FIELD OF INVENTION
[0001] The present disclosure relates to the field of immunology, in particular to the field of production of antibodies. The present disclosure specifically relates to the production of a monoclonal antibody against rabies virus.
BACKGROUND OF THE INVENTION
[0002] Rabies is one the invariably fatal viral infections of the nervous system that attacks all warm-blooded animals including humans. The virus is transmitted by the bite of an infected animal, usually from a dog (Cleaveland et al.,, Vaccine, 2003, 21 (17-18), 1965-1973; De Hoff et al., Md. State Med. J., 1981 30, 35-45; Jackson, A.C., J. Neurovirol., 2003, 9, 253-258). Every year, around 30,000 deaths are reported in India alone with 2.5-3 million people requiring post-exposure therapy, making rabies one of the major causes of mortality (Hemachuda et al., Curr Op Neurol., 1997, 10: 60-7). Despite world-wide research efforts, rabies remains a significant global disease world-wide.
[0003] Current pre- and post-rabies exposure management strategies in both humans and non-humans include injection of attenuated live rabies virus in to human or non-human subjects. A drawback of the current treatment paradigm is that the immune response mounted against the injected attenuated live virus is subject specific that is likely variable, and hence may not provide adequate predictable protection against rabies infection.
[0004] Additionally, while there are established standards for evaluating the potency of commercially manufactures vaccines, such methods include expensive in-vivo animal testing that is expensive, labor intensive and time consuming. Further, the use of live animals results in heterogeneity of results and lack of reproducibility, thus making qualitative comparison of rabies antisera all but impossible. Replacement of in-vivo test by a rapid and reliable in-vitro method that ranks high in reproducibility, practicality and costs is highly desirable. [0005] Current in-vitro rabies potency test rely on polyclonal or monoclonal antibodies derived from hybridomas. While this attempts to answer some of the concerns as mentioned earlier, hybridomas are eukaryotic expression platforms that are difficult to prepare and expensive to maintain.
[0006] To overcome the problems associated with in vivo testing methods while maintaining the advantages of an in vitro testing system, generation of single chain variable fragment (scFv)

antibodies through phage display technology has been developed as a method of choice (. Feldhaus et al.,, R. W. Siegel, L. K. Opresko et al., Nature Biotechnology, 2003, 21, 163–170; Hanes et al., “ Proceedings of the National Academy of Sciences of the United States of America, 1998, 95, 4130–4135). The field of antibody engineering has opened new avenues and gaining attention of large number of researchers as compared to conventional antibody technologies. Human antibody phage technology has more advantages in producing large number of antibodies which mimics the key features of the humoral immune system in-vitro than when produced in animals without prior immunization. This leads to the production of high affinity antibodies. The display of Fab and scFv antibody fragments on the surface of bacteriophage for generation of libraries provides an effective platform for isolation of diverse set of antibodies (Brekke et al., Curr Opin Pharmacol., 2003 ,3(5), 544-50. ScFv’s consists of a variable heavy (VH) and a variable light (VL) chain domain 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 demonstrates their structure-function relationship and antigen-antibody interactions, makes scFv antibodies useful in both clinical and medical applications (Huston et al., Proc. Natl. Acad. Sci.USA, 1988, 85, 5879–5883; Bird et al., Science, 1988, 242, 423–426; Condra et al., J Biol Chem., 1990 , 265(4), 2292-2295).
SUMMARY OF THE INVENTION
[0007] An aspect of the present disclosure relates to a monoclonal antibody or a fragment thereof having amino acid sequence as set forth in SEQ ID NO: 51, said monoclonal antibody or a fragment thereof comprising of (a) a heavy chain variable region having amino acid sequence as set forth in SEQ ID NO: 49; (b) a light chain variable region having amino acid sequence as set forth in SEQ ID NO: 50; and (c) linker region having amino acid sequence as set forth in SEQ ID NO: 27.
[0008] In an aspect of the present disclosure, there is provided a recombinant DNA construct comprising a nucleotide fragment encoding a monoclonal antibody or a fragment thereof having amino acid sequence as set forth in SEQ ID NO: 51, said monoclonal antibody or a fragment thereof comprising of (a) a heavy chain variable region having amino acid sequence as set forth in SEQ ID NO: 49; (b) a light chain variable region having amino acid sequence as set forth in

SEQ ID NO: 50; and (c) linker region having amino acid sequence as set forth in SEQ ID NO: 27.
[0009] In an aspect of the present disclosure, there is provided a recombinant DNA vector comprising a recombinant DNA construct, said recombinant DNA construct comprising of a nucleotide fragment encoding a monoclonal antibody or a fragment thereof having amino acid sequence as set forth in SEQ ID NO: 51, said monoclonal antibody or a fragment thereof comprising of (a) a heavy chain variable region having amino acid sequence as set forth in SEQ ID NO: 49; (b) a light chain variable region having amino acid sequence as set forth in SEQ ID NO: 50; and (c) linker region having amino acid sequence as set forth in SEQ ID NO: 27. [0010] In an aspect of the present disclosure, there is provided a host cell comprising a recombinant DNA construct, said recombinant DNA construct comprising of a nucleotide fragment encoding a monoclonal antibody or a fragment thereof having amino acid sequence as set forth in SEQ ID NO: 51, said monoclonal antibody or a fragment thereof comprising of (a) a heavy chain variable region having amino acid sequence as set forth in SEQ ID NO: 49; (b) a light chain variable region having amino acid sequence as set forth in SEQ ID NO: 50; and (c) linker region having amino acid sequence as set forth in SEQ ID NO: 27 or a recombinant DNA vector comprising a recombinant DNA construct, said recombinant DNA construct comprising of a nucleotide fragment encoding a monoclonal antibody or a fragment thereof having amino acid sequence as set forth in SEQ ID NO: 51, said monoclonal antibody or a fragment thereof comprising of (a) a heavy chain variable region having amino acid sequence as set forth in SEQ ID NO: 49; (b) a light chain variable region having amino acid sequence as set forth in SEQ ID NO: 50; and (c) linker region having amino acid sequence as set forth in SEQ ID NO: 27. [0011] In an aspect of the present disclosure, there is provided a pharmaceutical composition comprising of a monoclonal antibody or a fragment thereof comprising of (a) a heavy chain variable region having amino acid sequence as set forth in SEQ ID NO: 49; (b) a light chain variable region having amino acid sequence as set forth in SEQ ID NO: 50; and (c) linker region having amino acid sequence as set forth in SEQ ID NO: 27, further comprising of suitable carriers, diluents, and excipients.
[0012] In an aspect of the present disclosure, there is provided a method of detection of rabies CVS virus strain in a biological sample, wherein said method comprises of (i) contacting a monoclonal antibody or a fragment thereof comprising of (a) a heavy chain variable region

