# Patent

## Title
Newcastle disease virus monoclonal antibodies

## Abstract

Anti-peptide monoclonal antibodies (MAb's) specific for Exotic Newcastle Disease (END) are used for rapid diagnostic identification between poultry infected with vaccine strains of NDV (LaSota/B1) and END virus (ENDV). Exotic Newcastle Disease is a contagious and fatal viral disease of birds and poultry. The present invention provides for diagnostic detection of ENDV in commercial poultry.

## Background
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to monoclonal antibodies, the hybridomas producing the antibodies, and methods for identifying and distinguishing vaccine strains of Newcastle disease virus from highly virulent Exotic Newcastle Disease virus. It also relates to peptides used to make the monoclonal antibodies.
2. Description of the Related Art
Newcastle disease virus (NDV), a member of the Paramyxoviridae family, is designated avian paramyxovirus 1. The enveloped virus has a negative-sense single-stranded genome of approximately 15 kb which codes for six proteins, including an RNA-directed RNA polymerase, hemaglutinin-neuraminidase protein, fusion protein, matrix protein, phosphoprotein, and nucleocapsid protein (Alexander, In: B. W. Calnek, H. J. Barnes, C. W. Beard, W. M. Reid, and H. W. Yoder, Jr (Eds), Diseases of Poultry, 9th ed., Iowa State University Press, Ames, Iowa, p. 496-519, 1991). Outbreaks of Newcastle disease were first reported for poultry from Java, Indonesia, and Newcastle-upon-Tyne, England in 1926. The disease currently has a worldwide distribution with a wide host range in which all orders of birds have been reported to be infected by NDV. Infectious virus may be ingested or inhaled, which results in its transmission and is the basis for mass-application vaccination procedures for poultry (Diseases of Poultry, 1991, supra). Isolates of NDV are categorized into three main pathotypes depending on the severity of disease produced by the isolate in chickens (Alexander, In: H. G. Purchase, L. H. Arp, C. H. Domermuth, and J. E. Pearson (Eds.), A Laboratory Manual for the Isolation and Identification of Avian Pathogens, 3rd edition, American Association of Avian Pathologists, Inc., Kennet Square, Pa.; 114-120, 1989; Alexander, 1991, supra). Lentogenic isolates do not usually cause disease in adult birds and are considered avirulent. Viruses of intermediate virulence that cause respiratory disease are termed mesogenic, while virulent viruses that cause high mortality are termed velogenic. In the United States, disease outbreaks of velogenic NDV in poultry are also termed Exotic New Castle Disease (END) and isolates from those outbreaks are termed END Virus (ENDV). Neurotropic and viscerotropic forms of velogenic viruses have been reported worldwide (Alexander, 1991, supra). Viscerotropic velogenic viruses have entered the United States via importation of psittacines ( Bruning-Fann et al., J. Am. Vet. Med. Assoc., Volume 201, 1709-1714, 1992; Panigraphy et al., Avian Dis., Volume 37, 254-258, 1993; Senne et al., Avian Dis., Volume 27, 731-744, 1983) and were the causal agent of the major outbreak in southern California during the early 1970s (Schloer, ILnfect. Immun., Volume 10, 724-732, 1968; Utterback and Schwartz, J. Am. Vet. Med. Assoc., Volume 163, 1080-1088, 1973). Outbreaks of Newcastle disease in cormorants in the north central United States and southern Canada in the 1990s were attributed to neurotropic velogenic viruses (Bannerjee et al., Avian Dis., Volume 38, 873-878, 1994; Wobeser et al., Can. Vet. J., Volume 34, 353-359, 1993).
Differential diagnosis of NDV involves hemagglutination inhibition with polyclonal NDV-specific antisera (Alexander, 1989, supra; Alexander, 1991, supra), or use of the enzyme-linked imunosorbent assay (ELISA) (Jestin et al., Arch. Virol., Volume 105, 199-208, 1989; Miers et al., Avian Pathol., Volume 27, 1112-1125, 1983; Wilson et al., Avian Dis., Volume 29, 1070-1085, 1984). Oligonucleotide probes (Jarecki-Black et al., Avian Dis., Volume 36, 134-138, 1992; Jarecki-Black et al., Avian Dis., Volume 37, 724-730, 1993) and viral genomic RNA fingerprint analysis (McMillian and Hanson, Avian Dis., Volume 26, 332-339, 1982; Palmieri and Mitchell, Avian Dis., Volume 35, 384-388, 1991) have been-used to identify and differentiate NDV strains but with limited success. Monoclonal antibodies are now used to identify antigenic groups but pathotyping NDV isolates involves labor-intensive procedures. Pathotype prediction initially involves NDV inoculation of embryonated eggs to determine the mean time of death of the embryo. Further testing entails inoculation of chickens to determine the intracerebral pathogenicity index and the intravenous pathogenicity index. In the United States, the intracloacal inoculation pathogenicity test is used to distinguish viscerotropic velogenic NDV from neurotropic velogenic viruses. Additionally, virulent NDV can be differentiated by its ability to replicate in most avian and mammalian cell types without the addition of trypsin (Kaleta et al., Comp. Imm. Microbiol. Infect. Dis., Volume 2, 485-496, 1980; King, Avian Dis., Volume 37, 961-969, 1993; Nagai et al., Virology, Volume 72, 494-508, 1976). Although all NDV isolates can replicate in chicken embryo kidney cells, lentogenic viruses require trypsin for replication in avian fibroblasts or mammalian cells (King, supra).
