Patent Application
20240069022
The present invention relates a method and a kit for detecting the presence and/or amount of Enterobacteriaceae bacteria in a food or beverage sample, environmental sample, or biological sample.
In the field of testing various types of samples, such as food or beverage samples, environmental samples, and biological samples, there is a particular demand to detect the presence or amount of bacteria belonging to the Enterobacteriaceae family (hereinafter referred to as “Enterobacteriaceae bacteria”). Conventional methods for determining the degree of contamination of food or beverage samples or environmental samples by Enterobacteriaceae bacteria include the culture method (e.g., Patent Literature 1: JP2003-116594 A), in which a sample is cultured to check bacterial growth, and the ATP method (e.g., Patent Literature 2: JP-H11-239493 A; and Patent Literature 3: JP2009-136205 A), in which intracellular ATP (adenosine triphosphate) of bacteria in a sample is detected.
However, the conventional culture method requires culture facilities and takes several days to a week before detection, which presents challenges in terms of space, labor, and time. On the other hand, the conventional ATP method has poor selectivity for Enterobacteriaceae bacteria, because ATP is present not only in Enterobacteriaceae bacteria but also in other bacterial cells. It also has difficulty in identifying Enterobacteriaceae bacteria from eukaryotic cells derived from foods and beverages, environment, and living organisms, because ATP is also present in eukaryotic cells (e.g., animal cells and plant cells).
There are actual situations in which the presence and/or amount of Enterobacteriaceae bacteria in a food or beverage sample, environmental sample, or biological sample should be detected, such as in bacterial tests for food or beverage and environmental sanitation, where it is necessary to detect bacteria of plural genera comprehensively and rapidly, rather than detecting various bacteria individually. In particular, since many Enterobacteriaceae bacteria cause food poisoning, etc., there are cases where unique control standards are established for inspections such as food and beverage hygiene inspections, environmental hygiene inspections, and food worker inspections. Accordingly, there is a high demand for comprehensive and rapid detection of Enterobacteriaceae bacteria. Therefore, if a method is provided that can collectively detect plural genera of Enterobacteriaceae bacteria present in food or beverage, environment, and living organisms, it will be of high technical significance and utility.
An objective of an embodiment of the present invention is to provide a method and a kit capable of detecting the presence and/or amount of Enterobacteriaceae bacteria in food or beverage samples, environmental samples, and biological samples easily and efficiently in a short period of time.
As a result of intensive study, the present inventors have found that simultaneous detection of the presence and/or amount of different Enterobacteriaceae bacteria of plural genera in a sample based on antigen-antibody reaction makes it possible to detect the presence and/or amount of Enterobacteriaceae bacteria in a sample easily and efficiently in a short period of time. The present inventors have also found that by using a generic antibody that causes antigen-antibody reactions widely with a broad spectrum of Enterobacteriaceae bacteria in combination with one or more specific antibodies that cause antigen-antibody reactions specifically with particular Enterobacteriaceae bacteria, it is possible to distinguish plural species of Enterobacteriaceae bacteria from other components in a sample and detect the presence and/or amount of the Enterobacteriaceae bacteria quickly and conveniently. The present inventors have then actually produced antibodies that cause antigen-antibody reactions with plural genera of Enterobacteriaceae bacteria and have demonstrated that these antibodies can be used to detect the presence and/or amount of the Enterobacteriaceae bacteria in a sample easily and efficiently in a short period of time, thereby arriving at the present invention.
Specifically, some aspects of the present invention include the following.
[Aspect 1] A method for detecting the presence and/or amount of Enterobacteriaceae bacteria in a sample selected from food or beverage samples, environmental samples, and biological samples, the method comprising the step of simultaneously detecting the presence and/or amount of Enterobacteriaceae bacteria of two or more different genera in the sample based on antigen-antibody reactions.
[Aspect 2] The method according to Aspect 1, wherein the step of detecting includes simultaneously detecting Enterobacteriaceae bacteria of two or more different genera selected from the group consisting of the genus Escherichia, the genus Klebsiella, the genus Citrobacter, the genus Enterobacter, the genus Proteus, the genus Salmonella, and the genus Serratia.
[Aspect 3] The method according to Aspect 1 or 2, wherein the step of detecting includes simultaneously detecting Enterobacteriaceae bacteria of at least 3 or more, or 4 or more, or 5 or more, or 6 or more, or 7 or more different genera.
[Aspect 4] The method according to any one of Aspects 1 to 3, wherein the step of detecting comprises: contacting the sample with an antibody that causes antigen-antibody reactions with components derived from the Enterobacteriaceae bacteria of two or more genera; and measuring the presence and/or intensity of the antigen-antibody reaction that occurs in the sample after contact.
[Aspect 5] The method according to Aspect 4, wherein the antibody is an antibody that causes antigen-antibody reactions with ribosome proteins L7/L12 of the Enterobacteriaceae bacteria of two or more genera.
[Aspect 6] The method according to Aspect 4 or 5, further comprising the step of lysing Enterobacteriaceae bacteria in the sample before contacting the sample with the antibody.
[Aspect 7] The method according to any one of Aspects 4 to 6, wherein the antibody does not cause cross-reactions with components derived from one or more non-Enterobacteriaceae bacteria that may be present in the sample.
[Aspect 8] The method according to Aspect 7, wherein the one or more non-Enterobacteriaceae bacteria are one or more bacteria selected from the genus Pseudomonas, the genus Staphylococcus, the genus Bacillus, and the genus Enterococcus.
[Aspect 9] The method according to any one of Aspects 4 to 8, wherein the antibody does not cause cross-reactions with at least one non-bacterial component that may be present in the sample.
[Aspect 10] The method according to Aspect 9, wherein the non-bacterial component is an organic component derived from a virus, plant, and/or animal.
[Aspect 11] The method according to any one of Aspects 4 to 10, wherein the antibody is a monoclonal antibody or its fragment or a derivative thereof.
[Aspect 12] The method according to Aspect 11, wherein the monoclonal antibody or its fragment or the derivative thereof comprises:
[Aspect 13] The method according to Aspect 12, wherein the monoclonal antibody or its fragment or the derivative thereof has:
[Aspect 14] The method according to any one of Aspects 4 to 13, comprising detecting the presence and/or amount of Enterobacteriaceae bacteria in the sample by the steps of:
[Aspect 15] The method according to Aspect 14, wherein step (I) includes the steps of:
[Aspect 16] The method according to Aspect 14, wherein step (I) includes the steps of:
[Aspect 17] The method according to any one of Aspects 14 to 16, wherein the capture antibody is the generic antibody, and the detection antibody is the specific antibody.
[Aspect 18] The method according to any one of Aspects 14 to 16, wherein the detection antibody is the generic antibody, and the capture antibody is the specific antibody.
[Aspect 19] The method according to any one of Aspects 14 to 18, wherein the specific antibody causes antigen-antibody reactions with Enterobacteriaceae bacteria of two or more genera selected from at least the genus Escherichia, the genus Klebsiella, the genus Citrobacter, the genus Enterobacter, the genus Proteus, the genus Salmonella, and the genus Serratia.
[Aspect 20] The method according to any one of Aspects 14 to 19, wherein the generic antibody causes antigen-antibody reactions with bacteria of five or more genera selected from at least the genus Escherichia, the genus Klebsiella, the genus Citrobacter, the genus Enterobacter, the genus Proteus, the genus Salmonella, the genus Serratia, the genus Pseudomonas, the genus Staphylococcus, the genus Bacillus, and the genus Enterococcus.
[Aspect 21] The method according to any one of Aspects 14 to 20, wherein the specific antibody comprises:
[Aspect 22] The method according to Aspect 21, wherein the specific antibody comprises:
[Aspect 23] The method according to any one of Aspects 14 to 22, wherein the generic antibody comprises:
[Aspect 24] The method according to Aspect 23, wherein the generic antibody comprises:
[Aspect 25] A method for determining the degree of contamination by Enterobacteriaceae bacteria in a sample selected from food or beverage samples, environmental samples, and biological samples, comprising the step of simultaneously detecting the presence and/or amount of Enterobacteriaceae bacteria of two or more genera in the sample based on antigen-antibody reactions by the method according to any one of Aspects 1 to 24.
[Aspect 26] A kit for detecting the presence and/or amount of Enterobacteriaceae bacteria in a sample selected from food or beverage samples, environmental samples, and biological samples by the method according to any one of Aspects 4 to 13, comprising an antibody as recited in any one of Aspects 4 to 13.
[Aspect 27] A kit for detecting the presence and/or amount of Enterobacteriaceae bacteria in a sample selected from food or beverage samples, environmental samples, and biological samples by the method according to any one of Aspects 14 to 24, comprising a capture antibody and a detection antibody as recited in any one of Aspects 14 to 24, wherein one of the capture antibody and the detection antibody is the generic antibody and the other is the specific antibody.
[Aspect 28] The kit according to Aspect 27, further comprising an insoluble membrane carrier for developing the sample and contacting the sample with the capture antibody, wherein the insoluble membrane carrier has a detection line formed thereon and the capture antibody is immobilized on the detection line, whereby two or more species of bacteria in the sample are detected on the single detection line.
[Aspect 29] The kit according to Aspect 27 or 28, which is an immunochromatography kit.
[Aspect 30] The kit according to Aspect 29, wherein the capture antibody and the detection antibody are selected so as to cause antigen-antibody reactions with the ribosome protein L7/L12 of the Enterobacteriaceae bacteria to be detected in the sample to form a sandwich structure,
The method according to the present invention makes it possible to detect the presence and/or amount of Enterobacteriaceae bacteria in food or beverage samples, environmental samples, and biological samples easily and efficiently in a short period of time.
The present invention will be described in detail by referring to the specific embodiments below. However, the present invention should not be limited to the following embodiments, but can be implemented in any form to the extent that it does not depart from the gist of the present invention.
All documents cited herein, including patent gazettes, patent application publications, and non-patent documents, are incorporated herein by reference in their entirety for all purposes.
In the amino acid sequences described herein, each amino acid shall be represented by a single-letter code, unless otherwise specified.
