FIELD OF INVENTION

[0001] The present invention relates to the field of immunology, molecular biology, and genetic engineering, in particular to the field of production of recombinant antibodies. The present invention specifically relates to anti Brucella S-LPS antibody.

BACKGROUND OF THE INVENTION

[0002] Brucellosis represented by Brucella abortus, Brucella melitensis,

Brucella suis, Brucella ovis etc. is caused by facultative intracellular gram negative Brucella. In: Dworkin M., et al. (Ed.), The Prokaryotes. New York: Springer, 315— 456). It is a major bacterial zoonotic disease affecting a wide variety of mammals globally, and is considered as an emerging/reemerging disease (Corbel, 1997, Emerging Infectious Diseases Journal, 3(2): 213-221).The disease is characterized by abortion and infertility in domestic herds with concomitant loss in productivity. It causes a debilitating illness in humans, and is known to persist for a number of years (Lapaque N., et al., 2005, Current Opinion in Microbiology, 8(l):60-66). The currently available vaccines involve the use of Brucells abortus 19 (SI9), an attenuated smooth strain, to vaccinate cattle against Brucella infections (Nicoletti, P. et al., 1990, Vaccination. Nielsen K. (Ed.) Animal brucellosis. CRC Press, Inc., Boca Raton, 283-299). The vaccine elicits a sustained antibody response, which is mainly directed against the O chain of smooth lipopolysaccharide (S-LPS). The lipopolysaccharide (LPS) is the dominant virulence determinant, and several monoclonal antibodies (MAbs) directed towards the O-chain of LPS have been shown to confer protection against Brucella infection (Cloeckaert, A., et al., 1992, Infection and Immunity, 60: 312-315; Limet, J. N., et al, 1989, Journal of Medical Microbiology, 30: 37- 43; Limet, J., et al, 1987, Annales de llnstitut Pasteur Immunology, 138:417- 424). Though live attenuated vaccines confer a high degree of protection, they have several disadvantages which limit the efficacy of the vaccination only to female cattle or buffaloes between the age group of 4 to 12 months., Several subunit vaccines with limited efficacy have been developed against Brucellosis to overcome the limitations of the traditional vaccines.

[0003] Glycoconjugate vaccines have been developed against a number of gram-negative bacterial organisms and have been shown to render effective prophylaxis against various bacterial diseases (Stein, K. E. 1994, International Journal of Technology Assessment in Health Care, 10: 167-176; Jones, C, 2005, The Anais da Academia Brasileira de Ciencias, 77: 293-324). Studies with respect to Brucella have
indicated that. vaccination with- pdriri and ^smopth lipjfrblys&QKa Brucella abortus strain confers the level of protection7 which Ts""equivmemroThW^''5 produced by the live attenuated strain (Winter, A. J., et ah, 1988, Infection and Immunity, 56:2808-2817; Cloeckaert A., et al., 1996, Infection and Immunity, 64:2047-2055; Jacques L, et al., 1991, Vaccine, 9: 896-900; Mythili T., et al., 2010, Current Trends in Biotechnology and Pharmacy, 4:510-518). The methodology of production of glycoconjugate vaccines involves the purification of S-LPS using conventional methods from the organism (Beuvery E. C, et al., 1982, Infection and Immunity, 37: 15-22; Verstreate D. R., et al., 1982, Infection and Immunity, 35: 979-989; Yi E. C, et al., 2000, Analyst, 125: 651-656), its conjugation to a carrier protein, and its further purification. Quantification of S-LPS using ELISA based methods have been developed to test in-process control samples which enables reliable estimation of S-LPS. This would aid not only in the manufacture of good quality vaccines but also in reducing the cost, thereby making the vaccines more economically feasible, and affordable in developing countries. Use of monoclonal antibodies for estimation of Brucella S-LPS involves production of antibodies from conventional technologies which involve high cost, are time consuming, and laborious as opposed to production of recombinant antibodies (Nielsen K., et al., 2004, Revue scientifique et technique, 23(3): 979-87). The use of recombinant antibody fragments such as monovalent antibodies can help circumvent the limitations of traditional technologies, and provide homogenous, pure reagents that can be used for the development of quantitative assays.

[0004] The field of antibody engineering in recent times has gained attention by the display of Fab (Fragment, antigen binding) and scFv (single chain variable fragment) antibody fragments on the surface of bacteriophage for generation of libraries thereby acting as an effective platform for isolation of diverse set of antibodies (Brekke O. H., et al., 2003, Current Opinion in Pharmacology, 3: 544-50) against various disease targets from immune or naive samples which can be expressed in bacterial systems (Barbas III C.F., et al., 1992, Proceedings of the National Academy of Sciences, 89: 10164-8; Burton D.R., et al., 1991, Proceedings of the National Academy of- * Sciences, 88: 10134-7; Marks, J. D., et al., 1991, Journal of Molecular Biology, 222:. ■;' 581-597; Hoogenboom H.R., et al, 1992, Journal of Molecular Biology, 227: 381-388). ScFv consists of (VH and VL) domains tethered by a flexible peptide linker which retains the antigen binding site in a single linear molecule. The design, construction and expression of the scFv in Escherichia coli has demonstrated their structure-function relationship and antigen-antibody interactions, thus making the scFv useful in both clinical and medical application (Huston J.S., et al, 1988, Proceedings of the National Academy of Sciences, 85: 5879-83; Bird R.E., et al, 1988, Science, 242: 423-6; Condra J.H., etal., 1990, The Journal of Biological Chemistry, 5: 2292-5).

[0005] US 2011/0200596 discloses methods and compositions comprising a novel stabilized monovalent antibody fragments useful as therapeutics.

[0006] US 5190860 discloses an immunoassay procedure that allows the differentiation of animals infected with Brucella abortus from animals vaccinated with Brucella abortus Strain 19 by employing monospecific monoclonal antibodies having binding characteristics directed to B. abortus lipopolysaccharide antigen.

[0007] US 5188936 discloses diagnostic reagents comprising 20kd Brucella abortus CuZn superoxide dismutase protein and peptide segments thereof, effective as antigenic determinants. The reagents disclosed in the invention are useful in detection of antibody response to B. abortus CuZnSOD protein in bovine body fluids.

[0008] EP 0357642 discloses a vaccine against Brucella abortus in which specific antigens of Brucella abortus are combined to induce an immunological response which provides protective immunity and permits differentiation between field strain infected and vaccinated cattle.

[0009] EP 0627935 discloses a vaccine against Brucella abortus comprising cell envelopes isolated from an O polysaccharide antigen deficient, stable transposon mutant of B. abortus with a soluble carrier.

[00010] There is a therefore a need in the art for the development of an anti- Brucella antibody which is useful for the development of a rapid, reliable, and reproducible assay system for the detection and quantification of Brucella.

SUMMARY OF THE INVENTION

[00011] Accordingly it is an aspect of the present invention to provide a recombinant antibody that specifically binds to Brucella smooth lipopolysaccharide (S-LPS), wherein the amino acid sequence of single chain variable fragment of the recombinant antibody is as set forth in SEQ ID NO: 12.

