FIELD OF INVENTION

The present invention relates to the field of production of recombinant antibodies in particular, production of anti-bovine IgA antibody.

BACKGROUND OF THE INVENTION

A large number of mucosal infections continue to represent a challenge for the development of vaccines that either can prevent the pathogen from colonizing the surface epithelium (non-invasive bacterja), penetrate the surface barrier and replicate within the body (invasive bacteria and viruses), and/or block the binding of microbial toxins and neutralize them. In most cases, it would seem desirable to induce specific secretory IgA (slgA) antibodies associated with immunological memory, in addition to systemic immunity. Such infections include in the gastrointestinal tract: Helicobacter pylori, V. cholerae, Enterotoxigenic Escherichia coli (ETEC), Salmonella, Shigella spp., C. parvum, Campylobacter jejuni, C. difficile, rotaviruses and calici viruses; in the respiratory tract: Mycoplasma pneumoniae, influenza virus and respiratory syncytial virus; in the genital tract: HIV, Chlamydia, Neisseria gonorrhoeae and herpes simplex virus; and selected strains of E. coli in the urinary tract.

Immunoglobulin A (IgA) is a predominant class of immunoglobulin present on the mucosal surfaces, which constitutes the first line of defense against various infectious diseases and is one of the primary determinants that would indicate enhanced mucosal immune response/protection. Secretory IgA is transported into mucosal secretions and is resistant to proteases, prevents adhesion of bacteria/toxins to target cells, and neutralizes viruses and toxins, among other characteristics.

IgA being monovalent shows very high affinity in binding or neutralizing virus. It binds very poorly to the complement and therefore is less likely to initiate inflammatory reactions. Engineered IgA antibody can be secreted across the epithelium into the mucosal barriers of the body provides an external passive immunotherapy against viral, bacterial and eukaryotic pathogens.

US 4778751 describes a method of measuring circulating antigens or antibodies by using a ligand labeled specific antigen or ligand labeled specific antibody chemically attached to a soluble matrix or backbone, a differently labeled anti-antigen or anti-antibody and a solid phase anti-ligand directed at the ligand attached to the specific antigen or specific antibody. The antibody can be IgA. The process comprises reacting a sample with a ligand labeled specific antigen or a ligand labeled specific antibody in the liquid phase in the presence of a differently labeled specific anti-antigen or labeled specific anti-antibody. This immunological complex is reacted with an immobilized anti-ligand on a solid support which is directed against the ligand attached to the specific antigen or antibody through the liquid matrix. Subsequently the solid phase is washed and checked for the label on the anti-antigen or anti-antibody which is directly proportional to the concentration of specific antigen or antibody. However, the process of measuring the antibodies is time taking and tedious. Furthermore, the process requires a lot of reagents adding to the cost.

The presently available methods for the detection of IgA involve the use of monoclonal antibodies. These monoclonal antibodies are generated using conventional methods such as hybridoma technology, a process which is expensive and requires complex bio-processes. However, these monoclonal antibodies have limited utilization to clinical applications because of viral contamination and high cost involved in MAb preparations.

To overcome these problems, generation of single chain antibody fragments by utilizing recombinant DNA technology in the expression of antibody fragments has been widely used. These antibody fragments namely scFv's consign antigen binding sites within a single gene (McCafferty, J., A. D. Griffiths, G. Winter, and D. J. Chiswell. 1990. Phage antibodies: filamentous phage displaying antibody variable domains. Nature. 348:552-554; DalPAcqua, W., P. Carter. 1998. Antibody engineering. Curr. Opin. Struct. Biol. 8, 443-450; Begent, R.H., M.J. Verhaar, K.A. Chester, J.L. Casey, A.J. Green, M.P. Napier, L.D. Hope-Stone, N. Cushen, P.A.

Keep, C.J. Johnson, R.E. Hawkins, A.J. Hilson, L. Robson. 1996. Clinical evidence of efficient tumor targeting based on single-chain Fv antibody selected from a combinatorial library. Nat Med. 2(9), 979-84) can be readily produced from the genes encoding antibody variable domains, which can be derived either from hybridomas or from bacteriophage displaying antibody fragments (Orlandi, R., D. H. Gu'ssow, P. T. Jones, and G. Winter. 1989. Cloning immunoglobulin variable domains for expression by the polymerase chain reaction. Proc. Natl. Acad. Sci. U.S.A. 86(10):3833—3837; McCafferty, J., A. D. Griffiths, G. Winter, and D. J. Chiswell. 1990. Phage antibodies: filamentous phage displaying antibody variable domains. Nature. 348:552-554). ScFv consists of variable heavy and light chain domains tethered by a flexible peptide linker which retains the antigen binding site in a single linear molecule and their design, construction and expression in Escherichia coli demonstrated their structure-function relationship and antigen-antibody interactions makes scFv useful in both clinical and medical application (Huston JS, Levinson D, Mudgett-Hunter M, Tai MS, Novotny J, Margolies MN, Ridge RJ, Bruccoleri RE, Haber E, Crea R. Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proc Natl Acad Sci USA. 1988, 85(16): 5879-83 ; Bird, R. E., K.D. Hardman, J.W. Jacobson, S. Johnson, B.M. Kaufman, S.M. Lee,T. Lee, S. H. Pope, G.S. Riordan, and M. Whitlow. 1988. Single-chain antigen-binding proteins, Science. 242:423-426; Condra JH, Sardana VV, Tomassini JE, Schlabach AJ, Davies ME, Lineberger DW, Graham DJ, Gotlib L, Colonno RJ. Bacterial expression of antibody fragments that block human rhinovirus infection of cultured cells, J Biol Chem. 1990 265:2292-5).

US 6818748 describes a single chain variable fragment (ScFv) antibody from a monoclonal antibody (Mab) against Venezuelan equine encephalitis (VEE) virus, generated by cloning linked variable regions of the heavy (VH) and the light (VL) chain antibody genes. The document does not disclose the use of scFv for detecting IgA antibody.

US 6790937 describes variable regions of heavy and light chains of an antibody specific to a surface antigen in sporozoite of Eimeria spp. The patent further describes a recombinant scFV (single chain variable fragment) antibody prepared using the variable regions, a method for preparing the recombinant scFv antibody and an expression vector for expressing a recombinant scFv antibody.
There is no prior art associated with the detection of bovine IgA using scFv which has several advantages over the conventional monoclonal antibody used for detecting bovine IgA. Thus, there is a need for improved method for detection of IgA using anti-bovine antibody which is economically efficient and immunologically effective.

SUMMARY OF THE INVENTION

An aspect of the present invention relates to a recombinant monoclonal antibody that specifically binds to bovine IgA, wherein the amino acid sequence of the recombinant antibody is as set forth in SEQ ID NO: 5.

Another aspect of the present invention relates to a polynucleotide having the nucleotide sequence as set forth in SEQ ID NO: 4, wherein the polynucleotide encodes a recombinant monoclonal antibody having the amino acid sequence as set forth in SEQ ID NO: 5.
Yet another aspect of the present invention relates to a DNA expression cassette comprising a polynucleotide having the nucleotide sequence as set forth in SEQ ID NO: 4, wherein the polynucleotide encodes a recombinant monoclonal antibody having the amino acid sequence as set forth in SEQ ID NO: 5.

Still another aspect of the present invention relates to a composition comprising a recombinant monoclonal antibody that specifically binds to bovine IgA, wherein the amino acid sequence of the recombinant antibody is as set forth in SEQ ID NO: 5 and a pharmaceutically acceptable carrier.

An aspect of the present invention relates to a process for producing a recombinant monoclonal antibody that specifically binds to bovine IgA, wherein the process comprises introducing a recombinant vector comprising the polynucleotide having the nucleotide sequence as set forth in SEQ ID NO: 4 or the DNA expression cassette comprising a polynucleotide having the nucleotide sequence as set forth in SEQ ID NO: 4 into a host cell; and culturing the host cell under conditions which allow expression of the recombinant monoclonal antibody to obtain the recombinant monoclonal antibody.

Yet another aspect of the present invention relates to a process for detecting the presence of IgA in a sample, the method comprising contacting a sample with the recombinant monoclonal antibody that specifically binds to bovine IgA, wherein the amino acid sequence of the recombinant antibody is as set forth in SEQ ID NO: 5 and detecting formation of the complex between the recombinant monoclonal antibody and IgA using conventional methods, wherein the formation of complex confirms the presence of IgA in the sample.