having amino acid sequence as set forth in SEQ ID NO: 49; (b) a light chain variable region having amino acid sequence as set forth in SEQ ID NO: 50; and (c) linker region having amino acid sequence as set forth in SEQ ID NO: 27 with the biological sample, and (ii) detecting the presence of the immune complex formed between the said monoclonal antibody or a fragment thereof and the rabies CVS glycoprotein in the biological sample.
[0013] In an aspect of the present disclosure, there is provided a method of in-vitro ELISA based volumetric analysis of rabies CVS antibodies, wherein said method comprises of (i) immobilizing anti-rabies CVS sera on a micro titer plate; (ii) allowing the immobilized anti-rabies CVS sera to capture purified rabies glycoprotein by adding purified rabies; (iii) adding the monoclonal antibody or fragment thereof comprising of (a) a heavy chain variable region having amino acid sequence as set forth in SEQ ID NO: 49; (b) a light chain variable region having amino acid sequence as set forth in SEQ ID NO: 50; and (c) linker region having amino acid sequence as set forth in SEQ ID NO: 27 in amounts ranging from 2µg-0.25µg to the captured purified rabies glycoprotein; and (iv) assaying binding of the monoclonal antibody with purified rabies glycoprotein, wherein the binding ability of the said monoclonal antibody or a fragment thereof is indicative of the binding capacity of the anti-rabies CVS sera.
[0014] These and other features, aspects and advantages of the present subject matter will be better understood with reference to the following description and appended claims. This summary is provided to introduce a selection of concepts. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used for to limiting the scope of the claimed subject matter.
BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS
[0015] The following drawings form part of the present specification and are included to further
illustrate aspects of the present disclosure. The disclosure may be better understood by reference
to the drawings in combination with the detailed description of the specific embodiments
presented herein.
[0016] Figure 1 illustrates plasmid map showing cloned single chain variable fragment, in
accordance with an embodiment of the present subject matter.
[0017] Figure 2 shows vector construct comprising single chain variable fragment, in accordance
with an embodiment of the present subject matter.

[0018] Figure 3 shows electrophoretic analysis of PCR amplified variable heavy chain fragment and variable light chain fragment, according to an embodiment of the present subject matter. M-Molecular weight markers (showing 250, 500 and 750bp standards), VH-Variable heavy chain fragment (369bp), VL-Variable light chain fragment (324bp) and scFv - Assembled scFv product (738bp).
[0019] Figure 4 shows SDS-PAGE analysis of purified monovalent scFv, in accordance with an embodiment of the present subject matter. The purified protein was detected by staining with coomassie brilliant blue. The size of the corresponding molecular weight standards in kilo Daltons (kDa) are mentioned in the left.
[0020] Figure 5 shows western immunoblot analysis of purified scFv protein, in accordance with an embodiment of the present subject matter. The blot was reacted with His-probe specific for the histidine tag of the scFv. A protein band of ~30kDa was detected.
[0021] Figure 6 shows binding activity of scFv towards rabies CVS antigen of rabies virus by ELISA, in accordance with an embodiment of the present subject matter.
[0022] Figure 7 shows the specificity of scFv towards rabies CVS antigen of rabies virus by ELISA, in accordance with an embodiment of the present subject matter.
[0023] Figure 8 shows the Immuno-capture ELISA of the scFv towards rabies CVS antigen of rabies virus, in accordance with an embodiment of the present subject matter. [0024] Figure 9 shows the binding activity of the scFv towards WHO standard, in accordance with an embodiment of the present disclosure.
DETAILED DESCRIPTION OF THE INVENTION
[0025] Those skilled in the art will be aware that the disclosure described herein is subject to
variations and modifications other than those specifically described. It is to be understood that
the disclosure described herein includes all such variations and modifications. The disclosure
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
[0026] For convenience, before further description of the present disclosure, 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.
[0027] 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.
[0028] 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”.
[0029] 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.
[0030] The term “including” is used to mean “including but not limited to”. “Including” and
including but not limited to” are used interchangeably.
[0031] The term “Single-chain Fv” also abbreviated as “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.
[0032] The term “linker” used in the present disclosure 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.
[0033] The term “amino acid sequence” means the sequence of amino acids that characterizes a
given protein.
[0034] The term “polypeptide” means a polymer of amino acids joined together by peptide
bonds.
[0035] 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 10 to about 25 amino acids.
[0036] The term “polynucleotide” used in the present disclosure 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.
[0037] 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.
[0038] The term “nucleotide sequence” means the order in which nucleotides are situated in a chain relative to one another.
[0039] 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. [0040] The term “recombinant vector” means a vector carrying a foreign DNA fragment. [0041] The term “recombinant host cell” means a host cell carrying a recombinant vector. [0042] 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.
[0043] 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. [0044] Oligonucleotide sequences of forward primers for amplification of murine light chain variable domain are as set forth in:

[0045] SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID
NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ
ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID
NO: 17 .
[0046] Oligonucleotide sequences of reverse primers for amplification of murine light chain
variable domain are as set forth in:
[0047] SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21.
[0048] Oligonucleotide sequences of forward primers for amplification of murine heavy chain
variable domain are as set forth in:
[0049] SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO:
26,SEQ ID NO: 64, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID
NO: 32, EQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO:
37,SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40.
[0050] Oligonucleotide sequence of reverse primers for amplification of murine heavy chain
variable domain as set forth in:
[0051] SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 44.
[0052] Oligonucleotide sequence of heavy chain variable domain as set forth in: SEQ ID NO:
45.
[0053] Oligonucleotide sequence of light chain variable domain as set forth in: SEQ ID NO: 46.
[0054] Oligonucleotide sequence of linker as set forth in: SEQ ID NO: 47.
[0055] Oligonucleotide sequence of anti-rabies CVS scFv as set forth in: SEQ ID NO: 48.
[0056] Amino acid sequence of heavy chain variable domain as set forth in: SEQ ID NO: 49.
[0057] Amino acid sequence of light chain variable domain as set forth in: SEQ ID NO: 50.
[0058] Amino acid sequence of linker as set forth in: SEQ ID NO: 27.
[0059] Amino acid sequence of anti-rabies CVS scFv as set forth in: SEQ ID NO: 51.
[0060] Amino acid sequence of heavy variable chain CDR1 as set forth in SEQ ID NO: 52.
[0061] Amino acid sequence of heavy variable chain CDR2 as set forth in SEQ ID NO: 53.
[0062] Amino acid sequence of heavy chain variable chain CDR3 as set forth in SEQ ID NO:
54.
[0063] DNA sequence of heavy variable chain CDR1 as set forth in SEQ ID NO: 55.
[0064] DNA sequence of heavy variable chain CDR2 as set forth in SEQ ID NO: 56.

[0065] DNA sequence of heavy chain variable chain CDR3 as set forth in SEQ ID NO: 57. [0066] Amino acid sequence of light variable chain CDR1 as set forth in SEQ ID NO: 58. [0067] Amino acid sequence of light variable chain CDR2 as set forth in SEQ ID NO: 59. [0068] Amino acid sequence of light variable chain CDR3 as set forth in SEQ ID NO: 60. [0069] DNA sequence of light variable chain CDR1 as set forth in SEQ ID NO: 61. [0070] DNA sequence of light variable chain CDR2 as set forth in SEQ ID NO: 62. [0071] DNA sequence of light variable chain CDR3 as set forth in SEQ ID NO: 63. [0072] The recombinant murine anti-rabies CVS antibody as disclosed in the present disclosure comprises single chain variable (scFv) fragment and is generated from immunized mice spleenocytes using phage display technology. Biopanning was performed for selection of rabies CVS antigen specific antibody fragments. The eluted phages obtained after a third round of biopanning was used to infect Escherichia coli TG1 and 192 individual clones were tested for binding to target antigen. As indicated by phage ELISA, five specific clones showed positive binding to antigen out of which a single clone was selected for further characterization. Furthermore, PCR analysis and enzymatic analysis showed the presence of scFv antigen binding fragments. Selected scFv plasmid was transformed into Escherichia coli BL21 (DE3) for production of soluble scFv fragments. The purified scFv protein was checked for its antigen binding activity against the rabies CVS virus and found to be active.
[0073] An embodiment of the present disclosure provides an in-vitro ELISA based method for volumetric analysis of rabies CVS antisera by using the murine recombinant monoclonal anti-rabies CVS antibody generated from murine immunized antibody library.
[0074] In an embodiment of the present disclosure, there is provided a monoclonal antibody or a fragment thereof having amino acid sequence as set forth in SEQ ID NO: 51. [0075] In an embodiment of the present disclosure, there is provided a monoclonal antibody or a fragment thereof having amino acid sequence as set forth in SEQ ID NO: 51 encoded by a nucleotide sequence as set forth in SEQ ID NO: 48.
[0076] In an embodiment of the present disclosure, there is provided a monoclonal antibody or a fragment thereof having amino acid sequence as set forth in SEQ ID NO: 51, said monoclonal antibody or a fragment thereof comprising of (a) a heavy chain variable region having amino acid sequence as set forth in SEQ ID NO: 49; (b) a light chain variable region having amino acid