Makkay et al. (Veterinary Microbiology, Volume 66 (3), 209-222, 1999) report that with the advent of subunit vaccines for microbial diseases, it is becoming increasingly important to be able to differentiate naturally infected animals from those vaccinated. Makkay et al. disclose the development of an Enzyme-Linked Immunosorbent Assay (ELISA) using baculovirus-expressed nucleocapsid proteins from Newcastle Disease virus as the coating antigen for a diagnostic and differential ELISA in order to detect antibody to subunit vaccines to Newcastle Disease Virus.
Roehrig et al. (Journal of Clinical Microbiology, Volume 29 (3), 630-631, 1991) disclose the preparation of a murine monoclonal antibody which differentiates between vaccine and wild-type Venezuelan equine encephalomyelitis viruses when used in an ELISA or an ImmunoFlourescent Assay (IFA). The reference discloses that the monoclonal antibody was derived from immunization with a synthetic peptide corresponding to the first 19 amino acids of the E2 glycoprotein of Trinidad donkey Venezuelan equine encephalomyelitis virus.
While methods are available for detecting the presence of NDV in animals, there remains a need for monoclonal antibodies, the hybridomas that produce them, and methods for differentiating high virulent Exotic Newcastle Disease viruses from the low virulent NDV used to produce vaccines of the present invention which overcome some of the limitations of related art detection methods. The present invention described below are novel monoclonal antibodies, novel hybridomas producing the monoclonal antibody, novel peptide sequences used to produce the monoclonal antibodies and methods for using the antibodies and protein.
SUMMARY OF THE INVENTION
It is, therefore, an object of the present invention to provide novel monoclonal antibodies which differentiate between infected animals and vaccinated animals.
It is another object of the present invention to identify novel peptides for making the monoclonal antibodies which can differentiate between animals infected with high virulent Exotic NDV and animals vaccinated with low virulent NDV.
Another object of the present invention is to provide novel hybridomas producing novel monoclonal antibodies which differentiate between animals infected with high virulent Exotic NDV and animals vaccinated with low virulent NDV.
Another object of the present invention is to provide methods for detecting animals that are infected with Newcastle disease virus using a monoclonal antibody that differentiates between a wild type virulent Newcastle Disease Virus and a vaccine Newcastle Disease virus.
A still further object of the present invention is to provide reagents for a commercial diagnostic kit containing a monoclonal antibody capable of differentiating between an animal infected with virulent Newcastle Disease virus and an animal vaccinated for Newcastle Disease virus.
Further objects and advantages of the present invention will become apparent from following description.
DEPOSIT OF THE HYBRIDOMAS
Hybridoma Cell lines: 7F8.C7; 13F7.B6.A7; 10F11.B2.C3; 17F4.F4; 18E9.G3.D7 were submitted on Apr. 17, 2007 and 21A7.B12.F8 submitted on Sep. 20, 2007 for patent deposit purposes on behalf of USDA-ARS Southeast Poultry Research Laboratory. These materials have been deposited under the provisions of the Budapest Treaty with the American Type Tissue Culture Collection (ATCC, Manassas, Va. 20110, USA) on Apr. 17, 2007. The ATCC Patent Deposit Designation PTA-8331, PTA-8332, PTA-8333, PTA-8334, and PTA-8335 for MAb 13F7.B6.A7, 17F4.F4, 10F11.B2.C3, 18E9.G3.D7, and 7F8.C7, respectively. For 21A7.B12.F8, the Deposit Designation is PTA-8641. The deposit has been accepted by the International Depository Authority under the provisions of the Budapest Treaty and all restrictions upon public access to the deposit will be irrevocably removed upon the grant of a patent on this application.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph showing reactivity of the monoclonal antibodies to the LaSota vaccine strain of NDV and the END virus (California 2002: CA02). A cutoff of about 0.1 O.D. indicates a positive or negative reaction.
FIG. 2 shows amino acid alignment of the HN protein from LaSota, B1 and END (California 2002; CA02) viruses. Boxes indicate regions used to produce peptides utilized for immunization of mice.
FIG. 3 shows the amino acid alignment of the P protein from LaSota, B1 and END (California 2002; CA02) viruses. Boxed region indicated region of CA02 sequence used to produce peptides (No 7) for immunization of mice.
FIGS. 4a-f show Western blot analysis with anti-peptide monoclonal antibodies 7F8 (FIG. 4a), 10F11 (FIG. 4b), 13F7 (FIG. 4c), 17F4 (FIG. 4d), 18E9 (FIG. 4e) and 21A7 (FIG. 4f) reacting against vaccine NDV HN (SEQ ID NO 2), Exotic NDV HN (SEQ ID NO 5), NDV P (SEQ ID NO 7) or All NDV HN (SEQ ID NO 1) peptide conjugates.
FIG. 5 is a photograph of a Dot Blot reactivity of monoclonal antibodies 7F8, 10F11, 13F7, 17F4, 18E9, and 21A7 to NDV antigen. ENDV is Exotic Newcastle Disease Virus (Isolate CA02) and LaSota is a standard NDV vaccine strain.