Another aspect of the present invention relates to a kit for detecting the presence of IgA in a sample, the kit comprising the recombinant antibody having the amino acid sequence as set forth in SEQ ID NO: 5 and reagents

BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS

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.

Figure 1 shows the vector map showing cloned single chain variable fragment (scFv).

Figure 2 shows vector construct comprising single chain variable fragment (scFv).

Figure 3A shows electrophoretic analysis of PCR amplified heavy chain variable and light chain variable.

M represents molecular weight markers; VH represents heavy chain variable and VL represents light chain variable.

Figure 3B shows the electrophoretic analysis of scFv fragment generated by SOE PCR. M represents molecular weight markers; scFv represents single chain variable fragment.

Figure 3C shows restriction digestion analysis of recombinant plasmids with EcoRl and Noil. M represents molecular marker; Lane 1 and Lane 2 represents scfv fragment of 723 bp.

Figure 4 shows SDS-PAGE analysis of purified scFv. Lane M represents molecular marker; Lane 1 and Lane 2 represents purified scFv.

Figure 5 shows immunoblot analysis of purified monovalent scFv under reducing conditions which was probed with His probe. Lane M represents pre-stained molecular marker; Lane 1-3 represents fractions obtained from column chromatography for purification of scFv.

Figure 6 shows the binding affinity of scFv to bovine IgA by sandwich ELISA.

Figure 7 shows the binding of scFv to bovine IgA by competitive ELISA.

Figure 8A shows the reactivity of scFv with bovine IgA, Ig M, IgGl and IgG2.

Figure 8B shows the reactivity of scFv with IgA from various species such as goat, sheep, canine, buffalo and cattle. .

Figure 9A shows the SPR sensogram showing the binding profile of scFv with different concentrations of IgA.

Figure 9B shows the SPR sensogram showing residual plot.

Figure 10A shows the binding of scFv and commercial anti-bovine IgA to bovine IgA.

Figure 10B shows the binding of scFv and commercial anti-bovine IgA to FMDV specific bovine IgA.

Figure 11 shows the binding affinity of scFv stored at different temperatures to bovine IgA.

Figure 12 shows the sensitivity of scFv and commercial anti-bovine IgA antibody to bovine IgA.

BRIEF DESCRIPTION OF SEQUENCE LISTING

SEQ ID NO: 1 shows nucleotide sequence of Heavy chain variable (360 bp)

SEQ ID NO: 2 shows nucleotide sequence of Light chain variable (318 bp)

SEQ ID NO: 3 shows nucleotide sequence of linker (45 bp)

SEQ ID NO: 4 shows nucleotide sequence of single chain variable fragment (723 bp)

SEQ ID NO: 5 shows amino acid sequence of single chain variable fragment (241 a.a.)

SEQ ID NO: 6 shows amino acid sequence of Heavy chain variable (120 a.a.)

SEQ ID NO: 7 shows amino acid sequence of Light chain variable (106 a.a.)

SEQ ID NO: 8 shows amino acid sequence of Linker (15 a.a.)

DETAILED DESCRIPTION OF THE INVENTION

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 methods 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
For convenience, before further description of the present invention, certain terms employed in the specification, examples 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. The terms used throughout this specification are defined as follows, unless otherwise limited in specific instances.

As used in the specification and the claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

As used in this specification, the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

The terms "polynucleotide", "nucleotide", "DNA", "gene" and "nucleic acid" are used interchangeably.
The terms "primer" and "oligonucleotide" used herein are used interchangeably.

"Nucleotide" means a building block of DNA or RNA, consisting of one nitrogenous base, one phosphate molecule, and one sugar molecule (deoxyribose in DNA, ribose in RNA).

"Oligonucleotide" means a short string of nucleotides.

"Primer" means a short strand of oligonucleotides complementary to a specific target sequence of DNA, which is used to prime DNA synthesis.

The term "antibody" is used to refer to any antibody like molecule that has an antigen binding region and comprises at least the variable domain of a heavy chain, and normally comprises at least the variable domains of a heavy chain and a light chain.

Light chains are classified as either kappa or lambda. Each heavy chain class may be bound with either a kappa or lambda light chain. In general, the light and heavy chains are covalently bonded to each other, and the "tail" portions of the two heavy chains are bonded to each other by covalent disulfide linkages or on-covalent linkages when the immunoglobulins are generated either by hybridomas, B cells or genetically engineered host cells. In the heavy chain, the amino acid sequences run from an N-terminus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain.

Both the light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it will be appreciated that the variable domains of both the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CL) and the heavy chain (CHI, CH2 or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, and the like. By convention the numbering of the constant region domains increases as they become more distal from the antigen binding site or amino-terminus of the antibody. The N-terminal portion is a variable region and at the C-terminal portion is a constant region; the CH3 and CL domains actually comprise the carboxy-terminus of the heavy and light chain, respectively.

The invention also encompasses portions of antibodies that comprise sufficient variable region sequence to confer antigen binding. Portions of antibodies include, but are not limited to Fab, Fab', F(ab')2, Fv, SFv, ScFv, scFv (single-chain Fv), whether produced by proteolytic cleavage of intact antibodies, such as papain or pepsin cleavage, or by recombinant methods, in which the cDNAs for the intact heavy and light chains are manipulated to produce fragments of the heavy and light chains, either separately, or as part of the same polypeptide.

The term "antibody fragment" also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex. For example, antibody fragments include isolated fragments, "Fv" fragments, consisting of the variable regions of the heavy and light chains, recombinant single chain polypeptide molecules in which light and heavy chain variable regions are
connected by a peptide linker ("sFv proteins"), and minimal recognition units consisting of the amino acid residues that mimic the "hypervariable region".

"Single-chain Fv" also abbreviated as "ScFv" or "scFv" are antibody fragments that comprise the heavy chain variable region (VH) and light chain variable region (VL) 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.
The terms "scFv" and "antibody" used herein are used interchangeably.

"VLP" or "VLPs" mean(s) virus-like particle or virus-like particles.

As used herein, the term "linker" 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.

As used herein, the term "monovalent" means that a given domain antibody can bind only a single molecule of its target. Naturally-occurring antibodies are generally divalent, in that they have two functional antigen-binding loops, each comprising a VH and a VL domain. Where steric hindrance is not an issue, a divalent antibody can bind two separate molecules of the same antigen. In contrast, a "monovalent" antibody has the capacity to bind only one such antigen molecule. The antigen-binding domain of a monovalent antibody can comprise a VH and a VL domain.

The term "test sample" or "sample" refers to material obtained from a biological source, environmental source or a processed sample. The processed sample may include extraction of genetic material from the sample. The terms "test sample" or 'sample' are used interchangeably.

The term "vector", as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g.,. non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" may be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

The term "recombinant host cell" or "host cell", as used herein, is intended to refer to a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. Recombinant host cells include, for example, transfectomas, such as E. coli, yeast cells, plant cells or mammalian cells such as CHO cells, NS/0 cells, and lymphocytic cells.
The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a subject. Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions.

The term "treating" or treatment refers to the administration of an effective amount of a composition of the present invention to a subject, who has obesity, overweight and other metabolic disorders, or a symptom or a predisposition of such diseases, with the purpose to cure, alleviate, relieve, remedy, or ameliorate such diseases, the symptoms of them, or the predispositions towards them.

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.

The present invention provides a novel recombinant monovalent anti-bovine IgA antibody and a method for production of the antibody. The present invention further provides a recombinant vector and host cells comprising the recombinant anti-bovine IgA antibody. Further, the present invention provides a composition comprising the recombinant anti-bovine IgA antibody. The present invention also provides a method of detection of bovine IgA in a sample using the recombinant anti-bovine IgA antibody of the present invention. The recombinant anti-bovine IgA antibody disclosed in the present invention specifically recognizes bovine IgA.

An embodiment of the present invention provides a recombinant monoclonal antibody that specifically binds to bovine IgA, wherein the amino acid sequence of the recombinant antibody is as set forth in SEQ ID NO: 5.

In another embodiment of the present invention there is provided a recombinant monoclonal antibody that specifically binds to bovine IgA, wherein the amino acid sequence of the recombinant antibody is as set forth in SEQ ID NO: 5, wherein the antibody is encoded by a polynucleotide having the nucleotide sequence as set forth in SEQ ID NO: 4.

