WO2014188763A1 - Anticorps qui se lie à un antigène de leptospire - Google Patents

Anticorps qui se lie à un antigène de leptospire Download PDF

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WO2014188763A1
WO2014188763A1 PCT/JP2014/056384 JP2014056384W WO2014188763A1 WO 2014188763 A1 WO2014188763 A1 WO 2014188763A1 JP 2014056384 W JP2014056384 W JP 2014056384W WO 2014188763 A1 WO2014188763 A1 WO 2014188763A1
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Prior art keywords
antibody
antigen
leptospira
urine
leptospirosis
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PCT/JP2014/056384
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English (en)
Inventor
Shin-Ichi Yoshida
Mitsumasa Saito
Dian WIDIANTI
Sharon VILLANUEVA
Toshiyuki MASUZAWA
Takashi Fukui
Nobuo Koizumi
Original Assignee
Kyushu University, National University Corporation
Kake Educational Institution
Director-General, National Institute Of Infectious Diseases
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Application filed by Kyushu University, National University Corporation, Kake Educational Institution, Director-General, National Institute Of Infectious Diseases filed Critical Kyushu University, National University Corporation
Publication of WO2014188763A1 publication Critical patent/WO2014188763A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
    • C07K16/1207—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Spirochaetales (O), e.g. Treponema, Leptospira
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/20—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Spirochaetales (O), e.g. Treponema, Leptospira

Definitions

  • the present invention generally relates to a novel method of detecting infectious disease in animals and to a novel antibody that selectively binds to spirochete bacteria. More particularly, the present invention relates to a novel method of detecting infectious disease caused by the spirochete bacteria, Leptospira spp, and to a novel monoclonal antibody that binds to lipopolysaccharide common among Leptospira spp.
  • the disclosed antibody and method can be used to predict leptospirosis in animals. Therefore, the antibody of the present invention can be used to detect and diagnose a leptospirosis in humans and other animals.
  • the methods can also be useful for prescribing a treatment for an animal. Suitable treatment can be designed to delay or prevent the onset of the leptospirosis.
  • the present invention is also useful in monitoring the effectiveness of a prescribed treatment.
  • Leptospira belonging to the order Spirochaetales and family Leptospiraceae, is a spiral-shaped bacterium which is 0.1 ⁇ in diameter, 6-20 ⁇ in length and has hooks at its ends (1).
  • Leptospira organisms are Gram-negative, and obligate aerobic (2). Infection in humans or animals could happen by penetration of Leptospira, excreted by infected host animals into the environment, through a wound or mucous membrane. Signs and symptoms of leptospirosis in humans range from mild, flu-like symptoms to jaundice (hepatic dysfunction), oliguria- or anuria (renal failure) and hemoptysis (lung hemorrhage) which can lead to death (3).
  • ICG-based assay could become a solution because it is inexpensive, rapid, and easy to perform.
  • Development of an ICG-based assay for detection of bacterial antigen in the clinical sample has been performed on several bacteria such as Legionella pneumophila (13), 5 * . pneumoniae (14), and Neisseria meningitidis (15).
  • the present invention provides as follows:
  • the antibody of (1) wherein the antibody is selected from the group consisting of a Fab, F(ab') 2 , Fab', a diabody, dsFv, a linear antibody, scFv or a complementarity determining region as a part thereof.
  • An agent for detecting leptospirosis comprising the antibody according to any one of (l) to (8).
  • An agent for diagnosing leptospirosis comprising the antibody according to any one of (1) to (8).
  • a method of detecting leptospiral antigen that comprises:
  • a method of diagnosing leptospirosis that comprises:
  • test sample is urine
  • a kit for detecting leptospiral antigen or diagnosing leptospirosis comprising the antibody according to any one of (1) to (8).
  • FIG. 1 shows detection of of of MAb (lH6)-reactive antigen in Leptospira and other bacteria. SDS PAGE of bacterial LPS with proQ emerald staining (upper panel) and immunoblotting with mAb-lH6 (lower panel). Lane 1 : Legionella pneumophila; lanes
  • FIG. 2 shows Dipstick assay using hamster urine. (A) negative result; (B) positive result.
  • FIG. 3 shows ICG-based LFA using human urine. (A) negative result; (B) positive result.
  • Leptospirosis is an infectious disease caused by the spirochete bacteria, Leptospira spp, and commonly found throughout the world. Diagnosis of leptospirosis performed by culture and microscopic agglutination test are laborious and time-consuming. We, therefore, aimed to develop a novel immunochromatography(ICG)-based method to detect Leptospira antigen in urine of patients or animals.
  • ICG immunochromatography
  • the MAb was coupled to 40 nm-diameter colloidal gold and the amount of labeled antibody and immobilized antibody were 23 ⁇ g and 2 ⁇ g per test, respectively.
  • Several strains of Leptospira and other bacteria were used to evaluate the sensitivity and specificity of the assays developed.
  • the detection limit of the assays was 10 6 cells/ml when disrupted bacterial whole cells were used.
  • the assays were Leptospira-sipecific since they did not cross-react with other bacteria used.
  • Application of diagnostic assays was done on the urine of 46 Leptospira-infected hamsters, 44 suspected leptospirosis patients, and 14 healthy individuals.