[00012] Another aspect of the present invention relates to a recombinant vector comprising the polynucleotide, encoding the polypeptide as set forth in SEQ ID NQ: 12, having at least 90% sequence identity to the polynucleotide having nucleotide sequence as set forth in SEQ ID NO: 11.

[00013] In yet another aspect of the present invention, there is provided a method
for detecting the presence of Brucella S-LPS antigen, wherein the method comprises capturing Brucella S-LPS antigen using scFv antibody, wherein the amino acid sequence of scFv is as set forth in SEQ ID NO: 12.

[00014] Still another aspect of the present invention relates to a method for detecting the presence of Brucella S-LPS in a biological sample, the method comprising: coating a solid support with a primary antibody against Brucella S-LPS antigen to obtain coated support; adding a sample to the coated support to allow binding of Brucella S-LPS antigen present in the sample to the primary antibody on the coated support; screening for binding of Brucella S-LPS antigen to the primary antibody by adding scFv antibody conjugated to a label, wherein the amino acid sequence of scFv is as set forth in SEQ ID NO: 12; and detecting the label; wherein the detection of label indicates the presence of Brucella S-LPS.

[00015] This summary is provided to introduce concepts related to a recombinant antibody that specifically binds to Brucella smooth lipopolysaccharide (S-LPS). This summary is not intended to identify essential features of the claimed subject matter nor is it intended for use in determining or limiting the scope of the claimed subject matter.

BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS

[00016] The following drawings form part of the present specification and are included to further-illustrate aspects of the present invention. The invention may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.

[00017] Figure 1 shows the results of electrophoretic analysis of PCR amplified variable heavy and light chains; M: Molecular weight markers, VH: Variable heavy chain, VL: Variable light chain and scFv: Assembled scFv product.

[00018] Figure 2 shows vector map showing cloned single chain variable fragment (scFv)

[00019] Figure 3 shows vector construct comprising single chain variable fragment (scFv)

[00020] Figure 4 A shows the results of electrophoretic analysis of PCR amplified scFv with the restriction site primers to clone into pET 28a vector.

[00021] Figure 4B shows the results of restriction digestion analysis of Pet cloned plasmids with EcoRl and Notl.

[00022] Figure 5 shows the results of SDS-PAGE analysis of purified monovalent scFv. The purified protein was detected by staining with coomassie brilliant blue. The size of the corresponding protein molecular weight standards in KDa are denoted on the right.

[00023] Figure 6 shows the results of western blot analysis of purified scFv protein. The blot was reacted with His-probe specific for the histidine tag of the scFv. A protein band of 28 kDa was detected. Lanes: Lane 1 is eluted fraction of scFv obtained from affinity chromatography. Lane M: Prestained molecular weight marker.

[00024] Figure 7 shows the binding activity of purified scFv protein to Brucella S-LPSbyELISA.

[00025] Figure 8 shows the specificity of scFv towards Brucella S-LPS.

[00026] Figure 9 shows the binding activity of scFv to Brucella S-LPS by sandwich ELISA.

[00027] Figure 10 shows the results of determination of binding specificity of a mouse monoclonal antibody and scFv in a competitive ELISA.

[00028] Figure 11 shows the results of development of in-house immunocapture ELISA (IC-ELISA) format for detection of Brucella S-LPS antigen using scFv in in-process samples.

DETAILED DESCRIPTION OF THE INVENTION

[00029] Those skilled in the art will be aware that the invention described herein is subject to variations and modifications other than those specifically described. It is to be understood that the invention described herein includes all such variations and modifications. The invention also includes all such steps, features, compositions, and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

Definitions
[00030] For convenience, before further description of the present invention, certain terms employed in the specification, examples and appended claims are collected here. These definitions should be read in light of the remainder of the disclosure and understood as by a person of skill in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art.

[00031] The articles "a", "an", and "the" are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.

[00032] The terms "comprise" and "comprising" are used in the inclusive, open sense, meaning that additional elements may be included. It is not intended to be construed as "consists of only".

[00033] Throughout this specification, unless the context requires otherwise the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated element or step or group of element or steps but not the exclusion of any other element or step or group of element or steps.

[00034] The term "including" is used to mean "including but not limited to". "Including" and including but not limited to" are used interchangeably.

[00035] The term "antibody" refers to the Y-shaped protein on the surface of B-cells that is secreted into the blood or lymph in response to an antigenic stimulus, such as bacterium, vims, parasite, or transplanted organ, and that neutralizes the antigen by binding specifically to it.

[00036] The term "Single-chain Fv" also abbreviated as "sFv" or "scFv" refer to antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain. Preferably, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.

[00037] The term "linker" used in the present invention refers to a portion of or functional group on a building block that can be employed to or that does (e.g., reversibly) couple the building block to a support, for example, through covalent link, ionic interaction, electrostatic interaction, or hydrophobic interaction.

[00038] The term "amino acid sequence" means the sequence of amino acids that characterizes a given protein.

[00039] The term "polypeptide" means a polymer of amino acids joined together by peptide bonds.

[00040] The terms "single-chain variable fragment" and "scFv" are used interchangeably and refer to fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a short linker peptide of ten to about 25 amino acids.

[00041] The term "polynucleotide" used in the present invention refers to a DNA polymer composed of multiple nucleotides chemically bonded by a series of ester linkages between the phosphoryl group of one nucleotide and the hydroxyl group of the sugar in the adjacent nucleotide.

[00042] The polynucleotides described in the present description include "genes" and nucleic acid molecules described including "vectors" or "plasmids". Accordingly, the term "gene", also called a "structural gene" refers to a polynucleotide that codes for a particular sequence of amino acids, which comprise all or part of one or more proteins or enzymes, and may include regulatory (non-transcribed) DNA sequences, such as promoter sequences, which determine for example the conditions under which the gene is expressed.

[00043] The term "nucleotide sequence" means the order in which nucleotides are situated in a chain relative to one another.

[00044] The term "sequence identity" means the number (%) of matches in positions from an alignment of two molecular sequences.

[00045] A "vector" is any means by which a nucleic acid can be propagated and/or transferred between organisms, cells or cellular components. Vectors include viruses, bacteriophage, pro-viruses, plasmids, phagemids, transposons and artificial chromosomes such as YACs (yeast artificial chromosomes), BACs (bacterial artificial chromosomes), and PLACs (plant artificial chromosomes), and the like, that are "episomes", that is, that replicate autonomously or can integrate into a chromosome of a host cell. A vector can also be a naked RNA polynucleotide, a polynucleotide composed of both DNA and RNA within the same strand, a poly-lysine-conjugated DNA or RNA, a peptide conjugated DNA or RNA, a liposome-conjugated DNA, or the like, that are not episomal in nature, or it can be an organism which comprises one or more of the above polynucleotide constructs such as Agrobacterium or a bacterium.

[00046] The term "recombinant vector" means a vector carrying a foreign DNA fragment.

[00047] The term "recombinant host cell" means a host cell carrying a recombinant vector.

[00048] The term "monoclonal antibody" refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

[00049] The terms "test sample", "sample", and "biological sample" are used interchangeably and refer to materials obtained from a biological source, environmental source, or a processed sample. The processed sample may include extraction of genetic material from the sample.