In yet another embodiment of the present invention there is provided a recombinant monoclonal antibody that specifically binds to bovine IgA, wherein the amino acid sequence of the recombinant antibody is as set forth in SEQ ID NO: 5, wherein the antibody is a murine antibody.

In still another embodiment of the present invention there is provided a recombinant monoclonal antibody that specifically binds to bovine IgA, wherein the amino acid sequence of the recombinant antibody is as set forth in SEQ ID NO: 5, wherein the antibody is an antigen-binding antibody fragment.

In an embodiment of the present invention there is provided a recombinant monoclonal antibody that specifically binds to bovine IgA, wherein the amino acid sequence of the recombinant antibody is as set forth in SEQ ID NO: 5, wherein the antibody is a single chain antibody (scFv) comprising heavy chain variable region and light chain variable region operatively linked with a linker.

In another embodiment of the present invention there is provided a recombinant monoclonal antibody that specifically binds to bovine IgA, wherein the amino acid sequence of the recombinant antibody is as set forth in SEQ ID NO: 5, wherein the antibody optionally comprises a label.

An embodiment of the present invention provides a polynucleotide having the nucleotide sequence as set forth in SEQ ID NO: 4, wherein the polynucleotide encodes a recombinant monoclonal antibody having the amino acid sequence as set forth in SEQ ID NO: 5.

Another embodiment of the present invention provides a DNA expression cassette comprising a polynucleotide having the nucleotide sequence as set forth in SEQ ID NO: 4, wherein the polynucleotide encodes a recombinant monoclonal antibody having the amino acid sequence as set forth in SEQ ID NO: 5.

In still another embodiment of the present invention there is provided a DNA expression cassette comprising a polynucleotide having the nucleotide sequence as set forth in SEQ ID NO: 4, wherein the polynucleotide encodes a recombinant monoclonal antibody having the amino acid sequence as set forth in SEQ ID NO: 5, wherein the polynucleotide is operably linked to a promoter sequence.

Yet another embodiment of the present invention provides a recombinant vector comprising a DNA expression cassette, wherein the DNA expression cassette comprises a polynucleotide having the nucleotide sequence as set forth in SEQ ID NO: 4, wherein the polynucleotide encodes a recombinant monoclonal antibody having the amino acid sequence as set forth in SEQ ID NO: 5.

In an embodiment of the present invention, there is provided a recombinant vector comprising a DNA expression cassette, wherein the DNA expression cassette comprises a polynucleotide having the nucleotide sequence as set forth in SEQ ID NO:

4, wherein the polynucleotide encodes a recombinant monoclonal antibody having the amino acid sequence as set forth in SEQ ID NO: 5, wherein the polynucleotide is operably linked to a promoter sequence.

Another embodiment of the present invention provides a recombinant host cell comprising the DNA expression cassette, wherein the DNA expression cassette comprises a polynucleotide having the nucleotide sequence as set forth in SEQ ID NO: 4, wherein the polynucleotide encodes a recombinant monoclonal antibody having the amino acid sequence as set forth in SEQ ID NO: 5, wherein the polynucleotide is operably linked to a promoter sequence.

Yet another embodiment of the present invention provides a recombinant host cell comprising a recombinant vector, wherein the recombinant vector comprises a DNA expression cassette, wherein the DNA expression cassette comprises a polynucleotide having the nucleotide sequence as set forth in SEQ ID NO: 4, wherein the polynucleotide encodes a recombinant monoclonal antibody having the amino acid sequence as set forth in SEQ ID NO: 5, wherein the polynucleotide is operably linked to a promoter sequence.

In still another embodiment of the present invention, there is provided a recombinant host cell comprising the DNA expression cassette, wherein the DNA expression cassette comprises a polynucleotide having the nucleotide sequence as set forth in SEQ ID NO: 4, wherein the polynucleotide encodes a recombinant monoclonal antibody having the amino acid sequence as set forth in SEQ ID NO: 5, wherein the polynucleotide is operably linked to a promoter sequence, wherein the host cell is selected from the group consisting of E. coli, yeast cell, plant cell, insect cell and mammalian cell.

In an embodiment of the present invention, there is provided a recombinant host cell comprising a recombinant vector, wherein the recombinant vector comprises a DNA expression cassette, wherein the DNA expression cassette comprises a polynucleotide having the nucleotide sequence as set forth in SEQ ID NO: 4, wherein the polynucleotide encodes a recombinant monoclonal antibody having the amino acid sequence as set forth in SEQ ID NO: 5, wherein the polynucleotide is operably linked to a promoter sequence, wherein the host cell is selected from the group consisting of E. coli, yeast cell, plant cell, insect cell and mammalian cell.

An embodiment of the present invention provides a composition comprising a recombinant monoclonal antibody that specifically binds to bovine IgA, wherein, the amino acid sequence of the recombinant antibody is as set forth in SEQ ID NO: 5, and a pharmaceutical^ acceptable carrier.

Yet another embodiment of the present invention provides a process for producing a recombinant monoclonal antibody that specifically binds to bovine IgA, wherein the process comprises introducing a recombinant vector comprising the polynucleotide having the nucleotide sequence as set forth in SEQ ID NO: 4 or the DNA expression cassette comprising a polynucleotide having the nucleotide sequence as set forth in SEQ ID NO: 4 into a host cell; and culturing the host cell under conditions which allow expression of the recombinant monoclonal antibody to obtain the recombinant monoclonal antibody.

In still another embodiment of the present invention there is provided a process for producing a recombinant monoclonal antibody that specifically binds to bovine IgA, wherein the process comprises introducing a recombinant vector comprising the polynucleotide having the nucleotide sequence as set forth in SEQ ID NO: 4 or the DNA expression cassette comprising a polynucleotide having the nucleotide sequence as set forth in SEQ ID NO: 4 into a host cell; and culturing the host cell under conditions which allow expression of the recombinant monoclonal antibody to obtain the recombinant monoclonal antibody, wherein the process optionally comprises purifying the antibody using conventional method.

In another embodiment of the present invention there is provided a process for producing a recombinant monoclonal antibody that specifically binds to bovine IgA, wherein the process comprises introducing a recombinant vector comprising the polynucleotide having the nucleotide sequence as set forth in SEQ ID NO: 4 or the DNA expression cassette comprising a polynucleotide having the nucleotide sequence as set forth in SEQ ID NO: 4 into a host cell; and culturing the host cell under conditions which allow expression of the recombinant monoclonal antibody to obtain the recombinant monoclonal antibody, wherein the host cell is selected from a group consisting of E. coli, yeast cell, plant cell, insect cell and mammalian cell.

Another embodiment of the present invention provides a process for detecting the presence of IgA in a sample, the method comprising contacting a sample with the recombinant monoclonal antibody that specifically binds to bovine IgA, wherein, the amino acid sequence of the recombinant antibody is as set forth in SEQ ID NO: 5 and detecting formation of the complex between the recombinant monoclonal antibody and IgA using conventional methods, wherein the formation of the complex confirms the presence of IgA in the sample.

In another embodiment of the present invention there is provided a process for detecting the presence of IgA in a sample, the method comprising contacting a sample with the recombinant monoclonal antibody that specifically binds to bovine IgA, wherein, the amino acid sequence of the recombinant antibody is as set forth in SEQ ID NO: 5 and detecting formation of the complex between the recombinant monoclonal antibody and IgA using conventional methods, wherein the formation of the complex confirms the presence of IgA in the sample, wherein the sample is selected from the group consisting of serum sample, plasma, blood sample, milk, colostrum, saliva, tears, nasal secretion, uterine secretion, intestinal secretion and milk product.

Yet another embodiment of the present invention provides a kit for detecting the presence of IgA in a sample, the kit comprising the recombinant monoclonal antibody that specifically binds to bovine IgA, wherein the amino acid sequence of the recombinant antibody is as set forth in SEQ ID NO: 5 and reagents.

