL CDR2 amino acid sequence of antibody 4) (SEQ ID NO:41):
RASRLVH
VL CDR3 amino acid sequence of antibody 4) (SEQ ID NO:42):
LQYDEFPYT

The present invention also provides mutant antibodies. That is, the antibodies of the present invention may be modified in a number of ways, 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.

As used herein, the term "variant" refers to a polypeptide sequence that differs from that of a parent polypeptide by at least one amino acid modification. Amino acid modifications can include substitutions, insertions, and deletions, with substitutions, particularly conservative substitutions, being preferred in many cases.

In general, as described herein, variants may include any number of modifications, so long as the antibody's functionality remains present. That is, 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 CTx.

However, in general, the goal is often to alter function with the minimum number of modifications, so 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 permit the use of 1-2, 1-3, and 1-4 modifications.

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 1-5 modifications in the Fc region of a wild-type or variant protein, and 1-5 modifications in the Fv region, for example. The variant polypeptide sequence preferably 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 antibody group 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.

As used herein, "amino acid substitution" or "substitution" refers to the replacement of an amino acid at a particular 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 particular position in a parent polypeptide sequence. As used herein, "amino acid deletion" or "deletion" refers to the removal of an amino acid at a particular position in a parent polypeptide sequence.

As used herein, "parent polypeptide," "parent protein," "precursor polypeptide," or "precursor protein" refers to an unmodified polypeptide that is subsequently modified to create a variant. Typically, a parent polypeptide herein is any antibody selected from the group of antibodies of the present invention. A 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, a "parent Fc polypeptide" refers to an Fc polypeptide that is modified to create a variant, and a "parent antibody" refers to an antibody that is modified to create a variant antibody.

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.

As used herein, "variant Fc region" refers to an Fc sequence that differs from that of a wild-type Fc sequence by 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.

In some embodiments, one or more amino acid modifications are made in one or more CDRs of the antibody. Generally, only one or two amino acids are substituted in any single CDR, and usually no more than three, four, five, six, seven, eight, nine, or ten changes are made within a set of CDRs. However, any combination of none, one, two, or three or more substitutions in any CDR can be made, independently and optionally combined with any other substitution.

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

Affinity maturation can be performed to increase the binding affinity of an antibody for an antigen by at least about 10% to 50% to 100% to 150% or more, or 1 to 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) domain and light chain variable (VL) domain shuffling. Methods for random mutagenesis of CDR and/or framework residues are described in, for example, Barbas, et al., 1994, Proc. Nat. Acad. Sci. USA 91:3809-3813; Shier ... , 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 etc.

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 optimizing expression (which can be made to nucleic acids encoding an antibody of the invention).

It is well known in the art that certain amino acids can be substituted with other amino acids having a similar hydrophobicity index and still produce proteins with similar biological function (e.g., proteins equivalent in enzymatic activity; hereinafter, "bioisequences"). 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. Pat. No. 4,554,101, the following hydrophilicity indices have been 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.

Therefore, in the present invention, polypeptides may be subjected to mutations or conservative substitutions. Such mutations may be substitutions, insertions, or deletions. Methods for introducing 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.

Thus, variant CDRs and antibodies are included within the definition of the CDRs and antibodies of the present invention; i.e., antibodies of the present invention may contain 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).

In some embodiments, a mutant antibody specific for CTx is described. This antibody consists of six CDRs, where each CDR of this antibody can independently differ from each of the above amino acid sequences by 0, 1, or 2 amino acid substitutions. Conversely, in the FR region, the amino acid sequence does not significantly affect the binding specificity of the antibody, so more extensive mutations can be introduced, for example, mutations that result in approximately 80%, 85%, or 90% identity with the parent FR region sequence.

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). Additionally, there are a variety of covalent modifications of antibodies that can be made, as outlined below.

Covalent modifications of antibodies are included within the scope of the present invention and are usually, but not always, performed 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 N- or C-terminal residues.

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.

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.

Lysinyl and amino-terminal residues react with succinic or other carboxylic acid anhydrides. Derivatization with this agent has the effect of reversing the charge of the lysinyl residues. Other agents suitable 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.