[00050] The term "secondary antibody" refers to an antibody that binds to a primary antibody or antibody fragments. The secondary antibodies are typically labelled with probes which makes them useful for detection, purification, or cell sorting applications.

[00051] It is an object of the present invention to provide a recombinant antibody fragment capable of recognizing Brucella S-LPS.

[00052] It is yet another object of the present invention to provide a recombinant antibody fragment selected using bovine antibody library by phage display technology against Brucella S-LPS.

[00053] It is still another object of the present invention to provide a method of production of the recombinant antibody specific for Brucella S-LPS.

[00054] It is another object of the present invention to provide a composition comprising the recombinant antibody fragment capable of recognizing Brucella S-LPS and a pharmaceutically acceptable carrier.

[00055] It is yet another object of the present invention to develop an ELISA method for detection and quantification of Brucella S-LPS.

[00056] The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only. Functionally-equivalent products, compositions, and methods are clearly within the scope of the invention, as described herein.

[00057] The present invention provides a recombinant anti-Brucella S-LPS antibody and a method for production of the antibody. The invention disclosed in the present specification further provides a recombinant vector and host cells comprising the antibody. Further, the invention provides a composition comprising the antibody. The present invention also provides a method of detection of Brucella S-LPS using the recombinant anti- Brucella S-LPS antibody of the present invention. The anti-Brucella S-LPS antibody disclosed in the present invention is useful in the recognition of Brucella S-LPS.

[00058] In accordance with the present invention, there is provided a recombinant antibody that specifically binds to Brucella smooth lipopolysaccharide (S-LPS), wherein the amino acid sequence of single chain variable fragment of the recombinant antibody is as set forth in SEQ ID NO: 12.

[00059] In accordance with the present invention, total RNA was isolated from peripheral blood lymphocytes of non-immunized bovines and cDNA for the same was synthesized by reverse transcriptase polymerase chain reaction (RT-PCR). The RT-PCR amplified DNA was used as template for the amplification of variable domains of an antibody with universal primers. A fragment of 354 bp of heavy variable chain was obtained after amplification. The nucleotide sequence of heavy variable chain is as set forth in SEQ ID NO: 5. A second fragment of 309 bp of light variable chain was also obtained. The nucleotide sequence of light variable chain is as set forth in SEQ ID NO: 6. These amplified variable domains were assembled to form a single chain variable fragment by splicing by overlap extension polymerase chain reaction (SOE PCR). Figure 1 shows electrophoretic analysis of PCR amplified variable heavy and light chains. The molecular weights of the standards are denoted on the right side of the figure. The single chain variable fragment was cloned into phagemid vector to obtain recombinant phagemid vector. The recombinant phagemid vector thus obtained was transformed into E. coli TGI. The transformed E. coli cells comprising the recombinant phagemid vector were infected with M13K07 helper phage to yield recombinant phages.

[00060] In accordance with the present invention, Bovine phage displayed scFv library was constructed from non-immunised peripheral blood lymphocytes. scFv fragment comprises one variable heavy chain and one variable light chain linked by a peptide linker. The phage was produced and was used for biopanning after PEG precipitation.

[00061] In accordance with the present invention, phages displaying individual bovine scFv antibody fragments were selected by biopanning. Eluted phage from third round was used to infect Escherichia coli TGI and randomly selected 96 individual clones were tested by phage ELISA against Brucella S-LPS. Out of the 96 clones tested, thirty five clones showed positive binding to virus. Selected few clones, based on their high binding affinity, were used for further study.

[00062] In accordance with the present invention, the size of single chain variable fragment (scFv) is 717 bp. The nucleotide sequence of single chain variable fragment sequence is as set forth in SEQ ID NO: 11. The amino acid sequence of single chain variable fragment sequence is as set forth in SEQ ID NO: 12.

[00063] scFv (SEQ ID NO: 11) comprises 354 bp heavy variable chain (SEQ ID NO: 5); 309 bp light variable chain (SEQ ID NO: 6) and a 54 bp linker (Gly4Ser)3 (SEQ ID NO: 7) which is used to connect the heavy variable chain and light variable chain of antibody. The nucleotide sequence of 54 bp linker is as set forth in SEQ ID NO: 7. The amino acid sequence of heavy variable chain is as set forth in SEQ ID NO: 8. The amino acid sequence of light variable chain is as set forth in SEQ ID NO: 9. The amino acid sequence of the linker is as set forth in SEQ ID NO: 10. Figure 2 shows vector map showing cloned single chain variable fragment (scFv). Figure 3 shows vector construct comprising single chain variable fragment (scFv). The nucleotide sequence encoding monovalent anti- Brucella S-LPS antibody i.e. scFv (SEQ ID NO: 11) was cloned between the EcoRl and Notl sites of pET 28a bacterial expression vector. The pET 28a vector carries ribosome binding site, signal peptide sequence of bacterial pectate lyase, variable fragment of light chain, heavy chain, C-terminal His tag sequence and T7 promoter and T7 terminator. Figure 4A shows electrophoretic analysis of PCR amplified scFv with the restriction site primers to clone into pET 28a vector. Selected scFv plasmids were transformed into Escherichia coli BL21 (DE3) for production of soluble scFv fragments.

[00064] In accordance with the present invention, the transformed into Escherichia coli BL21 (DE3) cells were induced with IPTG. The purified protein eluted fractions of scFv fragment were analyzed by SDS-PAGE analysis under reducing conditions after staining with coomassie brilliant blue. Figure 5 shows the results of SDS-PAGE analysis of purified monovalent scFv. The purified protein was detected by staining with coomassie brilliant blue. Figure 6 shows the results of western blot analysis of purified scFv protein. A major band with an apparent molecular weight of about 28 kDa was detected.

[00065] In accordance with the present invention, the eluted fractions were screened to determine the ability of scFv to bind to Brucella S-LPS by indirect ELISA and to check the cross-reactivity of scFv with rough strain S-LPS. Titration of scFv against the Brucella S-LPS, revealed a concentration dependent reduction of the optical density values as shown in Figure 7. No reactivity was observed with a lysate of E.coli indicating the binding specificity of scFv towards S-LPS. Figure 8 shows the specificity of scFv towards Brucella S-LPS. The results showed reactivity of scFv with Brucella S-LPS and not with rough strain S-LPS, indicating that scFv could be used to detect Brucella S-LPS without any cross reaction with other strains of Brucella.

[00066] In accordance with the present invention, the recombinant anti-Brucella S-LPS antibody of the present invention is labeled. The term "label", "detectably labeled" or "labeled with a detectable marker" refer to an antibody composition capable of producing a detectable signal indicative of the presence of the labeled molecule. Suitable labels include radioisotopes, a dye, colloidal gold or a similarly detectable marker, nucleotide chromophores, enzymes, substrates, fluorescent molecules, chemiluminescent moieties, magnetic particles, bioluminescent moieties, and the like, including labels suitable for indirect detection, such as biotin. As such, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. A label may be attached by use of a chemical linker. Exemplary labels are those that produce a visible signal that can be detected by visual inspection.