In the present invention, total RNA was isolated from hybridoma cells lines secreting anti-bovine IgA antibody using Trizol reagent (Invitrogen), in accordance with the manufacturer's recommended protocol. The RNA was subjected to cDNA synthesis by RT-PCR using random hexamers. The RT-PCR amplified cDNA was used as template for amplification of VH and VL chain using commercially available primers from Amersham Biosciences. Briefly, 5ul of cDNA, lOpmol/ul of each forward and reverse primers, 1 unit of Taq DNA polymerase and buffer with lOmM dNTP's was pipetted into a clean PCR tube and placed in a thermal cycler and the reaction was carried out at 95°C for 30 seconds, 55°C for 30 seconds and 72°C for 60 seconds. PCR amplification was carried out for 34 cycles. Amplified DNAs of VH and VL were analyzed by agarose gel electrophoresis (Figure 3A). Distinct bands of 360 bp for VH and 318 bp for VL were detected. The PCR products were pooled and gel purified using the commercial kit from QIAGEN. The purified VH and VL DNAs were then mixed with linker DNA fragment. Assembly PCR was carried out for 34 cycles with the cycling parameters of 95°C for 30 seconds, 55°C for 60 seconds and 72°C for 60 seconds, thus connecting the two DNAs. The assembled fragment (recombinant scFv antibody of 723 bp; SEQ ID NO: 4) was analysed by gel electrophoresis (Figure 3B). Distinct band of 723 bp of scFv was detected.

The scFv (SEQ ID NO: 4) was cloned into pET 28a bacterial expression vector. The pET 28a vector carries T7 promotor, LacZ, ribosome binding site, N-terminal His Tag, T7 Tag, heavy chain variable, linker, light chain, C-terminal His tag sequence and T7 terminator. The vector pET 28a and insert scFv were digested with EcoRl and Notl, respectively by incubating at 37°C for 12 hours. The digested products were purified using the kit provided by QIAGEN and kept for various ratios of vector to insert (i.e., 1:3 and 1:6) for cohesive end ligation and incubated at 22°C for 2 hours. The ligation mixture for the recombinant ScFv antibody was transformed into XL-Blue strain of E. coli competent cells and the transformed cells were subsequently plated on Luria Bertani (LB) agar plates containing lOOug/mL ampicillin. The plates were incubated overnight at 30°C.

40 transformants of ScFv were picked and grown individually for screening by "miniprep" DNA analysis. In order to screen for the presence of full length 723 bp ScFv inserts, "miniprep" DNA was prepared from all 40 individual transformants and restriction enzyme analysis was performed. From the 40 transformants, a total of six were found to carry a full length 723 bp ScFv fragment. Plamids were isolated from these positive transformants and transformed into host cells for soluble expression of the antibody gene.

The host cells comprising the polynucleotide (SEQ ID NO: 4) of the present invention can be a prokaryotic host cell or an eukaryotic host cell. These host cells allow the production of the recombinant antibody. Methods for producing such antibody include culturing host cells transformed with the expression vectors comprising the polynucleotide sequence coding the recombinant antibody under conditions suitable for protein expression and recovering the protein from the host cell culture. The vectors include a promoter, a ribosome binding site (if needed), a selectable marker gene, and the start/stop codons. The vectors allow the expression of the recombinant antibody in the prokaryotic or eukaryotic host cells. For eukaryotic hosts (e.g. yeasts, plant cells or mammalian cells), different transcriptional and translational regulatory sequences are employed, depending on the nature of the host. They may be derived from viral sources, such as adenovirus, bovine Papilloma virus, Simian virus or the like, where the regulatory signals are associated with a particular gene which has a high level of expression. Examples are the TK promoter of the Herpes virus, the SV40 early promoter, the yeast gal4 gene promoter, etc. Transcriptional initiation regulatory signals may be selected which allow for the transient (or constitutive) repression and activation and for modulating gene expression.

In accordance with the present invention, host cells can be either prokaryotic or eukaryotic. Amongst prokaryotic host cells, the preferred one is E. coli. Amongst eukaryotic host cells, the preferred ones are yeast, insect, plant or mammalian cells. In particular, cells such as human, monkey, mouse, insect (using baculovirus-based expression systems) and Chinese Hamster Ovary (CHO) cells, provide post-translational modifications to protein molecules, including correct folding or certain forms of glycosylation at correct sites. Also yeast cells can carry out post-translational peptide modifications including glycosylation. Mammalian cell lines available as hosts for expression are known in the art and include many immortalized cell lines including, but not limited to, Chinese hamster ovary (CHO), HeLa, baby hamster kidney (BHK), monkey kidney (COS), CI27, 3T3, BHK, HEK 293, Per.C6, Bowes melanoma and human hepatocellular carcinoma (for example Hep G2) cells and a number of other cell lines.

In accordance with the present invention, the plasmids were isolated from positive transformants and transformed into E. coli BL21 (DE3) cells for soluble expression of the antibody gene. Individual clones of E. coli BL21 (DE3) cells were picked and grown overnight in LB broth containing lOO^g/mL ampicillin. Fresh 1.5 mL cultures were prepared the following morning at a starting A600nm of 0.05 and grown at 30°C with shaking to a density of 0.5. Cells were then pelleted by centrifugation and resuspended in 1.5 mL fresh LB broth containing lOOug/mL ampicillin and 1 mM iso-propyl P-D-thiogalactoside (1PTG). Subsequently, the cells were grown at 37°C to an A600nm of 0.6, pelleted again, resuspended in phosphate buffered saline (PBS), pH 7.5, and lysed by boiling. The boiled lysates were microfuged to remove cellular debris and the supernatants were frozen at -70°C. Three to six uL of the lysates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 12% gels (Figure 4). The separated proteins were transferred onto nitrocellulose membranes by use of buffer containing Tris (3.03 grams/liter), Glycine (14.4 grams/liter) and 20% Methanol. Membranes were blocked for one hour with blocking buffer (PBS containing 3% non-fat skim milk). The protein was probed using His probe specific for the histidine tage of the scFv (Figure 5). Figure 5 shows immunoblot analysis of monovalent scFv. As expected, ~ 32 KDa scFv product was detected.

The purification of the recombinant antibody of the invention can be carried out by any of the conventional methods known for this purpose, i.e. any procedure involving extraction, precipitation, chromatography, or the like. In particular, methods for antibody purification can make use of immobilized gel matrices contained within a column, exploiting the strong affinity of antibodies for substrates such Protein A, Protein G, or synthetic substrates, or for specific antigens or epitopes. After washing, the protein is eluted from the gel by a change in pH or ionic strength. Alternatively, HPLC (High Performance Liquid Chromatography) can be used. The elution can be carried out using a water-acetonitrile-based solvent commonly employed for protein purification.

To assess of the functionality of the scFv antibody, Sandwich ELISA was performed to determine the binding affinity with bovine IgA. Standard kit to detect IgA was used as positive control (Figure 6). The scFv shows strong binding towards IgA. Further, scFv of the present invention showed competition with the standard bovine IgA antibody for the same antigenic site on bovine IgA (Figure 7). The scFv specifically reacts with bovine IgA. Further, it does not exhibit any cross reactivity with other classes of bovine immunoglobulins such as IgGl, IgG2 and IgM and IgA of different species such as buffalo, goat, sheep and canine (Figure 8A and 8B). Moreover, the scFv has high affinity for IgA as determined using BIACore X-100. The association and dissociation rate constants for the interaction between scFv and IgA using the BIAEvaluation software was found to be 2.26xl07 M'1 s"1 and 1.87xl03 s"1, respectively. The affinity constant of the scFv to IgA was 8.1 xlO11 M'\ The residual distribution plot was linear with ±2 RU (response unit) deviation and no significant bulk contribution (RI) to the sensogram was found which validates the affinity measurement (Figure 9A and 9B). The scFv was able to detect bovine IgA in standard samples. Detection of bovine IgA using scFv and commercial anti-bovine IgA showed a good fit with r2 values of >0.9 in both the cases, indicating that detection of IgA using either of the antibodies remains linear over a wide concentration range (Figure 10A). The scFv was able to detect FMDV specific IgA in test saliva samples from infected and non-infected cattle (Figure 10B). The cutoff OD for positive samples using scFv was set at 0.520. FMDV specific bovine IgA was detected in 3 out of 18 animals which were infected and vaccinated and 1 out of 5 animals which were infected and unvaccinated. All four animals which were detected as FMDV specific IgA positive were FMDV carriers. Further, the scFv was very stable at temperatures below -20°C and there was no loss of binding activity over a long period of time.

