WO1997016537A1 - Stable chicken b-cell line and method of use thereof - Google Patents

Abstract

The invention relates to a stable chicken B-cell line which is suitable for use as a fusion partner to chicken splenocytes for the production of chicken monoclonal antibodies. The invention also relates to methods for the isolation of the specific antibody-producing cells, and to methods of using the monoclonal antibodies for diagnostic and therapeutic purposes.

Description

STABLE CHICKEN B-CELL LINE AND METHOD OF USE THEREOF

TECHNICAL FIELD OF THE INVENTION The present invention relates generally to a stable chicken B-cell line and its use for production of specific antibody secreting chicken hybrid cells.

BACKGROUND OF THE INVENTION Enzyme immunoassays (EIAs) have achieved broad application in clinical science and basic research. A feature common to most EIAs is the use of a monoclonal or polyclonal antibody.

Polyclonal antibodies have traditionally been produced in mammals such as rabbits, goats, sheep and pigs During the past decade, the use of chicken antibodies has gained increasing popularity in immunochemical assays. Recently, successful application of avian antibodies in passive vaccination in animals has been reported (Hamada et al ( 1 991 ) Infect Immun. 59:41 61 ; O'Farrelly et al ( 1 992) Infec. Immun 60.2593) .

The popularity of chicken antibodies is due to advantages the chicken offers over mammals. One of these advantages is that chicken polyclonal antibodies can be obtained from egg yolk without inhumane invasive collection. Another advantage is that chickens provide high specificity antibodies against highly conserved mammalian proteins, because mammalian proteins are in general more immunogenic in the phylogenetically distant chicken. Moreover, physicochemical properties of chicken immunoglobulins are slightly different from those of mammalian antibodies. Chicken immunoglobulins do not react with rheumatoid factor (Larsson et al ( 1 991 ) Clin. Chem 37:41 1 ) , therefore decreasing false-positive reactions often encountered in immunoassays. Furthermore, chicken IgG does not bind to protein A (Kronvall et al ( 1 970) J. Immunol. 104: 140) or protein G (Larsson and Lindahl ( 1 993) Hybridoma 12: 143), and does not react with mammalian Fc receptors or mammalian complement (Jensenius et al (1981 ) J. Immunol. Methods 46:63). Some of these features are obviously advantageous when chicken antibodies are applied in immunoassays.

Within a single immunoassay system, multiple pairs of antibody reagents are often utilized for detection of multiple analytes. In such systems, a commonly encountered problem is cross-reaction between antibody reagents, particularly among those derived from different species. A commonly known example of such a problem is the cross- reaction between mouse monoclonal and rabbit antibodies. Such cross reaction leads to high background signals in immunoassays. Chicken antibodies, on the other hand, have relatively low cross-reactivity with immunoglobulins derived from different mammalian species (Hadge et al ( 1984) Mol. Immunol. 21 :699) . Therefore, the use of chicken antibodies in combination with mammalian immunoglobulins in EIAs is practical and effective.

Monoclonal antibodies have particular advantages over polyclonal antibodies. Specifically, these advantages include the unlimited proliferative capacity of the hybridoma cells from which monoclonal antibodies are obtained, which allows for the production of unlimited quantities of the monoclonal antibody. Moreover, because monoclonal antibodies generated from a particular hybridoma consist of a single molecular entity, their biochemical characteristics, such as affinity and specificity, can be precisely determined. These advantages make monoclonal antibodies well suited as standard reagents for diagnostic and therapeutic purposes.

Murine hybridoma technology has demonstrated widespread applications of monoclonal antibodies in virtually every field While the starting material of polyclonal antibodies is variable, often resulting in difficulties in trouble-shooting processing problems with both the quantity and quality of the final products, one of the most advantageous aspects of monoclonal antibodies is such reproducibility

However, successful application of hybridoma technology has in the past been restricted mainly to murine systems This is primarily due to the lack of suitable cell fusion partners which are equivalent to Sp2/0 or NS- 1 murine myeloma cells lines.