One embodiment of the present invention relates to a method for detecting the presence and/or amount of Enterobacteriaceae bacteria in a food or beverage sample, environmental sample, or biological sample (hereinafter also referred to as “the method of the present invention”). The method of the present invention includes simultaneously detecting the presence and/or amount of Enterobacteriaceae bacteria of plural genera in the sample based on antigen-antibody reactions. According to one embodiment, such detection based on antigen-antibody reactions may be carried out by, e.g., contacting the sample with an antibody that causes antigen-antibody reactions with components derived from plural genera of Enterobacteriaceae bacteria in the sample (hereinafter also referred to as “the antibody of the present invention”), and measuring the presence and/or intensity of the antigen-antibody reactions that occur in the sample after contact. The method of the present invention makes it possible to determine the degree of contamination of, e.g., a food or beverage sample, environmental sample, or biological sample by Enterobacteriaceae bacteria easily and efficiently in a short period of time.
A kit comprising the antibody of the present invention for carrying out the method of the present invention (the kit of the present invention) constitutes another subject of the present invention.
In the following description, the method of the present invention will be explained first, followed by a description of the antibody of the present invention used in the method of the present invention, then by a description of a particularly preferred embodiment of the method of the present invention (the method (2) of the present invention), and lastly by a description of the kit for use in the method of the present invention.
[1. Method for Detecting the Presence and/or Amount of Enterobacteriaceae Bacteria in Sample (1)]
The method of the present invention is a method for detecting the presence and/or amount of Enterobacteriaceae bacteria in a sample selected from food or beverage samples, environmental samples, and biological samples. The method of the present invention includes simultaneously detecting the presence and/or amount of Enterobacteriaceae bacteria of plural genera in the sample based on antigen-antibody reactions. The method of the present invention may also be used for, e.g., determining the degree of contamination of the sample by Enterobacteriaceae bacteria.
Examples of samples include food or beverage samples, environmental samples, and biological samples (hereinafter, food or beverage samples and environmental samples may be referred to collectively as “food, beverage, or environmental samples,” and food or beverage samples, environmental samples, and biological samples may be referred to collectively as “food, beverage, environmental, or biological samples”). The types of food or beverage samples are not particularly limited, but examples include samples obtained from food ingredients such as meat, fish, vegetables, processed foods and beverages, and seasonings, and beverages such as water, tea, coffee, juice, and alcoholic beverages. The types of environmental samples are also not particularly limited, but examples include samples obtained by wiping surfaces such as fingers, work clothes, work shoes, nail brushes, cutting boards, knives, handles, conveyor belts, packaging materials, work desks, beds, faucets, showers, and medical equipment in environments such as food and beverage manufacturing facilities, food and beverage serving sites, medical devices, medical equipment, and medical care sites with a collection tool (swab) such as clean cotton or clean cloth impregnated with a liquid medium (e.g., water, physiological isotonic solution, ethanol, etc.), as well as liquid samples such as tap water, well water, and river or hot spring water. The types of biological samples are also not particularly limited, but examples include samples derived from human or non-human animals, such as whole blood, serum, plasma, urine, stool, hands, saliva, sputum, sweat, nasal discharge, throat swab, nasal aspirate, and lung lavage fluid. Such food, beverage, environmental, or biological samples can be contaminated by a wide variety of Enterobacteriaceae bacteria.
As mentioned above, the culture method and the ATP method have conventionally been used to detect contamination of such food, beverage, environmental, or biological samples by Enterobacteriaceae bacteria. However, the culture method requires culture facilities and takes several days to a week before detection. The ATP method has difficulty in identifying Enterobacteriaceae bacteria from other bacteria and also from eukaryotic cells derived from foods and beverages, environment, and living organisms, because ATP is present not only in Enterobacteriaceae bacteria but also in eukaryotic cells (e.g., animal cells and plant cells).
The method of the present invention includes simultaneously detecting the presence and/or amount of Enterobacteriaceae bacteria of plural genera in the sample based on antigen-antibody reactions. The term “antigen-antibody reaction” used herein refers to the specific binding of an antibody to its antigen. The use of antigen-antibody reactions makes it possible to detect detecting the presence and/or amount of Enterobacteriaceae bacteria in the sample immediately on site using simple equipment and operation. The phrase “simultaneous” detection of plural species or plural genera of bacteria herein refers to plural species or plural genera of bacteria at once, and should not necessarily be limited to simultaneous detection in time. According to the present invention, by appropriately selecting and using antibodies (antibodies of the invention) that specifically generate antigen-antibody reactions with components derived from plural genera of Enterobacteriaceae bacteria to be detected, it is possible to simultaneously detect the plurality of genera of Enterobacteriaceae bacteria to be detected, while reducing false positives caused by components other than Enterobacteriaceae bacteria, thereby improving detection sensitivity and detection accuracy. As a result, the degree of contamination of the sample by plural genera of Enterobacteriaceae can be determined easily and efficiently in a short period of time.
Examples of Enterobacteriaceae bacteria to be detected in the present invention include Enterobacteriaceae bacteria belonging to the genus Escherichia, the genus Klebsiella, the genus Citrobacter, the genus Enterobacter, the genus Proteus, the genus Salmonella, and the genus Serratia (hereinafter also referred to as “specific Enterobacteriaceae genera”), which are representative Enterobacteriaceae genera with particularly high demand for detection in food, beverage, environmental, or biological samples. According to the method of the present invention, it may be preferable to detect antigen-antibody reactions with Enterobacteriaceae bacteria of at least two or more of these specific Enterobacteriaceae genera (plural genera), and especially preferable to detect antigen-antibody reactions with Enterobacteriaceae bacteria of at least three, or at least four, or at least five, or at least six, or at least seven of these genera.
Examples of Enterobacteriaceae bacteria belonging to the genus Escherichia include Escherichia coli (E. coli, EC) and Escherichia albertii (E. albertii). When the method of the present invention is used for detecting antigen-antibody reactions with Enterobacteriaceae bacteria belonging to the genus Escherichia, it is sufficient to detect antigen-antibody reactions with any one or more of these Enterobacteriaceae bacteria, but it may be preferred to detect antigen-antibody reactions with at least Escherichia coli (E. coli).
Examples of Enterobacteriaceae bacteria belonging to the genus Klebsiella include Klebsiella pneumoniae (K. pneumoniae, KP), Klebsiella oxytoca (K. oxytoca), and Klebsiella aerogenes (K. aerogenes). When the method of the present invention is used for detecting antigen-antibody reactions with Enterobacteriaceae bacteria belonging to the genus Klebsiella, it is sufficient to detect antigen-antibody reactions with any one or more of these Enterobacteriaceae bacteria, but it may be preferred to detect antigen-antibody reactions with at least Klebsiella pneumoniae.
Examples of Enterobacteriaceae bacteria belonging to the genus Citrobacter include Citrobacter freundii (C. freundii, CF), Citrobacter amalonaticus (C. amalonaticus), and Citrobacter diversus (C. diversus). When the method of the present invention is used for detecting antigen-antibody reactions with Enterobacteriaceae bacteria belonging to the genus Citrobacter, it is sufficient to detect antigen-antibody reactions with any one or more of these Enterobacteriaceae bacteria, but it may be preferred to detect antigen-antibody reactions with at least Citrobacter freundii.
Examples of Enterobacteriaceae bacteria belonging to the genus Enterobacter include Enterobacter cloacae (E. cloacae, ECL), Enterobacter aerogenes (E. aerogenes), and Enterobacter sakazakii (E. sakazakii). When the method of the present invention is used for detecting antigen-antibody reactions with Enterobacteriaceae bacteria belonging to the genus Enterobacter, it is sufficient to detect antigen-antibody reactions with any one or more of these Enterobacteriaceae bacteria, but it may be preferred to detect antigen-antibody reactions with at least Enterobacter cloacae.
Examples of Enterobacteriaceae bacteria belonging to the genus Proteus include Proteus milabilis (P. milabilis, PM), Proteus morganii (P. morganii), Proteus vulgaris (P. vulgaris), and Proteus rettgeri (P. rettgeri). When the method of the present invention is used for detecting antigen-antibody reactions with Enterobacteriaceae bacteria belonging to the genus Proteus, it is sufficient to detect antigen-antibody reactions with any one or more of these Enterobacteriaceae bacteria, but it may be preferred to detect antigen-antibody reactions with at least Proteus milabilis.
Examples of Enterobacteriaceae bacteria belonging to the genus Salmonella include Salmonella enteritidis (S. enteritidis, SE), Salmonella infantis (S. infantis), and Salmonella typhimurium (S. typhimurium). When the method of the present invention is used for detecting antigen-antibody reactions with Enterobacteriaceae bacteria belonging to the genus Salmonella, it is sufficient to detect antigen-antibody reactions with any one or more of these Enterobacteriaceae bacteria, but it may be preferred to detect antigen-antibody reactions with at least Salmonella enteritidis.
Examples of Enterobacteriaceae bacteria belonging to the genus Serratia include Serratia liquefaciens (S. liquefaciens, SL), Serratia marcescens (S. marcescens), and Serratia fonticola (S. fonticola). When the method of the present invention is used for detecting antigen-antibody reactions with Enterobacteriaceae bacteria belonging to the genus Serratia, it is sufficient to detect antigen-antibody reactions with any one or more of these Enterobacteriaceae bacteria, but it may be preferred to detect antigen-antibody reactions with at least Serratia liquefaciens.
According to one embodiment, the plurality of genera of Enterobacteriaceae bacteria may preferably include both one or more Gram-negative bacteria and one or more Gram-positive bacteria. Because the cell membrane and cell wall structures are very different between these groups, it is difficult to detect both groups of bacteria simultaneously using conventional technology. On the other hand, the method of the present invention allows for simultaneous detection of Gram-negative Enterobacteriaceae bacteria and Gram-positive Enterobacteriaceae bacteria, provided that the antibodies used for detection are appropriately designed.
The method of the present invention may be implemented so as not only to detect antigen-antibody reactions with Enterobacteriaceae bacteria belonging to the specific Enterobacteriaceae genera explained above, but also to detect antigen-antibody reactions with Enterobacteriaceae bacteria belonging to other one or more Enterobacteriaceae genera. Examples of Enterobacteriaceae bacteria belonging to other Enterobacteriaceae genera than the specific Enterobacteriaceae genera include, although are not limited to, Enterobacteriaceae bacteria belonging to the genus Yersinia, the genus Erwinia, the genus Hafnia, the genus Morganella, the genus Obesumbacterium, the genus Providencia, the genus Shigella, the genus Aeromonas, and the genus Pectobacterium (hereinafter also referred to as “selective Enterobacteriaceae genera”). Among these selective Enterobacteriaceae genera, it may be preferable to detect Enterobacteriaceae bacteria belonging to at least 2 or more genera, more preferably at least 3 or more genera, or at least 4 or more genera, or at least 5 or more genera, or at least 6 or more genera, or at least 7 or more genera, especially at least 8 or more, most preferably all 9 genera, of these selective Enterobacteriaceae genera.
