TITLE OF THE INVENTION

An antibody that binds to leptospiral antigen

TECHNICAL FIELD

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.

BACKGROUND ART

Leptospira, belonging to the order Spirochaetales and family Leptospiraceae, is a spiral-shaped bacterium which is 0.1 μπι in diameter, 6-20 μηι 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).

Several assays could be applied for leptospirosis diagnosis. The World Health Organization (WHO) has specified standard requirements for leptospirosis patients, such as sufficient growth of leptospires from normally sterile organ, a clear amplified DNA band in PCR and a four-fold increase of titers between acute and convalescent sera in a microscopic agglutination test (MAT) (4). Unfortunately, MAT is laborious and time-consuming, and PCR is expensive due to a need for sophisticated equipment. Because leptospirosis is commonly found in developing or under-developed countries, there is a need for rapid, reliable, and inexpensive diagnostic kits. Development of diagnostic assay were achieved several years ago such as flow through assay (5), IgM dipstick (6), immunofluorescence assay (7), and latex agglutination (8), which detected the presence of antibodies in human sera. Nevertheless, the sensitivity and specificity of these methods were low when performed during the early stage of infection with Leptospira because the appropriate immune response may not have been elicited yet during the time of specimen collection. For example, the dipstick assay (6) which can detect the presence of IgM and often used in initial screening of leptospirosis has low sensitivity when applied to patient serum (9). Antigen detection assay might offer an effective solution to this difficulty, because antigen can be detected earlier after infection (10). Assays for detection of Leptospira antigen and DNA are still being developed in some studies (11, 12). 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).

SUMMARY OF THE INVENTION

In the present invention, we tried to develop an ICG-based assay for antigen detection of Leptospira, which could be applied in endemic areas of leptospirosis and applicable for detecting antigen in urine.

The present invention provides as follows:

(1) An antibody that binds to leptospiral antigen.

(2) The antibody of (1), wherein the antigen is a core lipopolysaccharide of Leptospira spp.

(3) The antibody of (1), wherein the antibody is a monoclonal antibody.

(4) The antibody of (1), wherein the antibody is coupled to colloidal gold. (5) The antibody of (1), wherein the antibody is coupled to a detectable label.

(6) The antibody of (1), wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody.

(7) 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.

(8) An antibody that binds to an antigen determinant to which the antibody according to any one of (1) to (7) binds.

(9) A hybridoma that produces the antibody according to (3).

(10) An agent for detecting leptospirosis, comprising the antibody according to any one of (l) to (8).

(11) An agent for diagnosing leptospirosis, comprising the antibody according to any one of (1) to (8).

(12) A method of detecting leptospiral antigen, allowing reaction between the antibody according to any one of (1) to (8) and a test sample.

(13) A method of detecting leptospiral antigen that comprises:

(a) reacting a test sample obtained from a subject with the antibody according to any one of (1) to (8); and

(b) detecting the complex formed by the antibody and leptospiral antigen

(14) A method of diagnosing leptospirosis that comprises:

(a) reacting a test sample obtained from a subject with the antibody according to any one of (1) to (8); and

(b) detecting the complex formed by the antibody and leptospiral antigen.

(15) The method of (12) or (13), wherein the test sample is urine.

(16) The method of (14), wherein the test sample is urine.

(17) A kit for detecting leptospiral antigen or diagnosing leptospirosis, comprising the antibody according to any one of (1) to (8).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows detection 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

2 and 3: Uropathogenic E. coli C16 & C17; lanes 4 and 5: Serratia marcescens J1& J5; lane 6: Streptococcus pyogenes; lane 7: Borrelia burgdorferi B31; lane 8: Borrelia afzelii P/Gau; lane 9: L. interrogans Hebdomadis; and lane 10 BSA.

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.