To date, two groups have succeeded in making avian monoclonals: Humphries (U .S Patent No 5,028, 540, issued July 2, 1 991 ) prepared specific antibody-producing chicken cell clones by first obtaining antibody-producing lymphocytes from immunized chicken spleen or bursa. The isolated lymphocytes were infected with transforming virus, followed by propagation of the same in the second chicken pretreated to remove normal B-cells After a period of approximately 2 weeks, lymphocytes from spleen, bursa or peripheral blood of the second chicken were isolated and subjected to cloning to select desired antibody producing clones.

A second group has also utilized chicken hybridoma technology (Matsuda et al, U.S.

Patent No. 5,41 1 ,881 , issued May 2, 1995) . Matsuda's group first established avian B- lymphocyte lines by transforming isolated B-lymphocytes in vitro with avian retrovirus. Such cell lines (TK /HAT sensitive) which are similar to murine myeloma cells, e.g., Sp2/0 cells, were used for PEG-mediated cell fusion with lymphocytes obtained from spleen of immunized chickens. The fused cells were grown in HAT selection medium and specific-antibody positive cultures were cloned using a soft-agar method to isolate hybrid clones

However, prior to the present invention, there had not been available a fusion partner for the production of chicken monoclonal antibodies which: ( 1 ) is stable but does not require retroviral transformation, (2) does not itself secrete antibody; and (3) has a short doubling time

SUMMARY OF THE INVENTION In view of the aforementioned deficiencies in prior art methods of producing monoclonal antibodies, in particular for therapeutic use in humans, it is apparent that there exists a need in the art for producing fusion partners and hybridomas for the production of chicken monoclonal antibodies, which fusion partners are stable, and can be used to produce hybridomas which secrete high levels of antibody suitable for diagnostic and therapeutic uses

In accordance with the present invention, a stable chicken B-cell line is provided, which is stable in cell culture, and can be used to produce hybridoma cells which secrete monoclonal antibodies of a particular specificity Advantages of the present stable chicken B-cell line for the production of monoclonal antibodies include the following ( 1 ) no viral transformation of the cells is necessary - the cells are spontaneous transformants demonstrating continuous growth, (2) the cells have a short doubling time, and (3) the cells themselves do not secrete any antibody, until fused to a splenocyte obtained from a chicken immunized with a particular antigen

Also provided by the invention are the hybridoma cells which are produced by fusing the stable chicken B-cell line with splenocytes from a chicken immunized with a particular antigen, and the monoclonal antibodies obtained therefrom.

In a further aspect, a method for the production of hybridoma cells is provided which includes fusion of a stable chicken B-cell line with splenocytes from a chicken immunized with a particular antigen, and isolation of monoclonal antibody-secreting hybridoma cells using a combination of panning and density gradient centrifugation to improve cell fusion efficiency, and isolation using, for example, antigen-coated magnetic beads.

In another aspect, a method for the production of monoclonal antibodies is provided, which includes the above-described steps for producing hybridoma cells, and the additional step of isolating secreted monoclonal antibody from the hybridoma cells.

In a particular embodiment, the stable chicken B-cell line, also referred to as a "fusion partner, " has a rapid doubling time, preferably less than 20 hours, more preferably less than 1 5 hours, and most preferably 10-1 3 hours

In another embodiment, the stable chicken B-cell line possesses a salvage pathway which makes the cell line resistant to high concentrations of 8-amιnoguanιdιne, or HAT medium.

The present invention naturally contemplates several means for preparation of the stable chicken B-cell line and hybridomas produced therefrom, including as illustrated herein known techniques, and the invention is accordingly intended to cover such preparations within its scope. However, the present invention particularly recognizes that in the process of isolating the B-cell line, additional steps may be required to remove contaminating cells particular to chicken isolates. More specifically, the method of the present invention optimally includes a step in which contaminating anchorage-dependent cells, in particular chicken nucleated red blood cells, are removed Such removal is preferably performed using a sacchaπde-based density gradient

In addition , the method of the present invention also contemplates an additional separation step, in which hybridoma cells specific for a particular antigen may be isolated As noted above, in a particular embodiment, the B-cell line of the invention may be resistant to drugs or other factors which have in the past been used to select for fused cells In particular, the present B-cell line may be resistant to 8- aminoguanidine, but is also resistent to HAT selection medium. Thus, recognizing this fact, a method of the present invention provides a step for the isolation of hybridoma cells which are secreting specific antibody by "rosetting, " using magnetic beads to which a specific antigen is attached (Figure 7) .