According to the method of the present invention, compared to the conventional methods such as the culture method and the ATP method, it is possible to detect the presence and/or amount of Enterobacteriaceae bacteria in food, beverage, environmental, or biological samples more easily and efficiently in a shorter period of time, by simultaneously detecting antigen-antibody reactions with these Enterobacteriaceae bacteria. The method of the present invention also makes it possible, for example, to determine the degree of contamination of food, beverage, environmental, or biological samples with Enterobacteriaceae bacteria much more quickly and easily.
[2. Antibodies that Cause Antigen-Antibody Reactions with Components Derived from Multiple Genera of Enterobacteriaceae Bacteria in Sample]
There are no restrictions to the means to be employed in the method of the present invention for simultaneously detecting the presence and/or amount of Enterobacteriaceae bacteria of plural genera in the sample based on antigen-antibody reactions. According to one embodiment, such detection based on antigen-antibody reactions may preferably be carried out by contacting the sample with an antibody that causes antigen-antibody reactions with components derived from Enterobacteriaceae bacteria (the antibody of the present invention) and measuring the presence and/or intensity of the antigen-antibody reactions that occur in the sample after contact. The antibody of the present invention will be explained below.
The term “antibody” used herein refers to a protein that recognizes and binds to a specific antigen or substance, which may also be referred to as an immunoglobulin (Ig). Common antibodies typically have two light chains (light chains) and two heavy chains (heavy chains) that are interconnected by disulfide bonds. There are two classes of light chains, called λ and κ chains, and five classes of heavy chains, called γ, μ, α, δ, and ε chains. Depending on the class of their heavy chains, antibodies are classified into five isotypes: IgG, IgM, IgA, IgD and IgE, respectively.
Heavy chains each include a heavy chain constant (CH) region and a heavy chain variable (VH) region. Light chains each include a light chain constant (CL) region and a light chain variable (VL) region. The light chain constant (CL) region consists of a single domain. The heavy chain constant (CL) region consists of three domains, namely CH1, CH2, and CH3. The light chain variable (VL) region and the heavy chain variable (VH) region each consist of four highly conserved regions called framework regions (FRs; FR-1, FR-2, FR-3, and FR-4) and three hypervariable regions called complementarity-determining regions (CDRs; CDR-1, CDR-2, and CDR-3). The heavy chain constant (CH) region consists of three CDRs (CDR-H1, CDR-H2, and CDR-H3) and four FRs (FR-H1, FR-H2, FR-H3, and FR-H4), which are arranged from the amino terminus to the carboxy terminus in the order of FR-H1, CDR-H1, FR-H2, CDR-H2, FR-H3, CDR-H3, and FR-H4. The light chain constant (CL) region has three CDRs (CDR-L1, CDR-L2, CDR-L3) and four FRs (FR-L1, FR-L2, FR L3, and FR-L4), which are arranged from the amino terminus to the carboxy terminus in the order of FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3, and FR-L4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen.
The antibody of the present invention may be either a polyclonal antibody or a monoclonal antibody, but a monoclonal antibody be preferred. A polyclonal antibody is usually prepared from the serum of an animal immunized with an antigen and is a mixture of various antibody molecules with different structures. A monoclonal antibody, on the other hand, is an antibody composed of a single type of molecules containing a combination of light chain variable (VL) and heavy chain variable (VH) regions having determined amino acid sequences. Monoclonal antibodies can be produced from clones derived from antibody-producing cells, or they can be produced using genetic engineering technique, by obtaining nucleic acid molecules having gene sequences encoding amino acids of antibody proteins. It is also a well-known technique to those skilled in the art to improve the binding and specificity of antibodies using genetic information of their heavy chains and light chains or their variable regions and CDRs.
The antibody of the present invention may be a fragment and/or derivative of an antibody. Fragments of antibodies include F(ab′)2, Fab, Fv, etc. Antibody derivatives include antibodies in which amino acid mutations have been artificially introduced into the constant region(s) of the light and/or heavy chains, antibodies in which the domain configuration of the constant region(s) of the light and/or heavy chains has been modified, antibodies with two or more Fc regions per molecule, glycosylated antibodies, bispecific antibodies, antibody conjugates in which an antibody or antibody fragment is bound to a protein other than the antibody, antibody enzymes, antibody conjugates in which an antibody or antibody fragment is bound to a protein other than an antibody, etc. Antibody conjugates, antibody enzymes, tandem scFv, bispecific tandem scFv, diabody, etc. Furthermore, when the aforementioned antibodies or their fragments or derivatives are derived from non-human animals, chimeric or humanized antibodies in which some or all of the sequences other than the CDRs thereof are replaced with the corresponding sequences of human antibodies are also included in the scope of the antibody of the present invention. When the simple term “antibodies” is used herein, it is intended to also encompass fragments and/or derivatives of antibodies, unless otherwise specified.
When the antibody of the present invention causes antigen-antibody reactions with certain bacteria, it means that they bind specifically to some components of the bacteria as antigens. The components of Enterobacteriaceae bacteria that serve as antigens for the antibody of the present invention are not limited. It may be a component contained in the cell walls or cell membranes that are exposed outside the bacterial cells, or it may be a component contained in the cytoplasm, cell organelles, or nucleus and not exposed outside the bacterial cells. When the antibody of the present invention causes antigen-antibody reactions with components of Enterobacteriaceae bacteria that are not exposed outside the cell surface of the bacteria, the food, beverage, environmental, or biological sample may be subjected to a treatment for lysing the bacteria before being brought into contact with the antibody of the present invention. Such bacterial lysis treatment will be described later.
The antibody of the present invention may preferably cause antigen-antibody reactions with Enterobacteriaceae bacteria belonging to at least 2 or more, or 3 or more, or 4 or more, or 5 or more, or 6 or more, or 7 or more genera selected from the group consisting of the genus Escherichia, the genus Klebsiella, the genus Citrobacter, the genus Enterobacter, the genus Proteus, the genus Salmonella, and the genus Serratia. The specific bacterial species belonging to these genera are described above.
According to one embodiment, the antibody of the present invention may preferably cause antigen-antibody reactions with ribosome proteins, especially ribosome proteins L7/L12, of Enterobacteriaceae bacteria belonging to any of the specific Enterobacteriaceae genera mentioned above. The antibody of the present invention may preferably cause antigen-antibody reactions also with ribosome proteins L7/L12 of Enterobacteriaceae bacteria belonging to any of the selective Enterobacteriaceae genera mentioned above. As used herein, the term “ribosome protein L7/L12,” or simply “L7/L12,” refers to a type of ribosomal protein essential for microbial protein synthesis and commonly possessed by various bacteria. For antibodies that cause antigen-antibody reactions against the bacterial ribosomal proteins L7/L12 and methods for producing the same, reference may be made to, e.g., to WO2000/006603A, the publication of an earlier patent application filed by the present inventors.
The components of Enterobacteriaceae bacteria that serve as antigens for the antibody of the present invention are not limited. They may be components contained in the cell walls or cell membranes that are exposed outside the bacterial cells, or they may be components that are contained in the cytoplasm, cell organelles, or nucleus and not exposed outside the bacterial cells. When the antibody of the present invention causes antigen-antibody reactions with components of Enterobacteriaceae bacteria that are not exposed outside the cell surface of the bacteria, the food, beverage, environmental, or biological sample may be subjected to a treatment for lysing the bacteria before being brought into contact with the antibody of the present invention. Such bacterial lysis treatment will be described later.
The degree of antigen-antibody reactions between the antibodies and Enterobacteriaceae bacteria is not particularly limited, but it is sufficient that at least antigen-antibody reactions occur to the extent that it can be detected by any known detection method. Methods for detecting antigen-antibody reactions between antibodies and Enterobacteriaceae bacteria are not limited, but include various known immunological assays as described below.
The antibody of the present invention may preferably not cause any cross-reactions with components derived from one or more bacteria other than Enterobacteriaceae bacteria (herein also referred to as “non-Enterobacteriaceae bacteria”) that may be present in the sample. Examples of such non-Enterobacteriaceae bacteria include, although are not limited to, one or more bacteria selected from the genus Pseudomonas, the genus Staphylococcus, the genus Bacillus, and the genus Enterococcus. The antibody of the present invention may preferably not cause any cross-reactions with components derived from at least one, or 2 or more, usually 3 or more, 4 or more, or 5 or more, or 6 or more, or 7 or more, or 8 or more, especially 9 or more, most preferably 10 or more, species selected from these non-Enterobacteriaceae bacteria.
The antibody of the present invention may also preferably not cause any cross-reactions with one or more non-bacterial components that may be present in the sample. Examples of such non-bacterial components include, although are not limited to, various bioorganic compounds derived from viruses, plants, and/or animals that are not present in Enterobacteriaceae bacteria. Specific examples of such bioorganic compounds include proteins, sugars, glycoproteins, lipids, complex lipids, and nucleic acids. The antibody of the present invention may preferably not cause any cross-reactions with components derived from at least one, or 2 or more, or 3 or more, or 4 or more, or 5 or more, or 6 or more, or 7 or more, or 8 or more, especially 9 or more, most preferably 10 or more, of these non-bacterial organic compounds.
Because antibodies are extremely antigen-specific, they are effective in specifically capturing certain antigens, but were considered unsuitable for, e.g., detecting plural different target substances. Moreover, since Enterobacteriaceae species that need to be detected in food or beverage, environmental, or biological tests are extremely diverse, it has conventionally been considered extremely difficult to simultaneously detect plural species of Enterobacteriaceae bacteria by antigen-antibody reactions. However, as will be described in the Examples below, the present inventors have succeeded in obtaining antibodies that cause antigen-antibody reactions with plural species of Enterobacteriaceae bacteria that can be the target for detection when testing food or beverage, environmental, or biological samples, and that can be used for simultaneous detection of these Enterobacteriaceae bacteria. This is an extremely surprising finding that goes against conventional technical knowledge.
The structure of the antibody of the present invention is not particularly limited but may preferably be as explained below. Incidentally, antibodies defined solely by the structural features described below shall also be included in the scope of the antibody of the present invention.
Specifically, the antibody of the present invention may have any of the amino acid sequences mentioned below as the amino acid sequences of each variable region of the heavy and light chains.