  • ICG-based lateral flow assay (LFA)
  • sensitivity and specificity of ICG-based lateral flow assay were 89% and 87%, respectively, which were higher than those of the dipstick assay, which were 80% and 74 %, respectively.
  • this ICG-based LFA could be used as an alternative diagnostic assay for leptospirosis.
  • an "antibody” is an immunoglobulin, a solution of identical or heterogeneous immunoglobulins, or a mixture of immunoglobulins.
  • the antibody of the present invention includes all known immunoglobulin forms and other protein scaffolds with antibody-like properties.
  • the antibody can be a murine antibody, a human antibody, a humanized antibody, or a chimeric antibody.
  • the antibody also can have any of the following isotypes: IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgAsec, IgD and IgE.
  • the antibody of the present invention comprises an antibody fragment.
  • antibody fragments include peptides containing at least Fab (antigen-binding fragment), F(ab') 2 , Fab', Fv, diabody (dibodies), dsFv, linear antibody, scFv (single chain Fv), or complementarity determining region (CDR) as a part thereof. Even if an amino acid sequence of the antibody is modified, such an antibody lies within the scope of the present invention as long as it can specifically bind to the above-described antigen. Also provided by the present invention is antibody that binds to the same or overlapping epitopes bound by any of the aforementioned antibody.
  • a “polyclonal antibody” is a mixture of heterogeneous antibodies. Typically, a polyclonal antibody will include myriad different antibodies molecules which bind a particular antigen or particular organism with at least some of the different antibodies immunoreacting with a different epitope of the antigen or organism. As used herein, a polyclonal antibody can be a mixture of two or more monoclonal antibodies.
  • monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. Monoclonal antibodies can be prepared using any art recognized technique and those described herein such as, for example, a hybridoma method, as described by Kohler et al.
  • chimeric antibody refers to an immunoglobulin whose variable regions derive from a first species and whose constant regions derive from a second species. Chimeric antibodies can be constructed, for example by genetic engineering, from immunoglobulin gene segments belonging to different species.
  • humanized antibody refers to an immunoglobulin that includes at least one humanized immunoglobulin chain (i.e., at least one humanized light or heavy chain).
  • humanized immunoglobulin chain refers to an immunoglobulin chain having a variable region that includes a variable framework region substantially from a human immunoglobulin and complementarity determining regions (CDRs) substantially from a non-human immunoglobulin, and further includes constant regions.
  • human antibody is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences, for example, by Kabat et al. (See Kabat, et al. (1991) Sequences of proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences.
  • the human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody”, as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • the invention provides fully human antibody (i.e., which contains human CDR and framework sequences) that bind to Leptospira spp.
  • Particular human antibody of the invention comprises a heavy chain variable region from a human VHl-24 or VH3-23 germline gene, and/or a light chain variable region from human V A26 or VK V2-17 germline gene.
  • the sequences of these and other human germline genes are publicly available and can be found, for example, in the "VBase" human germline sequence database (available on the Internet at http://www.vbase2.org/) and the "IMGT” database (available on the Internet at http://www.imgt.org/), and are hereby incorporated by reference.
  • epitope refers to a site on an antigen to which an immunoglobulin specifically binds.
  • Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents.
  • An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include techniques in the art and those described herein, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996).
  • antibodies that bind the same or an overlapping epitope as the particular antibodies described herein i.e., antibodies that compete for binding to core LPS of Leptospira, or bind to an epitope on LPS recognized by the particular antibodies described herein.
  • Antibodies that recognize the same or an overlapping epitope can be identified using routine techniques such as an immunoassay, for example, by showing the ability of one antibody to block the binding of another antibody to a target antigen, i.e., a competitive binding assay.
  • a competitive binding assay Numerous types of competitive binding assays are known, for example: solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay.
  • the term "subject” includes any human or non-human animal.
  • the methods of the present invention can be used to detect leptospiral antigen or to diagnose a subject having a leptospirosis.
  • the subject is a human.
  • non-human animal includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dog, cow, chickens, etc.
  • sample refers to tissue, urine, body fluid, or a cell from a patient or a subject. Normally, the tissue or cell will be removed from the patient, but in vivo diagnosis is also contemplated.
  • Other patient samples include tear drops, serum, cerebrospinal fluid, feces, sputum, cell extracts etc.
  • Leptospira spp generally have the following characteristics: (1) their cell wall only contains a few layers of peptidoglycan; and (2) the cells are surrounded by an outer membrane containing lipopolysaccharide (which consists of Lipid A, core polysaccharide, and O-polysaccharide) outside the peptidoglycan layer.
  • lipopolysaccharide which consists of Lipid A, core polysaccharide, and O-polysaccharide
  • Antigens useful for the production and testing of the antibodies of the invention can be obtained from commercial sources, or isolated from animals or humans harboring the bacteria of interest by conventional techniques in microbiology.
  • Leptospira spp are commercially available.
  • infectious bacteria such as Leptospira spp may be obtained from an infected host by isolation and culture.
  • Leptospira antigen Purified antigens derived from Leptospira spp (referred to as "Leptospira antigen") can be obtained commercially or isolated from whole bacteria by techniques known in the art. For example, core lipopolysaccharide contained in a sample of Leptospria spp can be separated according to size by SDS-polyacrylamide gel electrophoresis or by size and isoelectric point using two dimensional gel electrophoresis.