sequence as set forth in SEQ ID NO: 50; and (c) a linker region having amino acid sequence as set forth in SEQ ID NO: 27.
[0077] In an embodiment of the present disclosure, there is provided a monoclonal antibody or a fragment thereof as described herein, said monoclonal antibody or a fragment thereof comprising of (a) a heavy chain variable region having nucleotide sequence as set forth in SEQ ID NO: 45;
(b) a light chain variable region having nucleotide sequence as set forth in SEQ ID NO: 46; and
(c) a linker region having nucleotide sequence as set forth in SEQ ID NO: 47.
[0078] In an embodiment of the present disclosure, there is provided a monoclonal antibody or a fragment thereof as described herein, wherein said heavy chain variable region comprises a complementarity determining region (CDR1) having amino acid sequence as set forth in SEQ ID NO: 52, a complementarity determining region (CDR2) having amino acid sequence as set forth in SEQ ID NO: 53, a complementarity determining region (CDR3) having amino acid sequence as set forth in SEQ ID NO: 54, and wherein said light chain variable region comprises a complementarity determining region (CDR1) having amino acid sequence as set forth in SEQ ID NO: 58, a complementarity determining region (CDR2) having amino acid sequence as set forth in SEQ ID NO: 59, and a complementarity determining region (CDR3) having amino acid sequence as set forth in SEQ ID NO: 60.
[0079] In an embodiment of the present disclosure, there is provided a monoclonal antibody or a fragment thereof as described herein, wherein said heavy chain variable region CDR1 having nucleotide sequence as set forth in SEQ ID NO: 55, a heavy chain variable region CDR2 having nucleotide sequence as set forth in SEQ ID NO: 56, a heavy chain variable region CDR3 having nucleotide sequence as set forth in SEQ ID NO: 57, a light chain variable region CDR1 having nucleotide sequence as set forth in SEQ ID NO: 61, a light chain variable region having CDR2 having nucleotide sequence as set forth in SEQ ID NO: 62, and a light chain variable region having CDR3 having nucleotide sequence as set forth in SEQ ID NO: 63.
[0080] In an embodiment of the present disclosure, there is provided a monoclonal antibody or a fragment thereof as described herein, said monoclonal antibody or a fragment thereof comprising of a heavy chain variable region, said heavy chain variable region comprising of 3 complementarity determining regions, namely, CDR1, CDR2, and CDR3, wherein the amino acid sequence of CDR1, CDR2, and CDR3 are as set forth in SEQ ID NO: 52, SEQ ID NO: 53, and SEQ ID NO: 54 respectively.

[0081] In an embodiment of the present disclosure, there is provided a monoclonal antibody or a fragment thereof as described herein, said monoclonal antibody or a fragment thereof comprising of a heavy chain variable region, said heavy chain variable region comprising of 3 complementarity determining regions, namely, CDR1, CDR2, and CDR3, wherein the nucleotide sequence of CDR1, CDR2, and CDR3 are as set forth in SEQ ID NO: 55, SEQ ID NO: 56, and SEQ ID NO: 57 respectively.
[0082] In an embodiment of the present disclosure, there is provided a monoclonal antibody or a fragment thereof as described herein, said monoclonal antibody or a fragment thereof comprising of a light chain variable region, said light chain variable region comprising of 3 complementarity determining regions, namely, CDR1, CDR2, and CDR3, wherein the amino acid sequence of CDR1, CDR2, and CDR3 are as set forth in SEQ ID NO: 58, SEQ ID NO: 59, and SEQ ID NO: 60 respectively.
[0083] In an embodiment of the present disclosure, there is provided a monoclonal antibody or a fragment thereof as described herein, said monoclonal antibody or a fragment thereof comprising of a light chain variable region, said light chain variable region comprising of 3 complementarity determining regions, namely, CDR1, CDR2, and CDR3, wherein the nucleotide sequence of CDR1, CDR2, and CDR3 are as set forth in SEQ ID NO: 61, SEQ ID NO: 62, and SEQ ID NO: 63 respectively.
[0084] In an embodiment of the present disclosure, there is provided a monoclonal antibody or a fragment thereof as described herein, wherein said monoclonal antibody or a fragment thereof comprises of 6 complementarity determining regions having amino acid sequence as set forth in SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO: 59, and SEQ ID NO: 60.
[0085] In an embodiment of the present disclosure, there is provided a monoclonal antibody or a fragment thereof as described herein, wherein said monoclonal antibody or a fragment thereof comprises of 6 complementarity determining regions having nucleotide sequence as set forth in SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 61, SEQ ID NO: 62, and SEQ ID NO: 63.
[0086] In an embodiment of the present disclosure, there is provided a monoclonal antibody or a fragment thereof as described herein, wherein said monoclonal antibody or a fragment thereof binds glycoprotein encoded by rabies CVS virus strain.

[0087] In an embodiment of the present disclosure, there is provided a recombinant DNA
construct, said recombinant DNA construct comprising a nucleotide fragment encoding a
monoclonal antibody or a fragment thereof as described herein.
[0088] In an embodiment of the present disclosure, there is provided a recombinant DNA vector
comprising a recombinant DNA construct, said recombinant DNA construct comprising a
nucleotide fragment encoding a monoclonal antibody or a fragment thereof as described herein.
[0089] In an embodiment of the present disclosure, there is provided a host cell comprising a
recombinant DNA construct, said recombinant DNA construct comprising a nucleotide fragment
encoding a monoclonal antibody or a fragment thereof as described herein.
[0090] In an embodiment of the present disclosure, there is provided a host cell comprising a
recombinant DNA vector, said recombinant DNA vector comprising a recombinant DNA
construct, and wherein said recombinant DNA construct comprising a nucleotide fragment
encoding a monoclonal antibody or a fragment thereof as described herein.
[0091] In an embodiment of the present disclosure, there is provided a host cell as described
herein, wherein the nuclear genome of said host cell encodes a monoclonal antibody or a
fragment thereof as described herein.
[0092] In an embodiment of the present disclosure, there is provided a host cell as described
herein, wherein the nucleotide sequence encoding a monoclonal antibody or a fragment thereof
as described herein is encoded in extra-nuclear genome.
[0093] In an embodiment of the present disclosure, the host cell as described herein is a yeast
cell.
[0094] In an embodiment of the present disclosure, the host cell as described herein in a bacterial
cell.
[0095] In an embodiment of the present disclosure, the host cell as described herein is a
mammalian cell.
[0096] In an embodiment of the present disclosure, the host cell as described herein is a plant
cell.
[0097] In an embodiment of the present disclosure, there is provided a pharmaceutical
composition comprising a monoclonal antibody or a fragment thereof as described herein, further
comprising of suitable carriers, diluents, and excipients.