The invention also includes the use of the monoclonal antibodies produced as described above, for diagnostic and particularly for therapeutic uses. The diagnostic utility of the present invention extends to the use of the present stable chicken B-cell line as a fusion partner to establish chicken hybridomas which produce specific chicken mAbs which can be used in assays to screen for the particular antigen which has been used to immunize the chicken from which antibody-specific splenocytes are isolated Such antibodies could also be used to screen expression libraries to obtain the gene or genes that encode the antιgen(s)

Thus, the antibodies that may be raised to a particular antigen using the methods of the present invention, are capable of use in connection with various diagnostic techniques, including immunoassays, such as a radioimmunoassay, using for example, an antibody to the antigen that has been labeled by either radioactive addition, or radioiodination

In an immunoassay, a control quantity of the antibodies may be prepared and labeled with an enzyme, a specific binding partner and/or a radioactive element, and may then be introduced into a cellular sample After the labeled material or its binding partner(s) has had an opportunity to react with sites within the sample, the resulting mass may be examined by known techniques, which may vary with the nature of the label attached For example, antibodies against specific antigens may be selected and appropriately employed in the exemplary assay protocol, for the purpose of detecting antigen as described above

In the instance where a radioactive label, such as the isotopes ³H, ^, C, ³²P, ³⁵S, ³⁶CI, ⁵¹ Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁹⁰Y, ^{1 5}l, ^{1 3 ,} l, and ^{l β6}Re are used, known currently available counting procedures may be utilized In the instance where the label is an enzyme, detection may be accomplished by any of the presently utilized colorimetric spectrophotometπc, fluorospectrophotometπc, amperometric or gasometπc techniques known in the art

The present invention includes an assay system which may be prepared in the form of a test kit for the quantitative analysis for the presence of the antigen, or to identify drugs or other agents that may mimic or block the antigen's activity The system or test kit may comprise a labeled component prepared by one of the radioactive and/or enzymatic techniques discussed herein, coupling a label to the antigen, the antibody or binding partners thereto, and optionally one or more additional immunochemical reagents, wherein at least one of the antibody or additional immunochemical reagents is a free or immobilized monoclonal antibody produced by the method of the present invention. In a further embodiment, the present invention relates to certain therapeutic methods which would be based upon the activity of the antιbody(s), its (or their) subunits, or active fragments thereof, or upon agents or other drugs determined to possess the same activity. A first therapeutic method is associated with the prevention of the manifestations of conditions causally related to or following from the presence of the particular antigen or its subunits, and comprises administering an antibody capable of modulating the activity of the antigen or subunits thereof, either individually or in mixture with each other in an amount effective to prevent or diminish the development of those conditions in the host For example, the antibodies may be administered to potentiate the activity of another agent provided as therapy for conditions resulting from the presence of the antigen in a host.

More specifically, the therapeutic method generally referred to herein could include a method for the treatment of various pathologies or other cellular dysfunctions and derangements by the administration of pharmaceutical compositions that may comprise antibodies which are effective inhibitors or enhancers of the activity of the antigen, its subunits, or a larger entity, such as a virus, from which the antigen is derived, possibly in combination with other equally effective drugs developed for instance by a screening assay prepared and used in accordance with a further aspect of the present invention. In particular, the present antibodies, or active fragments thereof, could be prepared in pharmaceutical formulations for administration in a virally infected host, which administration may be in combination with other antiviral therapy including the use of interferon, interleukins, or nucleoside analogs, among others.