MODE FOR CARRYING OUT THE INVENTION

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. We used 1H6 monoclonal antibody (MAb) which is specific to lipopolysaccharide (LPS) common among Leptospira spp. The MAb was coupled to 40 nm-diameter colloidal gold and the amount of labeled antibody and immobilized antibody were 23 μg and 2 μg 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. Pre-treatment of urine sample by boiling and centrifugation (for ultrafiltration and concentration) eliminated non-specific reactions which occurred in the assay. The sensitivity and specificity of ICG-based lateral flow assay (LFA) were 89% and 87%, respectively, which were higher than those of the dipstick assay, which were 80% and 74 %, respectively. In summary, this ICG-based LFA could be used as an alternative diagnostic assay for leptospirosis.

1. Definition

As used herein, 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. For example, 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. Examples of 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.

The term "monoclonal antibody" as used herein 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. (1975) Nature, 256:495, a transgenic animal, as described by, for example, (see e.g., Lonberg, et al. (1994) Nature 368(6474): 856-859), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), or using phage antibody libraries using the techniques described in, for example, Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991).

The term "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.

The term "humanized antibody" refers to an immunoglobulin that includes at least one humanized immunoglobulin chain (i.e., at least one humanized light or heavy chain). The term "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.

The term "human antibody," as used herein, 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.

In one embodiment, 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.

The term "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).

Also encompassed by the present invention are 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. 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.

As used herein, the term "subject" includes any human or non-human animal. For example, the methods of the present invention can be used to detect leptospiral antigen or to diagnose a subject having a leptospirosis. In a particular embodiment, the subject is a human. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dog, cow, chickens, etc.

The term "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.

2. Methods for Producing Αη -Leptospira Antibody

(1) Bacteria and Bacterial Antigens

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.

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. For example, Leptospira spp are commercially available. Alternatively, infectious bacteria such as Leptospira spp may be obtained from an infected host by isolation and culture.

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.

(2) Polyclonal antibodies

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 μg with an adjuvant. Examples of 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. Preferably, 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. After confirming that the antibody titer has increased to a sufficient level, whole blood can be collected to separate and purify the antibody according to a general method. For example, 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.

(3) Monoclonal Antibodies 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. In particular embodiments, the antibodies are fully human monoclonal antibodies.

In one embodiment, a hybridoma method is used for producing an antibody that binds lipopolysaccharide of Leptospira spp. In this method, 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.

(i) Collection of antibody-producing cell

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 μg 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.

(ii) Cell fusion

In order to obtain a hybridoma, 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. As the myeloma cell to be fused to the antibody-producing cell, a generally available cell line from an animal such as a mouse may be used. Preferably, 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. Examples of myeloma cells 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. As a cell fusion promoter, an average molecular weight of 1000-6000 daltons of polyethylene glycol, Sendai virus or the like may be used. Alternatively, a commercially available cell fusion device employing electrostimulation (e.g., electroporation) may be used to fuse an antibody-producing cell with myeloma cells.

(iii) Selection and cloning of hybridoma

A hybridoma of interest is selected from the cells after the cell fusion treatment. According to such a method: 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. As a result, cells that have grown after about 10 days following the beginning of culture with the selection medium may be obtained as hybridomas.

Then, the grown hybridomas are further subjected to screening. 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. Then, 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.

(iv) Collection of monoclonal antibody

As a method for collecting a monoclonal antibody from the established hybridoma, general cell cultivation, ascites production or the like may be employed. In the case of cell cultivation, 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. 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. When purification of the antibody is required in the above antibody collection method, 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.

(v) Binding specificity

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). 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)).

(4) Preparation of recombinant antibody

In certain embodiments, partial antibody sequences derived from antibodies of the invention may be used for producing structurally and functionally related antibodies. For example, antibodies interact with target antigens predominantly through amino acid residues that are located in the six heavy and light chain complementarity determining regions (CDRs). For this reason, the amino acid sequences within CDRs are more diverse between individual antibodies than sequences outside of CDRs. Because 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. et al., 1998, Nature 332:323-327; Jones, P. et al., 1986, Nature 321 :522-525; Tamura et al., J Immunol., 2000 Feb. 1; 164(3): 1432-41; and Queen, C. et al., 1989, Proc. Natl. Acad. See. U.S.A. 86: 10029-10033). Such framework sequences can be obtained from public DNA databases that include germline antibody gene sequences.

