KR20120029137A - Antibody specifically binding to c. perfringens enterotoxin protein and composition for detecting said protein comprising the same - Google Patents

Abstract

PURPOSE: A composition containing an antibody for detecting clostridium perfringens enterotoxin(CPE) is provided to accurately detect CPE proteins. CONSTITUTION: An isolated antibody or antigen binding fragment thereof binds to N-terminal peptide having 1-87th amino acid sequences of sequence number 1; or CPE C-terminal peptide having 243-319th amino acid sequence of sequence number 1. The antibody is a monoclonal antibody. A composition for detecting CPE protein contains the antibody or antigen binding fragment thereof.

Description

Antibody Specific Binding to C. perfringens Enterotoxin Protein and Composition for Detecting Said Protein Antibody that specifically binds to Clostridium perfringens enterotoxin protein, and Clostridium perfringens enterotoxin detection Comprising the Same}

The present invention includes an isolated antibody or antigen-binding fragment thereof that specifically binds to the N-terminal site or C-terminal site of Clostridium perfringens enterotoxin (CPE) protein and an active ingredient thereof It relates to a composition for detecting CPE.

Clostridium perfringens, previously known as Clostridium welchii, was first described in detail in 1892 by Welch and Nuttall. The bacterium has been recognized as a cause of food poisoning since 1895, and its connection was firmly established in 1945 by an epidemiological study by McClung. In 1969 an enterotoxin was reported by Duncan and Strong. In addition to enteritis, C. perfringens causes tissue necrosis in humans and animals and severe intestinal intoxication in some animals. Clostridium perfringens is a gram-positive, anaerobic, apolytic bacillus that is unusually non-motile in clostria. Although it appears to be a natural colon of the human colon, it has a pathogenic potential in both humans and animals because it contains large amounts of deadly necrotic enzymes and toxins. Clostridium perfringens ( C. perfringens ) is divided into five types, A, B, C, D, E, depending on the presence of four major toxins that are fatal to rats. Enterotoxins that cause food poisoning in humans are produced by strains A. Very rarely, type C strains can cause even more severe necrotic enteritis (Pig-Bel), which can be fatal. The most representative type of diarrhea is a type A toxin known to secrete α toxin. However, the type A toxin type is known to be enterobacteriaceae also present in stool samples of normal people. Among the type A toxin types, the introduction of the cpe gene to secrete C. perfringens enterotoxin (CPE) causes severe diarrhea symptoms compared to the type A toxin type. Clostridium perfringens of the type A toxin type A that secrete CPE are not only food-borne diseases but also non-food-borne diseases and antibiotic-associated diarrhea (AAD). Is known to cause sporadic diarrhea (SD). In particular, CPE-secreting type A pathogens have been reported to cause AAD and SD when the cpe gene is present in the plasmid. Recently, there has been an increase in the number of infections caused by C. perfringens among AAD patients. According to a 2006 paper, 21% of AAD patients showed C. perfringens There are reports separated by causative organisms. CPE is not secreted in food or the natural environment, but it is known that toxins are secreted when the pathogen dissolves as it passes through the gastric mucosa and forms the apothelium after reaching an intestine. Toxins adhere to the mesothelial cell membranes, disrupting cell permeability and causing diarrhea symptoms.

In Korea, the separation of Clostridium perfringens, which secrete CPE annually, is increasing. Clostridium perfringens isolated from the laboratory monitoring of pathogens causing bacterial diarrheal diseases has been increasing every year, with 57 strains in 2005, 111 strains in 2006, and 178 strains isolated until October 2007. In particular, Clostridium perfringens is known as a major cause of group food poisoning due to group meals. As a diagnostic method for CPE toxin, PET-RPLA kit, which is widely used now, is used to diagnose the expression of toxin. However, there is a difficulty in accurately measuring the toxin expression of the strain in the use of the PET-RPLA kit.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

The present inventors have made extensive efforts to develop an effective Clostridium perfringens enterotoxin (CPE) detection kit, and for this purpose, we have developed an antibody that can specifically bind to CPE protein, and this antibody. The present invention was completed by experimentally confirming that the CPE protein can be accurately detected with a very high sensitivity by using an immunological detection method.

