FORM 2
THE PATENTS ACT, 1970
(Act 39 of 1970)
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(SECTION 10 and rule 13)
TITLE OF THE INVENTION
PROTEIN BIOMARKERS FOR LEPTOSPIROSIS
APPLICANT
Indian Institute of Technology, Bombay, Powai, Mumbai 400076
Maharashtra, India
INVENTORS
Prof. Sanjeeva Srivastava, Mr. Rajneesh Srivastava, Mr. Sandipan Ray
all Indian Nationals Of Indian Institute of Technology, Bombay
Powai, Mumbai-400076, Maharastra, India;
The following specification particularly describes the invention and the manner in which it is to be performed.

FIELD OF INVENTION
The present application is related to a panel of protein biomarkers for leptospirosis, and a kit for identification of these biomarkers in a sample.
BACKGROUND OF INVENTION
A biomarker, or biological marker, is a characteristic element that is objectively measured as an indicator of normal biological processes, pathogenic processes, or a pharmacological response to a therapeutic intervention. Biomarkers are endogenous elements, present in tissues or body fluids, whose amounts or modifications are indicative of disease state, progression characteristics, and/or response to therapies. During the last decade, improved understanding of mechanisms of many disease states has revealed a great number of potential effective biomarkers with a potential to reduce mortality rates by facilitating early diagnosis of diseases.
Leptospirosis is an infectious bacterial disease caused by gram negative type obligate, aerobic spirochete of genus Leptospira. It is the most common zoonotic disease of tropical or sub¬tropical countries with an occurrence found to be at an estimated range per 0.1 million cases of 0.1-1 in temperate climates to 10-100 in the humid tropics annually and occurs to its maximum viz. upto 100 per 0.1 million cases during outbreaks. Owing to the increasing number of severe leptospirosis infections in developed and developing countries, it has been recognized as a major health problem globally.
Till date very few studies on leptospiral infection were reported and most of the previously reported studies were focused on proteomic alteration /exploration of pathogen. Monahan et al, in Infect Immun 76:4952-8(2008) reported a proteomic study of leptospires excreted in urine of chronically infected hosts. No.proteome level analysis has been reported hitherto to describe alterations of human serum proteins and related biological pathways due to leptospiral infection. Investigation of the pathogen induced alteration in host serum proteome has immense clinical relevance and is one of the topics for research. Five strains of leptospira (four pathogenic and one non pathogenic) strains genome are sequenced [Vieira et al, The Open Microbiology Journal 3:69-74(2009)}.

Identification of the biomarkers for leptosporosis has potential uses in the management of patient care. Ideally, the uses would be for risk assessment, for early diagnosis, for establishing prognosis, for monitoring treatment, and for detecting relapse. Additionally, such markers could play a valuable role in developing therapeutic interventions.
Diagnosis of leptospirosis generally performed through the detection of IgM antibodies against the pathogen by rapid screening tests [Terpstra et al, J. Gen. Microbiol. 131:377-85(1985)]. Microscopic agglutination test (MAT) and ELISA-based immunoassays are the most common approaches for diagnosis of this acute bacterial infection and carried out at different health facilities. However misdiagnosis of leptospirosis sometime occurs due to its broad spectrum of symptoms, which may mimic the clinical signs of many related infectious diseases, such as dengue fever, hantavirus infection and malaria [Victoriano et al, BMC Infect Bis; 4:147(2009)]. Moreover, mixed infections with malaria or dengue along with leptospirosis also create complications and difficulties for diagnosis process. Establishment of a panel of serological markers that can confirm leptospiral infection with high accuracy and efficiently discriminate it from other related infectious diseases will be extremely precious from the diagnostic points of view. The present invention was carried out to provide better insight into the underlying molecular mechanisms of the disease pathogenesis and host immune response in leptospirosis, by establishment of early detection surrogates for the disease to meet the need for better diagnostics and effective therapy. The present application provides a simple and convenient process for the identification of potential serum based proteins involved in the pathogenesis and virulence of leptospora.
SUMMARY OF INVENTION
The present application is related to a panel of protein biomarkers for leptospirosis comprising three or more of proteins selected from alpha-1-antitrypsin precursor, vitronectin precursor, leucine-rich alpha-2-glycoprotein precursor, alpha-1B-glycoprotein precursor, complement component C9 precursor, alpha-1 -antichymotrypsin precursor, ceruloplasmin precursor, Inter-alpha-trypsin inhibitor heavy chain H4 precursor, complement factor B precursor, apolipoprotein E precursor, Ig alpha-1 chain C region, apolipoprotein A-I precursor, serum albumin precursor, Ig mu chain C region, complement C3 precursor,

clusterin precursor, apolipoprotein A-IV precursor, alpha-2-HS-glycoprotein precursor, complement factor H precursor, regulator of G-protein signaling 7, and Ig kappa chain C region.
In another aspect, the present invention provides a kit for identification of the biomarkers in a sample. The kit of the present invention comprises of:
a) A receptacle for receiving a sample;
b) one or more reagents for detecting three or more biomarkers selected from alpha-1-antitrypsin precursor, vitronectin precursor, leucine-rich alpha-2-glycoprotein precursor, alpha-1B-glycoprotein precursor, complement component C9 precursor, alpha-1-antichymotrypsin precursor, ceruloplasmin precursor, Inter-alpha-trypsin inhibitor heavy chain H4 precursor, complement factor B precursor, apolipoprotein E precursor, Ig alpha-1 chain C region, apolipoprotein A-I precursor, serum albumin precursor, Ig mu chain C region, complement C3 precursor, clusterin precursor, apolipoprotein A-IV precursor, alpha-2-HS-glycoprotein precursor, complement factor H precursor, regulator of G-protein signaling 7, and Ig kappa chain C region, and
c) a control sample.
Another aspect of the present invention relates to a method comprising:
a) measurement of the amount of three or more proteins biomarkers selected from alpha-1-antitrypsin precursor, vitronectin precursor, leucine-rich alpha-2-glycoprotein precursor, alpha-1 B-glycoprotein precursor, complement component C9 precursor, alpha-1-antichymotrypsin precursor, ceruloplasmin precursor, Inter-alpha-trypsin inhibitor heavy chain H4 precursor, complement factor B precursor, apolipoprotein E precursor, Ig alpha-1 chain C region, apolipoprotein A-I precursor, serum albumin precursor, Ig mu chain C region, complement C3 precursor, clusterin precursor, apolipoprotein A-IV precursor, alpha-2-HS-glycoprotein precursor, complement factor H precursor, regulator of G-protein signaling 7, and Ig kappa chain C region in a sample;
b) comparing the measured amount with a predetermined value from a control sample;
c) assessing patient status based on the difference between the measured value and the value obtained from the control sample.
Other objects and further scope of applicability of the present invention will become apparent

from the detailed description given hereinafter. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
BRIEF DESCRIPTION OF DRAWINGS
The following detailed description of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of assisting in the explanation of the invention, there are shown in the drawings embodiments which are presently preferred and considered illustrative. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown therein.
FIGURES
Fig. 1: A schematic work flow of the entire processes involved of the study. Flow chart
describing the entire processes of serum for profiling and exploring the differentially
expressed significant proteins identified in Leptospirosis patients using different proteomic
approaches.
Fig. 2: Trends of all differentially expressed serum proteins in leptospirosis patients identified in 2DE experiment. (A) Representative 2D gels of serum from healthy controls and leptospirosis patients. 600 µg of total serum proteins were focused on linear pH 4-7 IPG strips (18 cm) and then separated on 12.5% polyacrylamide gels, which were stained with Gel Code Blue Stain. Protein spots exhibiting altered expression levels are marked on the gels. Down (B) and up (C) -regulation of protein expression levels in leptospirosis patients. 3D views and bar diagrammatic representation of percentage volume of statistically significant (p < 0.05) differentially expressed spots were analyzed using IMP7 software. Data is represented as mean ± SEM (where n = 6 for leptospirosis (L) patients and n = 18 for HC).
Fig. 3: Regulation of all differentially expressed serum proteins in Leptospirosis (L) patients identified in 2D-DIGE gels. (A) Representative merged 2D-DIGE image of proteins from healthy controls (HC) (green) and Leptospirosis (L) (red), HC and L samples were labeled