Thus, in one embodiment, 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.

One of the preferable embodiments of an anti-Leptospira antibody of the present invention is a recombinant antibody. Examples of such 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.

In order to produce a humanized antibody, so-called CDR grafting (CDR implantation) technique may be employed. 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).

Techniques for preparing a human antibody are known. In addition, a method for preparing a gene sequence common with human by a genetic engineering procedure has been established. 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. Acids Res., (1998) 26, 3447-3448; Yoshida, H.et.al., Animal Cell Technology: Basic and Applied Aspects, (1999) 10, 69-73 (Kitagawa, Y, Matuda, T. and lijima, S. eds.), Kluwer Academic Publishers; Tomizuka, K.et.al., Proc. Natl. Acad. Sci. USA, (2000) 97, 722-727, etc.), or a method for obtaining a human antibody from a phage display selected from a human antibody library (see Wormstone, I. M.et.al, Investigative Ophthalmology & Visual Science., (2002) 43 (7), 2301-8; Carmen, S. et.al., Briefings in Functional Genomics and Proteomics, (2002) 1 (2), 189-203; Siriwardena, D. et.al., Opthalmology, (2002) 109 (3), 427-431, etc.). (5) Preparation of antibody fragment

Examples of the 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. The amino acid residues substituted with cysteine residues may be selected based on the conformational prediction of the antibody (Protein Engineering, 7, 697-704, 1994). 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 . 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).

According to the present invention, 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.

(6) Characteristics of the antibodies

Antibodies of the present invention generally are characterized as having one or more of the following properties:

(i) specific to lipopolysaccharide common among Leptospira spp; (ii) the detection limit of the assays was 10 ⁶ cells/ml when disrupted bacterial whole cells were used;

(iii) no cross-reacting with a broad spectrum of other bacteria;

3. Detection agent and method, and diagnosis agent and 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.

For detection or diagnosis of a leptospirosis, 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: *

(a) allowing reaction between an antibody of the present invention or a fragment thereof and a sample; and

(b) allowing reaction between the antigen-antibody complex formed in step (a) and an antibody labeled for detection.

At the end of the reaction, signals from the labeled antibodies are detected. 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.

A solid-phased antibody may be used for detecting a leptospiral antigen or diagnosing a leptospirosis in a sample. Also in the ICG-based method of the present invention, 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. These markers may be used in a form of beads, a filter, a membrane or the like, or as a carrier for affinity chromatography. In a preferred embodiment of the invention, colloidal gold-based immunoassay using a gold-conjugated monoclonal antibody may be employed.

When 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. In the case of 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. Specific examples of such labeling substances include enzymes, fluorescent substances, chemiluminescent substances, biotin, avidin and radioisotopes, more specifically, enzymes such as peroxidase, alkaline phosphatase, β-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. 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. According to the present invention, 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.

4. Kit for detection or diagnosis

Further provided by the invention is a kit 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.

For example, when an antibody of the present invention is used for a lateral flow colloidal gold immunoassay, 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. EXAMPLES

Other embodiments of the present invention are described in the following Examples. The present invention is further illustrated by the following examples which should not be construed as further limiting. The contents of Sequence Listing, figures and all references, patents and published patent applications cited throughout this application are expressly incorporated herein by reference.

MATERIALS AND METHODS

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).