Accordingly, an object of the present invention is to provide an antibody that specifically binds to the N-terminal site or C-terminal site of the Clostridium perfringens enterotoxin (CPE) protein.

Another object of the present invention is to provide a composition for detecting CPE protein comprising the antibody as an active ingredient.

The objects and advantages of the invention will become apparent from the following detailed description, claims and drawings.

According to one aspect of the invention, the invention is (i) CPE ( C. perfringens enterotoxin) N-terminal peptide having the amino acid sequence of the 1-87 th sequence of SEQ ID NO: 1; Or (ii) an isolated antibody or antigen-binding fragment thereof characterized by specifically binding to a CPE C-terminal peptide having amino acid sequences 243-319 of SEQ ID NO: 1.

According to another aspect of the present invention, the present invention provides a composition for detecting a CPE protein comprising an antibody or antigen-binding fragment thereof that specifically binds to an N-terminal peptide or C-terminal peptide of the CPE protein as an active ingredient. .

According to a preferred embodiment of the present invention, the N-terminal peptide in the antibody of the detection composition comprises the 1-87th amino acid sequence of SEQ ID NO: 1 sequence, the C-terminal peptide is 243 of SEQ ID NO: 1 The 319th amino acid sequence.

According to another preferred embodiment of the present invention, the antibody in the detection composition is a monoclonal antibody.

According to another preferred embodiment of the present invention, the detection composition is in the form of an Enzyme-Linked Immunosorbent Assay (ELISA) kit.

According to another preferred embodiment of the present invention, the ELISA kit comprises a capturing antibody and a detecting antibody.

According to another preferred embodiment of the present invention, the capture antibody is an antibody that specifically binds to the N-terminal peptide of the CPE protein, and the detection antibody is an antibody that specifically binds to the C-terminal peptide of the CPE protein.

Antibodies of the invention have specific binding capacity to the N-terminal peptide or C-terminal peptide of the CPE protein.

In the present invention, the term “CPE ( C. perfringens) endotoxin ”is a enterotoxin protein produced by Clostridium perfringens and is known to cause diarrhea symptoms. In the present invention, preferably, the CPE consists of the amino acid sequence of the first sequence of SEQ ID NO, more preferably encoded by the nucleotide sequence of the second SEQ ID NO.

As used herein, the term “antibody” as used to refer to a CPE is a specific antibody to a CPE protein, which includes antigen binding fragments of antibody molecules as well as complete antibody forms.

A complete antibody is a structure having two full length light chains and two full length heavy chains, each of which is linked by heavy and disulfide bonds. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ) and epsilon (ε) types and subclasses gamma 1 (γ1), gamma 2 (γ2), and gamma 3 (γ3). ), Gamma 4 (γ4), alpha 1 (α1) and alpha 2 (α2). The constant regions of the light chains have kappa (κ) and lambda (λ) types (Cellular and Molecular Immunology, Wonsiewicz, MJ, Ed., Chapter 45, pp. 41-50, WB Saunders Co. Philadelphia, PA (1991); Nisonoff, A., Introduction to Molecular Immunology, 2nd Ed., Chapter 4, pp. 45-65, sinauer Associates, Inc., Sunderland, MA (1984)).

The antigen binding fragment of an antibody molecule means the fragment which has antigen binding function, and includes Fab, F (ab '), F (ab') 2, Fv, etc. Fab in the antibody fragment has a structure having a variable region of the light and heavy chains, a constant region of the light chain and the first constant region (CH1) of the heavy chain has one antigen binding site. Fab 'differs from Fab in that it has a hinge region comprising at least one cysteine residue at the C-terminus of the heavy chain CH1 domain. F (ab ') 2 antibodies are produced by disulfide bonds of cysteine residues in the hinge region of Fab'. Recombinant techniques for generating Fv fragments with minimal antibody fragments in which Fv has only a heavy chain variable region and a light chain variable region are described in PCT International Publication Nos. WO88 / 10649, WO 88/106630, WO 88/07085, WO 88/07086 and WO. 88/09344. Double-chain Fv is a non-covalent bond in which a heavy chain variable region and a light chain variable region are linked, and a single-chain Fv generally has a variable region of a heavy chain and a short chain variable region through a peptide linker. Covalently linked or directly linked at the C-terminus can form a dimer-like structure, such as a double-chain Fv. Such antibody fragments can be obtained using proteolytic enzymes (e.g., restriction digestion of the entire antibody with papain can yield Fab and cleavage with pepsin can yield F (ab ') 2 fragment). Can be produced through genetic recombination techniques.