The heavy chain variable region (VH) may preferably have an amino acid sequence having a homology (preferably identity) of 80% or more, particularly 85% or more, more particularly 90% or more, especially 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more, most preferably 100%, with an amino acid sequence selected from SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5. The VH sequence may more preferably be an amino acid sequence selected from SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5.
The light chain variable region (VL) may preferably have an amino acid sequence having a homology (preferably identity) of 80% or more, particularly 85% or more, more particularly 90% or more, especially 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more, most preferably 100%, with an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6. The VL sequence may more preferably be an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6.
Preferred combinations of amino acid sequences of heavy chain variable (VH) and light chain variable (VL) regions include, although are not limited to, possible combinations of a heavy chain variable region (VH) having an amino acid sequence with 80% or more homology (preferably identity) with any one amino acid sequence selected from SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5 and a light chain variable region (VL) having an amino acid sequence with 80% or more homology (preferably identity) with any one amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6. However, any of the following combinations may particularly be preferred.
Combinations of a heavy chain variable region (VH) having a homology (preferably identity) of 80% or more, particularly 85% or more, more particularly 90% or more, especially 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more, most preferably 100%, with the amino acid sequence of SEQ ID NO:1 and a light chain variable region (VL) having a homology (preferably identity) of 80% or more, particularly 85% or more, more particularly 90% or more, especially 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more, most preferably 100%, with the amino acid sequence of SEQ ID NO:2.
Combinations of a heavy chain variable region (VH) having a homology (preferably identity) of 80% or more, particularly 85% or more, more particularly 90% or more, especially 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more, most preferably 100%, with the amino acid sequence of SEQ ID NO:3 and a light chain variable region (VL) having a homology (preferably identity) of 80% or more, particularly 85% or more, more particularly 90% or more, especially 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more, most preferably 100%, with the amino acid sequence of SEQ ID NO:4.
Combinations of a heavy chain variable region (VH) having a homology (preferably identity) of 80% or more, particularly 85% or more, more particularly 90% or more, especially 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more, most preferably 100%, with the amino acid sequence of SEQ ID NO:5 and a light chain variable region (VL) having a homology (preferably identity) of 80% or more, particularly 85% or more, more particularly 90% or more, especially 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more, most preferably 100%, with the amino acid sequence of SEQ ID NO:6.
As used herein, the term “homology” between two amino acid sequences refers to the ratio in which identical or similar amino acid residues appear in each corresponding position when these amino acid sequences are aligned, and the term “identity” between two amino acid sequences refers to the ratio of similar amino acid residues appearing in each corresponding position when these amino acid sequences are aligned. The “homology” and “identity” between two amino acid sequences can be determined using, for example, the BLAST (Basic Local Alignment Search Tool) program (Altschul et al., J. Mol. Biol., (1990), 215(3):403-10). (1990, 215(3):403-10).
Methods for identifying the sequence of each CDR from the respective variable sequences of the heavy and light chains of an antibody include, for example, the Kabat method (Kabat et al., The Journal of Immunology, 1991, Vol. 147, No. 5, pp. 1709-1719) and the Chothia method (Al Lazikani et al., Journal of Molecular Biology, 1997, Vol. 273, No. 4, pp. 927-948). These methods are common knowledge in this field, but it is possible to refer to, for example, the website of Dr. Andrew C. R. Martin's Group (http://www.bioinf.org.uk/abs/).
Similar amino acids include, for example, amino acids that are classified under the same group in the following classification based on the polarity, charge, and size of each amino acid (in any case, each amino acid type is indicated by a single-letter code).
Similar amino acids also include, for example, amino acids that are classified under the same group in the following classification based on the side chain of each amino acid (in any case, each amino acid type is indicated by a single-letter code).
The method of producing the antibody of the present invention is not restricted. In the case of polyclonal antibodies, they can be prepared using components derived from Enterobacteriaceae bacteria to be detected. The components derived from Enterobacteriaceae that can be used include bacterial cells, lysate made by lysing bacterial cells, and a fraction of lysate obtained via electrophoresis. When an electrophoretic fraction of bacterial lysate is used, there are no restrictions on which fraction should be used, but it is preferable to select a fraction that corresponds to a molecular weight of approximately 10 to 20 kDa, for example. In addition, it is preferable to use ribosomal proteins contained in Enterobacteriaceae bacteria as components derived from Enterobacteriaceae bacteria, especially ribosomal proteins L7/L12. These Enterobacteriaceae-derived components are inoculated into an animal, optionally together with adjuvants as necessary, and the serum is collected to obtain antiserum containing antibodies (polyclonal antibodies) that causes antigen-antibody reactions with the plural species of Enterobacteriaceae bacteria mentioned above. Examples of animals to be inoculated include sheep, horses, goats, rabbits, mice, rats, etc., of which sheep and rabbits are especially preferred for polyclonal antibody production. The antibodies may be purified and fractionated from the obtained antiserum and screened for antigen-antibody reactivity with the desired Enterobacteriaceae bacteria, and for no cross-reactivity with other components of food or beverage, environment, or biological origin, using known methods to obtain the desired antibodies with superior specificity. Furthermore, monoclonal antibodies can be obtained by isolating antibody-producing cells that produce desired antibody molecules and fusing them with myeloma cells to produce hybridomas capable of autonomous growth. As a method that does not require sensitization of animals, a phage library expressing heavy-chain variable (VH) regions or light-chain variable (VL) regions of antibodies or parts thereof can be screened for phage clones expressing specific amino acid sequences that specifically bind to components derived from Enterobacteriaceae bacteria to be detected, and antibodies can be produced using information of the screened phage clones.
Once the desired antibodies are obtained by the above procedure, the structure of each antibody, specifically part or all of the amino acid sequences of the heavy chain constant (CH) region, heavy chain variable (VH) region, light chain constant (CL) region, and/or light chain variable (VL) region, can be analyzed using known amino acid sequence analysis methods. It is also known to those skilled in the art to modify the amino acid sequences of the desired antibodies thus obtained to improve their binding ability and specificity. Furthermore, it is also possible to design other antibodies likely to have similar antigen specificity by using all or part of the amino acid sequences of each desired antibody (especially the amino acid sequences of all or part of the heavy chain variable (VH) region and the light chain variable (VL) region, especially the amino acid sequence of each CDR) and, if necessary, by combining them with part of amino acid sequences of known antibodies (especially the amino acid sequences of each FR of the heavy chain variable (VH) and light chain variable (VL) regions, and optionally of the heavy chain variable (VH) and light chain variable (VL) regions).
Once the amino acid sequences of a desired antibody are determined, a nucleic acid molecule having base sequences encoding all or part of the amino acid sequences of the desired antibody can be produced by a known method, and the antibody can be produced by genetic engineering using such a nucleic acid molecule. Furthermore, it is also possible to create a vector or plasmid for expressing each component of the desired antibody from such a nucleic acid sequence, and introduce it into a host cell (e.g., a mammalian cell, insect cell, plant cell, yeast cell, or microbial cell) to produce the antibody. In addition, modifications can be made to the structures of the constant regions of the antibody or to its sugar chains in order to improve the efficiency of the obtained antibody or to avoid its side effects, using techniques well known to those skilled in the art.
Those explained above, i.e., methods for producing the antibody of the present invention, nucleic acid molecules encoding the antibody of the present invention, vectors or plasmids containing such nucleic acid molecules, cells containing such nucleic acid molecules, vectors or plasmids, hybridomas producing the antibodies of the invention, etc., are also included in the scope of the present invention.
The techniques for making and modifying the antibodies described herein are all known to those skilled in the art, and may be carried out by referring to, e.g., Antibodies; A laboratory manual, E. Harlow et al. Likewise, the molecular biological techniques described herein (e.g., amino acid sequencing methods, methods for designing and producing nucleic acid molecules, methods for designing and producing vectors and plasmids, etc.) are also all known to those skilled in the art, and may be carried out by referring to, e.g., Molecular Cloning, A laboratory manual, Cold Spring Harbor Laboratory Press, Shambrook, NY. Harbor Laboratory Press, Shambrook, J. et al.
Thus, a preferred embodiment of the method of the present invention includes using an antibody that recognizes ribosomal proteins of each Enterobacteriaceae bacterium and causes antigen-antibody reactions (the antibody of the present invention), bringing them into contact with the sample, and detecting antigen-antibody reactions. In this embodiment, the detection sensitivity can be improved by exposing the ribosomal proteins of Enterobacteriaceae bacteria present in the sample to outside the bacterial cell membranes before bringing the sample into contact with the antibody of the present invention. Thus, in the preferred embodiment of the method of the present invention using the antibody of the present invention, the sample may preferably be subjected to treatment for lysing Enterobacteriaceae bacteria before being brought into contact with the antibody of the present invention. Examples of the lysis treatment of Enterobacteriaceae bacteria include, although are not limited to, heating treatment, ultrasonication, and chemical treatment using a surfactant. Conditions for the lysis treatment may be determined as appropriate depending on the bacteria contained in the sample. In the preferred embodiment of the method of the present invention using the antibody of the present invention, the antibody of the present invention may be brought into contact with the food, beverage, environmental, or biological sample by any arbitrary means.
In the preferred embodiment of the method of the present invention using the antibody of the present invention, immunological assays for detecting antigen-antibody reactions are not limited. Examples of immunological assays are not limited and may be either methods using a single antibody or those using two or more antibodies.
Examples of immunological assays using a single antibody include, although are not limited to, various known immunological assays such as ELISA (enzyme-linked immunosorbent assay) using microtiter plates loaded with bacterial antigens to detect antigen-antibody reactions with antibodies; and biosensors that have antibodies (or antigens) loaded on the sensor surface to detect antigen-antibody reactions with antigens (or antibodies) electrically (e.g., AC impedance method, FET (field effect transistor) method) or optically (SPR (surface plasmon resonance) method). Any of these assays can be used in the method of the present invention.
Examples of immunological assay methods using two or more antibodies include, although are not limited to, various known immunological assays such as ELISA using antibody-loaded microtiter plates; latex particle agglutination assay using latex particles (e.g. polystyrene latex particles) loaded with antibodies; immunochromatography using antibody-loaded membranes; and sandwich assay using a detection antibody labeled with colored or chromogenic particles, enzymes or fluorophores, etc., and a capture antibody immobilized on a solid phase carrier such as magnetic particles, etc. In the case of immunological assays that combine two or more antibodies, such as the sandwich assay, in which a detection antibody and a capture antibody are used, the antibody of the present invention may be used either as the capture antibody or as the detection antibody. Unless otherwise specified, the term “specific antibodies” as used herein refers to antibodies that cause antigen-antibody reactions with specific bacteria, including two or more Enterobacteriaceae species (bacteria to be detected) that are the final targets of detection, while the term “generic antibodies” as used herein refers to antibodies that cause antigen-antibody reactions with bacteria of five or more genera, including the aforementioned bacteria to be detected.