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 due to the high pKa of the guanidine functional group. Furthermore, these reagents may react with lysine groups and the arginine epsilon-amino group.

Specific modification of tyrosyl residues, where there is particular interest in introducing spectral labels into tyrosyl residues, may be achieved 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 using 125I or 131I to prepare labeled proteins for use in radioimmunoassay, the chloramine T method (described above as suitable).

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. Furthermore, aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

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) can be used for protein immobilization.

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 the present invention.

Other modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl group of seryl or threonyl residues, methylation of the α-amino group of lysine, arginine, and histidine side chains (T. E. Creighton, 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.

Furthermore, 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 present invention).

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 may be defined by a set of six CDRs, a variable region, or full-length heavy and light chains (including constant regions). Furthermore, as outlined above, amino acid substitutions may be made. Generally, in the context of changes within CDRs, 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 CDRs. While this is also applicable to discussing the number of amino acid modifications that can be introduced into variable, constant, or full-length sequences, it is also appropriate to define these changes in terms of "percent 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.

The present invention further provides methods for producing any antibody selected from the group of antibodies of the present invention. These methods involve 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 methods are performed under conditions that allow the antibody to be produced and isolated, for example.

Generally, nucleic acids encoding the antibodies of the invention are provided. Such polynucleotides encode both the variable and constant regions of each of the heavy and light chains, 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.

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.

In some embodiments, one or more nucleic acids encoding an antibody of the 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. The expression vector can include any number of appropriate regulatory sequences (including, but not limited to, transcriptional and translational regulatory sequences, promoters, ribosome 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., heavy chain in a first expression vector and light chain in a second expression vector) or they can be in the same expression vector. Those skilled in the art will understand 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.

Generally, nucleic acids and/or expression vectors are introduced into a suitable host cell using any method appropriate for the selected host cell (e.g., transformation, transfection, electroporation, infection, etc.) 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 host cell's genome) 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.) to produce 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.

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 or chickens.

General methods for 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.

The present invention also provides an immunochromatography kit for detecting cholera toxin, comprising any one antibody or combination of antibodies selected from the antibody group of the present invention. One embodiment of this kit comprises a detection section on a matrix made of a porous material such as a nitrocellulose membrane, where a capture antibody is immobilized linearly, and a labeled reagent zone upstream of the detection section, where 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, there is a development confirmation section where an anti-labeled antibody is immobilized linearly to confirm the development of the labeled antibody, and further downstream, there is a developer absorption section equipped with a porous absorption pad for absorbing the developer solution. Furthermore, if the label is an enzyme label, a substrate zone carrying a substrate for the labeled enzyme is provided upstream of the labeled reagent zone.

During use, the 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, supplying the developer 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 CTX, the CTX and labeled antibody bind via an antigen-antibody reaction. When they reach the detection zone, the immobilized antibody and CTX bind via an antigen-antibody reaction in the detection zone. As a result, the labeled antibody is immobilized in the detection zone via CTX. CTX is detected by measuring the label in the detection zone. If the sample does not contain CTX, 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 area downstream of the detection zone, the labeled antibody is fixed to the developer confirmation area. Therefore, if a label is detected in the developer confirmation area, it means that the developer has been developed correctly. The developer is ultimately absorbed by the absorbent pad downstream.

The present invention also provides a kit for use in the above-described method of the present invention. The kit of the present invention comprises at least a monoclonal antibody of the present invention or a combination thereof.

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, and preferably both, is a 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, the kit can include a labeled substance that binds to the detection antibody.

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 the 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.

The kit of the present invention may further include instructions for use of the kit.

The present invention will be explained in more detail below based on examples, but the present invention is not limited to the following examples.