[0098] In an embodiment of the present disclosure, there is provided a method of detection of
rabies CVS virus strain in a biological sample, wherein said method comprises (i) contacting a
monoclonal antibody or a fragment thereof as described herein with a biological sample; and (ii)
detecting the presence of the immune complex formed between the said monoclonal antibody or
a fragment thereof and the rabies CVS glycoprotein in the biological sample.
[0099] In an embodiment of the present disclosure, there is provided a method of in-vitro ELISA
based volumetric analysis of rabies CVS antibodies, wherein said method comprises (i)
immobilizing anti-rabies CVS sera on a micro titer plate; (ii) allowing the immobilized anti-
rabies CVS sera to capture purified rabies glycoprotein by adding purified rabies; (iii) adding the
monoclonal antibody or fragment thereof as described herein in amounts ranging from 2µg-
0.25µg to the captured purified rabies glycoprotein; and (iv) assaying binding of the monoclonal
antibody with purified rabies glycoprotein, wherein the binding ability of the said monoclonal
antibody or a fragment thereof is indicative of the binding capacity of the anti-rabies CVS sera.
[00100] In a preferred embodiment of the present disclosure, there is provided a method of
in-vitro ELISA based volumetric analysis of rabies CVS antibodies as described herein, wherein
the immobilizing anti-rabies CVS sera concentration per well of micro titer plate is 1:1000.
[00101] In a preferred embodiment of the present disclosure, there is provided a method of
in-vitro ELISA based volumetric analysis of rabies CVS antibodies as described herein, wherein
the purified rabies glycoprotein concentration per well of micro titer plate is 300ng.
[00102] The monoclonal anti-rabies CVS antibody as disclosed in the present disclosure
binds to rabies CVS virus and neutralizes rabies CVS virus. Further, the antibody can be used as a prophylactic and/or therapeutic agent.
[00103] The nucleic and amino acid sequence as disclosed or described in the present
disclosure are shown using the standard letter abbreviations for nucleotide bases and single letter codes for amino acid.
EXAMPLES
[00104] 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. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary.
Example 1
Identification of monoclonal antibody fragement
[00105] RNA extraction and Amplification of Antibody Variable domain sequences: Total
RNA was isolated from spleen cells of immunized mice and cDNA was synthesized by RT-PCR.
The cDNA was used as template for amplification with heavy/light chain variable domain
primers (Table 2). The amplified variable domains were assembled to form single chain variable
fragments by splicing by overlap extension PCR (Figure 3) and cloned into phagemid vector to
obtain recombinant phagemid vector. The recombinant phagemid vector thus obtained was
transformed into E. coli TG1. The transformed E. coli cells comprising the recombinant
phagemid vector were infected with M13KO7 helper phage to yield recombinant phages.
[00106] Phage display scFv library: Phage displayed scFv library was constructed from
immunized mice spleenocytes. The library comprises over 4 x 108 different scFv fragments cloned in to an ampicillin- resistant phagemid vector pCANTAB5E and transformed into TG1 E. coli cells. scFv fragment comprises one variable heavy chain and one variable light chain linked by a peptide linker.
[00107] Production of the phage library: Production of the phage was performed
essentially as described (Marks et al., 1991). Briefly, E. coli TG1 cultures in the exponential growth phase were infected with helper phage M13KO7 at a multiplicity of infection of 10. The bacterial culture infected with helper phage was grown over-night for phage production. The phages were PEG precipitated and used for biopanning.
[00108] Selection of recombinant antibodies against CVS antigen of rabies virus using
immunized antibody library: Phages displaying individual mice scFv antibody fragments were selected post three rounds of biopanning on immunotubes coated with decreasing amounts of purified rabies CVS glycoprotein for the first, second and third round of panning respectively. Eluted phage titers showed an increase after each of the three rounds of panning, indicating successful enrichment (Table 1). Eluted phage from round three was used to infect Escherichia

coli TG1 and 192 randomly selected individual clones were tested by phage ELISA against rabies CVS antigen. Of the 192 clones tested, five clones showed positive binding to rabies CVS glycoprotein. Out of five selected clones, one clone, designated as C12 was chosen for further characterization based on its high binding affinity.
[00109] Analysis of scFv sequences: The C12 clone was selected for sequencing analysis
based on its high binding affinity to rabies CVS glycoprotein. Plasmid DNA was purified
according to the manufacturer protocol and sequenced at Ocimum Biosolutions, India. The
sequencing primers, S1 and S6 sequences were used respectively for variable heavy chain (SEQ
ID NO: 45) and variable light chain (SEQ ID NO: 46) analysis. The entire sequences was
submitted to the International ImMunoGeneTics information system for immunoglobulins or
antibodies (IMGT) and verified as the variable domains of the antibody. The 738bp sequence
comprising the heavy and light chain regions and linker as set forth in SEQ ID NO: 48.
[00110] Cloning of scFv into pET vector: The schematic representations as depicted in
Figure 1 and Figure 2 show that the nucleotide sequence encoding monovalent mouse anti-rabies CVS antibody was cloned between the EcoRI and NotI sites of pET 28a(+) bacterial expression vector, wherein the size of scFv C12 is 738bp in which a 45bp linker (Gly4Ser)3 (SEQ ID NO: 47) was used to connect the variable regions of the heavy (369 bp, SEQ ID NO: 45) and light (324 bp, SEQ ID NO: 46) chains of antibody. The pET 28a(+) vector carries ribosome binding site, signal peptide sequence of bacterial pectate lyase, variable fragment of light chain, heavy chain, N- and C-terminal His tag sequence and T7 promoter and T7 terminator. Unique sites are shown on the circle map. ‘Kan’ denotes the kanamycin resistance gene. The vector pET 28a(+) and insert referred to as monovalent antibody fragment of size 738bp were digested with EcoRI and NotI from pET 28a(+) vector by incubating at 37oC for 12 hours. The digested products was 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 22oC for 2 hours. The ligated product was incubated for further 20 minutes at 65oC in order to inactive the enzyme. The final product was chemically transformed into XL-Blue strain of E. coli cells.
E. coli transformation
[00111] Preparation of Chemical competent XL-Blue strain of E.coli cells: Overnight
grown XL-Blue strain of E.coli was sub-cultured to 10mL of medium and grown at 37oC, shaking at 200RPM till the OD of the culture reached 0.6 measured at 600nm. The culture was