[3. Method for Detecting the Presence and/or Amount of Enterobacteriaceae Bacteria in Sample (2)]
According to the present invention, it may be preferred to use, as a sandwich assay, a method of detecting the presence and/or amount of the bacteria in the sample by capturing the bacteria in the sample and labeling the bacteria in the sample based on antigen-antibody reactions between the sample, a capture antibody bound to a solid-phase carrier, and a detection antibody having a detection label, and detecting the bacteria to be detected in the sample based on the detection label. This method (hereinafter also referred to as “the method (2) of the present invention”) will be explained below.
In the method (2) of the present invention, the specific antibody may preferably be an antibody that causes antigen-antibody reactions with bacteria of at least 1 or more, or 2 or more, or 3 or more, or 4 or more, or 5 or more, or 6 or more, or 7 or more genera selected from the genus Escherichia, the genus Klebsiella, the genus Citrobacter, the genus Enterobacter, the genus Proteus, the genus Salmonella, and the genus Serratia.
In the method (2) of the present invention, the generic antibody may preferably be an antibody that causes antigen-antibody reactions with bacteria of at least 5 or more, or 6 or more, or 7 or more, or 8 or more, or 9 or more, or 10 or more, or 11 or more genera selected from the genus Escherichia, the genus Klebsiella, the genus Citrobacter, the genus Enterobacter, the genus Proteus, the genus Salmonella, the genus Serratia, the genus Pseudomonas, the genus Staphylococcus, the genus Bacillus, and the genus Enterococcus.
In the method (2) of the present invention, the structures of the specific antibody and the generic antibody are not particularly restricted. However, these antibodies may have, as the amino acid sequence of each variable region of the heavy and light chains, any of the following amino acid sequences.
The heavy chain variable region (VH) of the specific antibody may preferably have an amino acid sequence with a homology (preferably identity) of 80% or more, particularly 85% or more, more particularly 90% or more, especially 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more, most preferably 100%, with an amino acid sequence selected from SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5. The VH sequence may more preferably be an amino acid sequence selected from SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5.
The light chain variable region (VL) of the specific antibody may preferably have an amino acid sequence with a homology (preferably identity) of 80% or more, particularly 85% or more, more particularly 90% or more, especially 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more, most preferably 100%, with an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6. The VL sequence may more preferably be an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6.
s
Preferred combinations of amino acid sequences of heavy chain variable (VH) and light chain variable (VL) regions of the specific antibody include, although are not limited to, possible combinations of a heavy chain variable region (VH) having an amino acid sequence with 80% or more homology (preferably identity) with any one amino acid sequence selected from SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5 and a light chain variable region (VL) having an amino acid sequence with 80% or more homology (preferably identity) with any one amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6. However, any of the following combinations may particularly be preferred.
Combinations of a heavy chain variable region (VH) having a homology (preferably identity) of 80% or more, particularly 85% or more, more particularly 90% or more, especially 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more, most preferably 100%, with the amino acid sequence of SEQ ID NO:1 and a light chain variable region (VL) having a homology (preferably identity) of 80% or more, particularly 85% or more, more particularly 90% or more, especially 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more, most preferably 100%, with the amino acid sequence of SEQ ID NO:2.
Combinations of a heavy chain variable region (VH) having a homology (preferably identity) of 80% or more, particularly 85% or more, more particularly 90% or more, especially 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more, most preferably 100%, with the amino acid sequence of SEQ ID NO:3 and a light chain variable region (VL) having a homology (preferably identity) of 80% or more, particularly 85% or more, more particularly 90% or more, especially 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more, most preferably 100%, with the amino acid sequence of SEQ ID NO:4.
Combinations of a heavy chain variable region (VH) having a homology (preferably identity) of 80% or more, particularly 85% or more, more particularly 90% or more, especially 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more, most preferably 100%, with the amino acid sequence of SEQ ID NO:5 and a light chain variable region (VL) having a homology (preferably identity) of 80% or more, particularly 85% or more, more particularly 90% or more, especially 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more, most preferably 100%, with the amino acid sequence of SEQ ID NO:6.
The heavy chain variable region (VH) of the generic antibody may preferably have an amino acid sequence with a homology (preferably identity) of 80% or more, particularly 85% or more, more particularly 90% or more, especially 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more, most preferably 100%, with an amino acid sequence selected from SEQ ID NO:7 and SEQ ID NO:9. The VH sequence may more preferably be an amino acid sequence selected from SEQ ID NO:7 and SEQ ID NO:9.
The light chain variable region (VL) of the generic antibody may preferably have an amino acid sequence with a homology (preferably identity) of 80% or more, particularly 85% or more, more particularly 90% or more, especially 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more, most preferably 100%, with an amino acid sequence selected from SEQ ID NO:8 and SEQ ID NO: 10. The VL sequence may more preferably be an amino acid sequence selected from SEQ ID NO:8 and SEQ ID NO:10.
Preferred combinations of amino acid sequences of heavy chain variable (VH) and light chain variable (VL) regions of the generic antibody include, although are not limited to, possible combinations of a heavy chain variable region (VH) having an amino acid sequence with 80% or more homology (preferably identity) with any one amino acid sequence selected from SEQ ID NO:7 and SEQ ID NO:9 and a light chain variable region (VL) having an amino acid sequence with 80% or more homology (preferably identity) with any one amino acid sequence selected from SEQ ID NO:8 and SEQ ID NO:10. However, any of the following combinations may particularly be preferred.
Combinations of a heavy chain variable region (VH) having a homology (preferably identity) of 80% or more, particularly 85% or more, more particularly 90% or more, especially 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more, most preferably 100%, with the amino acid sequence of SEQ ID NO:7 and a light chain variable region (VL) having a homology (preferably identity) of 80% or more, particularly 85% or more, more particularly 90% or more, especially 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more, most preferably 100%, with the amino acid sequence of SEQ ID NO:8.
Combinations of a heavy chain variable region (VH) having a homology (preferably identity) of 80% or more, particularly 85% or more, more particularly 90% or more, especially 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more, most preferably 100%, with the amino acid sequence of SEQ ID NO:9 and a light chain variable region (VL) having a homology (preferably identity) of 80% or more, particularly 85% or more, more particularly 90% or more, especially 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more, most preferably 100%, with the amino acid sequence of SEQ ID NO:10.
The method of the present invention (2) is a method for detecting the presence and/or amount of bacteria in the sample by (I) capturing the bacteria in the sample and detecting the bacteria in the sample based on antigen-antibody reactions between the sample, a capture antibody bound to a solid-phase carrier, and a detection antibody having a detection label, and (II) detecting the Enterobacteriaceae bacteria to be detected in the sample based on the detection label. This method is further characterized in that one of the capture antibody and the detection antibody is at least one specific antibody, which causes antigen-antibody reactions with two or more bacteria to be detected, and the other is bacteria of five or more genera including the bacteria to be detected causes antigen-antibody reactions with, at least one generic antibody.
One embodiment of the method (2) of the present invention (hereinafter referred to as “Embodiment A”) may be characterized in that Step (I) includes the steps of:
Another embodiment of the method (2) of the present invention (hereinafter referred to as “Embodiment B”) may be characterized in that Step (I) includes the steps of:
In either embodiment, the capture antibody may be the generic antibody and the detection antibody may be the specific antibody. Alternatively, the detection antibody may be the generic antibody and the capture antibody may be the specific antibody. In either case, the antibody of the present invention may be either the generic antibody or the specific antibody. According to one embodiment of the present invention, the antibody of the present invention may preferably be, although is not limited to, used as the specific antibody. Specifically, when the antibody of the present invention is used as the generic antibody, it is possible to prepare, as the generic antibody, an antibody of the present invention that has relatively low specificity, i.e., that causes antigen-antibody reactions with a wide variety of Enterobacteriaceae bacteria (usually 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or 11 or more genera), including the Enterobacteriaceae bacteria that are the final target of detection, and to use it in combination with, as the specific antibody, an antibody with relatively high specificity, i.e., that causes antigen-antibody reactions only with the Enterobacteriaceae bacteria that are the final target of detection, and also does not antigen-antibody reactions with bacteria of other genera. On the other hand, when the antibody of the present invention is used as the specific antibody, it is possible to prepare, as the specific antibody, an antibody of the present invention that has relatively high specificity, i.e., that causes antigen-antibody reactions only with the Enterobacteriaceae bacteria that are the final target of detection and does not cause antigen-antibody reactions with bacteria of other genera, and to use it in combination with, as the generic antibody, an antibody with relatively low specificity, i.e., that causes antigen-antibody reactions with a wide variety of Enterobacteriaceae bacteria (usually 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or 11 or more genera), including the Enterobacteriaceae bacteria that are the final target of detection.
In the method (2) of the present invention, either Embodiment A or Embodiment B may be chosen according to the type of immunoassay method actually used and the type of samples. Examples of immunoassay methods include, although are not limited to, various known immunological assays such as ELISA (enzyme-linked immunosorbent assay) using microtiter plates loaded with bacterial antigens to detect antigen-antibody reactions with antibodies; and biosensors that have antibodies (or antigens) loaded on the sensor surface to detect antigen-antibody reactions with antigens (or antibodies) electrically (e.g., AC impedance method, FET (field effect transistor) method) or optically (SPR (surface plasmon resonance) method).
The following explanation will be given based on an example where Embodiment A of the method (2) of the present invention is carried out using immunochromatography as the immunoassay. Each feature may be modified as appropriate when other types of immunoassay methods are used.
In Step (Ia-1) above, i.e., the step of contacting the sample with the detection antibody and labeling the bacteria in the sample based on antigen-antibody reactions between the detection antibody and bacteria, the detection antibody with the label for detection is brought into contact with the sample, so that the bacteria in the sample are labeled based on antigen-antibody reactions between the detection antibody and the bacteria. The method for bringing the sample into contact with the detection antibody is not limited, but this may typically be carried out by introducing the sample prepared as an aqueous sample onto a member area impregnated with the detection antibody, and maintaining it for a certain period of time. Although this can be achieved by a variety of specific embodiments depending on, e.g., the mode of capture in Step (a), one example includes preparing a solid-phase carrier (porous membrane) on which the capture antibody is immobilized, preparing a conjugate pad to which the detection antibody is attached, placing the conjugate pad upstream of the solid-phase carrier, and introducing the sample prepared as an aqueous sample onto the conjugate pad and allowing it to pass through, thereby bringing the sample into contact with the detection antibody. As another example, when a flow channel is used as the solid-phase carrier, the sample prepared as an aqueous sample may be brought into contact with the detection antibody either upstream of the position on the flow channel where the capture antibody is immobilized or before the introduction of the solid-phase carrier.