Table 1. List of organisms used in this stud

Species Strain Serovar or serogroup

Akiyami A Autumnalis

Hond Utrecht IV Canicola

K64 Manilae

Leptospira interrogans

K5 Grippotyphosa

Ictero No. 1 Icterohaemorrhagiae K37 Losbanos

Hebdomadis Hebdomadis

Poi Poi

Leptospira borgpetersenii Perepelitsin Tarassovi

6 Javanica

Leptospira biflexa Patoc 1 Patoc

K-12 MG 1655

Escherichia coli C16

C17

Philadephia- 1

Legionella pneumophila

(ATCC 33152)

Enterococcus faecalis Portland (ATCC 29212)

Pseudomonas aeruginosa PAGU 221

Streptococcus pneumoniae ATCC 1127

Jl

Serratia marcescens

J5

Borrelia burgdorferi B31

Borrelia afzelii P/Gau

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.

Antigen specificity of MAbs. Specificity of the generated MAbs, 1H6, was tested by immunoblotting against bacterial antigens including the lipopolysaccharide (LPS) of several bacterial species. The LPS was extracted using chloroform/methanol/extraction (17) followed by silica column chromatography (18) using iatrobeads 6RS-8060 (Iatron lab. Inc., USA). LPS was analyzed by SDS-PAGE with ProQ emerald staining (Invitrogen). Immunoblotting of LPS was performed using IH6 MAb.

Animal and human urine. Four-week old Golden Syrian hamsters (Japan SLC, Inc., Hamamatsu, Japan) were infected with i. interrogans serovars Manilae, Losbanos, Pyrogenes and Canicola. Seven to fourteen days after infection, urine was collected by aseptic aspiration from the urinary bladder of the dead or sacrificed hamsters. A part of the urine was then cultured in modified Korthof's medium and observed until one month of incubation at 30°C. Urine was also used to find the optimum condition for sample treatment. Forty-four urine samples of suspected leptospirosis patients and fourteen samples of healthy persons were obtained from the College of Public Health, University of Philippines Manila and Kyushu University, respectively. These urine samples were tested by dipstick, immunochxomatographic assay, and PCR.

Pre-treatment of urine. Optimization of urine treatment 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. Ultrafiltration and concentration of urine in (iii) and (iv) was performed two times by filtering the supernatant with AMICON Ultra 30K (Millipore, US), and collecting the filtrate. It was then filtered and concentrated using AMICON Ultra 10K device (Millipore, US). The concentrate was resuspended using 10 mM phosphate buffer pH 7.2. Centrifugation speed (14,000 X g) and time (i.e., 10 min and 15 min for AMICON 30K and 10K, respectively) were according to the manufacturer's instructions. As a result, urine samples were concentrated 10 times. Those samples were then tested using the dipstick to determine the best condition of urine treatment for analyzing urine samples from patients and hamsters.

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 μg/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).

Preparation of dipstick and immunochromatography(ICG)-based lateral flow assays (LFA).

(i) Membrane: Nitrocellulose membrane HF240 (Millipore, US) was cut into 0.5 cm width. 2 μg of 1H6 MAb in 2 μΐ was dropped on the test (T) area, while 2 μ of goat anti-mouse IgG antibody (Rockland, US) in 2 μΐ 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.

(ii) 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).

(iii) 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.

(iv) Dipstick assay: Forty microliters of pre-treated urine samples were put into a 96-well microliter plate. Twelve microliters of gold-conjugated antibodies were mixed with the sample. The mixture was incubated for 15 minutes at room temperature. The dipsticks were dipped into the mixture and the results were observed for a maximum of 15 min. Positive test results were indicated by two red spots (internal control (IC) area and test area), while negative test results were shown by only one red spot (internal control spot). The test was invalid if no red spot appeared in the IC spot.

(v) ICG-based LFA: One hundred microliters of samples were dropped onto the sample pad. The results were observed for a maximum of 15 min. The interpretation of results was the same as in the dipstick assay.

Sensitivity and specificity of the tests. 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.