  • the lipopolysaccharide prepared as described above is administered alone or together with a carrier or a diluent to an appropriate animal such as rabbit, dog, guinea pig, mouse, rat or goat for immunization.
  • a dosage of the antigen per animal is 1-10 mg without an adjuvant and 5-500 ⁇ g with an adjuvant.
  • adjuvants include Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA) and aluminum hydroxide adjuvant. Immunization is performed mainly by intravenous, subcutaneous or intraperitoneal injection or the like.
  • the intervals of immunizations are not particularly limited, and immunizations are performed at intervals of a few days to several weeks, preferably at intervals of 1 to 2 weeks, for 2-10 times, preferably for 3-5 times.
  • the intervals of immunizations may be determined by those skilled in the art by considering the resulting antibody titer.
  • blood is sampled at the end of 3-4 times of subcutaneous immunizations to measure an antibody titer.
  • the antibody titer in serum may be measured by ELIS A (enzyme- linked immunosorbent assay), EIA (enzyme immunoassay), radioimmuno assay ( IA) or the like.
  • whole blood can be collected to separate and purify the antibody according to a general method.
  • a serum containing the antibody of interest is passed through a column bound with core lipopolysaccharide from Leptospira spp, and the passed-through fraction is collected, thereby obtaining a polyclonal antibody having enhanced specificity to the antigen.
  • Monoclonal antibodies of the invention can be produced using a variety of known techniques, such as those described in the examples, as well as the standard somatic cell hybridization technique described by Kohler and Milstein (1975) Nature 256: 495, viral or oncogenic transformation of B lymphocytes or phage display technique using libraries of human antibody genes.
  • the antibodies are fully human monoclonal antibodies.
  • a hybridoma method is used for producing an antibody that binds lipopolysaccharide of Leptospira spp.
  • a mouse or other appropriate host animal can be immunized with Leptospira antigen in order to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to this antigen.
  • Suitable Leptospira antigen can be obtained using a variety of methods, purified from a source, produced recombinantly or chemically synthesized.
  • the Leptospira antigen prepared as described above is administered alone or together with a carrier and a diluent to appropriate animals for immunization.
  • a dosage of the antigen per animal is 1-10 mg without an adjuvant and 5-500 ⁇ g with an adjuvant.
  • the type of adjuvant, an immunization method and immunization intervals employed are the same as those for the case of preparing a polyclonal antibody.
  • One to thirty days, preferably 2-5 days after the final day of immunization, individuals with approved antibody titer are selected to collect antibody-producing cells. Examples of antibody-producing cells include spleen cells, lymph node cells and peripheral blood cells, but preferably spleen cells or lymph node cells.
  • an antibody-producing cell and a myeloma cell are fused.
  • the fusion process may be carried out by a known method, for example, by the method of Kohler et al.
  • a generally available cell line from an animal such as a mouse may be used as the myeloma cell to be fused to the antibody-producing cell.
  • the cell line used has drug selectivity, and cannot survive in a HAT selection medium (containing hypoxanthine, aminopterin and thymidine) in an unfused state but survives only when it is fused to the antibody-producing cell.
  • myeloma cells examples include mouse myeloma cell lines such as PAI, P3-X63-Ag8, P3-X63-Ag8-UI, P3-NSI/l-Ag4-l, X63-Ag8-6.5.3., SP2/0-Agl4, FO and NSO/1, and rat myeloma cell lines such as YB2/0.
  • the cell fusion between the above-described myeloma cell and an antibody-producing cell is performed by mixing 1 x 10 8 to 5 x 10 8 antibody-producing cells with 2 x 10 7 to 1 x 10 8 myeloma cells (cell ratio of antibody-producing cells to myeloma cells being 1: 1 to 1 : 10) in an animal cell culture medium such as a serum-free DMEM or an RPMI-1640 medium for fusion reaction in the presence of a cell fusion promoter.
  • an animal cell culture medium such as a serum-free DMEM or an RPMI-1640 medium for fusion reaction in the presence of a cell fusion promoter.
  • a cell fusion promoter an average molecular weight of 1000-6000 daltons of polyethylene glycol, Sendai virus or the like may be used.
  • a commercially available cell fusion device employing electrostimulation e.g., electroporation
  • electrostimulation e.g., electroporation
  • a hybridoma of interest is selected from the cells after the cell fusion treatment.
  • a cell suspension is appropriately diluted, for example, in a 10-20% fetal bovine serum-containing RPMI-1640 medium; the resultant is seeded onto a microtiter plate at approximately 5 x 10 7 cells/well; a selection medium such as a HAT medium is added to each well; and thereafter the selection medium is appropriately exchanged for cultivation.
  • a selection medium such as a HAT medium
  • the hybridomas may be screened according to a general method without particular limitation. For example, a part of the culture supernatant contained in the hybridoma-culturing wells can be collected and screened by enzyme-linked immunosorbent assay, radioimmuno assay or the like. Specifically, an antigen is adsorbed onto a 96-well plate, which is then blocked with a calf serum. The culture supernatant of the hybridoma cells is allowed to react with a solid-phased antigen at 37°C for an hour, followed by reaction with peroxidase-labeled anti-mouse IgG at 37°C for an hour.
  • ortho-phenylenediamine is used as a substrate for color development. After terminating the reaction with an acid, absorbance at a wavelength of 490 nm can be measured for screening.