[00067] The recombinant anti-Brucella S-LPS antibody of the present invention may also be conjugated by known methods and means to a solid phase support such as, but not limited to, glass, plastic, a synthetic membrane. Other non-limiting examples include beads, particles, dipsticks, fibers, filters, Petri dishes, ELISA (enzyme-linked immunosorbent assay) plates, microtiter plates, silane or silicate supports such as glass slides, and dishes, wells or containers, as well as the sides thereof. Such immobilized forms of the antibodies may be used in the detection methods disclosed herein.

[00068] The methods used to detect Brucella S-LPS are not limited by design. Non-limiting examples include methods utilizing the antibody of the invention, and alternative forms thereof as described herein, and based upon the principles of Western blotting or other immunoblotting, ELISA, lateral flow devices, sandwich assays, visual observation by microscopy, competitive and non-competitive immunoassays, immunoenzymetric assays, immunofluorescence, immunomagnetic selection, and flow cytometry (including detection by polychromatic flow cytometry). The methods of the invention are used to qualitatively or quantitatively detect the presence or absence of Brucella S-LPS in a sample.

[00069] The materials for use in the methods of the present invention are ideally suited for preparation of kits produced in accordance with well known procedures. The invention thus provides kits comprising agents for the detection and/or quantitation of Brucella S-LPS, or extracts or disrupted forms thereof, in a sample as described herein. Such kits optionally comprising the agents and/or reagents with an identifying description or label or instructions relating to the use of the kits, or the suitability of the kits, in the methods of the present invention, is provided. Such a kit may comprise containers, each with one or more of the various agents and/or reagents (optionally in concentrated form) utilized in the methods, including, for example, detection agents and/or pre-immobilized forms of capture reagents. A set of instructions or reagent identifiers will also typically be included. Other exemplary kits contain a device or solid phase supports, such as, but not limited to a lateral flow device, a test strip, beads, a membrane, or coated surfaces of a container, dish or well, for the practice of the invention.

[00070] An embodiment of the present invention provides a recombinant antibody that specifically binds to Brucella smooth lipopolysaccharide (S-LPS), wherein the amino acid sequence of single chain variable fragment of the recombinant antibody is as set forth in SEQ ID NO: 12.

[00071] In an embodiment of the present invention, there is provided a recombinant antibody that specifically binds to Brucella smooth lipopolysaccharide (S-LPS), wherein the amino acid sequence of single chain variable fragment of the recombinant antibody is as set forth in SEQ ID NO: 12 has at least 90% sequence identity to the polynucleotide having nucleotide sequence as set forth in SEQ ID NO: 11.

[00072] Another embodiment of the present invention provides a recombinant antibody that specifically binds to Brucella smooth lipopolysaccharide (S-LPS), wherein the polynucleotide encoding the amino acid sequence of single chain variable fragment of the recombinant antibody as set forth in SEQ ID NO: 12, is as set forth in SEQ ID NO: 11.

[00073] In yet another embodiment of the present invention, there is provided a recombinant antibody that specifically binds to Brucella smooth lipopolysaccharide (S-LPS), wherein the amino acid sequence of single chain variable fragment of the recombinant antibody is as set forth in SEQ ID NO: 12, wherein the recombinant antibody is a monovalent antibody.

[00074] In still another embodiment of the present invention, there is provided a recombinant vector comprising the polynucleotide, encoding the polypeptide as set forth in SEQ ID NO: 12, having at least 90% sequence identity to the polynucleotide having nucleotide sequence as set forth in SEQ ID NO: 11.

[00075] In yet another embodiment of the present invention, there is provided a recombinant vector comprising the polynucleotide, encoding the polypeptide as set forth in SEQ ID NO: 12, having the nucleotide sequence as set forth in SEQ ID NO: 11.

[00076] In an embodiment of the present invention, there is provided a recombinant host cell comprising a recombinant vector comprising the polynucleotide, encoding the polypeptide as set forth in SEQ ID NO: 12, having at least 90% sequence identity to the polynucleotide having nucleotide sequence as set forth in SEQ ID NO: 11.

[00077] An embodiment of the present invention provides a recombinant host cell comprising a recombinant vector comprising the polynucleotide, encoding the polypeptide as set forth in SEQ ID NO: 12, having the nucleotide sequence as set forth in SEQ ID NO: 11.

[00078] Another embodiment of the present invention provides a recombinant host cell comprising a recombinant vector comprising the polynucleotide, encoding the polypeptide as set forth in SEQ ID NO: 12, having at least 90% sequence identity to the polynucleotide having nucleotide sequence as set forth in SEQ ID NO: 11, wherein the recombinant host cell is either E. coli or yeast.

[00079] In yet another embodiment of the present invention, there is provided a recombinant host cell comprising a recombinant vector comprising the polynucleotide, encoding the polypeptide as set forth in SEQ ID NO: 12, having the nucleotide sequence as set forth in SEQ ID NO: 11, wherein the recombinant host cell is either E. coli or yeast.

[00080] In still another embodiment of the present invention, there is provided a method for detecting the presence of Brucella S-LPS antigen, wherein the method comprises capturing Brucella S-LPS antigen using scFv antibody, wherein the amino acid sequence of scFv is as set forth in SEQ ID NO: 12.

[00081] In another embodiment of the present invention, there is provided a method for detecting the presence of Brucella S-LPS antigen in bulk antigen, glycol-conjugate vaccine, and in process samples, wherein the method comprises capturing Brucella S-LPS antigen using scFv antibody, wherein the amino acid sequence of scFv is as set forth in SEQ ID NO: 12.

[00082] In an embodiment of the present invention, there is provided a method For detecting the presence of Brucella S-LPS antigen, wherein the method comprises capturing Brucella S-LPS antigen using scFv antibody having amino acid sequence as set forth in SEQ ID NO: 12, wherein the method is an immunoassay.

[00083] An embodiment of the present invention provides a method for detecting the presence of Brucella S-LPS antigen, wherein the method comprises capturing Brucella S-LPS antigen using scFv antibody having amino acid sequence as set forth in SEQ ID NO: 12, wherein the method is ELISA.

[00084] Another embodiment of the present invention provides a method for detecting the presence of Brucella S-LPS in a biological sample, the method comprising: coating a solid support with a primary antibody against Brucella S-LPS antigen to obtain coated support; adding a sample to the coated support to allow binding of Brucella S-LPS antigen present in the sample to the primary antibody on the coated support; screening for binding of Brucella S-LPS antigen to the primary antibody by adding scFv antibody conjugated to a label, wherein the amino acid sequence of scFv is as set forth in SEQ ID NO: 12; and detecting the label; wherein the detection of label indicates the presence of Brucella S-LPS.

[00085] In yet another embodiment of the present invention, there is provided a method for detecting the presence of Brucella S-LPS in a biological sample, the method comprising: coating a solid support with a primary antibody against Brucella S-LPS antigen to obtain coated support; adding a sample to the coated support to allow binding of Brucella S-LPS antigen present in the sample to the primary antibody on the coated support; screening for binding of Brucella S-LPS antigen to the primary antibody by adding scFv antibody conjugated to a label, wherein the amino acid sequence of scFv is as set forth in SEQ ID NO: 12; and detecting the label; wherein the primary antibody is mouse monoclonal antibody.