with Cy3 and Cy5 respectively, while the protein reference pool (internal standard) was labeled with Cy2. Differentially expressed significant spots located in DIGE gel. Bar-diagram and 3D views of fluorescence intensity of statistically significant (p < 0.05) down (B) and up (C) -regulated proteins identified in leptospirosis patients.
Fig. 4: Results obtained from PANTHER functional analysis. (A) Biological process (B) Cellular component (C) Molecular function (D) Protein class and (E) Pathway associated with the differentially expressed proteins in leptospirosis identified in this study.
Fig. 5: Different functional pathways obtained in DAVID analysis with the differentially expressed proteins identified in leptospirosis patients. Complement and coagulation cascades obtained in (A) KEGG-pathway and (B) Complement pathways (classical complement pathway, lectin induced complement pathway and alternative complement pathway) identified in DAVID analysis (Biocarta-Pathway Category). Proteins identified in our study are highlighted with red (A) and black (B) star marks.
DETAILED DESCRIPTION
In describing and claimed the invention, the following terminology will be used in accordance with the definitions set forth below. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein. As used herein, each of the following terms has the meaning associated with it in this section. Specific and preferred values listed below for individual process parameters, substituent, and ranges are for illustration only; they do not exclude other defined values or other values falling within the preferred defined ranges.
As used herein, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise.
The terms "preferred" and "preferably" refer to embodiments of the invention that may afford

certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.
When the term "about" is used in describing a value or an endpoint of a range, the disclosure should be understood to include both the specific value and end-point referred to.
As used herein the terms "comprises", "comprising", "includes", "including", "containing", "characterized by", "having" or any other variation thereof, are intended to cover a non-exclusive inclusion.
As used herein "biomarker" is an indicator of a biological state. It is a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention.
As used herein "proteome" is the entire set of proteins expressed by a genome, cell, tissue or organism at a certain time. It is the set of expressed proteins in a given type of cell or organism, at a given time, under defined conditions.
As used herein the term "control" is a reference value or range of values representing an amount of activity or expression determined to be representative of a given condition. Reference values can include a range of values, real or relative expected to occur under certain conditions. These values can be compared with experimental values to determine if a given protein molecule is up-regulated or down-regulated in a particular sample.
As used herein the term "detecting" intends to mean to measure or identify the existence, occurrence, presence, or fact of something. General methods of detecting are known to the person of ordinary skill in the art and may be supplemented with the protocols and reagents disclosed herein.
As used herein the term "diagnosis" intends to mean the process of identifying a disease by its signs, symptoms and/or results of various tests. The conclusion reached through that

process is also called "a diagnosis." Forms of testing commonly performed include blood tests, medical imaging, genetic analysis, urinalysis, biopsy and analysis of biological samples obtained from a subject.
As used herein the term "downregulated/upregulated" intends to mean a process by which a cell increases/decreases the quantity of a cellular component, such as a protein, in response to an external variable. A decrease of a cellular component is called down regulation, and an increase is called upregulation.
As used herein the term "sample" intends to mean biological specimens containing genomic DNA, cDNA, RNA, or protein obtained from the cells of a subject, such as those present in peripheral blood, urine, saliva, semen, tissue biopsy, surgical specimen, fine needle aspriates, amniocentesis samples and autopsy material. In one example, a sample includes plasma or serum obtained from a mammalian subject. In one example, the sample is a liquid sample.
As used herein, the term "protein" intends to mean biochemical compounds comprising one or more polypeptides. Proteins frequently exist in the body, and in samples derived there from, in a plurality of different forms. These forms can result from either or both of pre- and post-translational modification. When detecting or determining the level of a protein in a sample, the ability to differentiate between different forms of a protein depends upon the nature of the difference and the method used to detect or measure the protein level . For example, an immunoassay using a monoclonal antibody will detect all forms of a protein containing the epitope to which the antibody is raised and will not distinguish between different forms.
As used herein, the term "antigen" refers to a substance that evokes the production of one or more antibodies. Each protein is recognized by a particular antigen. These antigens are most commonly polypeptides or carbohydrates, but they can also be lipids, nucleic acids, or even small molecules like neurotransmitters. The protein can be quantitated by reacting it with a specific antigen.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains.

List of Units and Abbreviations used in the present application:
2DE: Two dimensional polyacrylamide gel electrophoresis
2D-DIGE: 2D- fluorescence difference gel electrophoresis
ANOVA: Analysis of variance
BPB: Bromophenol blue
DAVID: Database for annotation, visualization and integrated discovery
DTT: Dithiothreitol
GS: Genespring
IAA: Iodoacetamide
1EF; Isoelectric focusing
IP A: Ingenuity pathway analysis
IPG: Immobiline pH gradient
MALDI TOF: Matrix-assisted laser desorption ionization time of flight
PANTHER: Protein analysis through evolutionary relationships
PAGE: Polyacrylamide gel electrophoresis
RUT: rapid diagnostic test
SDS: sodium dodecyl sulphate
SEM: standard error of mean
TEMED: N, N, N\ N'-tetramethylethylenediamine
Tris: tris(hydroxymethyl)methylamine '
In an aspect, the present invention provides a panel of protein biomarkers for leptospirosis having clinically high specificity and sensitivity. Accordingly, the present invention also relates to a diagnostic kit which can diagnose leptospirosis early and predict the progress
stage.
The biomarker panels not only permit the distinction of patients with leptospirosis from normal subjects, but they also allow the identification of patients at early-stages of the disease from normal individuals.
More specifically, in one aspect, the present invention provides a panel of biomarkers indicative of leptospirosis comprising three or more proteins selected from upregulated proteins like alpha-1-antitrypsin precursor, vitronectin precursor, leucine-rich alpha-2-

glycoprotein precursor, alpha-IB-glycoprotein precursor, complement component C9 precursor, alpha-1-antichymotrypsin precursor, cerulopiasmin precursor, inter-alpha-trypsin inhibitor heavy chain H4 precursor, complement factor B precursor, apolipoprotein E precursor, Ig alpha-1 chain C region, and downregulated proteins like apolipoprotein A-I precursor, serum albumin precursor, Jg mu chain C region, complement C3 precursor, clusterin precursor, apolipoprotein A-1V precursor, Alpha-2-HS-glycoprotein precursor, complement factor H precursor, Regulator of G-protein signaling 7, Ig kappa chain C region.
As used herein a panel of protein biomarkers refers to a set of 3 to 22 biomarkers, preferably, a set of 5-10 biomarkers.
In certain embodiments, the biomarker panel provided by the present invention comprises of an inflammatory biomarker selected from apolipoprotein A-I, clusterin, α-1B-glycoprotein precursor, vitronectin precursor; a blood coagulant biomarker selected from complement component C9 precursor; an antioxidant biomarker selected from serum albuminprecursor, Apolipoprotein A-IV precursor, and cerulopiasmin precursor; peptidase/protease inhibitor biomarker selected from a-1-antitrypsin precursor, inter-alpha-trypsin inhibitor heavy chain H4 precursor; protein binding biomarkers selected from apolipoprotein Al precursor, apolipoprotein A-IV precursor, and regulator of G-protein signaling, or combinations thereof.
In a further preferred embodiment, the biomarker panel comprises of alpha-1-antitrypsin, vitronectin, cerulopiasmin, Regulator of G-protein signaling, and Apolipoprotein AIV precursors, analogs thereof and antibody-binding fragments thereof.
Appropriate sample specimens for use in the present invention for detecting the biomarkers include any conventional clinical samples, for instance blood or blood-fractions (such as serum or plasma). In a specific example, the sample is a human plasma or serum sample processed for detecting biomarkers. Techniques for acquisition of such samples are well known in the art (for example see Schluger et al. J. Exp. Med. 176:1327-33, 1992, for the collection of serum samples). Serum or other blood fractions can be prepared in the conventional manner. For example, 5 uL to 1000 µL of serum can be used to screen for the presence of proteins, depending on the detection method used.