  • Hybridomas that produce monoclonal antibodies that are positive in the above measurement are cloned by limiting dilution or the like. Eventually, a cell that produces a monoclonal antibody that specifically binds to Leptospira antigen, i.e., a hybridoma, is established.
  • the present invention also provides a hybridoma that expresss and/or produces the aforementioned antibody.
  • the hybridoma is cultured in an animal cell culture medium such as a 10% fetal bovine serum-containing RPMI-1640 medium, an MEM medium or a serum-free medium, under general culture conditions (for example, at 37°C, 5% C0 2 concentration) for 7-14 days, thereby harvesting an antibody from the resulting culture supernatant.
  • an animal cell culture medium such as a 10% fetal bovine serum-containing RPMI-1640 medium, an MEM medium or a serum-free medium, under general culture conditions (for example, at 37°C, 5% C0 2 concentration) for 7-14 days, thereby harvesting an antibody from the resulting culture supernatant.
  • hybridomas In the case of ascites production, about 2 x 10 hybridomas are administered intraperitoneally to an animal, for example, mice (BALB/c), syngeneic to the mammal from which myeloma cells are derived to proliferate the hybridomas in large amounts. After 1-2 weeks, ascites is collected.
  • purification may be performed by appropriately selecting a known method such as ammonium sulfate precipitation, ion-exchange chromatography, gel filtration or affinity chromatography, or by combining these methods.
  • the binding specificity of the antibodies of the present invention can be identified using any technique including those disclosed here, can be determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay ( IA) or enzyme-linked immunoabsorbent assay (ELISA).
  • IA radioimmunoassay
  • ELISA enzyme-linked immunoabsorbent assay
  • the binding affinity of a monoclonal antibody or portion thereof can be determined by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).
  • Art recognized techniques can also be used to alter or optimize particular binding specificities and/or affinities (see, for example, Carter P J, Nature Reviews Immunology 6: 343-357 (2006)).
  • partial antibody sequences derived from antibodies of the invention may be used for producing structurally and functionally related antibodies.
  • antibodies interact with target antigens predominantly through amino acid residues that are located in the six heavy and light chain complementarity determining regions (CDRs).
  • CDRs complementarity determining regions
  • the amino acid sequences within CDRs are more diverse between individual antibodies than sequences outside of CDRs.
  • CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of specific naturally occurring antibodies by constructing expression vectors that include CDR sequences from the specific naturally occurring antibody grafted onto framework sequences from a different antibody with different properties (see, e.g., Riechmann, L.
  • Such framework sequences can be obtained from public DNA databases that include germline antibody gene sequences.
  • one or more structural features of the particular anti-Leptospira antibodies of the invention are used to create structurally related anti-Leptospira antibodies that retain the functional properties of the parent antibodies of the invention, such as binding to the same epitope or overlapping epitopes bound by the anti-Leptospira antibodies exemplified herein, as well as cross-competing for antigen-binding with the anti-Leptospira antibodies exemplified herein.
  • an anti-Leptospira antibody of the present invention is a recombinant antibody.
  • recombinant antibodies include, without limitation, a chimeric antibody, a humanized antibody and a human antibody.
  • a chimeric antibody is an antibody in which a variable region of a mouse-derived antibody is linked (conjugated) to a human-derived constant region (see Proc. Natl. Acad. Sci. U.S.A. 81, 6851-6855, (1984), etc.).
  • a chimera may readily be constructed by genetic recombination technique for obtaining such a linked antibody.
  • CDR grafting is a method for producing a rearranged variable region including a human-derived framework region (FR) and a mouse-derived CDR, by implanting a complementarity determining region (CDR) of a variable region from a mouse antibody into a human variable region. Subsequently, the rearranged human variable region is linked to a human constant region.
  • a method for preparing such a humanized antibody is well known in the art (see Nature, 321, 522-525 (1986); J. Mol. Biol., 196, 901-917 (1987); Queen C et al., Proc. Natl. Acad. Sci. USA, 86: 10029-10033 (1989); Japanese Patent Publication No.2828340).
  • a human antibody may be obtained, for example, by a method that employs a human antibody-producing mouse having a human chromosome fragment containing a gene of H-chain and L-chain of human antibody (see Tomizuka, . et al., Nature Genetics, (1997) 16, 133-143; Kuroiwa, Y.et.al., Nuc.
  • antibody fragments of the present invention include peptides including at least Fab (antigen-binding fragment), F(ab') 2 , Fab', a diabody (dibodies), dsFv, a linear antibody, scFv (single chain Fv) or a complementarity determining region (CDR) as a part thereof.
  • Fab is an antibody fragment in which, among a fragment obtained by treating an antibody molecule with protease papain, about half of the N-terminal end of H-chain and the entire L-chain are linked via disulfide bond.
  • Fab may be generated by inserting DNA encoding Fab of the antibody into an expression vector, and introducing the vector into a host organism for expression.
  • F(ab') 2 is an antibody fragment which, among a fragment obtained by treating an antibody molecule with protease pepsin, is slightly larger than one linked with Fab via a disulfide bond at the hinge region.
  • F(ab') 2 may be generated by linking Fab via thioether bond or disulfide bond.
  • Fab' is an antibody fragment obtained by cleaving the disulfide bond at the above-mentioned hinge region of F(ab') 2 .