harvested by centrifuging at 3200RCF for 10 minutes at 4oC and resuspended in 15mL ice-cold
0.1mM CaCl2 and incubated overnight on the ice, before proceeding for transformation.
[00112] Transformation: 100µl of chemically competent XL-Blue cells were incubated
with 100ng of plasmid DNA for 30 minutes on ice. The cells were heat shocked for 90 seconds at 42oC in a water bath and immediately placed on ice for 2 minutes before Luria Bertani (LB) media was added to the cells. The cells were incubated for one hour at 37oC, 200RPM for recovery and antibiotic expression and plated on agar plates containing 50mg/mL kanamycin. The plates were incubated overnight at 37ºC and screened for positive clones by isolating plasmids and subjecting plasmids to double digestion with EcoR1 and Not1. The sequences of the positive clones were verified before the plasmid was transformed in to E.coli BL-21 DE3* cells for soluble expression of the antibody gene.
Example 2
Production of soluble scFv and reactivity against rabies CVS glycoprotein
[00113] Selected plasmid was transformed into non-suppressor Escherichia coli strain BL-
21 DE3* for production of soluble scFv. A signal colony was used to inoculate 10mL of LB medium and grown overnight at 37ºC, at 200RPM. Overnight grown culture was sub-cultured to 1L of LB medium containing 50ng/ml of kanamycin. The culture was allowed to grow at 37°C, 200RPM till the OD600 reached 0.6. Expression of His-tagged scFv was induced by addition of 1mM isopropyl-β-D- thiogalactopyranoside (IPTG) and the culture was allowed to grow for further 4 hours at 28°C, 200RPM. The cells were harvested by centrifugation at 3200RCF for 30minutes. The pelleted cells was resuspended in tris-buffer saline (TBS) and lysed by frequent freezing and thawing. The lysate was then loaded onto Ni-NTA affinity column and allowed to bind. Non-specific/host proteins were washed away by TBS containing 25mM imidazole and the specific protein/scFv was eluted using TBS containing 300mM imidazole. Purified scFv protein was analyzed by SDS-PAGE analysis under reducing conditions after staining with coomassie brilliant blue (Figure 4). Immunoblot analysis using anti-histidine probes under reducing conditions revealed a major band with an apparent molecular weight of about ~30 kDa (Figure 5). Indirect ELISA of purified scFv with rabies CVS antigen showed a concentration dependent signal (Figure 6).
Example 3
Specificity of soluble scFv against rabies CVS glycoprotein

[00114] The binding specificity of the soluble scFv C12 was evaluated by testing its
binding affinity with rabies Pasteur strain, CVS strain; Flury strain and SAD strain by indirect ELISA format. As shown in Figure 7 in graphical format, the scFv antibody binds specifically with rabies CVS strain and does not cross react with any other related strains.
Example 5
Sandwich ELISA for volumetric analysis of rabies CVS antisera
[00115] Wells in a micro titer plate were coated with anti-rabies CVS mouse sera (1:1000)
in carbonate buffer by incubating over-night at 4oC. The plate was washed thrice with phosphate buffer saline (PBS) containing 0.05% tween-20 PBST and any uncoated sites in the wells were blocked with 1% bovine serum albumin by incubating at 37°C for 1 hour. The plate was washed as above and 300ng of purified rabies CVS glycoprotein antigen was added to each well and allowed to react with the anti-rabies CVS mouse sera. The plate was washed as above and scFv C12 protein tagged with histidine was added to each well and allowed to react with the captured antigen. The concentration of scFv C12 added to each well was in the range of 0.25µg-2µg. The binding affinity of the scFv c12 antibody with rabies CVS glycoprotein was detected by added His-Probe and a chromogenic substrate TMB. The reaction was carried out for 10 min at 37°C and read at 450nm after the reaction was stopped with a 1.25M solution of sulphuric acid. The experiment was performed in duplicate and ELISA measurements were reported as average of two readings. The antigen binding affinity of scFv C12 was analyzed by sandwich ELISA. Titration of scFv C12 against the rabies CVS antisera revealed a concentration dependent increase in optical density values as shown in Figure 8.
Example 6
Immunocapture ELISA for Estimation of Rabies CVS Ag in Vaccine Samples
[00116] Maxisorp 96 well plate was (Nunc, Denmark) were coated with anti-rabies CVS
sera (500ng/well) diluted in 50 mM of sodium carbonate-bicarbonate buffer, pH 9.6, and
incubated overnight at 4ºC. The wells were washed 3 times with PBST and blocked with 5%
bovine serum albumin and incubated for one hour at 37ºC. The WHO standard (1:50 dilution)
100/well in the first well and diluted two folds in the subsequent wells. The wells were washed
five times with PBST and scFv was added constantly (7.5µg/well) to all the wells. The binding
activity of the scFv with WHO standard was detected by adding His-Probe and a chromogenic

substrate TMB. The reaction was stopped by addition of 1.25M H2SO4 and the absorbance was
measured at 450 nm using a microplate reader (BIO-TEK, US).
[00117] As seen in Figure 9, it can be inferred that the binding activity of the scFv towards
WHO standard as demonstrated by Immuno- capture ELISA using scFv, shows a good linear
graphical fit with R2 value of 0.9987 with the WHO standard.
[00118] Table 1: Enrichment of rabies virus specific scFv clones in three rounds of
biopanning
[00119] Table 2: Primers for Light and Heavy chain amplification