Once a sample has been obtained, multiple processing steps including sonication, depletion of high-abundance proteins for selective enrichment of low abundance targets, desalting for removal of salts and other interfering agents or combinations thereof were carried out. In particular examples, the sample is immunodepleted to remove proteins not of interest (e.g., proteins which need not be detected to practice the methods disclosed herein). In other particular examples, the sample is used without removing proteins not of interest (e.g., proteins which need not be detected to practice the methods disclosed herein), for example, the sample is not immunodepleted prior to analysis (e.g., no prior tryptic digestion of serum or plasma).
The biomarkers can be identified in a sample using combination of the techniques including gel-based techniques such as two dimensional polyacrylamide gel electrophoresis (2D), or 2D- fluorescence difference gel electrophoresis (2D-DIGE) in combination with mass spectrometry (Matrix-assisted laser desorption ionization time of flight tandem mass spectrometry; MALDI-TOF/TOF MS), and validation by western blot, or enzyme linked immunosorbent assay (ELISA). The preferred techniques are combination of 2D-fluorescence difference gel electrophoresis (2D-DIGE) and MALDI-TOF/TOF MS.
In an embodiment, the present invention involves identification of human serum proteome alterations in leptospirosis correlating host immune response and disease pathogenesis using 2DE, and 2DIGE techniques. A proteomic approach was applied to find out differential expressional changes and modulation of vital physiological pathways in leptospirosis patient serum proteome with respect to healthy control proteome. Human serum was processed and analyzed to explore much more about disease pathogenesis/ host immune response, and identification of serum protein markers of leptospirosis.
The present invention involves analysis of a comprehensive serum proteome profile of healthy subjects, and leptospirosis patients using two gel-based proteomic platforms classical 2DE (n = 32) and advanced 2D-DIGE (n = 18). In 2DE gels on an average 600 protein spots were detected by IMP7 software after staining with GelCode Blue Safe Protein Stain. 13 statistically significant (p < 0.05) differentially expressed, 7 down-regulated and 6 up-regulated (with fold changes ranging from -4.39 to fold to +2.7) proteins were identified in 2DE analysis (Fig, 2). Among the 6 up-regulated spots, 5 spots were between 1.1 and 2-fold,

and 1 spot exhibited over 2-fold increase in expression level; while in the case of down-regulation, 2 spots were between 1.1 and 2-fold, 1 spot was between 2- and 3-fold, and 4 spots found to have over 3-fold reduced expression levels. Representative 2DE images of serum proteome profile of leptospirosis patients and healthy individuals, and bar-diagrammatic representation of the fold change and 3D views of some selected differentially expressed proteins are shown in Fig. 2 (Tables 1, and 2).
Superior sensitivity of CyDyes compared to routinely used CBB stains, allows to visualize less abundance protein spots in 2D-gels , which were not detectable in classical 2DE profiling, and thereby increases the overall coverage of serum proteome. Approximately 1000 protein spots were detected on each 2D-DIGE gels in the Image Master 2D Platinum 7.0 DIGE software analysis. In 2D-DIGE profiling, total of 98 (around 10 % of the total detected spots) differentially expressed spots satisfied the statistical criteria (t-test and 1-way ANOVA; p < 0.05), among which, 48 were up-regulated (range from 1.15 to 2.71-fold) while, the remaining 50 protein spots showed reduced expression level (with changes from 1.19 to 7.5-fold) in the leptospirosis patients 37 up-regulated spots were between 1.1 and 2-fold change range, and for the remaining 11 spots the range was between 2 and 3-fold; while among the 50 down-regulated spots, 33 were between 1.1 and 2-fold, 12 were between 2 and 3-fold, and 5 spots exhibited over 3-fold reduced expression level. Owing to the superior sensitivity and reproducibility in DIGE technique, we obtained much higher numbers of differentially expressed protein spots from 2D-DIGE experiment. Fig 2 depicts 3D views and graphical representation of few selected differentially expressed protein spots
The differential expression of multiple serum proteins in leptospirosis patients detected by gel-based profiling was further set to identify by MALDI-TOF/TOF analysis. Among the several differentially expressed protein spots detected in 2DE, those 13 spots that satisfied the statistical criteria (p < 0.05) were subjected to in-gel trypsin digestion followed by MALDI-TOF/TOF analysis to establish protein identity using mass-spectrometry. MS and MS/MS analysis results revealed that 13 differentially expressed protein spots identified in regular 2DE experiment correspond to 6 proteins, among which 2 were up-regulated (α-1-antitrypsin and α-1B-glycoprotein precursor) and the remaining 4 were down-regulated (apolipoprotein A1 precursor, serum albumin precursor, apolipoprotein A-IV precursor, complement C4 precursor) (Table 1, Fig 2).

In 2D-DIGE, selected spots are excised manually from preparative 2D gels stained with GelCode Blue Safe Protein Stain (Thermo Scientific, USA) containing higher amount of serum protein.
Among the 98 differentially expressed protein spots, 57 spots that could be excised from the gels were subsequently subjected to MS and MS/MS analysis, which successfully established the identity of 42 spots (Fig. 3 and Table 2). For the remaining spots we were unable to establish MS identity most likely due to the very low intensity of those spots and insufficient amount of detectable peptides, probably beyond the sensitivity limit of the instrument. The 42 protein spots identified by MS correspond to 21 (9 down-regulated and 11 up-regulated) differentially expressed proteins in patients suffering from leptospiral infection (Fig. 3 and Table 2).
The number of identified proteins in 2DE and 2D-DIGE are 6 and 21 respectively, while 5 of the identified proteins (apolipoprotein A-I, serum albumin precursor, apolipoprotein A-IV, alpha-1-antitrypsin and alpha-1B-glycoprotein) found to be overlapping for 2DE and 2D-DIGE, and overall 22 differentially expressed proteins (p < 0.05) have been identified by the present process. Pre-electrophoresis labeling of protein samples with highly sensitive Cy dyes in 2D-DIGE attributed around 55% increase in spot number compared to the CBB stained 2DE gels, which certainly enhanced the overall coverage of the whole serum proteome. In 2D-DIGE experiment we obtained 16 more differentially expressed proteins including complement C3 precursor, clusterin precursor, complement factor H, regulator of G-protein signaling 7 (down-regulated) and vitronectin precursor, ceruloplasmin precursor, leucine-rich α-2-glycoprotein (up-regulated) (Table 4), which were not identified in classical 2DE due to lower sensitivity and reproducibility issues.
PANTHER analysis revealed involvement of identified proteins in blood coagulation system (33.3%), heterotrimeric G-protein signaling pathway-Gq alpha and Go alpha mediated pathway (33.3%) and heterotrimeric G-protein signaling pathway-Gi alpha and Gs alpha mediated pathway (33.3%) (Table 5).
The identified proteins were involved in 10 biological processes: the major five processes include metabolic process (17.3 %), immune system process (16%), response to stimulus

(13.30%), cell communication (13.3%) and cellular process (13.3%). Five major GO functions: binding (31.40%), receptor activity (17.10%), enzyme regulator activity (22.90%), catalytic activity (11.40%) and transporter activity (17.10%) were identified in PANTHER analysis for the differentially expressed proteins in leptospirosis patients (Fig. 4)
Several differentially expressed proteins were identified and modulation of multiple physiological processes and pathways, including inflammation mediated acute phase responses, complement pathways, heterotrimeric G-protein signaling pathway, coagulation cascade and hemostasis in patients suffering from leptospiral infection. Some of our identified proteins such as Ig mu chain C, clusterin precursor, different complement factors and associated pathways like acute phase response signaling, complement and coagulation cascades, even though not through proteomic studies, have been correlated with leptospiral infection earlier, supporting our findings and enhance the confidence in this study. Further, proteomic analysis revealed differential expression of few serum proteins, such as α-1-antitrypsin precursor (2.29 to 1.81-fold), vitronectin precursor (2.26-fold), α-1-antichymotrypsin precursor (1.40 to 1.68-fold), ceruloplasmin precursor (1.19 to 1.59-fold) up-regulated and regulator of G-protein signaling 7 (1.62-fold), apolipoprotein A-IV (1.91-fold) down-regulated, which have not been reported previously in the context of leptospirosis (Tables 3 and 4).
DAVID analysis, KEGG category revealed complement and coagulation cascades (1.01 E 09; 30.43 %) (Fig. 5). Reactome category revealed three biological pathways: signaling in immune system (p = 0.010079; 21.74 %), hemostasis (p = 0.035589; 17.39%), metabolism of lipids and lipoproteins (p = 0.01712; 17.39%), while, different complement cascades including classical complement pathway (p = 4.42E'06; 17.39%), lectin induced complement pathway (p = 4.42E"06; 17.39%) and alternative complement pathway (p = 4.3IE"04; 13.04%) were identified in Biocarta category.
Thrombocytopenia is found to be associated with majority of the leptospirosis patients. Bleeding tendency in leptospirosis may arise from an imbalance in the hemostatic equilibrium and toxic effect exerted by the pathogen on bone marrow leads to thrombocytopenia. The precise role of activation of coagulation system in the pathogenesis of leptospirosis needs to be established. In this context, our proteomic analysis reveals altered