[00086] In still another embodiment of the present invention, there is provided a method for detecting the presence of Brucella S-LPS in a biological sample, the method comprising: coating a solid support with a primary antibody against Brucella S-LPS antigen to obtain coated support; adding a sample to the coated support to allow binding of Brucella S-LPS antigen present in the sample to the primary antibody on the coated support; screening for binding of Brucella S-LPS antigen to the primary antibody by adding scFv antibody conjugated to a label, wherein the amino acid sequence of scFv is as set forth in SEQ ID NO: 12; and detecting the label; wherein the sample is either lysed cultures of Brucella, or purified LPS extracts of Brucella cultures.

[00087] In an embodiment of the present invention, there is provided a method for detecting the presence of Brucella S-LPS in a biological sample, the method comprising: coating a solid support with a primary antibody against Brucella S-LPS antigen to obtain coated support; adding a sample to the coated support to allow binding of Brucella S-LPSantigen present in the sample to the primary antibody on the coated support; screening for binding of Brucella S-LPS antigen to the primary antibody by adding scFv antibody conjugated to a label, wherein the amino acid sequence of scFv is as set forth in SEQ ID NO: 12; and detecting the label; wherein the label is selected from the group consisting of radioisotope, dye, colloidal gold, nucleotide chromophore, enzyme, substrate, fluorescent molecule, chemiluminescent moiety, magnetic particle, bioluminescent moiety, and biotin.

[00088] An embodiment of the present invention provides a method for detecting the presence of Brucella S-LPS in a biological sample, the method comprising: coating a solid support with a primary antibody against Brucella S-LPS antigen to obtain coated support; adding a sample to the coated support to allow binding of Brucella S-LPS antigen present in the sample to the primary antibody on the coated support; screening for binding of Brucella S-LPS antigen to the primary antibody by adding scFv antibody conjugated to a label, wherein the amino acid sequence of scFv is as set forth in SEQ ID NO: 12; and detecting the label; wherein the label is an enzyme.

[00089] Another embodiment of the present invention provides a method for detecting the presence of Brucella S-LPS in a biological sample, the method comprising: coating a solid support with a primary antibody against Brucella S-LPS antigen to obtain coated support; adding a sample to the coated support to allow binding of Brucella S-LPS antigen present in the sample to the primary antibody on the coated support; screening for binding of Brucella S-LPS antigen to the primary antibody by adding scFv antibody conjugated to a label, wherein the amino acid sequence of scFv is as set forth in SEQ ID NO: 12; and detecting the label; wherein the label is Horseradish peroxidase (HRP).

[00090] Although the subject matter has been described in considerable details with reference to certain preferred embodiments thereof, other embodiments are possible. As such, the spirit and scope of the appended claims should not be limited to the description of the preferred embodiment contained therein.

EXAMPLES

[00091] The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein.

Example 1
Preparation of antibody fragment
RNA extraction and amplification of Antibody Variable domain sequences

[00092] Total RNA was isolated from peripheral blood lymphocytes of non-immunized bovines using Trizol. 10 million Spleen cells were lysed with 1 ml Trizol for 5 minutes at room temperature and 200 ul of chloroform was added to it. The entire solution was added to 2 mL phase lock column (from Invitrogen) and centrifuged at

high speed for 15 minutes. The aqueous layer was pipetted out and RNA was precipitated with 100% Isopropanol. RNA was washed twice with 70% ethanol before proceeding to cDNA synthesis. According to manufacturer's instructions. cDNA for the isolated RNA was synthesized by reverse transcriptase polymerase chain reaction (RT-PCR) using RT-PCR kit from Invitrogen. The RT-PCR amplified DNA was used as template for the amplification of variable domains of an antibody with universal primers. Bovine variable heavy chain was amplified using forward and reverse primer sequences as set forth in SEQ ID NO: 1, and SEQ ID NO: 2 respectively. Bovine variable light chain was amplified using forward and reverse primer sequences as set forth in SEQ ID NO: 3, and SEQ ID NO: 4 respectively.

[00093] A fragment of 354 bp was obtained after amplification of the heavy variable chain was obtained. The nucleotide sequence of the heavy variable chain is as set forth in SEQ ID NO: 5. A second fragment of 309 bp was obtained on the amplification of the light variable chain was obtained. The nucleotide sequence of light variable chain is as set forth in SEQ ID NO: 6.

[00094] The amplified variable domains were assembled to form a single chain variable fragment by splicing by overlap extension polymerase chain reaction (SOE PCR). Figure 1 shows the results of the electrophoretic analysis of PCR amplified variable heavy and light chains; M-Molecular weight markers, VH -Variable heavy chain, VL -Variable light chain and scFv- Assembled scFv product. The single chain variable fragment was cloned into phagemid vector using Ligase (GeNei), according to manufacturer's instructions, to obtain recombinant phagemid vector. The recombinant phagemid vector thus obtained was transformed into E. coli TGI (Stratagene), according to manufacturer's instructions. The transformed E. coli cells comprising the recombinant phagemid vector were infected with M13K07 helper phage to yield recombinant phages. Bovine phage displayed scFv library was constructed from non-immunised peripheral blood lymphocytes. The library comprises over 3.4 x 108 different scFv fragments cloned in an ampicillin- resistant phagemid vector Pcantab 5E and transformed into E. coli TGI cells. scFv fragment comprises one variable heavy chain and one variable light chain linked by a peptide linker.
Production of the Phage Library

[00095] Production of the phage was performed essentially as described (Marks J. D., et al., 1991, Journal of Molecular Biology, 222: 581-597). Briefly, E. coli TGI cultures in the exponential phase were infected with helper phage M13K07 at multiplicity of infection of 10 to the bacteria. The bacteria infected with helper phage were grown overnight at 30°C for phage production. The phages were PEG precipitated and used for biopanning.

[00096] Selection of recombinant antibodies against Brucella S-LPS from an non-immunised antibody library by biopanning

[00097] Phages displaying individual bovine scFv antibody fragments were selected by three rounds of biopanning on immunotubes coated with a decreasing amount of purified antigen for the first, second and third round of panning. The results are provided in Table 1.

Table 1: Enrichment of Brucella S-LPS specific scFv clones in three rounds of panning.