In the present invention, the antibody is preferably in Fv form, Fab form, or intact antibody form.

As used herein, the term “heavy chain” refers to a variable region domain VH comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen and a full length heavy chain comprising three constant region domains CH1, CH2 and CH3 It means all fragments.

In addition, the term "light chain" as used herein refers to both the full-length light chain and fragments thereof including the variable region domain VL and the constant region domain CL comprising an amino acid sequence having sufficient variable region sequence to confer specificity to the antigen. .

Antibodies of the invention may be prepared by methods commonly practiced in the art, such as fusion methods (Kohler and Milstein, European Journal of Immunology, 6: 511-519 (1976)), recombinant DNA methods (US Pat. No. 4,816, 56) or phage antibody library methods (Clackson et al, Nature, 352: 624-628 (1991) and Marks et al, J. Mol. Biol., 222: 58, 1-597 (1991)). Can be. General procedures for antibody preparation are described in Harlow, E. and Lane, D., Using Antibodies: A Laboratory Manual, Cold Spring Harbor Press, New York, 1999; Zola, H., Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc., Boca Raton, Florida, 1984; And Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley / Greene, NY, 1991, which are incorporated herein by reference. For example, the preparation of hybridoma cells producing monoclonal antibodies is accomplished by fusing immortalized cell lines with antibody-producing lymphocytes, and the techniques required for this process are well known to those skilled in the art and can be readily implemented. Polyclonal antibodies can be obtained by injecting an antigen into a suitable animal, collecting antisera from the animal, and then isolating the antibody from the antisera using known affinity techniques.

According to a preferred embodiment of the invention, the antibody of the invention is a monoclonal antibody. The term “monoclonal antibody” refers to an antibody molecule of a single molecular composition obtained from substantially the same antibody population, wherein the monoclonal antibody exhibits single binding specificity and affinity for a particular epitope. As described in the Examples below, the antibodies of the invention isolated from a single hybridoma cell line may be referred to as monoclonal antibodies.

Antibodies of the invention are used for the detection or diagnosis of Clostridium perfringens Enterotoxin (CPE) in a "biological sample".

As used herein, the term “biological sample” refers to tissue, cells, whole blood, serum, plasma, tissue autopsy samples (brain, skin, lymph nodes, spinal cord, etc.), cell culture supernatants, ruptured eukaryotic cells, and bacterial expression systems. It is possible, but not limited to. CPE proteins can be detected by confirming antigen-antibody complex formation by reacting these biological samples with or without manipulation of the antibody of the invention.

The antigen-antibody complex is formed by colormetric method, electrochemical method, fluorimetric method, luminometry, particle counting method, visual assessment or flashing. It can be detected by a scintillation counting method.

Various markers may be used to detect the antigen-antibody complex in the present invention. Specific examples of the label include enzymes, fluorescent, ligands, luminescent, microparticles or radioisotopes. Enzymes used as detection markers include acetylcholinesterase, alkaline phosphatase, β-D-galactosidase, horseradish peroxidase and β-latamase and the like. Phosphorus, Eu ³⁺ , Eu ³⁺ chelate or cryptate and the like, and ligands include biotin derivatives and the like, luminescent materials include acridinium esters and isoluminol derivatives and the like, and microparticles include colloids Gold and colored latex, and the like, and radioisotopes include ⁵⁷ Co, ³ H, ¹²⁵ I and ¹²⁵ I-Bolton Hunter reagents, and the like.