  • Fab' may be generated by inserting DNA encoding Fab' fragment of the antibody into an expression vector, and introducing the vector into a host organism for expression.
  • scFv is a polypeptide in which a single H-chain V region (VH) and a single L-chain V region (V L ) are linked using an appropriate peptide linker, and an antibody fragment having an antigen-binding activity.
  • scFv may be generated by acquiring cDNA encoding VH and VL of the antibody, constructing DNA coding for scFv, introducing the DNA into an expression vector, and introducing the expression vector into a host organism for expression.
  • a diabody is an antibody fragment with dimerized scFv having a divalent antigen-binding activity.
  • the divalent antigen-binding activity may be identical or different from one another.
  • a diabody may be generated by acquiring cDNA encoding for VH and VL of the antibody, constructing DNA encoding scFv such that the length of the amino acid sequence of the peptide linker is 8 residues or less, inserting the DNA into an expression vector, and introducing the expression vector into a host organism for expression.
  • dsFv has polypeptides having an amino acid residue of each of VH and VL substituted with a cysteine residue, which are bound via a disulfide bond between the cysteine residues.
  • dsFv may be generated by acquiring cDNA coding for V H and VL of the antibody, constructing DNA encoding dsFv, inserting the DNA into an expression vector, and introducing the expression vector into a host organism for expression.
  • a peptide containing CDR is constructed to include at least one region of CDRs (CDRs 1-3) of VH or V L .
  • CDRs 1-3 CDRs 1-3
  • a peptide containing several CDRs may be bound directly or via an appropriate peptide linker.
  • a peptide containing CDR may be generated by constructing DNA coding for CDR of VH and V L of the antibody, inserting the DNA into an expression vector, and introducing the expression vector into a host organism for expression.
  • a peptide containing CDR may be generated by a chemical synthetic method such as an Fmoc method (fluorenylmethyloxycarbonyl method) or a Boc method (t-butyloxy carbonyl method).
  • an antibody fragment may be generated by using a hybridoma of the present invention (for example, hybridoma 1H6) or DNA or RNA extracted from said hybridoma as a raw material according to the above-described well-known method.
  • a hybridoma of the present invention for example, hybridoma 1H6
  • DNA or RNA extracted from said hybridoma as a raw material according to the above-described well-known method.
  • An antibody of the present invention may be used as a reagent for detecting or diagnosing a leptospirosis.
  • a sample subjected to a method of detection and/or diagnosis according to the present invention is not particularly limited as long as it is a biological sample that may possibly contain Leptospira, for example, urine, blood, etc.
  • an antibody that binds to a Leptospira antigen is preferably used in terms of detection or diagnosis sensitivity.
  • a method for detecting a leptospiral antigen or diagnosing a leptospirosis using an znti-Leptospira antibody may comprise, for example, the steps of: *
  • step (b) allowing reaction between the antigen-antibody complex formed in step (a) and an antibody labeled for detection.
  • a method for detecting or diagnosing using a detection or diagnosis agent of the present invention may be any method as long as it is an antibody-employing assay, i.e., dipstic assay; an immunochromatography(ICG)-based method such as lateral flow assay (LFA); an immunological assay such as enzyme-linked immunosorbent assay (ELISA), fluorescent immunoassay, radioimmuno assay (RIA), luminescent immunoassay; an enzyme antibody method; a fluorescent antibody method; latex agglutination reaction; a latex turbidimetry method, hemagglutination reaction, particle agglutination and western blot assay.
  • ICG immunochromatography
  • LFA lateral flow assay
  • an immunological assay such as enzyme-linked immunosorbent assay (ELISA), fluorescent immunoassay, radioimmuno assay (RIA), luminescent immunoassay
  • an enzyme antibody method a fluorescent antibody method
  • latex agglutination reaction
  • a solid-phased antibody may be used for detecting a leptospiral antigen or diagnosing a leptospirosis in a sample.
  • an insoluble granular marker can be preferably used, since a rapid and simple determination can be achieved by observing a color with the naked eyes.
  • the insoluble granular marker refers to a particle capable of coloring by itself among particles used as the labeling substance in the ICG. Examples are a colloidal metal particle such as gold colloid and a platinum colloid; a synthetic polymer particle such as a polystyrene colored by a pigment or the like (colored synthetic polymer particle); a polymerized-dye particle and the like.
  • markers may be used in a form of beads, a filter, a membrane or the like, or as a carrier for affinity chromatography.
  • colloidal gold-based immunoassay using a gold-conjugated monoclonal antibody may be employed.
  • a method of detection and/or diagnosis according to the present invention is carried out by enzyme-linked immunosorbent assay, fluorescent immunoassay, radioimmuno assay or luminescent immunoassay, it may be performed by a sandwich method or a competitive method.
  • a sandwich method at least a solid-phased antibody or a labeled antibody is an antibody of the present invention.
  • a labeled antibody refers to an antibody that is labeled with a labeling substance, such labeled antibodies may be used for detecting or quantifying an antigen contained in a sample.
  • a labeling substance that may be used with the present invention is not particularly limited as long as its presence can be detected through physical or chemical binding to the antibody.