In Step (Ia-2) above, i.e., the step of contacting the capture antibody with the sample containing the bacteria labeled by the detection antibody and capturing the bacteria in the sample based on antigen-antibody reactions between the capture antibody and the bacteria-detection antibody complex, the capture antibody is brought into contact with the sample, so that the bacteria in the sample is captured based on antigen-antibody reactions between the capture antibody and the bacteria. The method for bringing the sample into contact with the capture antibody is not limited, but this may typically be carried out by introducing the sample prepared as an aqueous sample onto an area where the capture antibody is present, and maintaining it for a certain period of time. Although this can be achieved by a variety of specific embodiments depending on, e.g., the type of solid-phase carrier of the capture antibody, one example includes using a porous membrane as the solid-phase carrier, and introducing the bacteria-detection antibody complex onto the porous membrane to which the capture antibody is immobilized and allowing it to permeate, such that the bacteria in the sample is captured by the capture antibody immobilized on the porous membrane. Another example includes immobilizing the capture antibody on an area of a flow channel used as the solid-phase carrier, and allowing the bacteria-detection antibody complex to pass through the flow channel to allow the capture antibody immobilized on the area of the flow channel to capture the bacteria in the sample.
In Step (II) above, i.e., the step detecting the bacteria to be detected in the sample based on the detection label, the bacteria to be detected captured by the capture antibody and labeled with the detection antibody is detected based on the detection label. The detection method is not restricted and may be selected depending on the type of detection label. For example, when a metallic colloid such as gold colloid is used as the detection label, the presence or amount of gold colloid bound to the target bacteria may be detected by any method such as visual inspection or using a camera.
According to the method (2) of the present invention described above, it is possible to distinguish the desired Enterobacteriaceae bacteria from other bacteria or other components in the sample and to detect them easily and efficiently, by appropriately combining a generic antibody, which causes antigen-antibody reactions widely with various species of bacteria, and a specific antibody, which causes antigen-antibody reactions specifically with a particular species of Enterobacteriaceae bacteria. Specifically, by using an appropriate combination of plural specific antibodies, it is possible to design a system for detecting various desired combinations of Enterobacteriaceae bacteria. Any immunoassay method can be used to perform method (2) of the present invention, as long as it employs capture and detection antibodies.
[4. Kit for Detecting the Presence and/or Amount of Enterobacteriaceae Bacteria in the Sample]
As mentioned above, a kit for use in the method of the present invention containing the antibody of the present invention (the kit of the invention) is also included in the subject of the present invention.
The kit of the present invention includes, in addition to the antibody of the present invention, one or more reagents and a device for detection or components thereof necessary to perform the method of the present invention using the antibody of the present invention, and/or instructions describing the procedure for performing the method of the present invention. The type of such reagents and instructions, as well as other components included in the kit of the present invention, may be determined according to the specific immunological assay used to detect Enterobacteriaceae bacteria of plural genera.
When the kit of the present invention contains a device for detection or components thereof, the device assembled from the kit is a device equipped with components necessary for performing the method of the present invention using the antibody of the present invention (hereinafter also referred to as “the device of the present invention”). The specific components of the device of the present invention may be determined according to the type of immunological assay as a specific embodiment of the method of the present invention. As explained above, examples of immunological assay methods using two or more antibodies include, although are not limited to, various known immunological assays such as ELISA using antibody-loaded microtiter plates; latex particle agglutination assay using latex particles (e.g. polystyrene latex particles) loaded with antibodies; immunochromatography using antibody-loaded membranes; and sandwich assay using a detection antibody labeled with colored or chromogenic particles, enzymes or fluorophores, etc., and a capture antibody immobilized on a solid phase carrier such as magnetic particles, ELISA using a single antibody, and biosensor methods. Devices equipped with components necessary to perform such various immunological assays may be used as the device of the present invention.
Specific examples of devices capable of simultaneously and simply detecting the presence and/or amount of Enterobacteriaceae bacteria of plural genera in the sample include devices of the lateral-flow type and those of the flow-through type. According to the lateral-flow devices, the analyte to be detected and the antibody to be detected are deployed parallel onto a membrane having a detection area on the surface of which the capture antibody is immobilized, and the target substance captured in the detection area of the membrane is detected. On the other hand, according to the flow-through devices, the analyte to be detected and the detection antibody are passed vertically through a membrane on which the capture antibody is immobilized on the surface, and the target substance captured on the membrane surface is detected. The method of the present invention can be applied to both lateral-flow devices and flow-through devices.
Both lateral-flow type and flow-through type immunochromatographic detection devices are well known, and the details of such devices can be designed by those skilled in the art based on their technical knowledge, other than those described herein. The following is a description of the schematic configuration of the detection mechanism of the lateral flow type immunochromatographic detection device, with reference to the drawings. The schematic configuration of the detection mechanism of the lateral-flow type immunochromatography detection system will be described below with reference to the FIGURE. However, these are only examples of the schematic configuration of the detection procedure, and the configuration of the lateral-flow type immunochromatographic detection system is not limited in any way to the embodiment illustrated in the FIGURE.
The types of the solid-phase carrier used for the capture antibody are not restricted, but specific examples include: porous membranes made of cellulose, nitrocellulose, cellulose acetate, nylon, PVDF (PolyVinylidene DiFluoride), glass fiber, etc.; flow channels made of glass, plastic, PDMS (Poly(dimethylsiloxane)), silicone, etc.; thread, paper, fiber, etc.
The method for attaching an antibody to the solid-phase carrier is also not restricted, but specific examples include fixation via physisorption using the hydrophobicity of the antibody and fixation via chemical bonding using a functional group of the antibody.
The type of detection label used for the detection antibody is not restricted and may be selected as appropriate in accordance with the detection method. Specific examples include: metal colloids such as gold colloids, platinum colloids, and palladium colloids; non-metal colloids such as selenium colloids, alumina colloids, and silica colloids; insoluble granular materials such as colored resin particles, dye colloids, colored liposomes, etc.; enzymes that catalyze chromogenic reactions such as alkaline phosphatase, peroxidase, luciferase, etc.; fluorescent dyes, radioisotopes; and chemiluminescent labels, bioluminescent labels, electrochemiluminescent labels, etc.
The method for attaching the label to the antibody is also not restricted, but specific examples of methods that can be used include physical adsorption using the hydrophobicity of the antibody, chemisorption using a functional group of the antibody, etc.
The detection antibody-impregnated member (conjugate pad) 2 and the sample addition member (sample pad) 3 may be optionally omitted. When the present mechanism lacks the detection antibody-impregnated member (conjugate pad) 2, the same test as above can be performed by applying the sample A and the detection antibody to one end on the insoluble membrane carrier 1 in a pre-mixed state or separately, either simultaneously or sequentially.
Even when the capture antibody and the detection antibody are interchanged, a detection kit can be constructed to enable the same detection. That is, the capture antibody may be the generic antibody and the detection antibody may be the specific antibody. Alternatively, the detection antibody may be the generic antibody and the capture antibody may be the specific antibody. In either case, either or both of the generic antibody and the specific antibody may be the antibody of the present invention, as described above.
According to the immunochromatographic detection method and device of the lateral-flow type according to one embodiment of the present invention mentioned above, the two or more genera of Enterobacteriaceae bacteria as the target for detection can be detected at once on a single detection line (in the example shown in
The present invention also provides a method for producing the above-mentioned immunochromatography kit. The method includes at least the steps of stacking the conjugate pad, to which the detection antibody is attached, on the insoluble membrane carrier, and immobilizing the capture antibody in the chromatographic development direction opposite to the conjugate pad on the insoluble membrane carrier.
Either the capture antibody or the detection antibody may be used as the specific antibody, and either may be used as the generic antibody. However, it is preferable to use the specific antibody as the capture antibody and the generic antibody as the detection antibody.
The specific antibody may preferably be an antibody that causes antigen-antibody reactions specifically with at least Enterobacteriaceae bacteria of two or more genera selected from the genus Escherichia, the genus Klebsiella, the genus Citrobacter, the genus Enterobacter, the genus Proteus, the genus Salmonella, and the genus Serratia.
The generic antibody may preferably be an antibody that causes antigen-antibody reactions specifically with at least 5 or more genera selected from the genus Escherichia, the genus Klebsiella, the genus Citrobacter, the genus Enterobacter, the genus Proteus, the genus Salmonella, the genus Serratia, the genus Pseudomonas, the genus Staphylococcus, the genus Bacillus, and the genus Enterococcus.
According to the method for producing an immunochromatography kit, it is possible to use the same generic antibody as the detection antibody for mass production of plural variations of immunochromatography kits, which is extremely effective in that plural types of immunochromatography kits can be manufactured by selecting only the capture antibody.
The present invention will be described in more detail with reference to the examples below. However, the present invention is not bound by the following examples, and can be implemented in any form within the scope that does not depart from the purpose of the present invention.
Production of Specific Antibodies A to C:
E. coli (EC) was used as an immunogen Enterobacteriaceae bacterium. Antibodies against ribosome protein L7/L12 of E. coli were produced by referring to the method described in WO2000/06603 A. Specifically, E. coli transformed with an expression vector incorporating DNA encoding the entire amino acid sequence of ribosomal protein L7/L12 of E. coli was cultured using, e.g., LB medium, and the ribosomal protein L7/L12 was purified by affinity column as a fusion protein using a tag sequence derived from the expression vector. The total length protein of E. coli L7/L12 was prepared as an immunogen in PBS according to the standard method for obtaining hybridomas, so that the concentration of the immunogen was 0.4 mg/mL, and Freund's adjuvant was added in the same volume. Mice were immunized four times with an immunogen level of 50 μg/time. After confirming the increase in serum antibody titer by test blood collection, mouse spleen cells were harvested. The excised mouse spleen cells were fused with myeloma cells, and various hybridomas were obtained.