1. Antibody Production The following media were used:
FBS (-) medium: DMEM (Nacalai) 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: FBS(+) medium 250 mL, HAT (×50 conc. MP Bio) 5 mL, IL-6 (PeproTech recombinant mouse IL-6) 10 ng/mL;
HT D-MEM medium: DMEM (Nacalai, high glucose) 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 (x50 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)

1-1. BALB/c mice were immunized by injecting a 1:1 mixture of cell fusion cholera toxin B subunit CTxB with Sigma Adjuvant system into the footpads of mice twice at two-week intervals (Figure 1). Lymph nodes and spleens harvested from mice were added to FBS(+) medium. The tissues were placed in a 5-cm-diameter Petri dish containing 5 mL of FBS(+) medium. After removing any attached fat, splenocytes or lymph nodes 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/well in a 96-well plate. The cells were cultured at 37°C in ₁₀ % CO , with 100 μL of HAT medium replaced every 2–3 days, until colonies appeared and grew. Ten hybridomas were obtained. The ten hybridomas were designated as 2G6, 3C3, 3F3, 3F7, 4E6, 5F2, 8A5, 8B3, 10B8, and 19F5, and the antibodies obtained from each hybridoma were designated as well. The scheme is shown in Figure 1.

1-2. Primary screening (ELISA)
The antigen, CTxB, was adjusted to a concentration using PBS, and lyophilized mouse intestinal bacteria (negative control) was dissolved in carbonate-bicarbonate buffer (killed mouse intestinal bacteria: 1.0 mg/mL, CTxB: 5.0 μg/mL). The antigen and killed mouse intestinal bacteria were each added to a 96-well plate at 100 μL/well and allowed to stand overnight at 4°C for immobilization. 170 μL/well of 1% BSA-PBS was added to the plate, which was then allowed to stand at room temperature for 2 hours for blocking. The plate was then washed three times with 250 μL PBS-T. 50 μL/well of each hybridoma culture supernatant was added to the plate, which was allowed to stand at room temperature for 2 hours and then washed three times with 250 μL 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 0.5 N HCl was added, and the plate was then plated. 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 CTxB, and the right bar indicates the absorbance of killed Enterobacterial cells. Compared with killed enterobacterial cells, five antibodies, 2G6, 3F7, 4E6, 5F2 and 8A5, showed extremely high reactivity to CTxB, with 19F5 being the next most positive.

1-3. Cloning (limiting dilution method)
Six 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 screening-positive well, the result was 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.

1-4. Monoclonal Antibody Purification: HT D-MEM medium was warmed to 37°C in an incubator, and the frozen hybridoma was thawed in the incubator at 37°C. The thawed hybridoma was added to 10 mL of HT D-MEM medium. After centrifugation (1,200 rpm, 3 minutes), the supernatant was removed, and the pellet was loosened and suspended in 10 mL of HT D-MEM 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 D-MEM medium and cultured at 37°C. After confirming hybridoma growth, the cells were suspended and 10 mL of the cell suspension was added to a flask containing 40 mL of HT D-MEM medium and cultured at 37°C. After confirming hybridoma growth, the supernatant was removed and the cells were washed twice with 15 mL of PBS, after which 50 mL of ADCF MAb was added and cultured at 37° C. After two 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.

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.

1-5. Purified antibody reactivity confirmation test (ELISA)
CTxB and CTx were dissolved in carbonate-bicarbonate buffer and adjusted to the desired concentrations (CTxB: 0.1 and 1.0 μg/mL, CTx: 0.1 and 1.0 μg/mL). Each antigen solution was added to a 96-well plate at 100 μL/well and incubated 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 incubated at room temperature for 2 hours for blocking. After washing with PBS-T, 50 μL of hybridoma culture supernatant was added to the plate at 50 μL/well and incubated at room temperature for 2 hours. The plate was then washed with PBS-T. Anti-mouse IgG (whole molecule)-HRP (1031-05, Southern Biotech) diluted in 1% BSA-PBS-T was added at 100 μL/well and incubated at 37°C for 1 hour. The plate was then washed 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 (CTxB) and Figure 4 (CTx). 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 CTxB at high concentrations, but overall reactivity decreased at low concentrations. In particular, 19F5 showed almost no reactivity with CTxB at low concentrations. Furthermore, the low reactivity of 19F5 with CTx was particularly notable.

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 in carbonate-bicarbonate buffer and adjusted to the desired concentration (CTxB: 1.0 μg/mL, CTx: 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 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. Anti-mouse IgG (whole molecule)-HRP (1031-05, Southern Biotech) diluted in 1% BSA-PBS-T was added to the plate at 100 μL/well, allowed to stand at 37°C for 1 hour, and then washed 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 CTxB and CTx, but no reactivity was observed with any of the above bacterial cells.