The recombinant antibody of the present invention has single antigen binding site that is reactive against bovine IgA. The antibody is devoid of constant regions and therefore provides high binding avidity and specificity to the bovine IgA similar to the parent antibody. ScFv of the present invention has several advantages over monoclonal antibodies generated by conventional methods, such as the scFv can be produced rapidly and economically. Furthermore, the expression of recombinant scFv in prokaryotic or eukaryotic cells and purification constitutes a cost-effective alternative approach for large-scale production of antibodies for use in diagnostic purpose against IgA. The scFv of the present invention is approximately five times smaller than a standard anti-bovine IgA monoclonal antibody in terms of molecular weight. Thus, at a given concentration of bovine IgA, there would be five times more molecules of scFv than a standard anti-bovine IgA monoclonal antibody. Thus, a lesser amount of scFv of the present invention is able to detect bovine IgA in a sample as compared to the amount of a standard anti-bovine IgA monoclonal antibody. Furthermore, the scFv of the present invention are functionally active and genetically stable over a long period of time. Functional analysis of the scFv antibody by Indirect ELISA against different classes of bovine immunoglobulins and different species of IgA showed that the scFv was functional in specifically recognizing only bovine IgA.

The scFv antibody of the present invention is used for detecting and quantifying bovine IgA in a sample. The sample is selected from the group consisting of serum sample, plasma, blood sample, milk, colostrum, saliva, tears, nasal secretion, uterine secretion and intestinal secretion. The method of detection comprises contacting a sample with the recombinant antibody of the present invention under conditions which allows formation of a complex between the recombinant antibody and the IgA. The formed complex is detected using conventional methods, wherein the formation of complex confirms the presence of IgA in the sample. The conventional methods include but are not limited to immunoassays, radio-immunoassays, competitive-binding assays, western blot analysis, ELISA (enzyme linked immunosorbent assay) assays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradiometric assays, immunofluorescence assays and protein A immunoassays. The process can be used for determining the amount of bovine IgA in a sample. The level of IgA is elevated in certain diseases such as Bovine Venereal Vibriosis and infection with FMDV. In certain infections such as Bovine Keratoconjunctivitis, the level of IgA decreases. Thus, the quantification of IgA in a sample can lead to preliminary detection of certain diseases such as Bovine Venereal Vibriosis, Bovine Keratoconjunctivitis, Bovine Tuberculosis and Bovine paratuberculosis in cattle. Further, the scFv of the present invention can be sued to detect the FMDV carrier status of bovine.

Although the subject matter has been described in considerable detail 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

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 this 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
Construction of scFv fragment
Hybridoma was generated by immortalizing immune mouse spleen cells mediated by a mouse X mouse hybridoma. The hybridoma was obtained from European Collection of Cell Cultures (ECACC) (Williams DJ, Newson J, Naessens J. 1990, Quantitation of bovine immunoglobulin isotypes and allotypes using monoclonal antibodies, Vet Immunol Immunopathol. Mar; 24(3):267-83). Total RNA was isolated from the hybridoma cell lines secreting anti-bovine IgA antibody using Trizol reagent (Invitrogen) and resuspended in nuclease free water. The RNA was subjected to cDNA synthesis by RT-PCR using random hexamers. The RT-PCR amplified DNA was used as a template for amplification of variable domains of heavy chain and light chain using commercially available primers from Amersham Biosciences. The primer sequences are proprietary material and owned by Enzon Inc. and licensed to Amersham Biosciences. However, a person skilled in the art can design the sequence of forward and reverse primers used for amplification of heavy chain variable and light chain variable based on the nucleotide sequence of heavy chain variable and light chain variable as set forth in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

Briefly, 5 μ l of cDNA, 10pmol/ μ l of each forward and reverse primers, 1 unit of Taq DNA polymerase and buffer with 10mM dNTP's was pipetted into a clean PCR tube and placed in a thermal cycler and the reaction was carried out at 95°C for 30 seconds, 55°C for 30 seconds and 72°C for 60 seconds. Amplifications were repeated for 34 cycles to obtain the final products i.e. a fragment of 360 bp (as set forth in SEQ ID NO: 1) which corresponds to DNA encoding heavy chain variable (VH) and another fragment of 318 bp (as set forth in SEQ ID NO: 2) which corresponds to DNA encoding light chain variable (VL). Figure 3A shows electrophoretic analysis of PCR amplified heavy chain variable and light chain variable. M represents molecular weight markers; VH represents heavy chain variable and VL represents light chain variable.

Assembly of variable heavy and light chain to form single chain variable fragment by SOE PCR
The final PCR products i.e. DNA encoding light chain variable (VL) and the DNA encoding heavy chain variable (VH) were pooled and gel purified using the commercial kit from QIAGEN. The DNA encoding light chain variable and heavy chain variable domains were assembled by linker DNA (as set forth in SEQ ID NO: 3) using splice overlap extension polymerase chain reaction (SOE PCR) to obtain a scFv fragment DNA of 723 bp using the commercially available forward light chain primer and commercially available reverse heavy chain primer. The primer sequences are proprietary material and owned by Enzon Inc. and licensed to Amersham Biosciences. However, a person skilled in the art can design the sequence of forward and reverse primers used for amplification based on the nucleotide sequence of single chain variable fragment as set forth in SEQ ID NO: 4. 2ul of each heavy chain variable and light chain variable, 10pmol/ul of each forward and reverse primers, 1 unit of Taq DNA polymerase and buffer with lOmM dNTP's was pipetted into a clean PCR tube and placed in a thermal cycler and the reaction was carried out at 95°C for 30 seconds, 55°C for 60 seconds and 72°C for 60 seconds. The nucleotide sequence of the scFv fragment is as set for the in SEQ ID NO: 4. The amino acid sequence of the scFv fragment is as set for the in SEQ ID NO: 5. Figure 3B shows the electrophoretic analysis of scFv fragment generated by SOE PCR. M represents molecular weight markers; scFv represents single chain variable fragment.
Result and inference: scFV fragment of size 723 bp was amplified which was confirmed by electrophoretic analysis of the PCR product obtained by SOE PCR reaction. The nucleotide sequence of the scFv fragment is as set for the in SEQ ID NO: 4.

Analysis of scFv sequence
The scFv fragment having nucleotide sequence as set forth in SEQ ID NO: 4 were cloned into TOPO-TA vector for sequence verification. The scFv amplified with Taq DNA polymerase was gel purified and incubated for 10 minutes with TOPO TA vector (commercially available from Invitrogen for TA cloning). Positive clones which showed release of 723 bp fragment after enzymatic analysis of the plasmid with EcoR1 were sequenced at Ocimum biosolutions, India and sequencing was done using the universal primers, namely Ml3 forward and reverse primers. The obtained sequence was confirmed using NCBI blast search. The NCBI blast search confirmed the presence of 360 bp of heavy chain variable (SEQ ID NO: 1), 318 bp of light chain variable (SEQ ID NO: 2) and 45 bp of linker DNA (SEQ ID NO: 3) which formed the single chain variable fragment of 723 bp (SEQ ID NO: 4).
Result and inference: The analysis of scFv fragment obtained by SOE PCR confirmed that the fragment was 723 bp in size and comprised of a heavy chain variable region of 360 bp, a light chain variable region of 318 bp connected via a linker DNA of 45 bp.

Example 2
Cloning of scFv into pET vector
The scFv (SEQ ID NO: 4) was cloned into pET 28a bacterial expression vector. The vector pET 28a and insert scFv referred to monovalent antibody fragment of size 723 bps were digested with EcoRl and Notl , respectively by incubating at 37°C for 12 hours. The digested products were purified using the kit provided by QIAGEN and kept for various ratios of vector to insert (i.e., 1:3 and 1:6) cohesive end ligation and incubated at 22°C for 2 hours. The ligated product was further incubated for 20 minutes at 65°C in order to inactive the enzyme. Figure 1 shows the vector map showing cloned single chain variable fragment (scFv). Figure 2 shows vector construct comprising single chain variable fragment (scFv). The pET 28a vector carries T7 promotor, LacZ, ribosome binding site, N-terminal His Tag, T7 Tag, heavy chain variable, linker, light chain, C-terminal His tag sequence and T7 terminator. The recombinant plasmids were chemically transformed into XL-Blue strain of E. coli cells and the scFv insert was verified by restriction digestion analysis with EcoRI and Notl. Figure 3C shows restriction digestion analysis of recombinant plasmids with EcoRI and Notl. M represents molecular marker; Lane 1 and Lane 2 represents scfv fragment of 723 bp.