expression of few members of the blood coagulation cascade such as alpha-1-anti-trypsin precursor, complement factor B and H etc. in the patients suffering from leptospiral infection.
Cytokines play a pivotal role in stimulating the production of APPs as a part of the host immune response against the infection. Altered expression of multiple acute phase proteins including ceruloplasmin, α-1-antichymotrypsin, α-1-antitrypsin, apolipoprotein A-I precursor, inter-alpha-trypsin inhibitor heavy chain H4 precursor have been identified in leptospirosis patients.
Among the identified differentially expressed serum proteins, apolipoprotein A-I, clusterin, α-IB-glycoprotein precursor, vitronectin precursor, and α -IB-glycoprotein precursor were found to be inflammation-related biomarkers of leptospiral infection.
In a further embodiment, the present invention provides a kit for detecting a panel of biomarkers of the present invention. More specifically, the present invention relates to a kit comprising:
a) A receptacle for receiving a sample;
b) one or more reagents for detecting one or more protein biomarkers selected from alpha-1-antitrypsin precursor, vitronectin precursor, leucine-rich alpha-2-glycoprotein precursor, alpha-1B-glycoprotein precursor, complement component C9 precursor, alpha-1-antichymotrypsin precursor, ceruloplasmin precursor, inter-alpha-trypsin inhibitor heavy chain H4 precursor, complement factor B precursor, apolipoprotein E precursor, Ig alpha-1 chain C region, apolipoprotein A-I precursor, serum albumin precursor, Ig mu chain C region, complement C3 precursor, clusterin precursor, apolipoprotein A-IV precursor, alpha-2-HS-glycoprotein precursor, complement factor H precursor, regulator of G-protein signaling, 7 Ig kappa chain C region, and
c) a control sample.
In an embodiment, the biomarkers are selected from three or more of alpha-1-antitrypsin, vitronectin, clusterin, ceruloplasmin, regulator of G-protein signaling, and apolipoprotein AIV precursors, analogs thereof and antibody-binding fragments thereof.
The receptacle is a sample-receiving container used to collect the sample. The primary

sample receiving container may be attached to the sampling equipment such as an injection, and sealed so as to prevent tampering or contamination of the sample throughout the analysis period.
The control is a sample of known composition that is analyzed along with test samples in order to evaluate the actual difference in expression of the protein biomarkers when compared with the healthy individual. In the present case, the control sample is taken from a healthy individual and the controlled experiment compares the results obtained from an experimental sample from diseased individual against a control sample, which is practically identical to the experimental sample except that the sample is taken from a healthy individual.
Potential panel of the identified biomarker can be detected using different technological approaches.
1. The reagents for detecting the protein biomarkers can be selected from antigens, or
antibodies which are specific to the selected protein biomarkers and which are immobilized.
Samples will be sandwiched by the immobilized antibody and biotinylated polyclonal
antibody specific for the target proteins, which will be subsequently recognized by a
streptavidin-peroxidase complex. Color development will be performed through the addition
of a peroxidase enzyme substrate and measured colorimetrically.
2. Alternatively, development of low-cost optical sensors for selective detection of a panel of
target marker proteins can be performed. Diffraction-based sensing could be used for label-
free quantification of multiple target proteins simultaneously.
3. Serum levels of the target proteins can also be measured by using mass spectrometry-based
multiple reaction monitoring (MRM) quantitation in a triple quadrupole (QQQ) MS
instrument. Using stable isotope-labeled internal standards (SISs) in MRM-based analysis
rapid, sensitive, and specific quantitation (absolute quantification) of multiple target protein
can be possible even in highly complex biological samples such as serum, plasma, or whole
blood.
In a preferred embodiment, the kit comprises one or more antigens that are specific for the biomarker and means for determining binding of the antigen to the diagnostic biomarker in the sample. The kit may also comprise packaging material comprising a label that indicates

that the one or more antigens of the kit can be used for the identification of leptospirosis. Other components such as buffers, controls, detection reagents, and the like known to those of ordinary skill in art may be included in such kits.
The kit of the present invention is manufactured by a conventional manufacturing method known in the art, and typically includes freeze-dried antibody/antigen and buffer, stabilizer, inactive protein, and the like. The antibody may be labeled with radioisotope, fluorescence, enzyme, or the like. The monoclonal antibody generated against the target antigens of the present invention may be variously used for an immunoassay kit (ELISA, antibody coated tube test, lateral-flow test, potable biosensor), and also may be used to develop a protein chip having a detection spectrum for various leptospirosis complications through development of an antibody showing higher specificity and sensitivity. Purified target proteins can be used as known standards for quantification of the candidates in control and diseased sera.
Monoclonal/polyclonal antibody against the target proteins in a format of a ELISA-based kit or as a chip-based biosensor can be used for detection and quantification of the potential biomarker candidates in patients' sera.
In yet another embodiment the present invention comprises a method comprising:
a) Measurement of the amount of three or more biomarkers selected from alpha-1-antitrypsin precursor, vitronectin precursor, leucine-rich alpha-2-glycoprotein precursor, alpha-lB-glycoprote in precursor, complement component C9 precursor, alpha-1-antichymotrypsin precursor, ceruloplasmin precursor, inter-alpha-trypsin inhibitor heavy chain H4 precursor, complement factor B precursor, apolipoprotein E precursor, Ig alpha-1 chain C region, apolipoprotein A-I precursor, serum albumin precursor, Ig mu chain C region, complement C3 precursor, clusterin precursor, apolipoprotein A-IV precursor, alpha-2-HS-glycoprotein precursor, complement factor H precursor, regulator of G-protein signaling, 7 Ig kappa chain C region, and
b) comparing the measured amount with a predetermined value from a control sample;
c) assessing patient status based on the difference between the measured value and the value obtained from control sample.
In an embodiment, the biomarkers are selected from three or more proteins selected from

alpha-1-antitrypsin, clusterin, vitronectin, ceruloplasmin, Regulator of G-protein signaling, and Apolipoprotein AIV precursors, analogs thereof and antibody-binding fragments thereof.
The assay method used to determine the level of the biomarker protein preferably detects all forms of the specific biomarker protein. Preferably at least all biologically active forms of the biomarker protein are detected. Preferably, all forms of the biomarker protein with at least 75% or more, preferably at least 80%, 85%, 90% or 95% or more, sequence identity with the published amino acid sequence of the biomarker will be detected in the method of the invention.
The level of the biomarker present in the sample may be determined by any suitable assay which may comprise the use of any of the group comprising immunoassays, spectrometry, , MS-based quantitative proteomics, ELISA-based immunoasays, western blot, quantitative RT-PCR, protein microarray, and antibody microarray, surface plasmon resonance-based label-free method or combinations thereof.
Establishment of a panel of serological markers that can confirm leptospiral infection with high accuracy and efficiency to discriminate it from other related infectious diseases will be extremely precious. While, the major emphasis of the process of the present invention is to provide better insight into the underlying molecular mechanisms of the disease pathogenesis and host immune response in leptospirosis, but, one of the possible outcomes of the present process could be establishment of early detection surrogates for the disease to meet the need for better diagnostics and effective therapy.
The following examples are provided to better illustrate the claimed invention and are not to be interpreted in any way as limiting the scope of the invention. All specific materials, and methods described below, fall within the scope of the invention. These specific compositions, materials, and methods are not intended to limit the invention, but merely to illustrate specific embodiments falling within the scope of the invention. One skilled in the art may develop equivalent materials, and methods without the exercise of inventive capacity and without departing from the scope of the invention. It is the intention of the inventors that such variations are included within the scope of the invention.