Sequences
[00120] SEQ ID NO: 45 Nucleotide sequence of heavy variable chain (369nts)
CAGGTCCAACTGCAGCAGTCTGGGGCAGAGCTTGTGAAGCCAGGGGCCTCAGTCAA
GTTGTCCTGCACAGCTTCTGGCTTCAACATTAAAGACTACTATATGCACTGGGTGAA

ACGGAGGACTGAACAGGGCCTGGAGTGGATTGGAGGGATTGATCCTGACAATGGCG
CAATTTACTATGCCCCGAAGTTCCAGGTCAAGGCCACTGTAACAGCAGACACATCCT
CCAACACAGCCTACCTGCAGCTCAGCAGCCTGACATCTGAGGACACTGCCGTCTATT
ACTGTTCTAGAGGGACGCCCCATTACTACGGCTACTACTATGCTTTGGACTACTGGG
GTCAAGGAACCTCAGTCACCGTCTCCTCG
[00121] SEQ ID NO: 46 Nucleotide sequence of light variable chain (324nts)
GATATTCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTC
ACCATCAGTTGCAGGGCAAGTCAGGACATTAGCAATTATTTAAACTGGTATCAGCAG
AAACCAGATGGAACTGTTAAACTCCTGATCTACTACACATCAAGATTACACTCAGGA
GTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGC
AACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATATGCTTCCG
TGGACGTTCGGTGGAGGCACCAAGCTGGAGCTGAAACGT
[00122] SEQ ID NO: 47 Nucleotide sequence of linker (45nts)
GGTGGTGGTGGTTCAGGTGGTGGTGGTTCTGGTGGTGGTGGTTCT
[00123] SEQ ID NO: 48 Nucleotide sequence of anti-rabies CVS scFv (738nts)
GATATTCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTC
ACCATCAGTTGCAGGGCAAGTCAGGACATTAGCAATTATTTAAACTGGTATCAGCAG
AAACCAGATGGAACTGTTAAACTCCTGATCTACTACACATCAAGATTACACTCAGGA
GTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGC
AACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATATGCTTCCG
TGGACGTTCGGTGGAGGCACCAAGCTGGAGCTGAAACGTGGTGGTGGTGGTTCTGG
TGGTGGTGGTTCTGGCGGCGGCGGCTCCCAGGTCCAACTGCAGCAGTCTGGGGCAG
AGCTTGTGAAGCCAGGGGCCTCAGTCAAGTTGTCCTGCACAGCTTCTGGCTTCAACA
TTAAAGACTACTATATGCACTGGGTGAAACGGAGGACTGAACAGGGCCTGGAGTGG
ATTGGAGGGATTGATCCTGACAATGGCGCAATTTACTATGCCCCGAAGTTCCAGGTC
AAGGCCACTGTAACAGCAGACACATCCTCCAACACAGCCTACCTGCAGCTCAGCAG
CCTGACATCTGAGGACACTGCCGTCTATTACTGTTCTAGAGGGACGCCCCATTACTA
CGGCTACTACTATGCTTTGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTC
G
[00124] SEQ ID NO: 49 Amino acid sequence of heavy variable chain (123aa)

QVQLQQSGAELVKPGASVKLSCTASGFNIKDYYMHWVKRRTEQGLEWIGGIDPDNGAI
YYAPKFQVKATVTADTSSNTAYLQLSSLTSEDTAVYYCSRGTPHYYGYYYALDYWGQ
GTSVTVSS
[00125] SEQ ID NO: 50 Amino acid sequence of light variable chain (108aa)
DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPS
RFSGSGSGTDYSLTISNLEQEDIATYFCQQGNMLPWTFGGGTKLELKR
[00126] SEQ ID NO: 27 Amino acid sequence of linker (15aa)
GGGGSGGGGSGGGGS
[00127] SEQ ID NO: 51 Amino acid sequence of anti-rabies CVS scFv (246aa)
DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPS
RFSGSGSGTDYSLTISNLEQEDIATYFCQQGNMLPWTFGGGTKLELKRGGGGSGGGGSG
GGGSQVQLQQSGAELVKPGASVKLSCTASGFNIKDYYMHWVKRRTEQGLEWIGGIDPD
NGAIYYAPKFQVKATVTADTSSNTAYLQLSSLTSEDTAVYYCSRGTPHYYGYYYALDY
WGQGTSVTVSS
[00128] SEQ ID NO: 52 Amino acid sequence of anti-rabies CVS scFv variable heavy
chain CDR1
GFNIKDYY
[00129] SEQ ID NO: 53 Amino acid sequence of anti-rabies CVS scFv variable heavy
chain CDR2
IDPDNGAI
[00130] SEQ ID NO: 54 Amino acid sequence of anti-rabies CVS scFv variable heavy
chain CDR3
SRGTPHYYGYYYALDY
[00131] SEQ ID NO: 55 DNA sequence of anti-rabies CVS scFv variable heavy chain
CDR1
GGCTTCAACATTAAAGACTACTAT
[00132] SEQ ID NO: 56 DNA sequence of anti-rabies CVS scFv variable heavy chain
CDR2
ATTGATCCTGACAATGGCGCAATT
[00133] SEQ ID NO; 57 DNA sequence of anti-rabies CVS scFv variable heavy chain
CDR3