Preferably, the CPE protein antigen-antibody complex can be detected using an ELISA (Enzyme-Linked Immunosorbent Assay) method. Enzyme immunosorbent (ELISA) involves (i) a direct ELISA using a labeled antibody that recognizes an antigen attached to a solid support, (ii) a labeled secondary that recognizes the antibody in a complex of antibodies that recognizes an antigen attached to a solid support. Indirect ELISA using antibodies, (iii) direct sandwich ELISA using another labeled antibody that recognizes the antigen in a complex of antibody and antigen attached to a solid support, (iii) antigen in a complex of antibody and antigen attached to a solid support And various ELISA methods, such as an indirect sandwich ELISA using a labeled secondary antibody that reacts with another antibody that recognizes the antibody and then recognizes the antibody. The antibody of the present invention may have a detection label, and when the antibody does not have a detection label, the antibody of the present invention may be captured and may be identified by treating another antibody having a detection label.

For example, when the assay method used in the present invention is carried out in an ELISA method, (i) coating an antibody against a CPE protein on a surface of a solid substrate as a capturing antibody; (Ii) reacting the capture antibody with a biological sample (eg, cell lysate); (Iii) reacting the product of step (ii) with a detecting antibody having a label that generates a signal and which specifically reacts with the CPE protein; And (iii) measuring the signal resulting from the label.

Suitable as the solid substrate are hydrocarbon polymers (eg polystyrene and polypropylene), glass, metal or gel, most preferably microtiter plates.

The detection antibody has a label that generates a detectable signal. Such labels may include chemicals (eg biotin), enzymes (alkaline phosphatase, β-galactosidase, horse radish peroxidase and cytochrome P450), radioactive substances (eg C ¹⁴ , I ¹²⁵ , P ^32). And S ³⁵ ), fluorescent materials (eg, fluorescein), luminescent materials, chemiluminescent, and fluorescence resonance energy transfer (FRET). Various labels and labeling methods are described in Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999.

Measurement of the final enzyme activity or signal in the ELISA method can be carried out according to various methods known in the art. Detection of this signal indicates the presence of CPE protein in the cell lysate. If biotin is used as a label, the signal can be easily detected with streptavidin and luciferin if luciferase is used. When alkaline phosphatase is used as a label, bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT) and naphthol-AS-B1-phosphate (naphthol-AS) are used as substrates of the enzyme. Chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis) if colorimetric substrates such as -B1-phosphate) and enhanced chemifluorescence (ECF) are used, and horse radish peroxidase is used -N-methylacridinium nitrate), resorupin benzyl ether, luminol, Amflex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine), TMB (3,3,5,5-tetramethylbenzidine) Substrates such as ABTS (2,2'-azine-di [3-ethylbenzthiazoline sulfonate]) and o-phenylenediamine (OPD) can be used.

The composition for detecting CPE proteins of the present invention may be provided in the form of a kit, and preferably, in the form of an Enzyme-Linked Immunosorbent Assay (ELISA) kit.

According to a preferred embodiment of the present invention, the ELISA kit comprises a capturing antibody and a detecting antibody.

According to another preferred embodiment of the present invention, the capture antibody uses an antibody that binds to the N-terminal peptide of the CPE protein, and the detection antibody uses an antibody that binds to the C-terminal peptide of the CPE protein.

The ELISA kit of the present invention may comprise instruments, reagents commonly used in the art for immunological analysis. Such instruments and reagents include, but are not limited to, suitable carriers, labeling materials capable of generating detectable signals, solubilizers, detergents, buffers and stabilizers, and the like. If the labeling substance is an enzyme, the kit may include a substrate for measuring enzyme activity and a reaction terminator.

The present invention relates to an antibody that specifically binds to an N-terminal peptide or C-terminal peptide of Clostridium perfringens enterotoxin (CPE) protein, and a composition for detecting CPE protein comprising the antibody as an active ingredient. According to the method of the present invention, the CPE protein can be accurately detected with very high sensitivity.