E. coli transformation
Overnight grown E. coli 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 5000 x rpm for 10 minutes at 4°C and re-suspended in ice-cold 0.1 mM CaCh and incubated overnight on the ice, before proceeding for transformation.

The chemically competent E. coli XL-Blue cells thus prepared were incubated with recombinant plasmid comprising the nucleotide sequence of the scFv as set forth in SEQ ID NO: 4 for 30 minutes on ice. The cells were transformed by heat shock treatment at 42°C for 90 seconds and immediately transferred 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 onto LB agar plates containing 100 mg/ml of ampicillin. The plates were incubated overnight and screened for positive clones by isolating the plasmids and subjecting to digestion with EcoRI and Notl. The positive clones were sequence verified before the plasmid was transformed into E. coli BL21 (DE3) cells for soluble expression of the antibody gene.

The recombinant plasmid from the transformed E. coli BL21 (DE3) was isolated and further transformed in E. coli BL21 (DE3) as described above. Cultures of E. coli BL21 (DE3) were grown till the OD reached 0.6 at 600 nm and induced by addition of ImM isopropyl-p-D-thiogalactopyranoside (IPTG) and allowed to grow at 28°C for 4 hours. Following completion of induction, the bacterial pellet was collected by centrifugation at 5000 X g for 20 minutes at 4°C. The bacterial pellet was resuspended in lysis buffer (50 mM Tris-HCl, 155 mM NaCl, pH 7.6) to prepare a 10% (w/v) suspension. Lysozyme was added to a final concentration of 50 u,g/10 ml of lysate and incubated overnight at -20°C. The sample was subjected to sonication and centrifuged at 9200 x g for 30 minutes at 4°C. The pellet was discarded and the supernatant was subjected to Immobilized metal affinity column chromatography (IMAC). An IMAC column (5 ml volume) was equilibrated with 10 column volumes of 50 mM Tris-HCl, 155 mM NaCl, pH 7.6 (equilibration buffer). The supernatant was loaded onto the column at a flow rate of lml/min and washed with 20 column volumes of washing buffer (equilibration buffer with 30 mM Imidazole, pH 7.6). Bound scFv was eluted with 5 column volumes of elution buffer containing equilibration buffer with 300 mM Imidazole, pH 7.6, as 1 ml fractions. The purified protein eluted fractions of scFv fragments were analyzed by SDS-PAGE analysis under reducing conditions after staining with coomassie brilliant blue. Figure 4 shows SDS-PAGE analysis of purified scFv. Lane M represents molecular marker; Lane 1 and Lane 2 represents purified scFv. The purified protein was detected by staining with coomassie brilliant blue. Figure 5 shows immunoblot analysis of purified monovalent scFv under reducing conditions which was probed with His probe. Lane M represents pre-stained molecular marker; Lane 1-3 represents fractions obtained from column chromatography for purification of scFv. The blot was reacted with His probe specific for the histidine tag of the scFv. A protein band 30 kDa was detected.
Result and inference: The scFv was successfully cloned into pET 28a bacterial expression vector and was getting expressed in E. coli BL21 (DE3) cells.

Example 3
Characterization of anti-bovine IgA scFv for activity
Sandwich ELISA
ELISA was performed with the kit provided by Bethyl Laboratories. Microtitre plates were pre-coated with anti-bovine IgA antibody and the un-reacted sites were blocked. 100 of standard bovine IgA (lug/ml) was added to the wells coated with anti-bovine IgA and incubated at room temperature for 1 hour. The microtitre wells were washed with wash buffer according to the manufacturer's instruction and 100 of recombinant scFv of the present invention was added at a concentration of lmg/ml and again incubated for 1 hour at room temperature. The binding affinity of the scFv with bovine IgA was detected by adding His-Probe and a chromogenic substrate TMB. The plate was read at 450nm after the reaction was stopped with 1.25M H2SO4. The result is provided in Figure 6. Figure 6 shows the binding affinity of scFv to bovine IgA by sandwich ELISA.

Result and Inference: Titration of purified scFv against bovine IgA revealed a concentration dependent increase of the optical density values. scFv of the present invention is biologically active and is able to recognize and bind to bovine IgA.

Competitive ELISA
A competitive ELISA was performed to identify the specificity of the scFv for bovine IgA. A microtiter plate was coated with 200 ng/well of bovine IgA 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 in phosphate buffer saline (PBS) containing 0.05% Tween 20 (PBS-T) followed by washing with PBS-T to remove the excess gelatin. scFv (1000 ng/100 ul) was added by serial dilution and incubated at 37°C for 1 hour. E. coli lysate was used as a negative control. A Mab IL-A71 specific for bovine IgA was added to each well containing scFv and E.coli lysate, incubated at 37°C for 1 hour. The plate was washed with PBS-T and dried by flicking. Goat anti-mouse IgG HRP conjugate (1:5000) was added to each well and the plate was incubated at 37°C for 1 hour. The plate was washed five times with PBS-T and 100 ul of H2O2 activated TMB (Sigma, USA) was added. The reaction was stopped after 10 minutes by addition of 100 ul of 1.25M H2SO4 to each well and absorbance was read at 450 nm using a micro plate reader (BIO-TEK, USA). The result is provided in Figure 7.

Figure 7 shows the binding of scFv to bovine IgA by competitive ELISA.
Result and Inference: Gradual increase in optical density values was observed following the dilution of the scFv. Competition was observed when the constant amount of mAb IL-A71 (Standard bovine IgA antibody) was allowed to compete with varying amount of the scFv. Further, the gradual increase in optical density values following the dilution of the scFv indicated that the scfv of the present invention has high specificity, thus even at low dilutions, it was competing with the standard bovine IgA antibody for the same antigenic site on bovine IgA.

Example 4
Characterization of anti-bovine IgA scFv for specificity
Indirect ELISA
Saliva samples were collected from cattle, buffaloes, sheep, goats and canines. Saliva samples were collected by placing a l/6th portion of a regular size cotton tampon (Tampax®, Kiev, Ukraine) pre-dampened by addition of 0.5 ml of PBS (pH 7.3-7.5) underneath the tongue and also from the vestibule. Approximately 1-2 ml of saliva was extracted from each tampon by compression within the barrel of a syringe or by centrifugation for 10 minutes at 1862xg before storage at -20 °C.

The binding specificity of scFv towards bovine IgA was evaluated by testing its reactivity with bovine IgGl, IgG2 and IgM by indirect ELISA. 100 ul of bovine IgA, IgGl, lgG2 and IgM was coated onto a microtiter wells at a concentration of 1 mg/ml. The wells were washed with PBS-T and blocked with 1% bovine gelatin by incubating at 37° C for 1 hour. The wells were washed as described above and scFv was serially diluted, added to the wells and incubated for 1 hour at 37°C. The wells were washed and scFv was detected by adding His-Probe (1:5000) followed by TMB substrate. The plate was incubated at 37°C for 10 minutes and the reaction was stopped by addition of 1.25M H2SO4 The absorbance was measured at 450 nm using a microplate reader (BIO-TEK, US). The results are provided in Figure 8. Figure 8A shows the reactivity of scFv with bovine IgA, Ig M, IgGl and IgG2. E.coli lysate was taken as negative control. Figure 8B shows the reactivity of scFv with IgA from various species such as goat, sheep, canine, buffalo and cattle.

Result: scFv of the present invention shows binding only towards bovine IgA. No binding was observed towards bovine IgGl, IgG2 and IgM. Further, no binding of scFv of the present invention was observed with IgA from different species of animals such as buffalo, goat, sheep and canine.
Inference: The scFv of the present invention is highly specific. The scFv shows reactivity only towards bovine IgA. It does not show any cross reactivity with other classes of bovine immunoglobulins such as IgGl, IgG2 and IgM and IgA of different species such as buffalo, goat, sheep and canine.