Polymerase Chain Reaction (PCR). Urine samples were centrifuged at 13,000 X g for 10 min. Genomic DNA was purified from the pellet using the Illustra bacteria genomicPrep mini spin kit (GE Healthcare, UK) according to the manufacturer's instructions. Two kinds of PCR assays were performed, which targeted flaB gene (793 bp), specific for pathogenic Leptospira (29), and rrl gene (482 bp), specific for Leptospira genus (30).

The primers used in this experiment were specific for flaB gene (L-flaB-Fl 5'TCTCACCGTTCTCTAAAGTTCAAC-3' (SEQ ID NO: 1), L-flaB-Rl

5 ' CTGAATTCGGTTTC ATATTTGCC-3 ' (SEQ ID NO: 2)) and rrl (rrlF

5 ' GACCCGA AGCCTGTCGAG-3 ' (SEQ ID NO: 3), rrlR 5 ' GCC ATGCTTAGTCCCG ATTAC-3 ' (SEQ ID NO: 4)) (30) of Leptospira spp. flaB was amplified under the following conditions: 40 cycles of denaturing at 94°C for 20 seconds; annealing at 50°C for 30 seconds; extension at 72°C for 60 seconds; and, final extension at 72 ^° C for 5 min. The PCR condition for rrl was according to reference 30. PCR products were electrophoresed using 0.7% agarose gel and visualized using ethidium bromide stain.

Statistical analysis. The minimum sample size required for the present study was estimated allowing for an error of 10% and for assumed sensitivity and specificity of 85% each. To test the difference between ICG-based LFA and dipstick, when compared with the gold standard, data were analyzed using the Wilcoxon test using SPSS 17.0.

Human and Animal Ethics. The Ethics Committee on Animal Experiment of the Faculty of Medical Sciences, Kyushu University reviewed and approved all the animal experiments in this study. These experiments were done based on the conditions stated in the Guideline for Animal Experiments of Kyushu University (Law 8 No. 105) and Notification No. 6 of the Government of Japan.

Human samples were obtained after verbal and written explanation of the study and procedures, and after consent of the subjects or their guardian (suspected leptospirosis patients) was obtained. The Ethics Committee of Kyushu University and University of the Philippines Manila approved the conduct of this study on samples from humans.

RESULTS

Characterization of monoclonal antibody. SDS-PAGE analysis of purified monoclonal antibodies was carried out. Under non-reducing condition (i.e., without DTT addition), a band of antibody was seen around 170 kD, while in reducing condition, a heavy chain was noted around 60 kD, and a light chain was seen at 25 kD (31). Based on these results, the anti-leptospiral LPS antibody was successfully purified by the method used here. LPS was extracted, purified, and electrophoresed as shown in Figure 1 (upper panel). Immunoblotting with 1H6 MAb to the purified LPS showed that the antibody was specific to LPS of L. interrogans (Figure 1, lower panel). We determined that purified MAb was class IgG3 based on typing result.

Optimization of conjugation of gold with monoclonal antibody. Preliminary experiments were performed to find the optimal combination of antibodies (Abs) using both polyclonal and monoclonal antibodies. When polyclonal antibodies purified from guinea pigs infected with five strains of Leptospira were used, we found many false-positive results. Therefore, we tried to use one monoclonal antibody as capture Ab and immobilized Ab, which showed better results compared to other combinations.

Preliminary experiments were also carried out to determine the optimum condition to conjugate antibodies with gold colloid. Prior to conjugation of gold colloid and monoclonal antibody, titration was performed to determine the least amount of antibody that could stabilize gold colloid. Using the method from the manufacturer (BB International Technical Note) with some modifications, the minimal concentration of antibody was determined (data not shown) and 23 μg/ml (four times of minimum concentration) of antibody was used for conjugation with gold colloid. The dose of immobilized antibody was determined to be 2 μg after preliminary experiments using different concentrations.

Sensitivity and specificity of the dipstick assay. Positive result in the dipstick assay was seen from 10 ⁷ to 10 ⁶ cells. Meanwhile, from 10 ⁵ cells, the antigen could not be detected anymore. Therefore, the detection limit of dipstick assay was determined at 10 ⁶ cells/ml when disrupted cells of Leptospira were used.