1 shows the C-terminal peptide ( cpe 234 bp: 243-319 amino acid sequence of SEQ ID NO: 1) and the N-terminal peptide ( cpe 261 bp: 1-87th amino acid sequence of SEQ ID NO: 1) SDS-PAGE photograph showing the expression of.
Figure 2 is an immunoblotting reaction of a cell line culture that generates monoclonal antibody (Mab) to the C-terminal peptide after Western blotting of the C-terminal peptide (234 bp portion) of the CPE protein. Shows the result. Of these, No. 6 (234-3G7) and No. 28 (234-2A4) showed strong antibody responses.
Figure 3 is Western blotting of the N-terminal peptide (261 bp portion) of the CPE protein, followed by reacting the monoclonal antibody (Mab) -producing cell line culture produced after immunization with the N-terminal peptide Show results. It was confirmed that the antibody reaction occurs at 6 (261-2D12), 7 (261-4E2), 8 (261-5A1), 9 (261-6C1) and 10 (261-3G7).
Figure 4 shows the ELISA measurement results for the capture antibody (capture antibody) selection. Panel (A) of FIG. 4 captures 261-3G7 antibody alone and a combination of 261-3G7 antibody and four other antibodies (261-6C1 antibody, 261-5A1 antibody, 261-4E2 antibody, 261-2D12 antibody). It shows the result of ELISA measurement. Panel (B) of FIG. 4 shows ELISA measurement results using a combination of 261-3G7 antibodies, 261-4E2, and 261-5A1 antibodies as capture antibodies.
Figure 5 shows the results of measuring the sensitivity of the ELISA kit of the present invention.
Figure 6 shows the results of measuring the amount of CPE toxin expressed per Clostridium perfringens bacteria using the ELISA kit of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example

Example  1: recombination CPE  Protein Purification and Monoclonal Antibody Preparation

In order to obtain base sequences for N-terminal and C-terminal peptide expression of the CPE protein, PCR was performed by constructing cpe-234 primers and cpe-261 primers for the CPE protein encoding cpe gene (ORF size 960bp) (see Table below). 1).

gene Primer sequence PCR product size PCR conditions
cpe-234 F; 5'-CACCGGAGAACAAAATACAACAGAA-3 ' 234 bp 95 ℃, 30sec 52 ℃, 30sec R; 5'-ACATCTTTCACCAGTTTCAAT-3 ' 72 ℃, 30sec
cpe-261 F; 5'-CACCATGCTTAGTAACAATTTA-3 ' 261 bp 95 ℃, 30sec 46 ℃, 30sec R; 5'-AAAATTTACATTTATAGATAC-3 ' 72 ℃, 30sec
cpe-ORF F; 5'-CACCATGCTTAGTAACAATTTAAAT-3 ' 960 bp 95 ℃, 45sec 55 ℃, 45sec R; 5'-TTAAAATTTTTGAAATAATAT-3 ' 72 ℃, 45sec

PCR had a final volume of 50 μl and 30 PCR cycles were performed using TaKaRa EX taq. Each PCR product was purified and inserted into the pET102 expression vector. The inserted plasmids were transformed into BL21 (DE3) E. coli and induced expression with IPTG to overexpress each protein. 234bp portion of the C-terminal (peptide of amino acid sequence 1-87 of SEQ ID NO: 1) of the CPE toxin protein, and 261bp portion of the N-terminal (peptide of amino acid sequence 243-319 of SEQ ID NO: 1) of the N-terminal Expression of the peptide corresponding to the above was confirmed (see FIG. 1).

C-terminal peptide (234bp) and N-terminal peptide (261bp) were injected into Bab / c mice by 50μg per animal three times to immunize three times, and serum was collected to check immunity. Finally after the third immunization, boosting was performed through the tail vein. After 3 days of boosting, mice were dissected to collect spleen cells and cell fused with the SP-2 myeloma cell line. Successfully cell fused cell lines were selected using HAT medium and HT medium. The cell line was finally cultured in a 90 mm cell culture dish, and some of the cells were collected and stored in LN2, and the remaining cells were overgrown and cultured, followed by immunoblotting.

C-terminal peptide (234 bp portion) and N-terminal peptide (261 bp portion) were electrophoresed on 12.5% polyacrylamide gel and then transferred to PVDF membrane. Twenty eight cultures of each cell producing monoclonal antibody (Mab) were selected by dot blotting and used for immunoblotting. 1 ml of cell culture was added for each reaction and then washed 5 times with PBS-T after 1 hour reaction at room temperature. AP-conjugated anti-mouse IgG Fc r in a secondary antibody reaction was diluted 1: 10,000, added 1 ml, and then washed 5 times with PBS-T after 1 hour reaction at room temperature. The color reaction was allowed to react for 10 minutes after adding 1 ml of NBT / BCIP.