  • labeling substances include enzymes, fluorescent substances, chemiluminescent substances, biotin, avidin and radioisotopes, more specifically, enzymes such as peroxidase, alkaline phosphatase, ⁇ -D-galactosidase, glucose oxidase, glucose-6-phosphate dehydrogenase, alcohol dehydrogenase, malate dehydrogenase, catalase, luciferase and acetylcholinesterase, fluorescent substances such as fluorescein isothiocyanate, dansyl chloride and tetramethyl rhodamine isothiocyanate, radioisotopes such as 3 H, l4 C, 125 I and 13 l l, biotin, avidin and chemiluminescent substances.
  • enzymes such as peroxidase, alkaline phosphatase, ⁇ -D-galactosidase, glucose oxidase, glucose-6-phosphate dehydrogena
  • a binding method between a labeling substance and an antibody may be a known method such as glutaraldehyde method, maleimide method, pyridyl disulfide method or periodic acid method.
  • the leptospirosis can be assessed utilizing detection results obtained by the detection method described above as indexes. When a detection result exceeds a predetermined reference value, it is regarded as leptospirosis-positive, and when a detection result is equal to or lower than the predetermined reference value, it is regarded as leptospirosis-negative. When a result is positive, it is judged that there is a possibility of leptospirosis. Thus, the state of cancer can be assessed.
  • the predetermined reference value is suitably set depending on the type of cancer.
  • kits comprising one or more of the aforementioned antibodies, optionally, with instructions for use in detecting or diagnosing bacterial diseases associated with Leptospira spp in humans, other animals and birds.
  • a kit for detecting a Ieptospiral antigen or a kit for diagnosing a leptospirosis according to the present invention comprises an antibody of the present invention.
  • An antibody used in this respect may be an immobilized antibody or a labeled antibody described above.
  • the kit of the present invention may comprise a gold-conjugated monoclonal antibody for detecting a complex formed via antigen-antibody binding reaction.
  • the kit of the present invention may comprise, other than these antibodies, various reagents in order to allow effective and simple use of the kit. Examples of such reagents include a phosphate buffer used for dissolving a test sample or washing an insolubilized carrier, a substrate for measuring an enzymatic activity when an enzyme is used as a labeling substance of the antibody, and a reaction terminator thereof.
  • Bacteria and culture Bacteria and culture. Bacteria used in this study are listed in Table 1. These bacteria were cultured in modified Korthof's medium (23) for Leptospira spp., Brain Heart Infusion (BHI) broth (Difco) for Streptococcus and Enterococcus, BCYEa for Legionella, and Luria Bertani (LB) medium for E.coli and Pseudomonas. These organisms were then used to examine the specificity and sensitivity of the assays developed or as infection agents to hamsters ⁇ Leptospira only).
  • BHI Brain Heart Infusion
  • LB Luria Bertani
  • mice Monoclonal antibody production. 6-week old BALB/c mice were primed intraperitoneally with 0.2 ml of a mixture of equal volume of 0.1 mg of the heat killed L. interrogans serovar Icterohaemorrhagiae strain RGA (1.0 X 10 8 cells/ml in PBS) and Freund's complete adjuvant. The mice were immunized two more times at 1-week intervals using the same immunogen and the same route, but with Freund's incomplete adjuvant. Three days after the last booster, the mice were sacrificed. Hybridomas were generated following fusion of splenocytes with P3-X63-Ag8.653 myeloma cells and selected cultures were grown following standard procedure (15).
  • Hybridomas were screened for secretion of the desired antibodies with ELISA and western blot using homologous sonicated antigen. Positive hybridoma cells were cloned using limiting dilution to obtain antibodies from a single cell. Hybridoma culture supernatants or ascitic fluid which were harvested after in vivo culture of hybridoma were used as 1H6 monoclonal antibody (MAb) source. Purification of protein from hybridoma was carried out by ammonium sulphate precipitation, followed by affinity chromatography (16) through HiTrap Protein G HP Column (GE healthcare) in the presence of 1.5 M glycine pH 9.0. Purified antibody was analyzed by SDS-PAGE, and quantitative measurement was determined by UV absorption (16). The immunoglobulin subclass was determined using a mouse monoclonal antibody isotyping kit (GE Healthcare) following the manufacturer's instructions.
  • LPS lipopolysaccharide
  • Pre-treatment of urine was performed using Leptospira -infected and non-infected hamster urine treated using several methods such as: (i) boiling for 5 minutes (20) ; (ii) centrifugation at 20,000 X g for 15 min (21) followed by resuspension of precipitate with phosphate buffer pH 7.2; (iii) ultrafiltration and concentration (22); and (iv) boiling for 5 min, centrifugation 1,000 X g for 15 min, and centrifugation (for ultrafiltration and concentration) of supernatant.
  • MAT Microscopic agglutination test (MAT). MAT of the sera of the same patients with urine samples was performed using the standard method (23, 24). The endpoint titer was the serum dilution which gave ⁇ 50% agglutination at a titer of > 1 :400.
  • Gold conjugation of MAbs Gold colloid with a 40 nm diameter (BB International, UK) was adjusted to pH 9 using 0.1 M K 2 C0 3 (25) and then mixed with 23 ⁇ g/ml purified MAbs. After one-hour incubation at room temperature with slow mixing, 0.1% of skim milk was added to block unconjugated sites and incubated for 10 minutes. Gold-conjugated antibodies were separated by centrifugation at 6,000 X g for an hour, washed two times with 2 mM borate buffer (pH 7.2), and kept in 10% initial volume of storage buffer (2 mM borate buffer pH 7.2; 0.1% skim milk) (26).