EXAMPLES
EXAMPLE 1
SUBJECT SELECTION AND SAMPLE COLLECTION
Blood samples were collected from leptospirosis patients enrolled in Seth GS Medical College & King Edward Memorial hospital, Mumbai with approval of the institutional ethics committee and written informed consent from each subject. On the basis of clinical symptoms blood samples were collected from patients and processed for further confirmation test by IgM antibodies to leptospira. Symptomatic patients with positive rapid immunodiagnostic test with no past history of autoimmune diseases or any significant systemic diseases were selected for this study. Demographic, epidemiological and clinical details, including past history of diseases of all leptospirosis patients (n = 6) selected for this proteomic study were tabulated. Blood specimens were also collected from age and sex matched healthy controls (n = 18) (voluntary blood donors) with written informed consent to perform comparative analysis. Serum separation tubes (BD Vacutainer®; BD Biosciences) were used to collect blood samples from the antecubital vein of leptospirosis and healthy subjects. Subsequent to blood collection, samples were allowed to clot by keeping the tubes in ice for 30 minutes. After clotting, the samples were centrifuged at 2500 rpm for 10 minutes at 20 C to separate serum from the clotted blood. Collected serum samples were labeled and stored in multiple aliquots at -80°C.,- Exactly similar collection; processing and storage conditions were maintained for each control and diseased samples in order to minimize any pre-analytical variations.
EXAMPLE 2
PROCESSING OF SERUM SAMPLES AND 2DE
Crude serum was diluted five times with phosphate buffer (pH 7.4) and subjected to mild
sonication in a Vibra cell sonicator using the following settings: 6 cycles of 5 sec pulse; 30
sec gap in between; at 20% amplitude. After sonication, level of top two high abundance
serum proteins (albumin and IgG) were reduced by using Albumin & IgG Depletion
SpinTrap (GE Healthcare) following manufacturer's instructions. Extraction of protein from
depleted serum samples was performed employing TCA/acetone precipitation method as
described by Chen et al with slight modifications [Chen et al., Electrophoresis, 26:2117-
2127(2005)]. In brief, depleted serum samples were diluted (1:4 ratio) with ice-cold acetone

containing 10% w/v TCA. Uniform mixing was performed using mild vortexing for 15 sec and the mixture was allowed to incubate at -20°C for 2 hrs for protein precipitation. After incubation, tubes were centrifuged at lOOOOg for 15 min at 4 C. Supernatants were separated and kept in fresh microcentrifuge tubes, and the pellets were dissolved in rehydration buffer (8 M urea, 2 M thiourea, 4% (w/v) CHAPS, 2% (v/v) IPG buffer (pH 4-7; Linear), 40mM DTT and traces of bromophenol blue). In order to precipitate the remaining amount of proteins present in the collected 10% TCA/acetone-containing supernatants, 1 mL ice-cold acetone was added to each tube and the samples were subjected one additional round of precipitation and extraction process. In all cases, prior to re-suspension in rehydration buffer, the pellet was briefly air-dried. Prior to further proteomic analyses, protein concentration in the samples was quantified using the 2D-Quant kit (GE Healthcare) following the manufacturer's instructions. A total of 600 µg of depleted serum protein extract dissolved in 350 µL of rehydration buffer was loaded on 18 cm (pH range 4-7) IPG strips and underwent passive rehydration for 14-16 hours. Isoelectric focusing (IEF) was performed on an Ettan IPGphoie 3 isoelectric focusing unit (GE Healthcare) for overall approximately 78kVh using the following voltage settings: 200 V for 4 h (step and hold), 500 V for 1 h (step and hold), 1000V for 1 h (step and hold), 8000V for 3 h (gradient), and 8000V for 7:30 h (step and hold). After completion of IEF, the focused IPG strips were stored at -20°C until the second dimensional analysis was performed. Preceding to the 2nd dimensional separation, each strip was equilibrated to reduce and alkylate the proteins (for 15 min each) using equilibration buffer contained 6 M Urea, 75 raM Tris-HCl pH 8.8, 29.3% (v/v) glycerol, 2% (w/v) SDS, and 0.002% (w/v) bromophenol blue. Just prior to use, 1% (w/v) DTT or 2.5 % (w/v) IAA was added in the first (reducing) and second (alkylating) equilibration buffer, respectively. The 2nd dimension was performed on 12.5% SDS polyacrylamide gels in an Ettan DALTsix electrophoresis unit (GE Healthcare). After electrophoresis GelCode Blue Safe Protein Stain (Thermo Scientific, USA) was applied for visualization of resolved protein spots. Proteins extracted from each of the subjects (L and HC) were run in duplicate to verify the reproducibility and curtail technical artifacts.
EXAMPLE 3
2D-DIGE
Each CyDye (Cy3, Cy5 and Cy2) was resuspended in anhydrous N, N-Dimethylforrnamide
(DMF) to prepare a stock dye concentration of 1 mM. A working solution of 400 pmol of

each CyDye was made by further dilution of the stock with DMF. Samples (test and control) were labeled with Cy3 and Cy5, while a mixture of equal amounts of all samples to be analyzed in the experiment, regarded as internal standard (IS), was labeled with the third fluorescent dye, Cy2, according to the manufacturer's instructions (GE Healthcare). In brief, the pH of each sample was adjusted to 8.5 using 100 mM NaOH. 50µg of each protein sample (L, FC/ HC and IS) were separately labeled with 400 pmol of CyDyes. After addition of CyDyes, samples were incubated on ice for 30 in the dark. Labeling reaction was stopped by addition of 10 mM lysine followed by incubation on ice for additional 10 min. Dye-swapping was executed while labeling the test and control samples for eliminating any type of dye effects. After labeling, samples labeled with Cy3, Cy5 and Cy2 were mixed, diluted with the rehydration buffer and loaded on 18 cm, 4-7 pH IPG strips. Subsequent IEF and SDS-PAGE separation were performed following the same protocol as described earlier in the regular 2DE section. Eight independent biological replicates of L and HC were prepared and resolved by 2D-DIGE.
EXAMPLE 4
IMAGE ACQUISITION AND SOFTWARE ANALYSIS
The 2D gel images were scanned by using LabScan software version 6.0 (GE Healthcare) and analysis was performed by using ImageMaster 2D Platinum 7.0 software (GE Healthcare). Comparative analysis of LS samples was performed by creating different "match sets" and using the HC samples as reference. Spot detection parameters like Smooth: 5, Saliency: 100 and Min Area: 5 were set and automatic detection of the spots through IMP7 was performed. A manual edition of the spots was applied to eliminate any contaminating artifacts, such as streaks or dust. Spot quantification was performed in % vol value using ImageMaster algorithm. It provided normalized value that remains relatively independent of variations due to staining or protein loading by considering the total volume over all the spots in the image. The gel analysis tables, histograms and 3D images generated by the software were used for further analysis.
2D-DIGE gels were scanned by using Ettan DIGE Imager scanner (GE Healthcare) employing suitable excitation/emission wavelengths and niters for CyDye [Cy3 (540/595nm), Cy5 (635/680nm), Cy2 (480/530 nm)] keeping the resolution at 40 urn. After scanning, the cropped gel images were imported to ImageMaster 2D Platinum 7.0 DIGE software (GE

Healthcare) and subjected to comparative analysis for relative protein quantification across all the leptospirosis and control samples. Statistical significance of the average ratios of expressions was analyzed by Student's t test and one-way ANOVA. Protein spots exhibiting differential expression with reproducibly and statistical significance (p < 0.05) were considered for further analysi s.
EXAMPLE 5 IN-GEL DIGESTION
Statistically significant (Student's t-test, p < 0.05) differentially expressed proteins spots identified in regular 2DE and 2D-D1GE experiments were selected for further MS analysis for protein identification. GelCode Blue stained preparative gels containing higher amount of protein (1 mg) were used for excision of spots of interest specified in the 2D-DIGE experiment. Spots were excised (small cubes ~1 x 1 mm) manually. Excised spots were washed with 50 uL of stain removal solution (25 mM ammonium bicarbonate buffer) for removal of CBB stain. After washing, 50 u.L of 25 mM ammonium bicarbonate/acetonitrile (1:1 v/v) was added followed by 5 min incubation with occasional vortexing at room temperature. After incubation, the solutions were removed. These two steps are repeated for three times. Then, 50 µL reduction solution (10 mM DTT in 100 mM ammonium bicarbonate) was added and the gel pieces were incubated for 60 mins at 56°C in an air thermostat. Tubes were allowed to cool to room temperature after incubation, and 50 uL of 25 mM ammonium bicarbonate buffer was added to wash the gel pieces followed by dehydration with 25 mM ammonium bicarbonate/acetonitrile (1:1, v/v). After this step, alkylation solution (50 mM 1AA in 100 mM ammonium bicarbonate) was added and the tubes containing the gel pieces were incubated for 30 mins at room temperature in dark. Rehydration and dehydration steps were followed twice and gel pieces were allowed to dry. Once the gel slices were properly dried, trypsin solution (Trypsin Gold; Promega, Madison, Wisconsin, United States) was added to the gel pieces and kept at ice for 30 mins for absorption of the solution. After this step, the tubes with gel pieces were incubated overnight at 37°C. Adequate amount of ammonium bicarbonate buffer was added to cover the gel pieces. Extraction of the digested peptides from the gel matrix was performed using 100 uL of extraction buffer (0.2% formic acid in 66% acetonitrile) after completion of the enzymatic reaction. Extraction step was repeated thrice to ensure the maximum amount of peptides. The collected supernatants were pooled in a single tube and concentrated using speed vac. After