Example 2: Antibody Selection for ELISA Kit Production

Monoclonal antibody (Mab) produced in each cell line was subjected to primary screening by collecting the cell culture in a 6-well plate and performing dot blotting. Immunoblotting was performed on 28 cell lines that showed a strong response in dot blotting. FIG. 2 shows that after Western blotting of the C-terminal peptide of the CPE (234 bp portion), the cell line (28) culture to generate monoclonal antibody (Mab) to the C-terminal peptide is immunized. This is the result of the blotting reaction. Of these, No. 6 (234-3G7) and No. 28 (234-2A4) showed strong antibody responses. The other 26 cell cultures showed a weak reaction, but the antibody for the ELISA kit preparation was excluded 234-2A4, which showed the cross reaction, and 234-2A4, which showed the strongest reaction, was selected. When using the selected antibody as a detection antibody, HRP was conjugated to the antibody.

In the case of FIG. 3, after Western blotting the N-terminal peptide (261 bp portion), the resultant was reacted with the culture solution of the monoclonal antibody (Mab) producing cell line produced after immunization with the N-terminal peptide. As shown in FIG. 3, antibody responses at 6 (261-2D12), 7 (261-4E2), 8 (261-5A1), 9 (261-6C1) and 10 (261-3G7) It confirmed that it occurred. Like the reaction in the C-terminal peptide (234 bp region) monoclonal antibody (Mab) -producing cell line culture, the remaining 23 antibodies were found to be weak. Therefore, as a capture antibody, antibodies produced in the above five cell lines were selected.

After incubating the selected total 6 monoclonal antibody cell lines (234-2A4, 261-2D12, 261-4E2, 261-5A1, 261-6C1, 261-3G7), 2 × 10 ⁶ cell lines were injected into the mouse abdominal cavity. To produce ascites fluid.

ELISA was performed on CPE-producing type A strains after attaching 261-3G7 antibody in monoclonal antibody (Mab) to N-terminal peptide (261 bp) and the other four in combination to a 96-well plate. As a result, as shown in panel (A) of FIG. 4, the combination of the 261-3G7 + 261-5A1 antibody and the 261-3G7 + 261-4E2 combination was 20-25% compared to the ELISA measurement in which only one 261-3G7 antibody was attached. The extent showed that the CPE protein capture ability was improved. In contrast, the 261-3G7 + 261-6C1 and 261-3G7 + 261-2D12 combinations showed the same results or a 20% reduction, respectively, compared to the case where only one 261-3G7 antibody was attached. In addition, the best results were obtained when ELISA was performed using a combination of three antibodies, 261-3G7, 261-4E2, and 261-5A1.

Example 3: Sensitivity of the ELISA Kit

To determine the limit of measurement of CPE toxin, the toxin was diluted and prepared at a concentration ranging from 1 pg-10 ng, and then used as an antigen. As a result, it was found that the measured value increased in proportion to the antigen, and when the cut-off value was set to 0,02, about 2.5 pg of toxin could be diagnosed (see FIG. 5). When the limit of measurement of the toxin was set to 2.5 pg, the expression amount of the toxin expressed per bacterial pathogen was measured. As a result, it was confirmed that CPE toxin expressed in 10 ⁴ CFU can be diagnosed (see FIG. 6).

Example 4 Comparison of ELISA Kits and PET-RPLA Kits

Comparison experiments were performed between the ELISA kit of the present invention and the PET-RPLA kit (Denka, Japan), which the present inventors used for diagnosing CPE toxin. The strains were subjected to CPE-producing type A strains, and the toxins in each of the cultures after the strains were cultured and the cultures that were heated by repeating the cultures in a 75 ° C. constant temperature bath for 1 to 4 times for 20 minutes were diagnosed.