  • (i) Membrane Nitrocellulose membrane HF240 (Millipore, US) was cut into 0.5 cm width. 2 ⁇ g of 1H6 MAb in 2 ⁇ was dropped on the test (T) area, while 2 ⁇ of goat anti-mouse IgG antibody (Rockland, US) in 2 ⁇ was dropped on the internal control (IC) area. The membrane was dried in a desiccator for 1-2 hours at 37°C. In order to block the unconjugated areas, the membrane was dipped in 10 mM phosphate buffer pH 7.2 containing 1% skim milk for 15 min, then washed two times in the same buffer to wash off any excessive blocking reagent. The membrane was then dried overnight at room temperature.
  • Conjugate pad Glass fiber conjugate pads (Millipore, US), size 1 X 0.5 cm, were dipped in gold-conjugated antibodies dissolved in 2 mM borate buffer pH 7.2 + 5% sucrose. The pad was then dried at 37°C for 2 hours (27).
  • Sample pad Sample pads were treated according to Shim et.al (28) with some modifications. Cellulose fiber sample pads (Millipore, US), size 1.5 X 0.5 cm, were dipped into the sample pad buffer (50 mM borate buffer pH 7.2; 5% sucrose, 0.5% Tween 20; 5% dextran; and, 0.1% skim milk), then dried at 50°C.
  • sample pad buffer 50 mM borate buffer pH 7.2; 5% sucrose, 0.5% Tween 20; 5% dextran; and, 0.1% skim milk
  • Leptospira strains were cultivated in modified Korthof's medium for several days and counted using a Thoma counting chamber. Leptospiral culture was centrifuged at 10,000 X g for 20 minutes. Cultures of Streptococcus, Enterococcus, Legionella, E. coli, and Pseudomonas were centrifuged at 9,000 X g for 20 min. The pellets were washed and resuspended in 10 mM phosphate buffer pH 7.2. The assay detection limit was tested using dilutions of 10 7 to 10 1 cells of I. interrogans serovar Manilae strain K64, a local isolate from the Philippines (24). The cells were sonicated in the same buffer and used for the sensitivity test. The specificity of the assays was tested using bacteria listed in Table 1.
  • PCR Polymerase Chain Reaction
  • the primers used in this experiment were specific for flaB gene (L-flaB-Fl 5'TCTCACCGTTCTCTAAAGTTCAAC-3' (SEQ ID NO: 1), L-flaB-Rl
  • Treatment of urine samples was carried out to determine the condition that could increase the sensitivity and specificity of the assays. Treatment was necessary because of non-specific bindings based on the immunoblotting results of urine from suspected leptospirosis patients.
  • the result of the dipstick assay showed that leptospiral antigen could be detected in the urine of Leptospira-mfected hamsters after using all treatments.
  • the dipstick assay became positive using urine of uninfected hamsters that was not treated, only boiled, or only centrifuged.
  • boiling urine eliminated non-specific reactions by substances in it.
  • boiling alone was not enough to eliminate it, probably due to heat-stable substance that bound to gold-conjugated antibody. Centrifugation at 20,000 X g was used to increase the sensitivity of the assay, but it could not eliminate the non-specific substances that interfered with the result.
  • Dipstick assay for Leptospira-infected hamster urine The minimum sample size calculated for this study was 13. Forty-six urine samples of Leptospira-infected hamsters were collected and stored in -20°C prior to testing. Optimum conditions of urine treatment mentioned above were used prior to analyzing the infected hamster urine using dipstick assay. Figure 2 shows the representative results of the dipstick assay.
  • results of the dipstick assay showed that 28 of 46 samples of hamster urine (60.9%) were positive, while 29 of 46 samples (63.1%) were positive in culture.
  • the sensitivity and specificity of the dipstick assay were calculated by comparing the results with gold standard (i.e., culture) (Table 2) and were found to be 76% and 65%, respectively. Some discrepancies between the dipstick assay and culture results were observed.
  • Urine from suspected leptospirosis patients was collected, stored in -20° C prior to test and then tested using dipstick assay, ICG-based LFA and PCR. Patients' sera were tested using MAT. For the human urine samples we performed dipstick, ICG-based LFA and PCR, because the amount of the samples was enough for performing all 3 methods. The representative results of ICG-based LFA are shown in Fig.3. For determining the specificity of the assays, we also tested the urine from healthy persons using dipstick and ICG-based LFA. PCR was used to confirm the results of the 2 assays.
  • flaB PCR was more sensitive in detecting Leptospira in urine than rrl. We found discrepancies between the results of MAT and PCR. Since these 2 methods were used as gold standards, to calculate for the sensitivity and specificity of the dipstick and ICG-based LFA, the gold standard was thought to be positive when either of these two methods was positive.
  • the assays developed in this study could detect the antigen from the first day after the onset of illness as shown in Table 3. Compared to the gold standard (PCR and/or MAT), different results of both assays were found mostly by the fifth day after onset in 19 samples, and became relatively consistent from the 6 th day and thereafter. By the fifth day after onset, we found 6 false negative and 3 false positive results for the dipstick assay and for ICG-based LFA, 3 were false negative and 2 were false positive. After the sixth day, we found only 1 false positive for ICG-based LFA, and 1 false negative for both assays with unknown time of disease onset.