DETAILED DESCRIPTION OF THE INVENTION
Exotic Newcastle disease (END) is a contagious and fatal viral disease of birds and poultry. On Oct. 1, 2002, END virus (ENDV) was confirmed in commercial poultry operations in California. A quarantine area was imposed over southern California and movement of all poultry and bird species was banned. The ENDV was also isolated in Nevada, Arizona, and Texas. More than 3.4 million birds were depopulated at a cost of more than 200 million dollars in state-federal task force funds. The END outbreak underscores the susceptibility of the poultry industry to Newcastle Disease virus and the need for rapid diagnostic reagents specific for END virus. The present invention is to anti-peptide monoclonal antibodies specific for all NDV and ENDV that can be used for rapid diagnostic identification and detection of END and to the hybridomas and antigens used to produce the monoclonal antibodies. Conventional diagnosis of NDV requires virus isolation and identification of the virus as NDV, which is very time consuming and laborious. The development of a rapid, single-use, field test for ENDV will remove the initial dependence of sample shipping, reduce expenses, and result in faster notification of state and federal officials to the positive identification of an END-infected flock.
Genetic sequence differences between velogenic (highly virulent) and lentogenic (low virulent) isolates were used to develop monoclonal antibodies that detect and differentiate high virulent (exotic) isolates from low virulent (vaccine) isolates.
Seven different peptides were identified to the hemagglutinin-neuraminidase (HN) and Phosphoprotein (P) that allow for specific identification and differentiation of ENDV. The peptides were synthesized, conjugated to Keyhole Limpet Hemocyanin (KLH) and injected into BALB/c mice. Monoclonal antibodies (MAbs) were isolated and tested by Enzyme-Linked Immunosorbent Assay (ELISA) and by Western Blot Analysis with both ENDV and LaSota virus. Final testing was performed using dot blot analysis with ENDV and LaSota virus.
Preparation of hybridomas was accomplished using conventional techniques such as described by Kohler and Milstein (Nature, Volume 256, 495-497, 1975) Koprowski et al. (U.S. Pat. No. 4,196,265) or Wands (U.S. Pat. No. 4,271,145), the contents of which are herein incorporated by reference. Generally, the process of preparation comprises the steps of immunizing an animal with an antigen of interest, recovering splenocytes or lymphocytes from the animal, fusing the splenocytes or lymphocytes with continuously replicating myeloma cells to produce hybrid cells, and screening the resultant hybrid cells for the production of antibodies to the antigen. However, because the peptides of the present invention are relatively small molecules, they are themselves incapable of stimulating the immune system to produce antibodies. To render the compound immunogenic, it must first be conjugated to an immunogenic carrier in such a manner that the resultant immunogen is capable of stimulating the immune system of an animal to produce specific antibodies that are capable of binding the unconjugated peptides of the present invention.
The amino acid sequences of the peptides of the present invention are as follows:











MAKSSYKPGRFGGK
SEQ ID NO 1




 



DGVREARSG
SEQ ID NO 2



 



EKITSTLG
SEQ ID NO 3



 



DRVTSLLN
SEQ ID NO 4



 



QRPTQQQGSQPSR
SEQ ID NO 5



 



QPLAKQHGNQLNH
SEQ ID NO 6



 



DRV
SEQ ID NO 7






SEQ ID NO 1 sequence is an HN amino acid sequence and is used to produce monoclonal antibodies to detect “all” NDV; SEQ ID NO 2 is an HN amino acid sequence and is used to produce monoclonal antibodies to detect vaccine strains (LaSota/B1) of NDV; SEQ ID NO 3 is an HN amino acid sequence based on LaSota/B1 isolates; SEQ ID NO 4 is an HN amino acid sequence and is used to produce monoclonal antibodies to detect only END viruses; SEQ ID NO 5 is a P protein amino acid sequence based on LaSota/B1 vaccine strains of NDV; and SEQ ID NO 6 is a P protein amino acid sequence used to produce monoclonal antibodies to detect ENDV/Ca02 isolate.

These peptides are rendered immunogenic by coupling them to an immunogenic carrier by the following procedure. The immunizing agent is constructed by covalently conjugating a peptide of the present invention to an immunogenic carrier protein, preferably by means of a crosslinker, such as a gluteraldehyde moiety. Immunogenic carriers are defined herein as any compound to which any one of the peptides of the present invention may be attached to render the peptide immunogenic. Suitable carriers are well known and may be readily determined by the skilled practitioner in the art. Without being limited thereto, preferred carriers include proteins such as, for example, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), ovalbumin (OVA) and human thyroglobulin. The function of the crosslinker is to introduce into the molecule a spacer of sufficient size to prevent the carrier protein from masking the peptide molecule.
To generate antibody-producing splenocytes or lymphocytes, an immunizing preparation comprising the antigen (peptide-carrier conjugate) is injected into an immunologically competent animal. The preparation may also contain other proteins, although pure or substantially pure compositions of the conjugate in a pharmaceutically acceptable carrier are preferred.
Without being limited thereto, rats and particularly mice are preferred hosts for raising antibodies because of ease of handling. BALB/c mice are most commonly used. Preparation of hybridomas using splenocytes from these animals fused to any of a variety of myeloma cell lines, such as SP2/0, is well-known in the literature.
The immunization protocol includes an injection schedule that is sufficient to allow for maturation of the immune response in the animal. Preferably, a series comprising an initial injection followed by about 3-4 boosters at approximately 30-day intervals should be given prior to fusion. The dose of antigen injected should be sufficient to stimulate the immune system. Any reasonable amount in excess of 25 μg per injection would be effective. Typically, the antigen is suspended in a physiological vehicle, such as isotonic saline or Phosphate Buffered Saline (PBS) buffer. In accordance with a preferred embodiment, an adjuvant may be incorporated into the immunizing preparation for enhanced stimulation of the immune system, particularly for the first 1-2 or 1-3 injections. A variety of adjuvants which are conventional in the art may be used herein, although Freund's or RIBI adjuvants are preferred. The route of injection is typically intraperitoneally, though other routes would also likely be effective in inducing an immune response.
Splenocytes or lymphocytes recovered from the immunized animal are fused with continuously replicating tumor cells, such as myeloma or lymphoma cells, cultured and hybridoma cells selected using techniques conventional in the art. Many continuously replicating tumor cell lines are available which may be used as fusion partners with the splenocytes. Without being limited thereto, preferred myeloma cells include P3, NS1, K653, and particularly SP2/0.
Fusion and culture of the cells can be performed using conventional techniques. In accordance with one well known effective procedure, the splenocytes and myeloma cells are fused by exposure to polyethylene glycol. Hybrid cells are selected by culture in hypoxanthine-aminopterin-thymidine (HAT) medium, whereby unfused myeloma cells are killed by HAT and splenocytes die out, leaving only the hybrid cells. The resultant hybridomas are then grown in RPMI or other suitable culture medium and assayed for antibody production.