[00098] Eluted phage titers showed an increase after each of the three rounds of panning, indicating successful enrichment. Eluted phage from third round was used to infect Escherichia coli TGI and randomly selected 96 individual clones were tested by phage ELISA against Brucella S-LPS (Analysis of phages for binding to Brucella SLP's virus by ELISA was performed on bacterial superaatants containing phages. Individual colonies after third round of biopanning were inoculated into 100 ul of 2xYT-AG (medium containing ampicillin (lOOug/ml) and 0.2% Glucose) and grown at 37°C overnight with shaking at 200 rpm. An aliquot of 10 ul from the overnight culture was sub-cultured into 100 ul of 2xYT media lOOug/ml and incubated at 37°C till the culture attained an O.D600 of 0.6 following which M13K07 helper phage of concentration 2.5xl010 pfu was added. Cultures were incubated with shaking at 37°C for 2 h at 200 rpm and centrifuged at 4000 rpm for 20 min. The bacterial pellet was resuspended in 2xYT containing lOOug/ml ampicillin, 50 mg/ml kanamycin and 2% w/v glucose, grown at 30°C overnight with shaking at 200 rpm. Cells were pelleted and the supernatant containing phages was collected for analysis in phage ELISA. Briefly, MaxiSorp ELISA plates were coated with Brucella SLP's antigen and incubated overnight at 4°C." Plates were blocked with 2% (w/v) milk in PBS for 1 h at 37°C. Undiluted phage was incubated with plate bound Ag for 2 h at 37°C. Plates were washed five times with PBS-T. Bound phage was detected using a 1:5000 dilution of horseradish peroxidase (HRP) conjugated anti-M13 mouse antibody (Pharmacia Biotech) in 2%(w/v) milk PBS. Plates were washed five times with PBS and bound phage was detected with TMB (3,3,5,5-tetramethylbenzidine) peroxidase substrate (Thermo Scientific, USA). OD was read at 450 nm after 30 min using a microplate reader (BIO-TEK, US).Out of the 96 clones tested, thirty five clones showed positive binding to virus. Selected few clones were used for further study based on their high binding affinity.

Analysis ofscFv sequences
[00099] Twelve clones from each round of biopanning were selected for sequencing analysis. Phagemid DNA was purified using Plasmid miniprep from QIAGEN) according to the manufacturer's protocol and sequenced at Ocimum Biosolutions, India. The sequencing primers, SI and S6 were used for variable heavy and light chain gene analysis respectively. The entire sequences was submitted to IMGT and verified as the variable domains of the antibody. The obtained 717 bp nucleotide sequence of single chain variable fragment sequence is as set forth in SEQ ID NO: 11. The amino acid sequence of single chain variable fragment sequence is as set forth in SEQ ID NO: 12.

Cloning ofscFv into pET vector

[000100] scFV is 717 bp (SEQ ID NO: 11) in size and it comprises 354 bp heavy variable chain (SEQ ID NO: 5); 309 bp light variable chain (SEQ ID NO: 6) and a 54 bp linker (GlytSer^ which is used to connect the heavy variable chain and light variable chain of antibody. The nucleotide sequence of 54 bp linker is as set forth in SEQ ID NO: 7. The amino acid sequence of heavy variable chain is as set forth in SEQ ID NO: 8. The amino acid sequence of light variable chain is as set forth in SEQ ID NO: 9. The amino acid sequence of the linker is as set forth in SEQ ID NO: 10.

[000101] Figure 2 shows vector map showing cloned single chain variable fragment (scFv). Figure 3 shows vector construct comprising single chain variable fragment (scFv). The schematic representations as depicted in Figure 2 and Figure 3 show that the nucleotide sequence encoding monovalent bovine anti- Brucella S-LPS antibody comprising scFv (SEQ ID NO: 11) was cloned between the EcoRl and Notl sites of pET 28a bacterial expression vector. scFv was PCR amplified with the restriction site primers to clone into pET 28a vector. The pET 28a vector carries ribosome binding site, signal peptide sequence of bacterial pectate lyase, variable fragment of light chain, heavy chain, C-terminal His tag sequence and T7 promoter and T7 terminator. Unique site are shown on the circle map. 'Ap' denotes the ampicillin resistance gene. Figure 4A shows the results of the electrophoretic analysis of PCR amplified scFv with the restriction site primers to clone into pET 28a vector.

[000102] The vector pET 28a and insert referred to monovalent antibody fragment scFv of size 717 bp (SEQ ID NO: 11) was digested with EcoRl and Notl, respectively by incubating at 37°C for 12 hours. The digested products was purified using the kit provided by QIAGEN and kept at various ratios of vector to insert (1:3 and 1:6) for cohesive end ligation and incubated at 22°C for 2 hours. The ligated product was incubated for further 20 minutes at 65°C in order to inactivate the enzyme. The final product was chemically transformed into XL-Blue strain of E. coli cells.

E. coli transformation
Preparation of Chemical competent XL-Blue strain ofE.coli cells

[000103] Overnight grown XL-Blue strain were sub-cultured and grown at 37°C with shaking until the OD of the culture reached to 0.6 at 600nm. The culture was harvested by centrifuging at 5000X rpm for 10 minutes at 4°C and resuspended in ice-cold 0.1 mM CaCb and incubated overnight on the ice, before proceeding for transformation.
Transformation
[000104] The chemically competent XL-Blue cells were incubated with plasmid
DNA for 30 minutes on ice. The cells were given heat shock at 42°C for 90 seconds and immediately placed on ice for 2 minutes before the media was added to cells. The cells were incubated for one hour at 37°C for recovery and plated on agar plates containing lOOmg/mL of ampicillin. The plates were incubated for overnight and screened for positive clones by isolating the plasmids and subjected to restriction digestion with EcoRl and Notl enzymes. Figure 4B shows the restriction digestion analysis of pET cloned plasmids with EcoRl and Notl. The positive clones were sequence verified before the plasmid was transformed into BL21 (DE3) cells of E.coli for soluble expression of the antibody gene.

Example 2
Production of soluble scFv fragments

[000105] Positive scFv clones identified by phage ELISA were transformed into non-suppressor Escherichia coli strain BL21 (DE3) for production of soluble scFvs. Individually selected clones were grown and production of scFv was induced by addition of ImM isopropyl-P-D- thiogalactopyranoside (IPTG). The purified protein eluted fractions of scFv fragment were analyzed by SDS-PAGE analysis under reducing conditions after staining with coomassie brilliant blue. Figure 5 shows the results of SDS-PAGE analysis of purified monovalent scFv. The purified protein was detected by staining with coomassie brilliant blue. Figure 6 shows the results of western blot analysis of purified scFv protein. A major band with an apparent molecular weight of about 28 kDa was detected. The blot was reacted with His-probe specific for the histidine tag of the scFv. The eluted fractions were screened for antigen binding by ELISA

Example 3
Determination of Binding Activity and Specificity of scFv
Determination of ability ofscFv to bind to Brucella S-LPS by indirect ELISA

[000106] The antigen binding activity of the purified scFv was measured by an indirect ELISA. Titration of scFv against the Brucella S-LPS, revealed a concentration dependent reduction of the optical density values as shown in Figure 7. No reactivity was observed with a lysate of E.coli indicating the binding specificity of scFv towards S-LPS. A microtiter plate was coated with SLP's (300ng/well) in 50mM carbonate-bicarbonate buffer (pH 9.6) and incubated at 4°C overnight. The plate was washed thrice with PBS-T and the unbound sites in the wells were blocked with 1% bovine gelatin. The plate was washed with PBS-T and dried. scFv (3ug) was added two-fold serially diluted to the ELISA plate and incubated at 37°C for lhrs. The plate was washed with PBS-T, 5 times and dried. The binding of the scFv with SLP's was detected by addition of His-Probe and a chromogenic substrate TMB. The plate was incubated at 37°C in dark for lOmin. The reaction was stopped by addition of 1.25M H2SO4 and the absorbance was measured at 450nm using a microplate reader (BIO-TEK, US). The experiment in triplicates to evaluate the concentration dependent binding activity of scFv towards SLP's antigen.