  • Patoc (1 :6400), Canicola (1 :800), Ratnapura (1 :800), Semaranga (1 :800)
  • Leptospirosis is an infectious disease prevailing around the world.
  • the diagnosis of leptospirosis is mainly carried out by MAT, culture, and PCR methods (23).
  • the limitations of these assays i.e., laborious, time-consuming, and expensive) brought out the need for a simple, fast, and inexpensive diagnostic method.
  • a rapid diagnostic assay i.e., dipstick and ICG-based LFA
  • ICG assay for leptospirosis which is used as one of the diagnostic methods, is mainly used for detection of antibodies (9, 34, 35).
  • the monoclonal antibody 1H6 which was used in this study, has been characterized to be against the lipopolysaccharide of Leptospira. This antibody was tested for purified LPS of several bacteria prior to the development of the diagnostic kit. The monoclonal antibody was reactive to 12 kDa LPS of Leptospira as seen in Figure 1. Leptospiral LPS is known to have high antigenicity and therefore, anti-LPS antibodies are found in human and animal sera (37).
  • Dipstick assay was applied to the urine samples of 46 infected hamsters.
  • the culture method was used as a comparison method and a gold standard for calculation of sensitivity and specificity.
  • the culture method is known to be one of the reference diagnostic tests for leptospirosis (4).
  • the sensitivity and specificity of the dipstick assay were 76% and 65%, respectively. These results were quite low when comparing dipstick assay with detection of Leptospira antibody in serum (41, 42), which showed >90% sensitivity and specificity. This might be caused by the relatively low amount of Leptospira antigens in the sample.
  • the detection limit for the dipstick assay was 10 6 cells of Leptospira.
  • the concentration of Leptospira that is usually found in urine of dogs ranges from 10 1 - 10 6 cells/ml (43). Monahan et.al. (10) reported that rats could excrete high concentrations of Leptospira (10 7 cells/ml) after three weeks of infection.
  • the sensitivity and specificity of the dipstick assay used for human urine were 80% and 74%, respectively, higher than those (76% and 65%) of hamster urine. Although we do not have evidence yet, we think that this is because the concentration of Leptospira antigen in hamster urine was mostly below the detection limit and less than in human urine. However the sensitivity and specificity of dipstick assay for human urine were lower than ICG-based LFA which were 89% and 87%, respectively. This might be caused by the treatments of the sample pad and the conjugate pad.
  • flaB-polymerase chain reaction flaB-PCR
  • RFLP restriction fragment length polymorphism

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Abstract

La présente invention concerne un anticorps monoclonal qui se lie à un antigène de leptospire.
PCT/JP2014/056384 2013-05-24 2014-03-05 Anticorps qui se lie à un antigène de leptospire WO2014188763A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001064753A1 (fr) * 2000-02-29 2001-09-07 Roselieb, Markus Diagnostic de leptospirose par detection d'antigenes a partir d'anticorps monoclonaux
JP2005027652A (ja) * 2003-06-17 2005-02-03 Japan Science & Technology Agency レプトスピラタンパク質、これをコードする遺伝子、レプトスピラ病に対する感染防御活性を有する薬剤、及び診断キット

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001064753A1 (fr) * 2000-02-29 2001-09-07 Roselieb, Markus Diagnostic de leptospirose par detection d'antigenes a partir d'anticorps monoclonaux
JP2005027652A (ja) * 2003-06-17 2005-02-03 Japan Science & Technology Agency レプトスピラタンパク質、これをコードする遺伝子、レプトスピラ病に対する感染防御活性を有する薬剤、及び診断キット

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
CHALLA S. ET AL.: "Passive immunization with Leptospira LPS-specific agglutinating but not non-agglutinating monoclonal antibodies protect guinea pigs from fatal pulmonary hemorrhages induced by serovar Copenhageni challenge", VACCINE, vol. 29, no. 27, 2011, pages 4431 - 4434 *
CHIRATHAWORN C. ET AL.: "Detection of Leptospira in urine using anti-Leptospira-coated gold nanoparticles", COMP. IMMUNOL. MICROBIOL. INFECT. DIS., vol. 34, no. 1, 2011, pages 31 - 34 *
MURRAY G. ET AL.: "Mutations affecting Leptospira interrogans lipopolysaccharide attenuate virulence", MOL. MICROBIOL., vol. 78, no. 3, 2010, pages 701 - 709 *
SAENGJARUK P. ET AL.: "Diagnosis of human leptospirosis by monoclonal antibody-based antigen detection in urine", J. CLIN. MICROBIOL., vol. 40, no. 2, 2002, pages 480 - 489 *
WIDIYANTI D. ET AL.: "Development of Immunochromatography-Based Methods for Detection of Leptospiral Lipopolysaccharide Antigen in Urine", CLIN. VACCINE IMMUNOL., vol. 20, no. 5, 2013, pages 683 - 690 *
ZAPATA S. ET AL.: "Characterization of a lipopolysaccharide mutant of Leptospira derived by growth in the presence of an anti-lipopolysaccharide monoclonal antibody", FEMS MICROBIOL. LETT., vol. 309, no. 2, 2010, pages 144 - 150 *

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