Specificity of the dipstick assay was tested against several bacteria listed in Table 1. Results showed that the assay has a high specificity for all strains of Leptospira used in this study whether pathogenic or not (L. interrogans serovars Autumnalis, Canicola, Manilae, Gryppotyphosa, Icterohaemorrhagiae; and, L. borgpetersenii serovars Poi, Tarassovi, Javanica; and, L. biflexa serovar Patoc). However, the assay was negative when other bacteria were used such as those whose antigens are known to be excreted in urine (L. pneumophila & S. pneumoniae), and uropathogenic bacteria (E.coli, E. faecalis, and P. aeruginosa). The results showed that this assay could discriminate Leptospira from other bacteria, but could not discriminate pathogenic from non-pathogenic Leptospira.

Determination of the best condition for sample treatment. 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. However, the dipstick assay became positive using urine of uninfected hamsters that was not treated, only boiled, or only centrifuged. In a study by Doskeland and Berdal (32), boiling urine eliminated non-specific reactions by substances in it. In our study, however, 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. A combination of boiling and centrifugal filtration, however, could eliminate the non-specific reactions in uninfected hamster urine. Based on the results of our study and that of Cinco et al (33), lipopolysaccharide of Leptospira has a size between 10 and 30 kD; therefore, we used two kinds of ultrafilters to obtain the antigen with a size between 10 and 30 kD. Using this method, we successfully eliminated the non-specific substance in uninfected hamster urine. These methods, therefore, could be used to pre-treat urine samples of infected hamsters and suspected leptospirosis patients prior to analysis using the two diagnostic methods.

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.

Table 2. Comparison of dipstick assay results with culture method using infected hamster urine

Culture method

Positive Negative Total

Positive 22 6 28

Dipstick

Negative 7 11 18

Total 29 17 46

Sensitivity: 0.76 (76%); 95% CI:0.63-0.89

Specificity: 0.65 (65%); 95% CI: 0.51-0.79

Dipstick and ICG-based LFA for urine of suspected (eptospirosis humans.

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. From 44 samples of suspected leptospirosis patients, 4 samples were negative for all assay, therefore we could deduce that these patients were not infected by Leptospira. The result from the urine of suspected leptospirosis patients and healthy persons showed that 35 of 58 samples (60.3%) were positive for MAT or PCR. Thirty- four of the 58 samples (58.7%) were positive in the dipstick assay-and ICG-based LFA as shown in Tables 4 and 5. The sensitivity and specificity of dipstick assay were 80% and 74%, respectively. The results of ICG-based LFA and gold standard are shown in Table 5, and the sensitivity and specificity were 89% and 87%, respectively. The sensitivity and specificity of ICG-based LFA were higher than the dipstick. The Wilcoxon analysis also showed that ICG-based LFA was not significantly different from the gold standard (P = 0.3557, a = 0.05). The efficiency of dipstick and ICG-based LFA were 78% and 88%, respectively. These results showed that ICG-based LFA was better than dipstick due to its higher sensitivity, specificity, and efficiency.

Table 3. Results of MAT, PCR, Dipstick and ICG-based LFA on suspected leptospirosis patient urine.

Patient Day of Dipstick ICG-based PCR MAT MAT serovars (titers) no. urine LFA

collection

after onset

of illness

1 1 + + - + Copenhageni (1:400)

2 2 + - + -

3 2 + + + + Patoc (1:3200)

4 2 + + + + Poi (1:400)

5 3 - - - -

6 3 + - - -

7 3 + - 8 3 + +

9 3 + + + + Patoc (1:1600), Poi (1:400)

10 3 + + + + Patoc (1:400)

11 4 - - - + Manilae (1:800), Patoc (1:800)

12 4 - + - + Poi (1:400)