extraction trypsin digested samples were further processed using ZipTip C18 pipette tips (Millipore, USA) according to the manufacturer's protocol for salt removal and enrichment of the peptides.
EXAMPLE 6
MALDI-TOF/TOF ANALYSIS AND PROTEIN IDENTIFICATION
Subsequent to enrichment and purification through the Zip-Tip pipette tips, peptide mixtures were dissolved in 0.5 µL of CHCA matrix solution (5 mg/mL CHCA in 50% ACN/0.1% TFA) and spotted onto a freshly cleaned MALDI target plate. Spots were allowed to dry for 30mins at room temperature. After air drying, the crystallized spots were analysed using a 4800 MALDI-TOF/TOF mass spectrometer (AB Sciex, Framingham, MA) linked to 4000 series explorer software (v.3.5.3). All mass spectra were recorded in a reflector mode within a mass range from 800 to 4000 Da, using a Nd:YAG 355 nm laser. The acceleration voltage and extraction voltage were kept 20 kV and 18 kV respectively. Six point calibration of the instrument was automatically performed by a peptide standard Kit (AB Sciex) that included des-Argl-bradykinin (m/z 904.468), Angiotensin I (m/z 1296.685), Glul-fibrinopeptide B (m/z 1570.677), ACTH (18-39, m/z 2465.199), ACTH (1-17, m/z 2903.087), and ACTH (7-38, m/z 3657.923). All the MS spectra were obtained from accumulation of 900 shots. MS/MS spectra were acquired for the 15 most abundant precursor ions, with a total accumulation of 1500 laser shots and collision energy of 1 kV. Once the MS survey scans were completed, the data were processed to generate a list of precursor ions for interrogation by MS/MS. The combined MS and MS/MS peak lists were searched using the GPSTM Explorer software version 3.6 (AB Sciex). Protein identification was performed by MS/MS ion search using MASCOT version12.1 (http://www.martixscience.com) search engine against the Swiss-Prot database. Searches were carried out with the following parameters; all entries taxonomy, trypsin digestion with one missed cleavage, fixed modifications: carbamidomethylation of cysteine residues, variable modifications: oxidation of methionine residues, mass tolerance 75 ppm for MS and 0.4 Da for MS/MS. Identified proteins having at least two unique matched peptides were selected for further analysis. As above, we have reported only those proteins with a protein identification confidence interval of > 95%.
EXAMPLE 7
WESTERN BLOT ANALYSIS

Serum proteins extracted from healthy and leptospirosis patients were resolved in 12% SDS-PAGE (50 µg per track) and then transferred onto PVDF membranes under semidry conditions by using ECL semi-dry transfer unit (GE Healthcare). Western blot was performed by using monoclonal/polyclonal antibody against ceruloplasmin (Santacruz biotechnology, sc-365206) and clusterin (Santacruz biotechnology, sc-8354) and appropriate secondary antibody conjugated with HRP (GeNei (MERCK)-621140380011730 or 621140680011730). Quantitation of protein bands in western blot was performed by using ImageQuant software version 5.0 (GE Healthcare).
EXAMPLE 8
PROTEIN NETWORKS AND FUNCTIONAL ANALYSIS
Differentially expressed proteins in leptospirosis patients were analyzed using PANTHER (Protein Analysis Through Evolutionary Relationships) system, version 7 (http://www. pantherdb.org) and DAVID (Database for Annotation, Visualization and Integrated Discovery) database version 6.7 (http://david.abcc.ncifcrf.gov/home.jsp) to determine the association of identified proteins with various physiological pathways and disease pathogenesis. The significance of association between our dataset and identified networks/pathways was considered on basis of two parameters, ratio and p values. The list of UniProt Accession from each dataset were uploaded in tab delimited text format at once which was mapped against reference Homo sapiens dataset to extract and summarize functional annotation associated with individual or group of genes and proteins.

Table 1. Serum proteins identified by MALDI-TOF/TOF identified proteins in serum of
Leptospirosis patients from classical 2DE experiment
[Analysis Type: Combined (MS+MS/MS); Database: SwissProt; Taxonomy: Human (Homo
sapiens)]

s. Spot Fold Name of protein with MW Protein Total No. of
No ID change UniProt accession (kDa) score ion matched
1 D-l number score peptides

-4.39 (P02647) 30.75 530 394 19
Apolipoprptein A-I precursor (Apo-AI)
2 D-2 -3.41 (P02647) Apolipoprotein A-I precursor (Apo-AI) 30.75 227 169 13
3 D-3 -3.4 (P02768) Serum albumin precursor 69.32 456 386 30
4 D-4 -3.36 (P02647) Apolipoprotein A-I precursor (Apo-AI) 30.75 815 628 24
5 D-5 -2.96 (P06727) Apolipoprotein A-IV precursor (Apo-AIV)(ApoA-IV) 45.34 56 16
6 D-6 -1.91 (P02647) Apolipoprotein A-I precursor (Apo-AI) 30.76 163 137 8
7 D-7 -1.9 (P01028) Complement C4 192.65 492 483 19
precursor
8 U-l 2,71 (PO1009) Alpha-1-antitrypsin precursor (Alpha-1 protease 46.7 223 54 23

inhibitor)
9 U-2 1.75 (P01009) Alpha-1-antitrypsin precursor (Alpha-1 protease inhibitor) 46.7 670 538 23
10 U-3 1.63 (P04217) Alpha-1B-glycoprotein precursor (Alpha-1-B glycoprotein) 54.24 776 724 13
11 U-4 1.62 (P04217) Alpha-1B-glycoprotein precursor (Alpha-1-BN glycoprotein) 54.3 900 821 16
12 U-5 1.6 (P01009) Alpha-1-antitrypsin precursor (Alpha-1 protease inhibitor) 46.7 151 66 16
13 U-6 1.47 (P01009)Alpha-l-antitrypsin precursor (Alpha-1 protease inhibitor) 46.7 152 84 14
Table 2. Serum proteins identified by MALDI-TOF/TOF in Leptospirosis patients from 2D-DIGE experiment. [Analysis Type: Combined (MS+MS/MS); Database: SwissProt; Taxonomy: Human {Homo sapiens)]

S. Spot Fold Name of protein with MW Protein Total No. of
No ID change UniProt accession number (kDa) score ion
score matched peptides
1 58 1.19 (P00450) Ceruloplasmin precursor (EC 1.16.3.1)
(Ferroxidase) 122.13 437 264 35

2 60 1.24 (P00450) Ceruloplasmin precursor (EC 1.16.3.1 )(Ferroxidase) 122.98 29 200 181 13
3 74 -1.34 (P08603) Complement factor H precursor 139.03 701 494 40
4 75 -1.27 (P08603) Complement factor H precursor (H factor 1) 143.71 778 572 40
5 83 1.59 (P04217) Alpha-1B-glycoprotein precursor (Alpha-1-B glycoprotein) 54.24 110 101 8
6 84 1.43 (P02649) Apolipoprotein E precursor (Apo-E) 36.13 669 573 18
7 85 1.84 (P02748) Complement component C9 precursor 63.13 73 73 11
8 86 1.36 (Q14624)Inter-alpha-trypsin inhibitor heavy chain H4 precursor (ITI heavy chain H4) 103.29 230 153 21
9 88 1.59 (P00450) Ceruloplasmin precursor (Ferroxidase) 122.13 246
205 18
10 106 1.17 (P00751) Complement factor B precursor 85.4 694 513 30
11 117 -1.76 (P02768) Serum albumin precursor 69.32 1370 1213 25

12 135 1.89 (P04217) Alpha-1B-glycoprotein precursor (Alpha-1-BN glycoprotein) 54.3 900 821 16
13 146 1.29 (P04217) Alpha-1B-glycoprotein precursor (Alpha-1-BN glycoprotein) 54.8 787 721 15
14 171 -3 (P02768) Serum albumin precursor 69.32 456 386 30
15 175 1.43 (P01011)Alpha-l-antichymotrypsin precursor (ACT) 47.62 375 220 27
16 176 1.4 (P01011)Alpha-l-antichymotrypsin precursor (ACT) 47.62 184 118 14
17 185 2.29 (P01009) Alpha-1-antitrypsin precursor (Alpha-1 protease inhibitor) 46.7 277 195 16
18 188 2.26 (P04004) Vitronectin precursor (Serum spreading factor) (S-protein) 55.06 173 164 5
19 198 2.21 (P01009) Alpha-1-antitrypsin precursor (Alpha-1 protease inhibitor) 46.7 532 394 22
20 201 1.41 (P01009) Alpha-1-antitrypsin precursor (Alpha-1 protease inhibitor) 46.7 223 54 23