Samples of the supernatant culture fluid from the hybridomas are screened using a plurality of techniques such as RIA and Enzyme-Linked immunosorbent assay (ELISA) , in accordance with the preferred embodiment of the invention, an indirect ELISA is employed. Generally, solid substrates, such as beads or the wells of a microtiter plate, which are coated with the antigen or antigen-conjugate, are used to bind the anti-peptide antibody in the supernatant culture fluid. A preferred coating is BSA-coupled peptide . Detection of bound antibody may be accomplished by addition of enzyme-labeled anti-immunoglobulin antibodies followed by enzyme substrate. Horseradish peroxidase and its substrate, 2,2′-azinobis-3-ethylbenthiazoline-sulfonic acid (ABTS) are preferred enzyme/substrate labels. However, it is understood that other enzyme/substrate labels or non-enzyme labels such as radiolabels or chromophores may also be used.
Cloning of hybridomas which are positive for desired antibody production can be carried out as soon as they are detected by any method known in the art. Hybridomas having a positive response in the ELISA screen are preferably expanded and subcloned one or more times by limiting dilution to assure monoclonality.
The supernatant culture fluid from the cloned hybridomas may also be screened to select for those producing antibodies having a high affinity for the peptides of the present invention. Affinity may be measured using a variety of well known techniques, such as ELISA or RIA. An indirect ELISA was utilized to detect monoclonal binding in these studies. Briefly, the cell-culture supernatant containing monoclonal antibody is added to the wells of an assay plate containing the peptide, conjugated to a different carrier protein than used for injection into the animal. This application ensures antibody reactivity to the peptide and not the carrier protein molecule used for injection. For these studies, KLH was coupled to the peptide to inject into mice, and BSA was coupled to the peptide for testing by ELISA. Following incubation and washing, bound antibody in the wells is detected by the addition of an enzyme-labeled secondary antibody which reacts to the primary monoclonal antibody. The enzyme substrate is added to the wells of the plate and antibody detection is determined colormetrically.
Once hybridomas producing and secreting the desired anti-peptide antibodies are identified, large quantities of the antibody(ies) may be produced in tissue culture using well-known techniques. Alternatively, antibody may be produced within host animals, such as by ascites formation in syngenic mice. Monoclonal antibodies so produced may be purified, for example, by affinity chromatography on protein A or G resin, or using peptide bound to a resin.
The antibodies of the present invention may be used to detect exotic Newcastle Disease virus (ENDV) in commercial poultry, as well as low virulent vaccine strains. The antibodies can used to detect ENDV using well known immunoassay procedures including but no limited to radioimmunoassay, ELISA, lateral flow assay, etc.
Lateral flow tests are also known as “dip-stick” or immunochromatographic strip tests. They are a popular platform for rapid tests since their introduction in the late 1980s, and have been designed to detect other virus (e.g. influenza), as well as for home pregnancy tests. Lateral flow tests are used for the specific qualitative or semi-quantitative detection of many analytes including antigens from pathogens, or antibodies against pathogens. Single or multi-analytes can be tested for simultaneously on the same strip. In the case of poultry testing for avian influenza, oral or cloacal swabs are the normal sample applied to the strip, however, in human applications, urine, saliva, serum, plasma, or whole blood can be used as specimens. Test sensitivity and specificity can vary depending on the affinity and avidity of reagents produced. In the case of hepatitis B surface antigen, tests have claimed a sensitivity of 1.0 ng or less. The tests generally use colloidal gold, dye, or latex bead conjugates to generate a signal detectable by the user. An advantage of these types of tests in a diagnostic setting is that they are self contained and do not require specific skills or training to perform or interpret. The assembled strips are prepared, dried and packaged and have a stable shelf-life when properly stored. To perform a test, the sample is placed in extraction or running buffer and placed on the sample pad at one end of the strip. The capture reagent binds to the antigen or antibody in the sample and moves through the strip by capillary action. If specific analyte is present, the signal reagent binds to it, and a second antibody or antigen-immobilized as a line in the nitrocellulose-then captures the complex. If the test is positive, a pink/purple line develops depending on the chromatogen employed in the test strip. Results are generally observed in 5 to 20 minutes. All tests include an internal positive control line that is used to validate the test result. The appearance of two lines, therefore, indicates a positive result, while a negative test produces only a single line.