13 4 - + - + Copenhageni (1:800), Patoc (1:400)

14 4 + + + + Patoc (1:1600)

15 4 + + - + Patoc (1:400)

16 4 + + - -

17 5 - + + -

18 5 + + + + Pyrogenes (1:400)

19 ^* 5 - + + -

20 6 + + + + Patoc (1:400)

21 6 + + + + Canicola (1:800)

22 6 + + + -

23 7 - - - -

24 7 + + - + Patoc (1:6400)

25 8 - - . - -

26 8 + + + + Patoc (1:6400)

27 8 + + - + Patoc (1:800)

28 8 + + + + Patoc (1:1600), Copenhageni

(1:800), Semaranga (1:800)

29 9 + + - + Poi (1:800), Patoc (1:400)

30 10 - + - -

31 12 + + - + Losbanos (1:400)

32 13 + + + + Copenhageni (1:400), Losbanos

(1:400)

33 14 - - - -

34 15 + + + + Patoc (1:3200)

35 18 + + - + Ratnapura (1:1600), Poi (1:800),

Patoc (1:800), Semaranga (1:400)

36 21 - - - -

37 22 + + + + Patoc (1:3200), Pyrogenes (1:800)

38 52 + + - + Ratnapura (1:1600), Copenhageni

(1:400), Patoc (1:400), Semaranga (1 :400)

Patoc (1 :6400), Canicola (1 :800), Ratnapura (1 :800), Semaranga (1 :800)

40 unknown + + + + Patoc (1 :800)

41 unknown + + - + Patoc (1 :800), Losbanos (1 :400)

42 unknown + + + + Semaranga (1 : 1600), Patoc (1 :400)

43 unknown - - + -

44 unknown + + + -

Table 4. Comparison of dipstick assay results with PCR (human urine) and/or MAT (human sera)

PCR and/or MAT

Positive Negative Total

Positive 28 6 34

Dipstick

Negative 7 17 24

Total 35 23 58

Sensitivity: 0.8 (80%); 95% CI: 0.7-0.9

Specificity: 0.74 (74%); 95% CI: 0.63-0.85

Table 5. Comparison of ICG-based LFA results with PCR (human urine) and/or MAT (human sera)

PCR and/or MAT

Positive Negative Total

ICG-based Positive 31 3 34

LFA Negative 4 20 24

Total 35 23 58

Sensitivity: 0.89 (89%); 95% CI: 0.78-0.96

Specificity: 0.87 (87%); 95% CI: 0.8-0.96

DISCUSSION

Leptospirosis is an infectious disease prevailing around the world. The diagnosis of leptospirosis is mainly carried out by MAT, culture, and PCR methods (23). However, the limitations of these assays (i.e., laborious, time-consuming, and expensive) brought out the need for a simple, fast, and inexpensive diagnostic method. In this study, we tried to develop a rapid diagnostic assay (i.e., dipstick and ICG-based LFA) for leptospirosis, which might fulfill these requirements and be applicable in all countries. Nowadays, ICG assay for leptospirosis, which is used as one of the diagnostic methods, is mainly used for detection of antibodies (9, 34, 35). However, these assays are difficult to use for early diagnosis, because of low sensitivity and the fact that the immune response might not have developed enough to be detected during early stages of infection. The antigen of Leptospira could become a good target for detection, particularly during the early phase of infection, because it is excreted in the urine from day 6 post infection (10, 36). Therefore, we tried to develop ICG-based methods which aimed to detect the antigen of Leptospira using mti-Leptospira antibodies.

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).