As shown in the comparison results of the ELISA kit and PET-RPLA kit of Table 2 below, the PET-RPLA kit diagnosis result was negative in the 24-hour culture medium and the heated culture medium once, but was diagnosed with the ELISA kit of the present invention. In one case, the mean value was 0.0868 for a 24 hour culture and 0.2952 for a single heated culture. The RPLA kit showed positive results at two or more heatings, and the ELISA kit showed positive results even in a 24-hour culture when the cut-off value was set to 0,02.

Example 5: Comparison in Strains Isolated from Diarrhea Patients

We compared and analyzed the detectability of RPLA kits and ELISA kits in 2007 isolated strains and Major Toxin type A ~ E reference strains. (3) cpe as three groups Group with RPLA positive strain, (ii) cpe gene but RPLA negative, and (i) RPLA negative without cpe gene, 32, 33 and 10 weeks respectively. Was performed. The experiment was carried out according to the manual of RPLA, and was performed by performing the apoptosis method three times. In the first group, RPLA and ELISA were positive in 32 strains of human isolates and in type A and type reference strains (see Table 3).

In contrast, in the second group with cpe gene but RPLA-negative, all ELISA measurements were valid at 32 weeks except 1 of 33 (see Table 4 below). However, the ELISA value was about 50% compared to that of the first group, and it is considered that there is a difference in the amount of toxin expression in each isolate.

The third negative control group was RLPA negative for 10 strains of human isolates and 3 strains for reference strains, and the ELISA values were also measured to be below cut-off values (see Table 5).

According to the experimental results, it was confirmed that the use of the ELISA kit using the monoclonal antibody of the present invention significantly improved the detection capacity for the CPE toxin, compared to the PET-RPLA kit used in the prior art.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