Accordingly, it is a principal object of the present invention to provide a method for preparing chicken hybridoma cells which secrete a monoclonal antibody specific for a particular antigen including the steps of: a isolating a splenocyte suspension from a chicken immunized with the particular antigen; b. isolating splenocytes from the splenocyte suspension by removing large nucleated erythrocytes and nonviable cells from the splenocyte suspension via buoyant density centrifugation; c. fusing the isolated splenocytes with a stable chicken B-cell line; d. incubating the cells fused in step (c) with antigen-coated immunomagnetic beads which are coupled to the particular antigen, such that the hybridoma cells which secrete the monoclonal antibody specific for the particular antigen form rosettes around the immunomagnetic beads; and e. isolating the fused cells which have formed rosettes.

In particular embodiments, the stable chicken B-cell line is not transformed with a virus, does not secrete antibody, and has a doubling time of less than 13 hours. The fact that the stable chicken B-cell line is not transformed with a virus means that no viral particles will be produced by the cell line, making the antibody samples isolated from the cell line safe for administration to an animal. Moreover, the fact that the stable chicken B-cell line does not itself secrete antibody (i e., until it is fused with a suitable cell to produce a hybridoma) , means that there are essentially no non-functional hybrid immunoglobulins produced by the hybridoma cells.

In a specific embodiment, the cell line is cB-6, deposited with the American Type Culture Collection under ATCC accession number CRL-1 1 984.

Another object of the invention is to provide a method for preparing chicken monoclonal antibodies specific for a particular antigen including the steps of: a isolating a splenocyte suspension from a chicken immunized with the particular antigen, b isolating splenocytes from the splenocyte suspension by removing large nucleated erythrocytes and nonviable cells from the splenocyte suspension via buoyant density centrifugation, c fusing the isolated splenocytes with a stable chicken B-cell line, d incubating the cells fused in step (c) with antigen-coated immunomagnetic beads which are coupled to the particular antigen, such that the hybridoma cells which secrete the monoclonal antibody specific for the particular antigen form rosettes around the immunomagnetic beads, e isolating the fused cells which have formed rosettes; f culturing the fused cells in a suitable medium; and g isolating monoclonal antibody secreted by the cultured fused cells.

Yet another object of the invention is to provide a stable chicken B-cell line suitable as a fusion partner for the production of monoclonal antibody-secreting hybridoma cells, which stable chicken B-cell line is not transformed with a virus.

Other objects and advantages will become apparent to those skilled in the art from a review of the ensuing description which proceeds with reference to the following illustrative drawings BRIEF DESCRIPTION OF THE DRAWINGS FIGURE 1 shows a single cell suspension from chicken spleen.

FIGURE 2 shows chicken lymphocytes from spleen after Ficoll-Paque gradient centrifugation.

FIGURE 3 shows fused cB-6/chιcken immunocytes, 30 minutes post-cell fusion.

FIGURE 4 shows antigen-specific chicken hybridoma cells rosetted with immunomagnetic beads.

FIGURE 5 shows the growth curve of the cB-6 cell line.

FIGURE 6 is a comparison of mRNA levels of immunoglobulin heavy chain and light chain transcripts between chicken cB-6⁺ cells and chicken B cells. Lane 1 is a DNA marker ( 100 base pair ladder) . DNA was amplified from control RNA (lanes 3 and 5) or RNA isolated from cB-6⁺ cells (lanes 2 and 4) . In lanes 2 and 3, light chain variable region primers were used to amplify a 430 base pair DNA fragment. In lanes 4 and 5 heavy chain variable region primers were used to amplify a 410 base pair DNA fragment. Fifteen 1 5 μ\ of PCR product was analyzed with 1 .2% TBE agarose gel electrophoresis.

FIGURE 7 is a schematic illustration of steps for selecting hybridoma cells produced using the stable chicken B-cell line.