21 217 -1.65 (P02765) Alpha-2-HS-glycoprotein precursor (Fetuin-A) 39.3 405 369 9
22 225 1.88 (P01009) Alpha-1-
antitrypsin precursor
(Alpha-1 protease
inhibitor) (Alpha-1-
antiproteinase) 46.7 63 49 6
23 228 1.81 (P01009) Alpha-1-anritrypsin precursor (Alpha-1 protease inhibitor) 46.8 667 520 22
24 234 2.05 (P02750) Leucine-rich alpha-2-glycoprotein precursor 38.15 132 132 1
25 238 2.28 P01871)Igmu chain C region 49.53 391 352 11
26 241 1.54 (P02750) Leucine-rich alpha-2-glycoprotein precursor (LRG) 38.15 573 522 11
27 244 1.55 (P02750) Leucine-rich alpha-2-glycoprotein precursor (LRG) 38.15 634 568 13
28 250 -1.73 (P01871)Ig mu chain C region 49.53 153 133 8
29 252 -1.91 P06727) Apolipoprotein A-IV precursor (Apo-AIV)(ApoA-IV) 45.34 56 16
30 257 -2.21 (P01024) 187.05 207 165 24

Complement C3
precursor
31 275 -2.16 (P10909)Clusterin precursor 52.46 241 211 10
32 312 -1.2 (P01834)Ig kappa chain C region 11.6 68 54
33 322 1.31 (P01834)Ig kappa chain C region 11.6 70 56 3
34 329 -3.01 (P02647) Apolipoprotein A-I precursor (Apo-AI) 30.75 815 628 24
35 331 -2.83 (P02647) Apolipoprotein A-I precursor (Apo-AI) 30.75 227 169 13
36 332 -1.35 (P01834)Ig kappa chain C region 11.6 213 191 4
37 389 1.41 (P04217) Alpha-1B-giycoprotein precursor (Alpha-1-B glycoprotein) 54.24 92 73 8
38 499 1.68 (POlOll)Alpha-I-antichymotrypsin precursor (ACT) 47.62 208 92 20
39 500 1.66 (P01011) Alpha-1-antichymotrypsin precursor (ACT) 47.62 193 108 17
40 504 1.15 (P01876)Ig alpha-1 chain C region 37.63 612 560 11
41 514 -1.62 (P49802) Regulator of G-protein signaling 7 75.79 28 12
42 516 -2.21 (P02768) Serum albumin precursor 69.32 72 27 14

Table 3. List of significant differentially expressed proteins identified in the Leptospirosis patients using 2DE
# For proteins with multiple spots in the 2D gels, representative spot detail is provided. Exact values for each spot are provided in supplementary
information
*A-protein binding; B-DNA binding; C-metal binding; D-antioxidant activity; E-drug binding; F-peptidase/protease inhibitor activity; G- Plasma
Glyco protein, function unknown; H-lipid binding; I-lipid metabolism/transport; J-brain development; K- enzyme regulator activity; L-
complement activation; M-biood coagulation; N-antigen/antibody binding; O-catalytic activity; P-cell surface binding; Q- chaperone binding; R-
enzyme inhibitor activity.

S.No Name of protein with
UniProt
accession number Gene name Fold change MW (kDa) No of spots (spot numbers) p value (t-test) Protein score Total ion
score No. of matched peptides# Funct ions*
Down-regulated proteins
1 (P02647) Apolipoprotein Al precursor APOA1 4.39-1.91 30.75 4(D-l,D-2, D-4.D-6) 2.30E-07 815 628 24 A,H,I
2 (P02768) Serum albumin precursor ALB 3.4 69.32 1 (D-3) 0.00018 456 386 30 B,D, E,P,Q
3 (P06727) Apolipoprotein A-IV precursor APOA4 2.96 45.34 1 (D-5) 5.80E-05 56 - 16 A,C,
D,H,1
4 (PO1028) Complement C4 precursor C4 1.90 192.65 1 (D-7) 0.02288 492 483 19 J,L
Up-regulated proteins
5 (P01009) Alpha-1-anti trypsin precursor SERPIN Al 2.71 - 1.47 46.71 4(U-l,U-2, U-5,U~6) 0.0029 670 538 23 A,F
6 (P04217) Alpha-1B-glycoprotein precursor A1BG 1.63-1.62 54.3 2 (U-3.U-4) 0.0405 900 821 16 G

Table 4. List of significant differentially expressed proteins identified in the Leptospirosis patients using 2D-D1GE

SI no Name of protein with
UniProt
accession number Gene name Fold chang
e MW (kDa) No of
spots
(spot
number
s) p value (t-test) Protein score Tota 1 ion
scor
e# No. of matche
d peptide
s# Functions
*
Down- regulated proteins
1 (P02647) Apolipoprotein A-I precursor (Apo-AI) APOA 1 3.01 -
2.83 30.75 2 (329, 331) 0.00284 815 628 24 A,H,I
2 (P02768) Serum albumin precursor ALB 3-1.76 69.32 3
(117,171
,516) 0.03566 1370 1213 25 B,D,E,P,Q
3 (P01871)Igmu chain C region IGHM 2.28-1.73 49.53 1 (238, 250) 0.00521 391 352 11 N
4 (P01024) Complement C3 precursor C3 2.21 187.05 1 (257) 0.00123 207 165 24 J,L
5 (P10909)Clusterin precursor CLU 2.16 52.46 1 (275)
0.00168 241 211 10 A,0

6 (P06727)
Apolipoprotein A-IV
precursor APOA
4 1.91 45.43 1 (252) 0.03608 56 - 16 A,C,D,H,I
7 (P02765) Alpha-2-HS-
glycoprotein precursor
(Fetuin-A) AHSG 1.65 39.3 1(217) 0.0276 405 369 9 A,K,R
8 (P08603) Complement factor H precursor CFH 1.27-1.34 143.71 2(74, 75) 0.04136 778 572 40 L
9 (P49802) Regulator of G-protein signaling 7 RGS7 1.62 75.79 1(514) 0.02774 28 - 12 A
10 (P01834)Ig kappa chain C region IGKC 1.35-1.2 11.6 3 (312,
322, 332) 0.03339 213 191 4 N
Up-reg ulated proteins
11 (P01009) Alpha-1-
antitrypsin precursor
(Alpha-1 protease
inhibitor) SERPl
NAl 2.29 to 1.81 46.7 5(185, 198, 201,
225, 228) 0.0233 667 520 22 A,F
12 (P04004) Vitronectin precursor (Serum VTN 2.26 54.27 1 (188) 0.01282 173 164 5 A

spreading factor) (S-protein)



634
13 (P02750) Leucine-nch
alpha-2-glycoprotein
precursor LRG1 2.05 to 1.54 38.15 3 (234, 241, 244) 0.04313
568
821
73 13
A
(P04217) Alpha-1B-glycoprotein precursor A1BG 1.89 to 1.30 A 900


14


54.3 (83,135 ,146,
389) 0.01302

16 G
15 (P02748) Complement
component C9
precursor C9 1.84 63.13 1(85) 0.03195 73
11 L,M,O
16 (P01011) Alpha-1-
antichymotrypsin
precursor
SERPI
NA3 1.68 to 1.40 15.88 4(175,
176,
499,
500) 0.01572 375
220 27 A,B,F
17 (P00450)
Ceruloplasmin
precursor (EC
1.16.3.1) (Ferroxidase) CP 1.59 to 1.19 122.13 3(58, 60, 88) 0.02839 437 264 35 C,D,Q

(Q14624) Inter-alpha-
18 trypsin inhibitor heavy chain H4 precursor ITIH4 1.36 103.29 1(86) 0.01552 230 153 21 F
(P00751)
19 Complement factor B precursor CFB 1.17 85.4 1 (106) 0.04961 694 513 30 A
(P02649)
20 Apolipoprotein E precursor Apo-E 1.43 36.13 1(84) 0.00758 669 573 18 A,H
21 (P01876)lg alpha-1 chain C region IGHA 1 1.15 37.63 1 (504) 0.02851 612 560 11 A,H
For proteins with multiple spots in the 2D gels, representative spot detail is provided. Exact values for each spot are provided in supplementary
information
*A-protein binding; B-DNA binding; C-metal binding; D-antioxidant activity; E-drug binding; F-peptidase/protease inhibitor activity; G- Plasma
Glycoprotein, function unknown; H-lipid binding; I-lipid metabolism/transport; J-brain development; K- enzyme regulator activity; L-
complement activation; M-blood coagulation; N-antigen/antibody binding; O-catalytic activity; P-cell surface binding; Q- chaperone binding; R-
enzyme inhibitor activity.