[000107] ELISA to check the specificity and cross-reactivity of scFv with rough strain S-LPS

[000108] Indirect ELISA was performed to determine the specificity of scFv. The results are provided in Figure 8. Figure 8 shows the specificity of scFv towards Brucella S-LPS. The results showed reactivity of scFv with Brucella S-LPS and not with rough strain S-LPS, indicating that scFv could be used to detect Brucella S-LPS without any cross reaction with other strains of Brucella.

[000109] A microtiter plate was coated with Brucella S-LPS, Brucella R-LPS, Salmonella LPS and E.coli LPS (300ng/well) in 50mM carbonate-bicarbonate buffer (pH 9.6) and incubated at 4°C overnight. The plate was washed thrice with PBS-T and the unbound sites in the wells were blocked with 1% bovine gelatin. The plate was washed with PBS-T and dried. scFv (3ug)) was serially tow fold diluted and added to the ELISA plate and incubated at 37°C for lhrs. The plate was washed with PBS-T, 5 times and dried. The binding of the scFv with SLP's was detected by addition of His-Probe and a chromogenic substrate TMB. The plate was incubated at 37°C in dark for lOmin. The reaction was stopped by addition of 1.25M H2SO4 and the absorbance was measured at 450nm using a microplate reader (BIO-TEK, US). The experiment in triplicates to evaluate the concentration dependent binding activity of scFv towards SLP's antigen and not RLP's.

Binding activity ofscFv by Sandwich ELISA

[000110] Sandwich ELISA was performed to determine the reactivity of the scFv
towards brucella S-LPS. The Mab (5C3) was coated to microtiter plate at a concentration of 200ng/well and was incubated at 4°C overnight. The plate was blocked with 1% bovine gelatin in PBS-T (phosphate buffer saline containing 0.05% Tween-20) at 37°C for 1 hour. The plate was washed thrice with PBS-T, S-LPS was added to all the wells of the plate, except negative wells, at a concentration of 300ng/well. The plate was incubated at 37°C for 1 hour and was washed thrice with PBS-T. scFv was added in the first well and was two-fold serial diluted at a concentration of 3 fig/well, incubated further for lhour at 37°C. The plate was washed thrice with PBST and bound scFv was detected by adding His-Probe and the chromogenic substrate TMB at a concentration of lmg/mL. The reaction was stopped by addition of 1.25M H2SO4. The absorbance of the reaction products was read at 450 run using a microplate reader.

[000111] Titration of S-LPS against a constant dilution of scFv revealed a concentration dependent reduction of the binding signal (Figure 9). The control showed no binding signal in ELISA indicating the specificity of scFv towards Brucella S-LPS.

Determination of binding specificity of a mouse monoclonal antibody and scFv in a competitive ELISA

[000112] Competitive ELISA was performed to determine the competition between scFv and in house mouse monoclonal antibody towards the affinity on brucella S-LPS. A microtiter plate was coated with 300ng/well of S-LPS in 50mM carbonate-bicarbonate buffer (pH 9.6) and incubated overnight at 4°C. The plate was washed thrice with PBS-T and blocked with 1% bovine gelatin followed by washing with PBS-T. ScFv was added into the first well at a concentration of 3ng and was serially two-fold diluted . The plate was then incubated at 37°C for 30 minutes. E. coli lysate was used as negative control. The plates were washed with PBS-T and a constant amount of mouse Mab (5C3) was added to each well containing scFv and incubated at 37°C for 1 hour. The plate was then washed with PBS-T and goat anti-mouse IgG HRP conjugate (1:5000) was added to each well. The plate was incubated further at 37°C for 1 hour. The plate was washed five times with PBS-T and the chromogenic substrate TMB was added to the microtiter wells at a concentration of lmg/mL. The reaction was stopped by the addition of 1.25M H2S04. The absorbance of the reaction product was recorded at 450 nm using a microplate reader.

[000113] No competition was detected between scFv and in house mouse monoclonal antibody when constant amount of Mab was allowed to compete with varying two fold dilutions of scFv. There was no change in the optical density following the dilution of the scFv indicating that it did not compete for the same antigenic site on S-LPS (Figure 10).

Example 4
Development of in-house immunocapture ELISA (IC-ELISA) for detection of Brucella S-LPS antigen
[000114] An in-house immunocapture ELISA format was developed to detect the
brucella S-LPS antigen content in vaccine preparations using the in-house mouse monoclonal antibody against brucella S-LPS in combination with the scFv. The mouse Mab (5C3) was coated to microtiter plate at a concentration of 200ng/well. The plate was incubated overnight at 4°C.The plate was blocked using 1% bovine gelatin in PBS-T (phosphate buffer saline containing 0.05% Tween-20) at 37°C for 1 hour. The plate was washed thrice with PBS-T and samples from different time intervals of brucella S-LPS in process samples were added to the first well and were serially two-fold diluted. The plate was incubated at 37°C for 1 hour and washed thrice with PBS-T. scFv was added to the plate at a concentration of 3[ig/well and was incubated further at 37°C for 1 hour. Bound scFv was detected by addition of His-Probe and the chromogenic substrate TMB at a concentration of lmg/mL. The reaction was stopped by the addition of 1.25M FkSC^.Absorbance of the reaction products was recorded at 450 nm using a microplate reader.

[000115] Titration of the test sample against a constant dilution of scFv revealed a concentration dependent reduction of the binding signal (Figure 11). The control showed no binding signal in ELISA. The results clearly indicate the ability of scFv to detect S-LPS content in in-process samples.

SEQ ID NO: 1 Forward Primer Sequence for amplification of Bovine variable heavy chains
GGCGGCGGCGGCTCCAGCCTGGTGAAGCCCTCACAGACC
SEQ ID NO: 2 Reverse Primer Sequence for amplification of Bovine variable heavy chain
TAGCGGCCGCTGAGGAGACGGTGACCAGGAG
SEQ ID NO: 3 Forward Primer Sequence for amplification of Bovine variable light chain
TACCATGGCCGTGTCCGTSWMYCTGGG
SEQ ID NO: 4 Reverse Primer Sequence for amplification of Bovine variable light chain
GGAGCCGCCGCCGCCAGAACCACCACCACCAGAACCACCACCACCGAAGGTGGGGACT TG
SEQ ID NO: 5 Nucleotide Sequence of Heavy Variable Chain
AGCCTGGTGAAGCCCTCACAGACCCTCTCCCTCACCTGCACGGTCTCTGGGCTCTCAT TGAGCAGCTATAACGTAGGCTGGGTCCGCCAGGCTCCGGGGAAGGCACTGGAGTGGAT TGGTAGTATTAGGCCTAGTGGAAACACATGCTATAACCCAGCCCTGAAATCCCGACTC AGCATCACCAAGGACAACTCCAAGAACCAAGTCTCTCTGTCACTGAGCAGCGTGACAA CTGAGGACACGGCCACATACTACTGTGCGAAATGTAATAGTGGTAATCGTAATCTTTG CTTACTTGATAATTCCAAATACGTCGATGCCTGGGGCCAAGGACTCCTGGTCACCGTC TCCTCA
SEQ ID NO: 6 Nucleotide Sequence of Light Variable Chain
GTCGTGTCCGTGTCCCTGGGCCAGAGGGTCTCCATCACCTGCTCTGGAAGCAGCGGCA ATGTTGGAAATGGATATGTGAGCTGGTACCAACTGATCCCAGGATCGGCCCCCAGAAC CCTCATCTATGGTGACACCAGTCGAGCCTCGGGGGTCCCCGACCGATTCTCCGGCTCC AGGTCTGGGAACACAGCCACCCTGACCATCAGCTCGCTCCAGGCTGAGGACGAGGCAG