1-7. Sandwich ELISA
A sandwich ELISA test for detecting CTxB was performed using various combinations of the six positive antibodies (2G6, 3F7, 4E6, 5F2, 8A5, and 19F5) obtained in sections 1-4 above. First, the antibodies were labeled. The modifier solution in 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 for 3 hours to overnight at 20-25°C in the dark. Quencher solution was added, and the reaction was allowed to proceed for 30 minutes at 20-25°C in the dark (1 μL quencher/10 μL antibody).

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 allowed to stand overnight at 4°C. The plate was washed three times with 300 μL of PBS-T, and the capture antibody solution was removed. 200 μL/well of 1% BSA-PBS was added to the 96-well plate and allowed to stand at room temperature for 2 hours or overnight at 4°C. 100 μL/well of an antigen CTxB (0.1 μg/mL) solution diluted with 1% BSA-PBST was added, and the plate was allowed to react 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 allowed to react for 2 hours at room temperature. After removing the liquid, the plate was washed three times with 300 μL of PBS-T. 100 μL of TMB was added per well, and after exactly 2 minutes at room temperature, 50 μL of 0.5 N HCl was added per well. The absorbance at 450 nm was measured using a spectrophotometer.

When 5F2-HRP and 19F5-HRP were used as the labeled antibody, the antigen was not detectable regardless of the immobilized antibody. Furthermore, when 5F2 was used as the immobilized antibody or in the negative control, the antigen was not detectable at all regardless of the labeled antibody. When 19F5 was used as the immobilized antibody, the antigen was detectable when 3F7-HRP or 8A5-HRP was used as the labeled antibody, but was not detectable with other labeled antibodies. Among 2G6, 3F7, 4E6, and 8A5, the antigen was detectable in all combinations, including when the labeled antibody and the immobilized antibody were the same antibody.

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-CTx monoclonal antibodies were captured. A running buffer containing CTxB (0.1 M HEPES, 1.5 M NaCl, 30 mM EDTA, 0.5% v/v Surfactant P 20) was added at a flow rate of 30 μL/min (association phase), and the running buffer was allowed to flow through 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. All four antibodies, 2G6, 3F7, 4E6, and 8A5, exhibited extremely low KD.

3. Immunochromatography 3-1. Preparation of an Immunochromatography Kit Four antibodies, 2G6, 3F7, 4E6, and 8A5, were prepared to the determined concentrations of coating protein. _KH2PO4 at the optimal pH was dispensed, and _a 40 nm diameter colloidal gold solution (BB International) was added, stirred, and allowed to stand. After adding 1% PEG 20,000 and 10% BSA, the mixture was centrifuged, the supernatant removed, and the precipitate was dispersed ultrasonically. Gold colloid storage buffer was added to the mixture, and the centrifugation procedure was repeated to obtain an antibody-sensitized colloidal gold solution.

Four antibodies, 2G6, 3F7, 4E6, and 8A5, were prepared for use in the test line and each was applied to a nitrocellulose membrane (150CNPH-N-SS40). After application, the membrane was heat-dried at 50°C for 30 minutes. After drying, blocking and stabilization treatments were performed and the membrane was dried again to obtain an antibody-immobilized membrane. 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.

100 μL each of water alone or CTxB 0.1, 1, 10, and 100 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 observed at 10 to 100 ng/mL, and slight color development was also observed even at 1 ng/mL.

3-2. Verification of immunochromatography using CTxB and LTB CTxB and Escherichia coli heat-labile enterotoxin B subunit (LTB) were each prepared and suspended in 1% NP40-PBS at 10 ng/mL, and 100 μL of each was dropped onto the full strip obtained by preparing the immunochromatography kit described above. The color development of the line after approximately 20 minutes was confirmed. The results are shown in Figure 7. CTxB was detected in both full strips, but LTB was not.