Table 5. Details of the pathways defined by PANTHER and DAVID Analysis Table 5.1. Interaction networks defined by PANTHER Analysis

SNo. Pathways % Proteins involved Protein ID GO Molecular Function GO Biological Process Lep
protein
IDs
(expected) Lep protein
IDs (over/
under) P-value
1 Helerotrimeric G-
protein signaling
pathway-Gq
alpha and Go
alpha mediated
pathway 33.3% Regulator
ofG-
protein
signaling
7;RGS7 P49802 • protein binding
• small GTPase
regulator activity • cell surface receptor linked signal transduction
• signal transduction
• dorsal/ventral axis
specification 0.15 + 0.138
2 Heterotrimeric G-protein signaling pathway-Gi alpha
and Gs alpha mediated pathway 33.3% Regulator
ofG-
protein
signaling
7;RGS7 P49802 • protein binding
• small GTPase
regulator activity • cell surface receptor linked signal transduction
• signal transduction
• dorsal/ventral axis
specification 0.18 + 0.168
3 Blood coagulation 33.3% Alpha-1-
antitrypsin
;SERPIN
Al P01009 • protein binding
• peptidase inhibitor activity • protein metabolic process 0.05 + 0.051
7

Table 5.2. Interaction networks defined by DAVID Analysis

Category Term Count % P-value Accession no. List Total Pop Hits Pop Total Fold Enrichment Bonferroni Benja-mini FDR
REACT-OME
PATH-WAY REACT6 900: Signal
i-ng in Immune
system 5 21.7 3 0.01 P01834, P00751, P02748, P01028, P01024 12 286 3398 4.95046 0.0493898 0.0493 89 4.3100 6
REACT-OME PATH-WAY REACT_6
04:Hemost
-asis 4 17.3 9 0.03 P02768, P10909, P01009,
P02647 12. 235 3398 4.81985 0.1657227 0.0586 08 14.580 9
REACT-OME PATH-WAY REACT_6
02:Metabo
1-ism of
lipids and
lipoprotein
-s 4 17.3 9 0.01 P02649, P02768, P06727, P02647 12 150 3398 7.55111 0.0524247 0.0265 65 4.5758
4
KEGG PATH-WAY hsa04610: Compleme
nt and coagulatio n cascades 7 30.4
3 1.01E-09 P00751, P02748, P08603, P01028, P01009, P01024 11 69 5085 46.89723 8.06E-09 8.06E-09 5.52E'
07
BIOCART A h_compPat
hway:Com
plement
Pathway 5 21.7 4 6.68E-08 P00751, P02748, P01028, P01024 6 17 1437 70.44118 4.01 E-07 4.01E-07 3.20E'
05
BIOCART A h_classicP athway:Cla
ssical Compleme 4 17.3 9 4.42E-06 P02748, P010285 P01024 6 4 12 1437 79.83333 2.65E-05 1.32E-05 0.0021 1

nt Pathway
BIOCART A h_lectinPat hway:Lecti n Induced Compleme nt Pathway 4 17.3 9 4.42E-06 P02748, P01028, P01024 6 12 1437 79.83333 2.65E-05 1.32E-05 0.0021 1
BIOCART A h_alternati vePathway :Alternativ
e Compleme nt Pathway 3 13.0 4 4.31E-04 P00751, P02748, P01024 6 10 1437 71.85 0.002585 8.62E-04 0.2062
4

We Claim:
1. A biomarker panel for diagnosing leptospirosis, the biomarker panel comprising three or more proteins selected from alpha-1-antitrypsin precursor, vitronectin precursor, leucine-rich alpha-2-glycoprotein precursor, alpha-1 B-glycoprotein precursor, complement component C9 precursor, alpha-1 -antichymotrypsin precursor, ceruloplasmin precursor, inter-alpha-trypsin inhibitor heavy chain H4 precursor, complement factor B precursor, apolipoprotein E precursor, Ig alpha-1 chain C region, apolipoprotein A-I precursor, serum albumin precursor, Ig mu chain C region, complement C3 precursor, clusterin precursor, apolipoprotein A-IV precursor, alpha-2-HS-glycoprotein precursor, complement factor H precursor, regulator of G-protein signaling, 7 Ig kappa chain C region wherein trends of differential expression (up/ down-regulations) of these identified proteins and fold-change values compared to the healthy controls are specific for leptospiral infection.
2. A biomarker panel as claimed in Claim 1, comprising of an inflammatory biomarker selected from apolipoprotein A-I, clusterin, α-1 B-glycoprotein precursor, vitronectin precursor; a blood coagulant biomarker selected from complement component C9 precursor; an antioxidant biomarker selected from serum albuminprecursor, Apolipoprotein A-IV precursor, and ceruloplasmin precursor; peptidase/protease inhibitor biomarker selected from α-1-antitrypsin precursor, inter-alpha-trypsin inhibitor heavy chain H4 precursor; protein binding biomarkers selected from apolipoprotein Al precursor, apolipoprotein A-IV precursor, and regulator of G-protein signaling.
3. A biomarker panel as claimed in Claim 1, wherein the panel comprises: alpha-1-antitrypsin, vitronectin, clusterin, ceruloplasmin, Regulator of G-protein signaling, and Apo AIV.
4. A kit for detecting a panel of biomarkers as claimed in claim 1 comprising:
a) a receptacle for receiving a sample;
b) one or more reagents for detecting three or more biomarkers selected from alpha-1-
antitrypsin precursor, vitronectin precursor, leucine-rich alpha-2-glycoprotein precursor,
alpha-1 B-glycoprotein precursor, complement component C9 precursor, alpha-1-
antichymotrypsin precursor, ceruloplasmin precursor, inter-alpha-trypsin inhibitor heavy

chain H4 precursor, complement factor B precursor, apolipoprotein E precursor, Ig alpha-1 chain C region, apolipoprotein A-I precursor, serum albumin precursor, Ig mu chain C region, complement C3 precursor, clusterin precursor, apolipoprotein A-IV precursor, alpha-2-HS-glycoprotein precursor, complement factor H precursor, regulator of G-protein signaling, 7 Ig kappa chain C region, and c) a reference sample.
5. A kit as claimed in claim 4, wherein the biomarker panel comprises: alpha-1-antitrypsin, clusterin, vitronectin, ceruloplasmin, regulator of G-protein signaling, and apo AIV.
6. A kit as claimed in Claim 4, wherein the reference sample is collected from a healthy individual.
7. A kit as claimed in claim 4, wherein the reagents for detecting the biomarkers are selected from antigens that are specific for the selected protein biomarkers.
8. A method comprising:

a) Measurement of the amount of three or more protein biomarkers selected from alpha-1-antitrypsin precursor, vitronectin precursor, leucine-rich alpha-2-glycoprotein precursor, alpha-1B-glycoprotein precursor, complement component C9 precursor, alpha-1-antichymotrypsin precursor, ceruloplasmin precursor, inter-alpha-trypsin inhibitor heavy chain H4 precursor, complement factor B precursor, apolipoprotein E precursor, Ig alpha-1 chain C region, apolipoprotein A-I precursor, serum albumin precursor, Ig mu chain C region, complement C3 precursor, clusterin precursor, apolipoprotein A-IV precursor, alpha-2-HS-glycoprotein precursor, complement factor H precursor, regulator of G-protein signaling, 7 Ig kappa chain C region in a sample;
b) comparing the measured amount with a predetermined value for control;
c) assessing patient status based on the difference between the measured value and value for the control sample.
9. A method as claimed in Claim 8, wherein the sample is a serum, plasma, or whole
blood sample.

10. A method as claimed in Claim 8, wherein the protein biomarker panel comprises of alpha-1-antitrypsin, vitronectin, ceruloplasmin, regulator of G-protein signaling, and apo
A1V.