ATTATTTCTGTGCATCTGCTGAGGATAGTAGCAGTAATGCTGTTTTCGGCAGCGGGAC CACACTGACCGTCCTGGGT
SEQ ID NO: 7 Nucleotide Sequence of Linker
GGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGT
SEQ ID NO: 8 Amino Acid Sequence of Heavy Chain
SLVKPSQTLSLTCTVSGLSLSSYNVGWVRQAPGKALEWIGSIRPSGNTCYNPALKSRL SITKDNSKNQVSLSLSSVTTEDTATYYCAKCNSGNRNLCLLDNSKYVDAWGQGLLVTV SS
SEQ ID NO: 9 Amino Acid Sequence of Light Chain
VVSVSLGQRVSITCSGSSGNVGNGYVSWYQLIPGSAPRTLIYGDTSRASGVPDRFSGS RSGNTATLTISSLQAEDEADYFCASAEDSSSNAVFGSGTTLTVLG
SEQ ID NO: 10 Amino Acid Sequence of Linker
GGGGSGGGGSGGGGSGGG
SEQ ID NO: 11 Nucleotide Sequence of Single Chain Variable Fragment
GTCGTGTCCGTGTCCCTGGGCCAGAGGGTCTCCATCACCTGCTCTGGAAGCAGCGGCA ATGTTGGAAATGGATATGTGAGCTGGTACCAACTGATCCCAGGATCGGCCCCCAGAAC CCTCATCTATGGTGACACCAGTCGAGCCTCGGGGGTCCCCGACCGATTCTCCGGCTCC AGGTCTGGGAACACAGCCACCCTGACCATCAGCTCGCTCCAGGCTGAGGACGAGGCAG ATTATTTCTGTGCATCTGCTGAGGATAGTAGCAGTAATGCTGTTTTCGGCAGCGGGAC CACACTGACCGTCCTGGGTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGGCGGCGGC GGCTCCGGTGGTGGTAGCCTGGTGAAGCCCTCACAGACCCTCTCCCTCACCTGCACGG TCTCTGGGCTCTCATTGAGCAGCTATAACGTAGGCTGGGTCCGCCAGGCTCCGGGGAA GGCACTGGAGTGGATTGGTAGTATTAGGCCTAGTGGAAACACATGCTATAACCCAGCC CTGAAATCCCGACTCAGCATCACCAAGGACAACTCCAAGAACCAAGTCTCTCTGTCAC TGAGCAGCGTGACAACTGAGGACACGGCCACATACTACTGTGCGAAATGTAATAGTGG

TAATCGTAATCTTTGCTTACTTGATAATTCCAAATACGTCGATGCCTGGGGCCAAGGA CTCCTGGTCACCGTCTCCTCA
SEQ ID NO: 12 Amino Acid Sequence of Single Chain Variable Fragment
VVSVSLGQRVSITCSGSSGNVGNGYVSWYQLIPGSAPRTLIYGDTSRASGVPDRFSGS RSGNTATLTISSLQAEDEADYFCASAEDSSSNAVFGSGTTLTVLGGGGGSGGGGSGGG GSGGGSLVKPSQTLSLTCTVSGLSLSSYNVGWVRQAPGKALEWIGSIRPSGNTCYNPA LKSRLSITKDNSKNQVSLSLSSVTTEDTATYYCAKCNSGNRNLCLLDNSKYVDAWGQG LLVTVSS

I/We claim:
1. A recombinant antibody that specifically binds to Brucella smooth lipopolysaccharide (S-LPS), wherein the amino acid sequence of single chain variable fragment of the recombinant antibody is as set forth in SEQ ID NO: 12.

2. The recombinant antibody as claimed in claim 1, wherein the polynucleotide, encoding the polypeptide as set forth in SEQ ID NO: 12, has at least 90% sequence identity to the polynucleotide having nucleotide sequence as set forth in SEQ ID NO: 11.

3. The recombinant antibody as claimed in claim 1, wherein the polynucleotide, encoding the polypeptide as set forth in SEQ ID NO: 12, is as set forth in SEQ ID NO: 11.

4. The recombinant antibody as claimed in claim 1, wherein the recombinant antibody is a monovalent antibody.

5. A recombinant vector comprising the polynucleotide, encoding the polypeptide as set forth in SEQ ID NO: 12, having at least 90% sequence identity to the polynucleotide having nucleotide sequence as set forth in SEQ ID NO: 11.

6. The recombinant vector as claimed in claim 5, wherein the polynucleotide has the nucleotide sequence as set forth in SEQ ID NO: 11.

7. A recombinant host cell comprising the recombinant vector as claimed in claim 5 or 6.

8. The recombinant host cell as claimed in claim 7, wherein the recombinant host cell is either E. coli or yeast.

9. A method for detecting the presence of Brucella S-LPS antigen, wherein the method comprises capturing Brucella S-LPS antigen using scFv antibody, wherein the amino acid sequence of scFv is as set forth in SEQ ID NO: 12.

10. The method as claimed in claim 9, wherein the method is an immunoassay.

11. The method as claimed in claim 10, wherein the immunoassay is ELISA.

12. A method for detecting the presence of Brucella S-LPS in a biological sample, the method comprising:

coating a solid support with a primary antibody against Brucella S-LPS antigen to obtain coated support;
adding a sample to the coated support to allow binding of Brucella S-LPS antigen present in the sample to the primary antibody on the coated support;

screening for binding of Brucella S-LPS antigen to the primary antibody by adding scFv antibody conjugated to a label, wherein the amino acid sequence of scFv is as set forth in SEQ ID NO: 12; and
detecting the label;

wherein the detection of label indicates the presence of Brucella S-LPS.

13. The method as claimed in claim 12, wherein the primary antibody is mouse monoclonal antibody.

14. The method as claimed in claim 12, wherein the sample is either lysed cultures of Brucella, or purified LPS extracts of Brucella cultures.

15. The method as claimed in 12, wherein the label is selected from the group consisting of radioisotope, dye, colloidal gold, nucleotide chromophore, enzyme, substrate, fluorescent molecule, chemiluminescent moiety, magnetic particle, bioluminescent moiety, and biotin.

16. The method as claimed in claim 12, wherein the label is an enzyme.

17. The method as claimed in claim 16, wherein the label is Horseradish peroxidase (HRP).