Example 5
Affinity measurement of scFv
Affinity of scFv was determined using BIACore X-100 following ligand capture protocol. NTA sensor chip (GE, USA) was used to capture His6-tagged scFv on to the chip surface. Ligand capture and analyte binding conditions were optimized prior to performing the kinetic analysis, using BIACore Applications wizard (GE, USA). ScFv at a concentration of 50 ng/ml (2 nM) was used for affinity measurement. Standard IgA was diluted to concentrations ranging from 20-125 ng/ml (12-78 nM) in lx HBS-EP buffer (10 raM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005%, v/v polysorbate 20; GE, USA). Affinity measurement was performed using multiple injection workflows with contact time of 180 seconds for binding and dissociation time of 400 seconds for each concentration of IgA. Sensor chip regeneration and kinetic analysis workflow was setup as suggested by the BlAcore Wizard template (GE, USA). Surface plasmon resonance (SPR) data was evaluated, assuming 1:1 interaction, using BLAEvaluation software (GE, USA). The kinetic measurements were verified with internal consistency tests.

Since the standard monoclonal antibody, IL-A71, reacts to the Fc portion of IgA, immobilization of IgA or scFv could potentially interfere with the three dimensional structure of the molecule. Therefore, ligand capture method was employed for measuring affinity of scFv. The interaction between scFv and purified IgA standards were studied at 25°C over a concentration range of 12-78 nM of purified IgA. The binding responses in resonance units (RUs) were continuously recorded and presented graphically as a function of time. The association and dissociation rate constants for the interaction using the BIAEvaluation software was found to be 2.26x107 M"1 s"1 and 1.87xl03 s"1 respectively. Figure 9A shows the SPR sensogram showing the binding profile of scFv with different concentrations of IgA. Figure 9B shows the SPR sensogram showing residual plot.

Result: The affinity constant of the scFv towards IgA using BIACore X-100 was found to be 8.1xlOn M"1. The residual distribution plot as obtained in SPR sensogram was linear.

Inference: The scFv of the present invention has high affinity for IgA. Further, as the residual distribution plot was linear with ±2 RU (response unit) deviation and no significant bulk contribution (RI) to the sensogram was found, these quality parameters validate affinity measurement.

Example 6
Characterization of anti-bovine IgA scFv for stability
Sandwich ELISA
Sandwich ELISA was performed to determine the stability of the scFv stored at different aliquots at different temperatures of 37°C, 22°C, 4°C, -20°C, and -70°C and assayed for its functional activity by sandwich ELISA to detect bovine IgA at different time points of 1, 2,4 weeks and 2, 3 and 6 months respectively. ELISA was performed with the kit provided by Bethyl Laboratories. Microtiter plates were pre-coated with anti-bovine IgA antibody and the un-reacted sites were blocked. 100ul of standard bovine IgA (lug/ml) was added to the wells coated with anti-bovine IgA and incubated at room temperature for 1 hour. The microtiter wells were washed with wash buffer according to the manufacturer's instruction and 100 of recombinant scFv incubated at different temperature and time points was added at a concentration
of lmg/ml respectively, and again incubated for 1 hour at room temperature. The binding affinity of the scFv with bovine IgA was detected by adding His-Probe and a chromogenic substrate TMB. The plate was read at 450nm after the reaction was stopped with 1.25M H2S04. The results are provided in Figure 11.

Figure 11 shows the binding affinity of scFv stored at different temperatures to bovine IgA.

Result: The scFv of the present invention stored at -20°C and -70°C was active even after 6 months and was able to bind IgA. No binding was observed for scFv stored at 4°C and above.

Inference: scFv was stable at temperatures below -20°C and there was no loss of binding activity. There was a loss of binding activity when scFv was stored at temperature above 4°C post 1 - 2 weeks. Thus, the scFv of the present invention is highly stable.

Example 7
Characterization of anti-bovine IgA scFv for sensitivity
Sandwich ELISA
Sandwich ELISA was performed to compare the sensitivity of scFv and commercial anti-bovine IgA antibody (kit Mab from Bethyl laboratories). Titration of scFv and Kit Mab was performed against varying concentrations of bovine IgA (800 to 6.25ng/well) in a precoated mAb strips as described by the manufacturer instructions. The binding of the scFv and Kit Mab towards bovine IgA was detected by addition of His-Probe/Kit conjugate HRPO followed by 3,3', 5,5'-tetramethylbenzidine (TMB) substrate. The plate was incubated at 37°C for 10 minutes and the reaction was stopped by addition of 1.25M H2SO4. The absorbance was measured at 450 nm using a microplate reader (BIO-TEK, US). The reading of absorbance obtained in provided in Table 1. The result is provided in Figure 12. Figure 12 shows the sensitivity of scFv and commercial anti-bovine IgA antibody to bovine IgA.

Result: The scFv was able to detect lower concentration of IgA compared to Kit Mab.
Inference: The scFv of the present invention is able to detect lower concentration of IgA confirming that the scFv has high sensitivity towards IgA.

The commercial anti-bovine IgA is a standard monoclonal antibody known in the art which has been used along with scFv of the present invention for the detection of IgA. Since the scFv of the present invention is high sensitivity than the commercial anti-bovine IgA used in the experiment, the scFv has high sensitivity than all the other monoclonal antibody in the art.

Example 8
Detection of bovine IgA in a sample
Indirect ELISA
Indirect ELISA was performed for the qualitative analysis of bovine IgA in standard sample (Purified bovine IgA provided in the commercial kit from Bethyl laboratories) using scFv and commercial anti-bovine IgA. Standard IgA was coated onto microtiter wells by incubating overnight at 4°C. ScFv (250ng/well) and commercial anti-bovine IgA were serially diluted and added to separate coated well and incubated for 1 hour at 37 °C. E. coli lysate was taken as negative control. The binding of scFv and commercial anti-bovine IgA towards bovine IgA was detected by addition of His-Probe and anti-mouse HRPO, respectively followed by addition of 3,3',5,5'-tetramelhylbenzidine (TMB) substrate. The plate was incubated at 37 °C for 10 minutes and the reaction was stopped by addition of 1.25M H2S04. The absorbance was measured at 450 nm using a microplate reader (BIO-TEK, US). The result is provided in Figure 10A. Figure 10A shows the binding of scFv and commercial anti-bovine IgA to bovine IgA.

Result: Both scFv and anti-bovine IgA were able to detect bovine IgA in standard samples. Detection of bovine IgA using scFv and commercial anti-bovine IgA showed a good fit with r2 values of >0.9 in both the cases, indicating that detection of IgA using either of the antibodies remains linear over a wide concentration range.

Inference: The scFv of the present invention is able to detect IgA over a wide range of concentration confirming that the scFv has high affinity and high sensitivity towards IgA.

Detection of FMDV specific bovine IgA in saliva sample
immunocapture ELISA
Saliva samples were collected from both naive cattle that were neither vaccinated nor infected with FMDV and also from cattle that had been experimentally infected with FMDV. Saliva samples were collected by placing a l/6th portion of a regular size cotton tampon (Tampax®, Kiev, Ukraine) pre-dampened by the addition of 0.5 ml of PBS (pH 7.3-7.5) underneath the tongue and also from the vestibule. Approximately 1-2 ml of saliva was extracted from each tampon by compression within the barrel of a syringe or by centrifugation for 10 minutes at 1862xg before storage at -20 °C.

An immunocapture ELISA was performed for detection of IgA antibodies raised against the structural proteins of FMDV. A polyclonal rabbit anti-FMDV antibody was coated to the microtiter wells at a dilution of 1:6000 in carbonate buffer pH 9.6 and blocked using blocking buffer (3% w/v skim milk containing 0.05% v/v Tween 20) by incubating at 37°C for 1 hour. The wells were washed with PBS-T. Concentrated inactivated FMDV antigen (1:10 in blocking buffer) was added to the wells and incubated for 1 hour. After washing the microtiter wells, the saliva samples were added (1:5 in blocking buffer) and further incubated for 1 hour at 37 °C. The microtiter wells were washed and FMDV specific IgA in saliva samples was detected using anti-bovine IgA scFv (375 ng/well) and anti-bovine IgA HRPO (Commercial). scFv was detected by adding His-Probe at a dilution of 1:5000 in PBS-T. The reactions were developed by addition of substrate (OPD: Sigma, USA and H2O2: Merck, Germany) and stopped using 1M H2SO4.
The microtiter plate was read on a multi-channel spectrophotometer (Versamax, Molecular device, USA) at 492 nm (A492). A sample was considered negative when the Optical density (OD) value was less than the mean value for the negative control plus two standard deviations. The sample was considered positive when the OD value was above mean plus two standard deviations. The result is provided in Figure 10B. Figure 10B shows the binding of scFv and commercial anti-bovine IgA to FMDV specific bovine IgA.