The monoclonal antibodies and/or peptides of the present invention may be incorporated into lateral flow kits alone, or together with any other necessary reagents. Such a kit for use comprises a first container comprising the monoclonal antibody, a second container comprising a detection reagent effective for detecting bound antibody, and a peptide conjugate of the present invention bound to a solid support.
Detection and differentiation of ENDV from NDV in animal tissue samples, body fluids, excretions or feeds may be performed using the above-described assay with minimal sample preparation and without extensive extraction procedures. Samples need only be placed in appropriate extraction or running buffer and applied to the lateral flow strip .
In an alternative embodiment of this invention, it is understood that antibody fragments having the epitope for recognizing and/or binding a peptide of the present invention could be substituted for the intact immunoglobulin. In illustration, the Fab monovalent fragment can be generated by digestion of the immunoglobulin with papain. The Fab fragments each have a single antigen-binding site. The (Fab′)2 bivalent fragment can be generated by digestion of immunoglobulin with pepsin wherein each fragment contains two antigen-binding sites and is still capable of cross-linking antigen. The Fv (variable fragment) can be used for the same purpose as the intact IgG for binding a peptide of the present invention.
Fv is the minimum antibody fragment which contains a complete antigen-recognition and -binding site. In a two-chain Fv species, this region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. In a single-chain Fv species (scFv), one heavy- and one light-chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a dimeric structure analogous to that in a two-chain Fv species.
The following examples are intended only to further illustrate the invention and are not intended to limit the scope of the invention which is defined by the claims.
EXAMPLE 1
To identify specific peptide sequences, embryo-propagated Newcastle Disease Virus (NDV) for the California 2002-2003 END outbreak isolate (CA02) was purified by density gradient centrifugation and genomic RNA extracted with a commercial RNA extraction kit (Qiagen RNeasyTM). Degenerate oligonucleotide primers that recognize NDV genomic sequences were used for RT-PCR amplification of the F and HN genes (Seal et al., J. Clin. Microbiol., Volume 33, 2624-2630, 1995; herein incorporated by reference). Synthesized cDNA was cloned into plasmid vectors (Mead et al., Biotechnology, Volume 9, 657-663, 1991; herein incorporated by reference) and replicated in bacteria (Hanahan, Techniques for transformation of E. coli, IRL Press, Oxford, 1985; herein incorporated by reference). Double-stranded DNA sequencing (Sanger et al., Proc Natl Acad Sci USA, Volume 74, 5463-5467, 1977; herein incorporated by reference) with fluorescently-labeled dideoxynucleotides and Taq polymerase (Applied Biosystems Inc., Foster City, Calif.) was performed with an automated sequencer (Smith et al., Nature, Volume 321, 674-679, 1986) and M13 universal forward and reverse primers. Nucleotide, predicted amino acid sequence analysis, and multiple alignments were performed with the CLUSTAL VTM (Higgins et al., Comput Appl Biosci, Volume 8, 189-191, 1992; herein incorporated by reference) method using LASERGENETM software version 1.03 (DNASTAR, Madison, Wis.). The HN amino acid sequences from the commonly used NDV vaccine strains, LaSota/B1, were obtained form GenBank (LaSota-AY845400; B1-NC—002617) and aligned (MEGALIGNTM) with the CA02 isolate. Peptides specific for CA02 and vaccine strains were identified and analyzed for predicted antigenicity. Peptides were synthesized and coupled to Keyhole Limpet Hemocyanin (KLH) using standard procedures (Sigma-GenoSysTM). Based on amino acid alignment (FIGS. 1 and 2) and antigenic index analysis, three different peptides were identified to the HN (Table 1) protein and one to the P protein (Table 2) that would allow for specific detection and differentiation of ENDV from LaSota/B1 vaccine strains.