3-3. Verification of the Developer CTxB, CTx, and LTB were prepared and suspended in 1% NP40-PBS developer solution at various concentrations, and 100 μL of each was applied to an immunochromatographic reagent (colloidal gold: 8A5, test line: 8A5). 20 minutes later, the results were analyzed. The results are shown in Figure 8. Furthermore, healthy human stool was suspended in 1% NP40-PBS, and 100 μL of each pseudo-sample was prepared by preparing and suspending CTxB, CTx, and LTB at various concentrations. 100 μL of each was applied to an immunochromatographic reagent (colloidal gold: 8A5, test line: 8A5). 20 minutes later, the results were analyzed. The results are shown in Figure 9. Since bands were observed in both the test line and the control, it was determined that the development had been carried out without any problems.

3-3. Immunochromatographic Reagent Cross-Reactivity Test The following bacteria were prepared with 1% NP40-PBS to a McFarland concentration of 0.5 (approximately 1.0 x 10 ⁸ cfu/mL), and 100 μL was applied to the immunochromatographic reagent (colloidal gold: 8A5, test line: 8A5). After 20 minutes, the results were evaluated to verify cross-reactivity. The immunochromatographic reagent (colloidal gold: 8A5, test line: 8A5) 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

4. Amino Acid and Gene Sequence Analysis RNA was extracted from various hybridomas ( ¹⁰ cells), and cDNA was synthesized. Using the cDNA as a template, VH and VL genes 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 (21)

A method for detecting cholera toxin in a sample by immunochromatography, characterized in that two identical or 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: 9 as the VH CDR3 region and the amino acid sequence of SEQ ID NO: 12 as the VL CDR3 region;
2) an 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) an 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) an 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) 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 4) above. The method of claim 1, wherein the antibody is an isolated antibody. The method of claim 1 or 2, wherein the antibody is a monoclonal antibody. The method according to any one of claims 1 to 3, wherein the antibody has an equilibrium dissociation constant KD of 5.0 x 10 ^-9 M or less with respect to cholera toxin. 5. The method of claim 4, wherein the antibody has an equilibrium dissociation constant KD of 5.0× ^{10 −10} M or less for cholera toxin. The method according to any one of claims 1 to 5, wherein the amino acid mutation in the antibody of 5) above is a substitution. The method of claim 6, wherein the substitution is a conservative amino acid substitution. The method according to any one of claims 1 to 7, wherein the antibody further has the following characteristics:
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;
5) 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 4) above. The method according to any one of claims 1 to 8, 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 a sequence identity of 80% or more with the antibodies of 1) to 4) above. The method according to any one of claims 1 to 9, wherein the antibody is an antibody of 4) or a combination of antibodies of 5) derived from the antibody of 4). An antibody for detecting cholera toxin,
1) an 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 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) an 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) an 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; and 5) 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 4) above, or a combination of two or more identical or different antibodies selected from the group consisting of: The antibody or antibody combination of claim 11, wherein the antibody is an isolated antibody. The antibody or antibody combination of claim 11 or 12, wherein the antibody is a monoclonal antibody. The antibody or antibody combination according to any one of claims 11 to 13, characterized in that said antibody has an equilibrium dissociation constant KD of 5.0 x 10 ^-9 M or less with respect to cholera toxin. 15. The antibody or antibody combination of claim 14, wherein said antibody has an equilibrium dissociation constant KD for cholera toxin of 5.0×10 ⁻¹⁰ M or less. The antibody or antibody combination according to any one of claims 11 to 15, wherein the amino acid mutation in the antibody of 5) above is a substitution. The antibody or antibody combination of claim 16, wherein the substitution is a conservative amino acid substitution. The antibody or antibody combination according to any one of claims 11 to 17, characterized in that said antibody further has the following characteristics:
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;
5) 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 4) above. An antibody or antibody combination according to any one of claims 11 to 18, 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 a sequence identity of 80% or more with the antibodies of 1) to 4) above. An immunochromatography kit for detecting cholera toxin, comprising the antibody or antibody combination described in any one of claims 11 to 19. The kit of claim 20, wherein the combination of antibodies is a combination of antibodies 4), in which the antibody 4) is sensitized with gold colloid to form a labeled antibody, and the antibody 4) is immobilized linearly on a solid phase to form a capture antibody.