The various hybridomas obtained were cultured in HAT medium, and screened based on antibodies in the culture supernatant. The screening was performed by ELISA using solid-phase lysates of several genera of Enterobacteriaceae as antigens, to select hybridomas producing antibodies simultaneously reactive with lysates of seven species of Enterobacteriaceae bacteria: Escherichia coli (EC), Klebsiella pneumoniae (KP), Citrobacter freundii (CF), Enterobacter cloacae (ECL), Proteus mirabilis (PM), Salmonella enterica (SE), and Serratia liquefaciens (SL). According to the established method of monoclonal antibody production, the selected hybridomas were cultured in TIL Medial medium supplemented with 10% fetal bovine serum (FBS), injected into the abdominal cavity of a mouse, and ascites was collected. Antibodies were purified from the collected ascites by centrifuging the ascites to remove suspended solids and erythrocytes, filtering the supernatant through a filter with a mesh size of 0.45 m, and pass the filtrate through a Protein G column to adsorb antibodies.
Through the above procedure, the specific antibodies A (EC50C1), B (EC149C3), and C (EC162A3) were obtained.
Production of Generic Antibodies A and B:
Pseudomonas aeruginosa (PA) was used as an immunogen bacterium. An antibody against ribosome protein L7/L12 of Pseudomonas aeruginosa was produced by referring to the method described in WO2000/06603 A. Specifically, E. coli transformed with an expression vector incorporating DNA encoding the entire amino acid sequence of ribosomal protein L7/L12 of Pseudomonas aeruginosa was cultured using, e.g., LB medium, and the ribosomal protein L7/L12 was purified by affinity column as a fusion protein using a tag sequence derived from the expression vector. The total length protein of Pseudomonas aeruginosa L7/L12 was prepared as an immunogen in PBS according to the standard method for obtaining hybridomas, so that the concentration of the immunogen was 0.4 mg/mL, and Freund's adjuvant was added in the same volume. Mice were immunized four times with an immunogen level of 50 μg/time. After confirming the increase in serum antibody titer by test blood collection, mouse spleen cells were harvested. The excised mouse spleen cells were fused with myeloma cells, and various hybridomas were obtained.
The various hybridomas obtained were cultured in HAT medium, and screened based on antibodies in the culture supernatant. The screening was performed by ELISA using solid-phase lysates of several genera of bacteria including non-Enterobacteriaceae bacteria as antigens, to select hybridomas producing antibodies simultaneously reactive with lysates of these several genera of bacteria. According to the established method of monoclonal antibody production, the selected hybridomas were cultured in TIL Medial medium supplemented with 10% fetal bovine serum (FBS), injected into the abdominal cavity of a mouse, and ascites was collected. An antibody was purified from the collected ascites by centrifuging the ascites to remove suspended solids and erythrocytes, filtering the supernatant through a filter with a mesh size of 0.45 m, and pass the filtrate through a Protein G column to adsorb the antibody, to thereby produce a generic antibody A (PA51B2).
The same procedure as that for producing the generic antibody A was carried out except that ribosome protein L7/L12 of Chlamydia pneumoniae (LP) was used as an immunogen, to thereby produce a generic antibody B (CP141A190.1).
Amino Acid Sequencing of Each Variable Region of the Heavy and Light Chains of Specific Antibodies A to C and Generic Antibodies A and B:
The amino acid sequences of each variable sequence of the heavy and light chains for the specific antibodies A to C (antibodies of the invention) and the generic antibodies A and B produced by the above procedure were determined according to the usual method. The correspondence between the amino acid sequences and the sequence identification numbers is shown below.
Specific Antibody a (EC50C1):
Specific Antibody B (EC149C3):
Specific Antibody C (EC162A3):
Generic Antibody A (PA51B2):
Generic Antibody B (CP141A190.1):
The thus-produced Specific Antibodies A to C (antibodies of the present invention) and Generic Antibodies A and B were used to obtain data on the reactivity with plural genera of Enterobacteriaceae bacteria and bacteria other than Enterobacteriaceae (non-Enterobacteriaceae bacteria) by means of ELISA. Seven species of Enterobacteriaceae bacteria were selected for the study: Escherichia coli (EC), Klebsiella pneumoniae (KP), Citrobacter freundii (CF), Enterobacter cloacae (ECL), Proteus mirabilis (PM), Salmonella enterica (SE), and Serratia liquefaciens (SL), which are frequently detected in food, beverage, environmental, or biological samples. Four species of bacteria other than Enterobacteriaceae (non-Enterobacteriaceae bacteria) were also selected for the study: Pseudomonas aeruginosa (PA), Staphylococcus aureus (SA), Bacillus subtilis (BS), and Enterococcus faecalis (EF), which are frequently detected in food, beverage, environmental, or biological samples. The seven species of Enterobacteriaceae bacteria and four species of non-Enterobacteriaceae bacteria described above were purchased and cultured from ATCC, prepared at 1×e8 cfu/mL each, and suspended in PBS. Each bacterium was lysed by sonication, and debris was removed by filtration through a filter with a 0.45-μm mesh opening to obtain the bacterial lysate of each bacterium.
50 μL of the above bacterial lysate was dropped into each well of a 96-well polystyrene plate for ELISA and solidified on the bottom of the plate. After washing each well three times with PBS-T (PBS with Tween 20), blocking treatment was performed with PBS containing 1% BSA (Bovine Serum Albumin). After blocking, the wells were washed three times with PBS-T, and 50 μL of 10 μg/mL solution of each of the Specific Antibodies A to C and Generic Antibodies A and B was dropped into each well for 1 hour for antigen-antibody reactions. After the reactions, the samples were washed three times with PBS-T, and then 0.5 μg/mL of HRP (Horse Radish Peroxidase) labeled secondary antibody (Goat Anti-mouse IgG: Goat Anti-mouse IgG), which is the enzyme for detection, was added dropwise. After the reaction, the wells were washed five times with PBS-T, and 100 μL of a mixture of tetramethylbenzidine (TMB) and hydrogen peroxide, the chromogenic substrate, was added dropwise to each well for the chromogenic reaction. After 10 minutes, hydrochloric acid, the reaction stopping solution, was dropped into each well, and the absorbance of each well at 450 nm was measured with a plate reader.
The measurement results are shown in Table 1 below. The results shown in this table indicate that the Specific Antibodies A to C reacted more sensitively (with an absorbance of 0.3 or more for each bacterium) with the samples of seven Enterobacteriaceae species, compared to the sample without bacteria, and did not react (with an absorbance of less than 0.3 for each bacterium) with the samples of four non-Enterobacteriaceae species. In other words, it was confirmed that the Specific Antibodies A to C can selectively cause (detect) antigen-antibody reactions with the Enterobacteriaceae bacteria. On the other hand, the Generic Antibodies A and B showed antigen-antibody reactivity with all Enterobacteriaceae and non-Enterobacteriaceae bacteria.
[3. Detection of Antigen-Antibody Reactions between Antibodies and Enterobacteriaceae Bacteria 2—Recombinant Antigen Solid-Phase ELISA]
The thus-produced Specific Antibodies A to C (antibodies of the present invention) and Generic Antibodies A and B were used to obtain data on the reactivity with ribosome protein L7/L12 of plural genera of Enterobacteriaceae bacteria by means of ELISA. Seven species of Enterobacteriaceae bacteria were selected for the study: Escherichia coli (EC), Klebsiella pneumoniae (KP), Citrobacter freundii (CF), Enterobacter cloacae (ECL), Proteus mirabilis (PM), Salmonella enterica (SE), and Serratia liquefaciens (SL), which are frequently detected in food, beverage, environmental, or biological samples. Four species of bacteria other than Enterobacteriaceae (non-Enterobacteriaceae bacteria) were also selected for the study: Pseudomonas aeruginosa (PA), Staphylococcus aureus (SA), Bacillus subtilis (BS), and Enterococcus faecalis (EF), which are frequently detected in food, beverage, environmental, or biological samples. E. coli transformed with expression vectors incorporating DNA encoding the amino acid sequence of ribosomal protein L7/L12 of each of the above seven Enterobacteriaceae and four non-Enterobacteriaceae species were cultured using, e.g., LB medium, and the ribosomal protein L7/L12 was purified as a fusion protein by affinity columns using tag sequences derived from the expression vector.
50 μL of 10 ng/mL solution of the ribosomal protein L7/L12 of each bacterial species was dropped into each well of a 96-well polystyrene plate for ELISA and solidified on the bottom of the plate. After washing each well three times with PBS-T (PBS with Tween 20), blocking treatment was performed with PBS containing 1% BSA (Bovine Serum Albumin). After blocking, the wells were washed three times with PBS-T, and 50 L of 10 μg/mL solution of each of the Specific Antibodies A to C and Generic Antibodies A and B was dropped into each well for 1 hour for antigen-antibody reactions. After the reactions, the samples were washed three times with PBS-T, and then 0.5 μg/mL of HRP (Horse Radish Peroxidase) labeled secondary antibody (Goat Anti-mouse IgG: Goat Anti-mouse IgG), which is the enzyme for detection, was added dropwise. After the reaction, the wells were washed five times with PBS-T, and 100 μL of a mixture of tetramethylbenzidine (TMB) and hydrogen peroxide, the chromogenic substrate, was added dropwise to each well for the chromogenic reaction. After 10 minutes, hydrochloric acid, the reaction stopping solution, was dropped into each well, and the absorbance of each well at 450 nm was measured with a plate reader.
The measurement results are shown in Table 2 below. The results shown in this table indicate that the Specific Antibodies A to C reacted more sensitively (with an absorbance of 0.3 or more for each bacterium) with the samples of seven Enterobacteriaceae species, compared to the sample without bacteria, and did not react (with an absorbance of less than 0.3 for each bacterium) with the samples of four non-Enterobacteriaceae species. In other words, it was confirmed that the Specific Antibodies A to C can selectively cause (detect) antigen-antibody reactions with the ribosomal protein L7/L12 of each Enterobacteriaceae bacteria. On the other hand, the Generic Antibodies A and B showed antigen-antibody reactivity with all Enterobacteriaceae and non-Enterobacteriaceae bacteria.