DETAILED DESCRIPTION In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g ., e.g., Sambrook et al, "Molecular Cloning: A Laboratory Manual" ( 1 989); "Current Protocols in Molecular Biology" Volumes l-lll [Ausubel, R. M., ed. ( 1994)]; "Cell Biology: A Laboratory Handbook" Volumes l-lll [J. E. Cehs, ed . ( 1 994))]; "Current Protocols in Immunology" Volumes l-lll [Cohgan, J. E., ed. ( 1994)]; "Oligonucleotide Synthesis" (M.J . Gait ed. 1 984); " Nucleic Acid Hybridization" [B.D. Hames & S.J. Higgins eds. ( 1 985)]; "Transcription And Translation" [B.D. Hames & S.J. Higgms, eds. ( 1 984)]; "Animal Cell Culture" [R.I . Freshney, ed. (1986)]; "Immobilized Cells And Enzymes" [IRL Press, ( 1 986)]; B. Perbal, "A Practical Guide To Molecular Cloning" ( 1 984) .

RECTIFIED SHEET (RULE 91) ISA/EP Therefore, if appearing herein, the following terms shall have the definitions set out below.

The terms "stable B-cell line," "fusion partner, " "cB cell," and any variants not specifically listed, may be used herein interchangeably, and as used throughout the present application and claims refer to stable cell lines isolated from chicken, which are suitable as partners for fusion to splenocytes in the production of monoclonal antibodies.

A " DNA molecule" refers to the polymeric form of deoxyπbonucleotides (adenine, guanine, thymine, or cytosine) in its either single stranded form, or a double-stranded helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double- stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments) , viruses, plasmids, and chromosomes. In discussing the structure of particular double- stranded DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA) .

The term "oligonucleotide" , as used herein in referring to the probe of the present invention, is defined as a molecule comprised of two or more πbonucleotides, preferably more than three Its exact size will depend upon many factors which, in turn, depend upon the ultimate function and use of the oligonucleotide

The term " primer" as used herein refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand, is induced, i.e., in the presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH. The primer may be either smgle-stranded or double-stranded and must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent. The exact length of the primer will depend upon many factors, including temperature, source of primer and use of the method. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide primer typically contains 1 5- 25 or more nucleotides, although it may contain fewer nucleotides. The primers herein are selected to be "substantially" complementary to different strands of a particular target DNA sequence. This means that the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primer sequence need not reflect the exact sequence of the template. For example, a non- complementary nucleotide fragment may be attached to the 5' end of the primer, with the remainder of the primer sequence being complementary to the strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence of the strand to hybridize therewith and thereby form the template for the synthesis of the extension product.

As used herein, the terms "restriction endonucleases" and "restriction enzymes" refer to bacterial enzymes, each of which cut double-stranded DNA at or near a specific nucleotide sequence.

Two DNA sequences are "substantially homologous" when at least about 75 % (preferably at least about 80%, and most preferably at least about 90 or 95%) of the nucleotides match over the defined length of the DNA sequences. Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system Defining appropriate hybridization conditions is within the skill of the art. See, e.g . , Maniatis et al. , supra, DNA Cloning, Vols. I & II, supra; Nucleic Acid Hybridization, supra.

A "heterologous" region of the DNA construct is an identifiable segment of DNA within a larger DNA molecule that is not found in association with the larger molecule in nature Thus, when the heterologous region encodes a gene, the gene will usually be flanked by DNA that does not flank the genomic DNA in the genome of the source organism . Another example of a heterologous coding sequence is a construct where the coding sequence itself is not found in nature (e.g., a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene) . Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA as defined herein.

An "antibody" is any immunoglobulin, including antibodies and fragments thereof, that binds a specific epitope. The term encompasses polyclonal, monoclonal, and chimeric antibodies, the last mentioned described in further detail in U.S. Patent Nos. 4,81 6,397 and 4,81 6,567. However, the methods of the present invention are directed in particular to the production of monoclonal antibodies.