We hypothesized that there are multiple epitopes in one molecule of LPS, and that the capture antibody bound to one of the epitopes, while the immobilized antibody bound to unoccupied epitopes. Usage of single antibody for capture and immobilized antigen were also reported in ICG-based methods, which were successful in detecting Campylobacter antigens (39) and botulinum neurotoxin (40). This single antibody was adapted for our dipstick and ICG-based LFA, which were tested for sensitivity and specificity against Leptospira and other bacteria. For sensitivity, we have tested different concentrations of Leptospira culture, and the detection limit was 10 ⁶ cells/ml. This result was almost the same as the detection limit of immunochromatographic assay for other bacteria (26, 38). For specificity assay, we tested this single antibody system against several strains of Leptospira and bacteria which are commonly found in urine or are known to be the causative agents of urinary infection. Results showed that all Leptospira could be detected but the non-Leptospira bacteria used in this study could not, which means this assay could discriminate Leptospira from other bacterial antigen.

The method was applied to infected and non-infected hamster urine and compared with cultures. Direct applications of the hamster urine always gave false-positive results for non-infected hamster urine. This might be caused by non-specific reactions with unknown substances in the urine. Therefore, pre-treatment of the urine was necessary to eliminate these substances. Boiling urine for three minutes could cause the liberation of LPS antigen from naturally formed antigen-antibody complex and increase specific antigen-antibody reaction (32). However, in this study, pre-treatment of urine by boiling only was not enough to eliminate non-specific reactions. A combination of boiling and concentrating (through ultrafiltration) urine could eliminate non-specific reactions in uninfected hamster urine in our study. Concentration and filtration of urine were believed to increase the sensitivity and specificity of diagnostic assays (20). This result was consistent when using human urine.

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. We have estimated that 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.

Urine samples from 44 suspected leptospirosis patients and fourteen healthy humans were tested by dipstick and ICG-based LFA. PCR and MAT were also performed and were used as gold standards because we found several discrepancies between PCR and MAT results as shown in Table 3. Some samples were positive in PCR but negative by MAT. This might be because some of the serum samples were obtained during the acute phase of illness; therefore, the immune response elicited was not enough to be detected using MAT. We also found that some PCR-negative samples were diagnosed as positive by MAT. We think it might be caused by Leptospira, which is not always excreted in the urine of leptospirosis patients, or the number of Leptospira in the urine was below the detection limit of PCR. Another possible cause of this discrepancy is that antibiotics, used by patients, eradicated the Leptospira in the kidneys, making them undetectable by PCR. Recently, it has been hypothesized that MAT is an imperfect gold standard for leptospirosis (44). Therefore, a combination of PCR and MAT as gold standard might more precisely predict the true sensitivity and specificity of the diagnostic assays that we developed.

The result in Table 3 showed that both of our assays (dipstick and ICG-based LFA) could detect the leptospiral antigen in urine from the first day after the onset of illness. Saengjaruk et.al. (9) also showed varying results of antigen detection in urine collected consecutively, caused by the intermittent shedding of Leptospira in urine. Results of urine from patients, tested after the sixth day post onset, showed relatively consistent results with the gold standard which might be caused by an increasing number and continuous shedding of Leptospira or its antigen. Prolonged shedding of leptospires in the urine (collected at the 52" and 68 days after onset of illness) was also found in two patients who tested positive in the two assays. For urine of healthy persons, ICG-based LFA showed consistent results with the gold standard.

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. The presence of dextran and non-ionic detergent, Tween 20, at low concentrations in the sample pad, might enhance resolubilization of the conjugate, reduce non-specific reactions and minimize adsorption of analyte on membrane (27). The addition of sucrose, which is known as a preservative and resolubilization agent (27) in the conjugate pad, made the gold conjugate more stable and flow better. Even though the overall sensitivity and specificity of ICG-based LFA are still below 90%, but it is still higher than IgM dipstick (9), which is often used in initial screening for leptospirosis. In summary, we have developed assays that could detect the presence of Leptospira antigen in the urine of humans and animals, and discriminate it from other bacterial antigens. We have also developed new approaches for eliminating non-specific reactions and concentrating urine. This is the first study that could detect Leptospira antigen in human and hamster urine using immunochromatography-based assays, with good sensitivity and specificity.

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