TABLE 1







Amino acid sequences of HN protein from differ-



ent NDV strains. Peptides produced to detect


all NDV vaccine strains (LaSota/B1) and Exotic


strains specific are shown in bold.













To detect






NDV strains
To detect END



To detect All
Specific
strain Speci-



NDV Amino Acid
Amino Acid
fic Amino



position
position
Acid Position


NDV Strain
354-367
572-580
68-75





LaSota-Vac-
MAKSSYKPGRFGGK
DGVREARSG
EKITSTLG



cine type
SEQ ID NO 1
SEQ ID NO 2
SEQ ID NO 3


 


B1-Vaccine
MAKSSYKPGRFGGK
DGVREARSG
EKITSTLG


type
SEQ ID NO 1
SEQ ID NO 2
SEQ ID NO 3


 


END-Exotic
MAKSSYKPGRFGGK
DRV
DRVTSLLN


NDV
SEQ ID NO 1
SEQ ID NO 7
SEQ ID NO 4


 


PEPTIDE
MAKSSYKPGRFGGK
DGVREARSG
DRVTSLLN


PRODUCED
SEQ ID NO 1
SEQ ID NO 2
SEQ ID NO 4

















TABLE 2







Amino acid sequences of P protein from differ-



ent NDV strains. Peptides produced to detect


ENDV specific are shown in bold.












To detect All NDV





Amino Acid position



NDV Strain
145-157







LaSota-Vaccine type
QRPTQQQGSQPSR





SEQ ID NO 5



 



B1-Vaccine type
QRPTQQQGSQPSR




SEQ ID NO 5



 



END-Exotic NDV
QPLAKQHGNQLNH




SEQ ID NO 6



 



PEPTIDE
QPLAKQHGNQLNH



PRODUCED
SEQ ID NO 6










EXAMPLE 2
Monoclonal antibodies were produced by immunizing Balb/c mice with an emulsion prepared from KLH-coupled peptide in Freud's complete and incomplete adjuvant for priming and boosting, respectively. Mice were injected intra-peritoneally with 0.1 mg of peptide coupled to KLH in complete adjuvant on day 1, 21 and 35. The mice were boosted at day 49 with incomplete adjuvant. Monoclonal antibodies were prepared by fusing spleen cells from peptide-immunized mice with the mouse myeloma cell line SP2/0 in the presence of about 50% polyethylene glycol 1000 (Sigma, St. Louis, Mo.) (Harlow and Lane, Antibodies: a Laboratory Manual, Cold Spring Harbor Laboratory, NY, 1988; herein incorporated by reference). Fused hybridoma cells were resuspended in Iscove's Modified Dulbecco's Medium (Sigma, St. Louis, Mo.) containing hypoxanthine, aminopterin, and thymine (HAT) supplemented with about 20% fetal calf serum in 96-well flat-bottom microtiter plates.
Following growth to about 80% confluency, hybridomas were analyzed for the presence of peptide-specific antibodies by indirect enzyme-linked immunosorbent assay (ELISA) using Bovine Serum Albumin-coupled peptides as antigen. Approximately 50 μl aliquots of cell culture supernatants were transferred to individual wells of a 96-well microtiter plate coated with about 10 μg/ml peptide. Detection was carried out using rabbit anti-mouse immunoglobulin (Ig)G or M conjugated to alkaline pohosphatase (Sigma, St. Louis, Mo.) and substrate containing SigmaFast pNPP tablets(Sigma, St. Louis, Mo.). Individual microtiter plate wells were read at the dual wavelengths of about 405 nm and about 490 nm. Selected monoclonal antibody-secreting hybridoma cell lines were cloned by limiting dilution in microtiter plates.
Isotypes of the monoclonal antibodies were determined using IsoStrip Mouse Monoclonal Antibody Isotyping Kit according to the manufacturer's instructions (Roche Applied Science, Indianapolis, Ind.).
Characterization of monoclonal antibody specificity was examined using CA02 and LaSota virus as antigen. Wells of microtiter plates (Immunlon 2; Dynex, Chantilly, Va.) were coated with approximately 10 μg/ml of betapropiolactone (BPL)-inactivated Newcastle Disease Virus in carbonate coating buffer (pH about 9.6) for approximately 3 hours at room temperature and held overnight at about 4° C. Hybridoma supernatants were diluted 2-fold and reactivity performed in the indirect ELISA as described above in this example. All incubation times were approximately 1 hour and volumes were approximately 50 μl.
In addition, characterization of monoclonal antibody specificity was performed using Western blot analysis. The BSA-coupled NDV amino acid peptides were separated by SDS-PAGE in a 10% polyacrylamide gel using the Bio-Rad Criterion system (Bio-Rad Laboratories, Inc., Hercules, Calif.) (Laemmli et al., J. Mol. Biol., Volume 47, 69-85, 1979; herein incorporated by reference). The separated proteins were transferred onto a 0.45 μM nitrocellulose membrane by semi-dry blot with a Bio-Rad Trans-Blot SD Semi-Dry Cell (Towbin et al., Proc. Natl. Acad.: Sci., USA, Volume 76, 4350-4354, 1979; herein incorporated by reference). Transfer was performed at a constant voltage of approximately 15 volts for about 20 minutes. Following transfer, unbound protein binding sites were blocked by incubation with about 5% skim milk in PBS-T (about 0.05% TWEEN 20 in Phosphate Buffer Solution) for about 1 hour at room temperature. Longitudinal strips were cut and reacted with undiluted hybridoma culture supernatant. Each membrane strip was reacted with secondary antibody (goat anti-mouse IgG or M conjugated to horseradish peroxidase, Southern Biotech Associates, Inc., Birmingham, Ala.) at about a 1:2000 dilution for about 1 hour. The membranes were reacted with ECL Western Blotting Detection Reagents (Amersham Biosciences, Piscataway, N.J.) according to manufacturers' recommendation and exposed to Hyperfilm ECL (Amersham Biosciences). The film was developed using Kodak GBX developing reagents (Eastman Kodak Company, Rochester, N.Y.) according to the manufacturers' recommendations.
The results from the ELISA studies using whole inactivated NDV isolates, ENDV or LaSota, are shown in FIG. 1. Results indicate that all MAb's produced reacted against either ENDV alone or both isolates. MAb's 10F11, 13F7, 18E9 and 21A7 reacted against ENDV and LaSota, while MAb's 7F8 and 17F4 only detected ENDV. Likewise, western blot analysis of the MAb's produced in this invention confirmed reactivity against the peptide used for injection in mice (FIG. 4). MAb 13F7 and 18E9 react against the HN protein to amino acid sequences found in “all” NDV, and were identified as IgG and IgM isotypes, respectively (Table 3). MAb 10F11 reacted against the P protein of NDV, and is an IgM isotype. MAb's 7F8 (IgM) and 17F4 (IgG) reacted against the HN of ENDV only, while 21A7 (IgM) appeared to against peptide SEQ ID NO 2.