The thus-produced Specific Antibodies A to C (antibodies of the present invention) and Generic Antibodies A and B were used to obtain data on the reactivity with non-bacterial components in various food, beverage, or environmental samples (food, beverage, or environmental components) by means of ELISA. Raw fish (yellowtail and horse mackerel), raw noodles (yakisoba), raw egg, prepared food (potato salad), vegetables (cucumber (fruit vegetable), carrot (root vegetable), and lettuce (leaf vegetable)), and meats and processed meats (beef ribs, beef short ribs, pork loin, chicken breast, and ham) were purchased at a supermarket and used as food samples. Each of these foodstuffs was weighed 25 g, placed in a commercial stomacher bag, and stomached with 225 ml of PBS. A portion of the stomacher-treated solution was processed through a filter with a mesh aperture of 0.45 m to remove solids containing bacteria, thereby preparing a bacteria-free food sample for ELISA. Milk and tea were purchased at a supermarket and used as beverage samples. Each of these beverages was suspended in PBS to a concentration of 1/10, and solids containing bacteria were removed with a filter with a 0.45 m mesh opening, thereby preparing a bacteria-free beverage sample for ELISA. As environmental samples, hand fingers, cutting boards, kitchen knives, and refrigerator handles were wiped with a commercially available wiping kit (ELMEX Pro-mediaST-25, PBS), suspended in PBS provided with the kit, and solids containing bacteria were removed with a filter with a 0.45 m mesh opening, thereby preparing a bacteria-free environmental sample for ELISA.
50 μL of each of the food, beverage, and environment samples was dropped into each well of a 96-well polystyrene plate for ELISA and solidified on the bottom of the plate. After washing each well three times with PBS-T (PBS with Tween 20), blocking treatment was performed with PBS containing 1% BSA. After blocking, the wells were washed three times with PBS-T, and 50 μL of 10 μg/mL solution of each of the Specific Antibodies A to C and Generic Antibodies A and B was dropped into each well for 1 hour for reactions. After the reactions, the samples were washed three times with PBS-T, and then 0.5 μg/mL of HRP (Horse Radish Peroxidase) labeled secondary antibody (Goat Anti-mouse IgG: Goat Anti-mouse IgG), which is the enzyme for detection, was added dropwise. After the reaction, the wells were washed five times with PBS-T, and 100 μL of a mixture of TMB and hydrogen peroxide, the chromogenic substrate, was added dropwise to each well for the chromogenic reaction. After 10 minutes, hydrochloric acid, the reaction stopping solution, was dropped into each well, and the absorbance of each well at 450 nm was measured with a plate reader.
The measurement results are shown in Table 3 below. The results shown in this table indicate that the Specific Antibodies A to C did not react with any of the above non-bacterial components in the food, beverage, or environmental samples (with an absorbance of less than 0.3 for each sample). Combined with the results of the aforementioned examples, it was confirmed that all of the Specific Antibodies A to C do not react with any of the non-bacterial components in the food, beverage, or environmental samples (food, beverage, or environmental components), but only with the specific plural genera of Enterobacteriaceae bacteria to be detected. In other words, it was confirmed that these antibodies can detect with high selectivity the presence and/or amount of the specific plural genera of Enterobacteriaceae bacteria to be detected in the food, beverage, or environmental samples. On the other hand, the Generic Antibodies A and B did not show cross-reactivity with any of the non-Enterobacterial components in the food, beverage, or environmental samples.
Three types of immunochromatographic detection kits (a) to (c) were prepared by using the Specific Antibodies A to C (antibodies of the present invention) as primary antibodies (capture antibodies) and the Generic Antibodies A and B as secondary antibodies (detection antibodies) in the combinations shown in (a) to (c) of Table 4 below. In addition, additional three types of immunochromatographic detection kits (d) to (f) capable of similar detection were prepared by changing the antibodies used for the primary and secondary antibodies in the combinations listed in (d) to (f) of Table 4 below.
Production of Membrane Carrier for Immunochromatography Development:
A solution was prepared that contain 1.5 mg/mL of each primary antibody (capture antibody) as listed in (a) to (f) of Table 4 above and 3% (v/v) of trehalose in 10 mM sodium phosphate buffer solution. The resulting solution was applied to a commercially available nitrocellulose membrane cut to 2.5 cm wide and 15 cm long at a volume of 1 μL solution per cm2 and dried to make a membrane carrier for immunochromatography development.
Preparation of Antibodies for Gold Colloid Labeling Detection and Antibody-Impregnated Members for Gold Colloid Labeling Detection:
A solution with an antibody concentration of 0.1 mg/mL was prepared by mixing a commercially available gold colloid solution (60 nm particle size) with 1/10 volume of each secondary antibody (detection antibody) listed in (a) to (f) of Table 4 above. The solution was allowed to stand at room temperature for 30 minutes to allow the antibody to bind to the surface of the gold colloidal particles. The antibody solution was then blocked by adding BSA solution so that the final concentration in the gold colloid solution was 0.1%, whereby the antibody solution for gold colloid labeling detection was prepared. This antibody solution was soaked into commercially available glass fiber sheets and dried to prepare an antibody-impregnated member for gold colloid labeling detection.
Assembly of Immunochromatographic Detection Kits:
In addition to the membrane carrier for immunochromatographic development and the antibody-impregnated member for gold colloid labeling detection prepared by the procedures described above, a cotton cloth for adding a sample and a filter paper for absorbing a sample were also prepared for each kit. These components were laminated onto a commercially available polyethylene substrate, cut into 5 mm widths, to prepare an immunochromatographic detection kit with a detection mechanism as shown in
[6. Detection of Bacteria using the Immunochromatographic Detection Kits]
The thus-obtained immunochromatographic detection kits (a) to (f) were used to detect plural genera of Enterobacteriaceae bacteria. Seven species of Enterobacteriaceae bacteria were selected for the study: Escherichia coli (EC), Klebsiella pneumoniae (KP), Citrobacter freundii (CF), Enterobacter cloacae (ECL), Proteus mirabilis (PM), Salmonella enterica (SE), and Serratia liquefaciens (SL), which are frequently detected in food, beverage, environmental, or biological samples. Four species of bacteria other than Enterobacteriaceae (non-Enterobacteriaceae bacteria) were also selected for the study: Pseudomonas aeruginosa (PA), Staphylococcus aureus (SA), Bacillus subtilis (BS), and Enterococcus faecalis (EF), which are frequently detected in food, beverage, environmental, or biological samples. The seven species of Enterobacteriaceae bacteria and the four species of non-Enterobacteriaceae bacteria were each prepared at 1×e8 cfu/mL and suspended in PBS. Each bacterium was lysed by sonication to obtain bacterial lysates (bacterial samples for immunochromatographic assay) of the seven Enterobacteriaceae bacteria and the four non-Enterobacteriaceae bacteria. A PBS solution without bacterial lysates was also prepared as a bacteria-free sample. Tween 20 was added to each of these samples at a final concentration of 1%, whereby samples for immunochromatographic development were prepared.
The prepared samples (one bacteria-free sample and 11 bacterial lysate samples) were each added to the sample addition member area of each of the immunochromatographic detection kits having the configurations of (a) through (f) above, and 30 minutes later, the line coloration of the capture-antibody applied area of the membrane carrier was visually confirmed.
The results of the detection kits (a) through (c) are shown in Table 5 below. These results indicate that as intended, line coloration was confirmed for all Enterobacteriaceae bacteria in all of the detection kits (a) through (c), indicating that antigen-antibody reactions occurred. On the other hand, no line coloration was confirmed for the bacteria other than Enterobacteriaceae bacteria (non-Enterobacteriaceae bacteria) in all of the detection kits (a) through (c), indicating that no antigen-antibody reactions occurred. In other words, each immunochromatographic detection kit constructed by combining a specific antibody, that causes antigen-antibody reactions only with Enterobacteriaceae bacteria, and a generic antibody, that causes antigen-antibody reactions widely with bacteria of many genera, was shown to be able to detect the Enterobacteriaceae bacteria easily and rapidly. The detection kits using combinations of (d) through (f) in Table 4 above were also tested for detection of the plural genera of Enterobacteriaceae, and similar results were obtained.
The thus-obtained immunochromatographic detection kits (a) to (f) were used to obtain data on the reactivity with non-bacterial components in various food, beverage, or environmental samples (food, beverage, or environmental components). Raw fish (yellowtail and horse mackerel), raw noodles (yakisoba), raw egg, prepared food (potato salad), vegetables (cucumber (fruit vegetable), carrot (root vegetable), and lettuce (leaf vegetable)), and meats and processed meats (beef ribs, beef short ribs, pork loin, chicken breast, and ham) were purchased at a supermarket and used as food samples. Each of these foodstuffs was weighed 25 g, placed in a commercial stomacher bag, and stomached with 225 ml of PBS. A portion of the stomacher-treated solution was processed through a filter with a mesh aperture of 0.45 m to remove solids containing bacteria, thereby preparing a sample for immunochromatography. Milk and tea were purchased at a supermarket and used as beverage samples. Each of these beverages was suspended in PBS to a concentration of 1/10, and solids containing bacteria were removed with a filter with a 0.45 m mesh opening, thereby preparing a sample for immunochromatography. As environmental samples, hand fingers, cutting boards, kitchen knives, and refrigerator handles were wiped with a commercially available wiping kit (ELMEX Pro-mediaST-25, PBS), suspended in PBS provided with the kit, and solids containing bacteria were removed with a filter with a 0.45 m mesh opening, thereby preparing a sample for immunochromatography.
The samples prepared for immunochromatography from various food, beverage, or environmental samples were each added to the sample addition member area of each of the immunochromatographic detection kits having the configurations of (a) through (f) above, and 30 minutes later, the line coloration of the capture-antibody applied area of the membrane carrier was visually confirmed. The results for the detection kits (a) through (c) are shown in Table 6. As intended, none of the detection kits (a) through (c) showed cross-reactivity with the components in the above food, beverage, or environmental samples. In combination with the aforementioned examples, it was confirmed that the immunochromatographic detection kits (a) through (c) prepared by combining the aforementioned generic and specific antibodies do not react with non-bacterial components (food, beverage, or environmental components) in the food, beverage, or environmental samples, and makes it possible to detect only the specific Enterobacteriaceae bacteria to be detected easily and rapidly with high selectivity. The detection kits using combinations of (d) through (f) in Table 4 above were also tested for cross-reactivity with non-bacterial components in the food, beverage, or environmental samples, and similar results were obtained.
The present invention can be widely used in fields where simultaneous and simple detection of plural genera of Enterobacteriaceae bacteria in food, beverage, environmental, or biological samples is required, mainly in the fields of medicines and foods, and therefore has extremely high industrial usefulness.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2021-004806 | Jan 2021 | JP | national |
| 2021-004860 | Jan 2021 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2022/001206 | 1/14/2022 | WO |