<110> Republic of Korea (Management: Korea Center for Disease Control and Prevention) <120> Antibody Specifically Binding to C. perfringens Enterotoxin          Protein and Composition for Detecting Said Protein Comprising the          Same <160> 8 <170> KopatentIn 1.71 <210> 1 <211> 319 <212> PRT <213> Clostridium perfringens <400> 1 Met Leu Ser Asn Asn Leu Asn Pro Met Val Phe Glu Asn Ala Lys Glu   1 5 10 15 Val Phe Leu Ile Ser Glu Asp Leu Lys Thr Pro Ile Asn Ile Thr Asn              20 25 30 Ser Asn Ser Asn Leu Ser Asp Gly Leu Tyr Val Ile Asp Lys Gly Asp          35 40 45 Gly Trp Ile Leu Gly Glu Pro Ser Val Val Ser Ser Gln Ile Leu Asn      50 55 60 Pro Asn Glu Thr Gly Thr Phe Ser Gln Ser Leu Thr Lys Ser Lys Glu  65 70 75 80 Val Ser Ile Asn Val Asn Phe Ser Val Gly Phe Thr Ser Glu Phe Ile                  85 90 95 Gln Ala Ser Val Glu Tyr Gly Phe Gly Ile Thr Ile Gly Glu Gln Asn             100 105 110 Thr Ile Glu Arg Ser Val Ser Thr Thr Ala Gly Pro Asn Glu Tyr Val         115 120 125 Tyr Tyr Lys Val Tyr Ala Thr Tyr Arg Lys Tyr Gln Ala Ile Arg Ile     130 135 140 Ser His Gly Asn Ile Ser Asp Asp Gly Ser Ile Tyr Lys Leu Thr Gly 145 150 155 160 Ile Trp Leu Ser Lys Thr Ser Ala Asp Ser Leu Gly Asn Ile Asp Gln                 165 170 175 Gly Ser Leu Ile Glu Thr Gly Glu Arg Cys Val Leu Thr Val Pro Ser             180 185 190 Thr Asp Ile Glu Lys Glu Ile Leu Asp Leu Ala Ala Ala Thr Glu Arg         195 200 205 Leu Asn Leu Thr Asp Ala Leu Asn Ser Asn Pro Ala Gly Asn Leu Tyr     210 215 220 Asp Trp Arg Ser Ser Asn Ser Tyr Pro Trp Thr Gln Lys Leu Asn Leu 225 230 235 240 His Leu Thr Ile Thr Ala Thr Gly Gln Lys Tyr Arg Ile Leu Ala Ser                 245 250 255 Lys Ile Val Asp Phe Asn Ile Tyr Ser Asn Asn Phe Asn Asn Leu Val             260 265 270 Lys Leu Glu Gln Ser Leu Gly Asp Gly Val Lys Asp His Tyr Val Asp         275 280 285 Ile Ser Leu Asp Ala Gly Gln Tyr Val Leu Val Met Lys Ala Asn Ser     290 295 300 Ser Tyr Ser Gly Asn Tyr Pro Tyr Ser Ile Leu Phe Gln Lys Phe 305 310 315 <210> 2 <211> 960 <212> DNA <213> Clostridium perfringens <400> 2 atgcttagta acaatttaaa tccaatggtg ttcgaaaatg ctaaagaagt atttcttatt 60 tctgaggatt taaaaacacc aattaatatt acaaactcta actcaaattt aagtgatgga 120 ttatatgtaa tagataaagg agatggttgg atattagggg aaccctcagt agtttcaagt 180 caaattctta atcctaatga aacaggtacc tttagccaat cattaactaa atctaaagaa 240 gtatctataa atgtaaattt ttcagttgga tttacttctg aatttataca agcatctgta 300 gaatatggat ttggaataac tataggagaa caaaatacaa tagaaagatc tgtatctaca 360 actgctggtc caaatgaata tgtatattat aaggtttatg caacttatag aaagtatcaa 420 gctattagaa tttctcatgg taatatctct gatgatggat caatttataa attaacagga 480 atatggctta gtaaaacatc tgcagatagc ttaggaaata ttgatcaagg ttcattaatt 540 gaaactggtg aaagatgtgt tttaacagtt ccatctacag atatagaaaa agaaatcctt 600 gatttagctg ctgctacaga aagattaaat ttaactgatg cattaaactc aaatccagct 660 ggtaatttat atgattggcg ttcttctaac tcataccctt ggactcaaaa gcttaattta 720 cacttaacaa ttacagctac tggacaaaaa tatagaatct tagctagcaa aattgttgat 780 tttaatattt attcaaataa ttttaataat ctagtgaaat tagaacagtc cttaggtgat 840 ggagtaaaag atcattatgt tgatataagc ttagatgctg gacaatatgt tcttgtaatg 900 aaagctaatt catcatatag tggaaattac ccttattcaa tattatttca aaaattttaa 960                                                                          960 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 3 caccggagaa caaaatacaa cagaa 25 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 4 acatctttca ccagtttcaa t 21 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 5 caccatgctt agtaacaatt ta 22 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 6 aaaatttaca tttatagata c 21 <210> 7 <211> 25 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 7 caccatgctt agtaacaatt taaat 25 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 8 ttaaaatttt tgaaataata t 21

Claims (8)

(i) CPE ( C. perfringens enterotoxin) N-terminal peptide having amino acid sequence 1-87 of SEQ ID NO: 1; Or (ii) an isolated antibody or antigen-binding fragment thereof, characterized in that it specifically binds to a CPE C-terminal peptide having amino acid sequences 243-319 of SEQ ID NO: 1.
The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody is a monoclonal antibody.
A composition for detecting a CPE protein comprising an antibody or antigen-binding fragment thereof that specifically binds to an N-terminal peptide or C-terminal peptide of CPE protein as an active ingredient.
The amino acid sequence of claim 3, wherein the N-terminal peptide comprises the 1-87th amino acid sequence of SEQ ID NO: 1, and the C-terminal peptide comprises the 243-319th amino acid sequence of SEQ ID NO: 1 Characterized by a composition.
The composition of claim 3, wherein the antibody is a monoclonal antibody.
The composition of claim 3, wherein the composition for detection is in the form of an Enzyme-Linked Immunosorbent Assay (ELISA) kit.
7. The composition of claim 6, wherein the ELISA kit comprises a capturing antibody and a detecting antibody.
8. The composition according to claim 7, wherein the capture antibody is an antibody that specifically binds to the N-terminal peptide of the CPE protein, and the detection antibody is an antibody that specifically binds to the C-terminal peptide of the CPE protein. .

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