Result: The FMDV specific IgA was detected using scFv and anti-bovine IgA in test saliva samples from infected and non-infected cattle. The cutoff OD for positive samples using scFv was set at 0.520. FMDV specific bovine IgA was detected in 3 out of 18 animals which were infected and vaccinated and 1 out of 5 animals which were infected and unvaccinated. All four animals which were detected as FMDV specific IgA positive were FMDV carriers. Their carrier status was confirmed by isolation of virus after probing the samples and detecting NSP antibodies at 35 days post challenge. No FMDV specific IgA was detected in 17 vaccinated and protected animals and in 52 naive animals. The scFv of the present invention has high affinity and sensitivity for IgA and is able to FMDV specific IgA in a sample.

Table 1: Reading of absorbance obtained for scFv and kit Mab (commercial antibody)

Any publications discussed above and throughout the text are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior disclosure.

SEQ ID NO: 1 shows nucleotide sequence of Heavy chain variable (360 bp)
CATGTGCAACTGCAGCAGTCAGGGGAAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCA CTCTGGATTCACTTTCAGTAGCTATGCCATGTCTTGGGTTCGCCTGACTCCAGAGAAGAGGCTGGAG TGGGTTGCATACATTAGTAGTGGCAGTAGTACCATCTACTATGCAGACACAGTGAAGGGCCGATTCACC TCTCCAGAGACAATGCCAAGAACACCCTGTTCCTGCAAATGACCAGTCTAAGGTCTGAGGACACAGCC ATGTATTACTGTGCAAGGCAGGCAGCTCGGGCTACCTACTACTTTGACTCCTGGGGCCAAGGCACCACG GTCACCGTCTCCTCA

SEQ ID NO: 2 shows nucleotide sequence of Light chain variable (318 bp)
GACATCGAGCTCACTCAGTCTCCAACAATCATGTCTGCATCTCTAGGGGAGGAGATCACCCTAACCTGC AGTGCCAGCTCGAGTGTAAGTTACATGCACTGGTACCAGCAGAAGTCAGGCACTTCTCCCAAACTCTTG ATTTATAGCACATCCAACCTGGCTTCTGGAGTCCCTTCTCGCTCCAGTGGCAGTGGGTCTGGGACCTTT TATTCTCTCACAATCAGCAACATGGAGGCTGAAGATGCTGCCACTTATTACTGTCAACAGAGGAGTAGT TACCCGCTCACGTTCGGTGCTGGGACAAAGTTGGAAATAAAA

SEQ ID NO: 3 shows nucleotide sequence of linker (45 bp)
GGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCG

SEQ ID NO: 4 shows nucleotide sequence of single chain variable fragment (723 bp)
CATGTGCAACTGCAGCAGTCAGGGGAAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCA GCCTCTGGATTCACTTTCAGTAGCTATGCCATGTCTTGGGTTCGCCTGACTCCAGAGAAGAGGCTGGAG TGGGTTGCATACATTAGTAGTGGCAGTAGTACCATCTACTATGCAGACACAGTGAAGGGCCGATTCACC ATCTCCAGAGACAATGCCAAGAACACCCTGTTCCTGCAAATGACCAGTCTAAGGTCTGAGGACACAGCC ATGTATTACTGTGCAAGGCAGGCAGCTCGGGCTACCTACTACTTTGACTCCTGGGGCCAAGGCACCACG GTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGGACATCGAG CTCACTCAGTCTCCAACAATCATGTCTGCATCTCTAGGGGAGGAGATCACCCTAACCTGCAGTGCCAGC TCGAGTGTAAGTTACATGCACTGGTACCAGCAGAAGTCAGGCACTTCTCCCAAACTCTTGATTTATAGC ACATCCAACCTGGCTTCTGGAGTCCCTTCTCGCTCCAGTGGCAGTGGGTCTGGGACCTTTTATTCTCTC ACAATCAGCAACATGGAGGCTGAAGATGCTGCCACTTATTACTGTCAACAGAGGAGTAGTTACCCGCTC ACGTTCGGTGCTGGGACAAAGTTGGAAATAAAA

SEQ ID NO: 5 shows amino acid sequence of single chain variable fragment (241 a.a.)
HVQLQQSGEGLVKPGGSLKLSCAASGFTFSSYAMSWVRLTPEKRLEWVAYISSGSSTIYYADTVKGRFT ISRDNAKNTLFLQMTSLRSEDTAMYYCARQAARATYYFDSWGQGTTVTVSSGGGGSGGGGSGGGGSDIE LTQSPTIMSASLGEEITLTCSASSSVSYMHWYQQKSGTSPKLLIYSTSNliASGVPSRSSGSGSGTFYSL TISNMEAEDAATYYCQQRSSYPLTFGAGTKLEIK

SEQ ID NO: 6 shows amino acid sequence of Heavy chain variable (120 a.a.)

HVQLQQSGEGLVKPGGSLKLSCAASGFTFSSYAMSWVRLTPEKRLEVWAYISSGSSTIYYADTVKGRFT ISRDNAKNTLFLQMTSLRSEDTAMYYCARQAARATYYFDSWGQGTTVTVSS

SEQ ID NO: 7 shows amino acid sequence of Light chain variable (106 a.a.)
DIELTQSPTIMSASLGEEITLTCSASSSVSYMHWYQQKSGTSPKLLIYSTSNLASGVPSRSSGSGSGTF YSLTISNMEAEDAATYYCQQRSSYPLTFGAGTKLEIK

SEQ ID NO: 8 shows amino acid sequence of Linker (15 a.a.)
GGGGSGGGGSGGGGS

We claim:

1. A recombinant monoclonal antibody that specifically binds to bovine IgA, wherein, the amino acid sequence of said recombinant antibody is as set forth inSEQIDNO:5.

2. The recombinant antibody as claimed in claim 1, wherein the antibody is encoded by a polynucleotide having the nucleotide sequence as set forth in SEQ ID NO: 4.

3. The recombinant antibody as claimed in claim 1, wherein the antibody is a murine antibody.

4. The recombinant antibody as claimed in claim 1, wherein the antibody is an antigen-binding antibody fragment.

5. The recombinant antibody as claimed in claim 1, wherein the antibody is a single chain antibody (scFv) comprising heavy chain variable region and light chain variable region operatively linked with a linker.

6. The recombinant monoclonal antibody as claimed in claim 1, wherein the antibody optionally comprises a label.

7. A polynucleotide having the nucleotide sequence as set forth in SEQ ID NO: 4, wherein the polynucleotide encodes a recombinant monoclonal antibody having the amino acid sequence as set forth in SEQ ID NO: 5.

8. A DNA expression cassette comprising the polynucleotide as claimed in claim 7, wherein the polynucleotide is operably linked to a promoter sequence.

9. A recombinant vector comprising the DNA expression cassette as claimed in claim 8.

10. A recombinant host cell comprising the DNA expression cassette as claimed in claim 8 or a recombinant vector as claimed in claim 9.

11. The recombinant host cell as claimed in claim 10, wherein the host cell is selected from a group consisting of E. coli, yeast cell, plant cell, insect cell and mammalian cell.

12. A composition comprising the antibody as claimed in claim 1 and a pharmaceutically acceptable carrier.

13. A process for producing a recombinant monoclonal antibody that specifically binds to bovine IgA, wherein said process comprises introducing a recombinant vector comprising the polynucleotide as claimed in claim 7 or the DNA expression cassette as claimed in claim 8 into a host cell; and culturing said host cell under conditions which allow expression of the recombinant monoclonal antibody to obtain said recombinant monoclonal antibody.

14. The process as claimed in claim 13, wherein said process optionally comprises purifying said antibody using conventional method.

15. The process as claimed in claim 13, wherein said host cell is selected from a group consisting of E. coli, yeast cell, plant cell, insect cell and mammalian cell.

16. A process for detecting the presence of IgA in a sample, said method comprising contacting a sample with the recombinant antibody as claimed in claim 1 and detecting formation of the complex between the recombinant antibody and IgA using conventional methods, wherein the formation of
complex confirms presence of the IgA in the sample.

17. The process as claimed in claim 16, wherein the sample is selected from the group consisting of serum sample, plasma, blood sample, milk, colostrum, saliva, tears, nasal secretion, uterine secretion and intestinal secretion.

18. A kit for detecting the presence of IgA, said kit comprising the recombinant antibody of claim 1 and reagents.