TCTAGAGGGACGCCCCATTACTACGGCTACTACTATGCTTTGGACTAC
[00134] SEQ ID NO: 58 Amino acid sequence of anti-rabies CVS scFv variable light
chain CDR1
QDISNY
[00135] SEQ ID NO: 59 Amino acid sequence of anti-rabies CVS scFv variable light
chain CDR2
YTS
[00136] SEQ ID NO: 60 Amino acid sequence of anti-rabies CVS scFv variable light
chain CDR3
QQGNMLPWT
[00137] SEQ ID NO: 61 DNA sequence of anti-rabies CVS scFv variable light chain
CDR1
CAGCAGAAACCAGATGGA
[00138] SEQ ID NO: 62 DNA sequence of anti-rabies CVS scFv variable light chain
CDR2
TCAAGGTTC
[00139] SEQ ID NO: 63 DNA sequence of anti-rabies CVS scFv variable light chain
CDR3
CAACAGGGTAATATGCTTCCGTGGACG
[00140] Overall, the present disclosure relates to a monoclonal antibody or a fragment
thereof, specifically a single chain variable fragment (scFv) comprising of a variable heavy chain
domain and a variable light chain domain attached via a linker region. The variable heavy chain
domain, and the variable light chain domain are defined by three complementarity determining
regions (CDR), 3 CDRs each for the variable heavy chain and the variable light chain
respectively. The scFv as disclosed herein binds to glycoprotein of rabies CVS strain, and is
useful for detection of rabies CVS virus in a biological sample, and is also useful as a
prophylactic agent.

I/We claim:
1. A monoclonal antibody or a fragment thereof having amino acid sequence as set forth in
SEQ ID NO: 51, said monoclonal antibody or a fragment thereof comprising:
a. a heavy chain variable region having amino acid sequence as set forth in SEQ ID
NO: 49;
b. a light chain variable region having amino acid sequence as set forth in SEQ ID
NO: 50; and
c. a linker region having amino acid sequence as set forth in SEQ ID NO: 27.
2. The monoclonal antibody or a fragment thereof as claimed in claim 1 having nucleotide
sequence as set forth in SEQ ID NO: 48, said monoclonal antibody or a fragment thereof
comprising:
a. a heavy chain variable region having nucleotide sequence as set forth in SEQ ID
NO: 45;
b. a light chain variable region having nucleotide sequence as set forth in SEQ ID
NO: 46; and
c. a linker region having nucleotide sequence as set forth in SEQ ID NO: 47.
3. The monoclonal antibody or a fragment thereof, as claimed in claim 1, wherein said heavy chain variable region comprises a complementarity determining region (CDR1) having amino acid sequence as set forth in SEQ ID NO: 52, a complementarity determining region (CDR2) having amino acid sequence as set forth in SEQ ID NO: 53, a complementarity determining region (CDR3) having amino acid sequence as set forth in SEQ ID NO: 54, and wherein said light chain variable region comprises a complementarity determining region (CDR1) having amino acid sequence as set forth in SEQ ID NO: 58, a complementarity determining region (CDR2) having amino acid sequence as set forth in SEQ ID NO: 59, and a complementarity determining region (CDR3) having amino acid sequence as set forth in SEQ ID NO: 60.
4. The monoclonal antibody or fragment thereof as claimed in claim 3, wherein said heavy chain variable region CDR1 having nucleotide sequence as set forth in SEQ ID NO: 55, a heavy chain variable region CDR2 having nucleotide sequence as set forth in SEQ ID NO: 56, a heavy chain variable region CDR3 having nucleotide sequence as set forth in SEQ ID NO: 57, a light chain variable region CDR1 having nucleotide sequence as set forth in SEQ ID NO: 61, a light

chain variable region having CDR2 having nucleotide sequence as set forth in SEQ ID NO: 62, and a light chain variable region having CDR3 having nucleotide sequence as set forth in SEQ ID NO: 63.
5. The monoclonal antibody or a fragment thereof as claimed in claim 1, wherein said monoclonal antibody or a fragment thereof binds glycoprotein encoded by rabies CVS virus strain.
6. A recombinant DNA construct comprising a nucleotide fragment encoding a monoclonal antibody or a fragment thereof as claimed in claim 1.
7. A recombinant DNA vector comprising a recombinant DNA construct as claimed in claim 6.
8. A host cell comprising a recombinant DNA construct as claimed in claim 6 or a recombinant DNA vector as claimed in claim 7.
9. The host cell as claimed in claim 8, wherein said host cell is a bacterial cell or a fungal cell.
10. A pharmaceutical composition comprising of a monoclonal antibody or a fragment thereof as claimed in claim 1, further comprising of suitable carriers, diluents, and excipients.
11. A method of detection of rabies CVS virus strain in a biological sample, wherein said method comprises:
a. contacting a monoclonal antibody or a fragment thereof as claimed in claim 1
with the biological sample; and
b. detecting the presence of the immune complex formed between the said
monoclonal antibody or a fragment thereof and the rabies CVS glycoprotein in the
biological sample.
12. A method of in-vitro ELISA based volumetric analysis of rabies CVS antibodies, wherein
said method comprises:
a. immobilizing anti-rabies CVS sera on a micro titer plate;
b. allowing the immobilized anti-rabies CVS sera to capture purified rabies
glycoprotein by adding purified rabies;
c. adding the monoclonal antibody or fragment thereof as claimed in claim 1 in
amounts ranging from 2µg-0.25µg to the captured purified rabies glycoprotein; and
d. assaying binding of the monoclonal antibody with purified rabies glycoprotein,

wherein the binding ability of the said monoclonal antibody or a fragment thereof is indicative of the binding capacity of the anti-rabies CVS sera.