TABLE 3







Monoclonal antibodies produced in this invention react against different


NDV peptides. Hybridomas were tested and identified by indirect


ELISA and Western blot to BSA coupled peptide.


Isotypes were determined based on reactivity in a commercial kit.












MAb




React against
Identification















ALL NDV HN peptide
13F7
18E9



SEQ ID NO 1



MAb Isotype
IgG
IgM



Vaccine strains HN
21A7



SEQ ID NO 2



MAb Isotype
IgM



ENDV HN
7F8
17F4



SEQ ID NO 4



MAb Isotype
IgM
IgG



NDV P peptide
10F11



SEQ ID NO 6



MAb Isotype
IgM










EXAMPLE 3
Based on the MAb reactivity profile established above, the reagents were tested on live NDV viruses for detection via Dot Blot analysis (FIG. 5). NDV viruses were grown in 9-11 day-of-embryonation SPF chicken eggs and harvest after 72 hour. Viruses were partially purified (Kapczynski, 2003) and 5 μl spots applied onto nitrocellulose membranes. After drying, the spots were reacted against the MAb's reported here. Results indicate MAb's 7F8 and 17F4 react only against END and not the vaccine strain of NDV. In contrast, MAb's 10F11, 13F7, 18E9 and 21A7 reacted with both ENDV and the LaSota vaccine isolate, indicating they could be used to detect all NDV's in a lateral flow assay. The reaction of MAb 21A7 against ENDV was unexpected based on amino acid sequence, however, a small degree of homology is observed between the NDV isolates, and thus the MAb is believed to be recognizing that conserved epitope. Combining ENDV specific MAb's 7F8 or 17F4 with MAb specific for all strains of NDV would results in a test that could distinguish vaccine strains of NDV from ENDV infected birds.
The foregoing detailed description is for the purpose of illustration. Such detail is solely for that purpose and those skilled in the art can make variations therein without departing from the spirit and scope of the invention.

## Claims

1. An isolated monoclonal antibody or fragment thereof that binds to a peptide consisting of SEQ ID NO 1.

2. An isolated monoclonal antibody or fragment thereof that binds to a peptide consisting of SEQ ID NO 2.

3. An isolated monoclonal antibody or fragment thereof that binds to a peptide consisting of SEQ ID NO 4.

4. An isolated monoclonal antibody or fragment thereof that binds to a peptide consisting of SEQ ID 6.