The phrase " monoclonal antibody" in its various grammatical forms refers to an antibody having only one species of antibody combining site capable of immunoreacting with a particular antigen A monoclonal antibody thus typically displays a single binding affinity for any antigen with which it immunoreacts. A monoclonal antibody may therefore contain an antibody molecule having a plurality of antibody combining sites, each immunospecific for a different antigen, e.g , a bispecific (chimeric) monoclonal antibody

An " antibody combining site" is that structural portion of an antibody molecule comprised of heavy and light chain variable and hypervariable regions that specifically binds antigen

The phrase "antibody molecule" in its various grammatical forms as used herein contemplates both an intact immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule

Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and those portions of an immunoglobulin molecule that contain the paratope, including those portions known in the art as Fab, Fab' , F(ab')₂ and F(v) , which portions are preferred for use in the therapeutic methods described herein

Fab and F(ab ) ₂ portions of antibody molecules are prepared by the proteolytic reaction of papain and pepsin, respectively, on substantially intact antibody molecules by methods that are well-known See for example, U S Patent No 4,342,566 to Theofilopolous et al Fab' antibody molecule portions are also well-known and are produced from F(ab')₂ portions followed by reduction of the disulfide bonds linking the two heavy chain portions as with mercaptoethanol, and followed by alkyiation of the resulting protein mercaptan with a reagent such as iodoacetamide An antibody containing intact antibody molecules is preferred herein

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 human The phrase "therapeutically effective amount" is used herein to mean an amount sufficient to prevent, and preferably reduce the S phase activity of a target cellular mass, or other feature of pathology such as for example, elevated blood pressure, fever or white cell count as may attend its presence and activity.

The term " standard hybridization conditions" refers to salt and temperature conditions substantially equivalent to 5 x SSC and 65°C for both hybridization and wash

In its primary aspect, the present invention concerns the isolation and identification of a stable chicken B-cell line which is a suitable fusion partner with chicken splenocytes for the production of chicken monoclonal antibodies

The possibilities both diagnostic and therapeutic that are raised by the ability to prepare chicken monoclonal antibodies are based in part on the fact that mammalian antigens are highly immunogenic in chickens, and the resulting antibodies demonstrate little cross-reactivity with antibodies from other species. This lack of cross-reactivity makes the present chicken monoclonal antibodies particularly well suited for diagnostic purposes, where they may be used in combination with other antibodies Additionally, the chicken monoclonal antibodies may be directly administered to a patient, i.e. , providing passive immunity, in which the antibody reacts with an antigen associated with a particular disease state Thus, in instances where it is desired to reduce or inhibit the activity resulting from a particular antigen, an appropriate antibody specific for the particular antigen could be introduced to block that activity

The monoclonal antibodies used to provide passive immunity may be administered by any method known in the art, but particularly preferable is administration intramuscularly The monoclonal antibodies of the present invention may be pooled, i.e. , a mixture of monoclonal antibodies with various specificities, may be mixed together to provide immunity to multiple epitopes of a given antigen. The monoclonal antibodies may be of any isotype, but preferably for passive transfer of immunity, IgG, which has the longest serum half-life, is preferable.

As discussed earlier, the antibodies exhibiting binding activity to the particular antigen, may be prepared in pharmaceutical compositions, with a suitable carrier and at a strength effective for administration by various means to a patient experiencing an adverse medical condition associated with the specific antigen for the treatment thereof. A variety of administrative techniques may be utilized, among them parenteral techniques such as subcutaneous, intravenous and intraperitoneal injections, catheteπzations and the like. Average quantities of the antibodies or their subunits may vary and in particular should be based upon the recommendations and prescription of a qualified physician or veterinarian.

Also, the antibodies may possess certain diagnostic applications and may for example, be utilized for the purpose of detecting and/or measuring conditions such as viral infection or the like. Thus, the present invention advantageously provides for sensitive, specific, and rapid diagnostic tests to permit prompt selection of treatment protocols. The tests according to the invention can be performed at the point of care by medically trained personnel . For example, emergency medical service workers can perform a test of the invention at the site of a medical emergency or in the ambulance on the way to the hospital . Similarly, medical personal in the emergency room, cardiac care facility or other point of care location at a hospital can perform a test of the invention themselves. Naturally and where clinically appropriate, the patient sample such as blood, plasma, or serum, may be provided to a hospital laboratory to perform the test. The test results indicate to a medical practitioner, e.g. , a physician, whether the chest pain is cardiac in nature or whether the patient is suffering from an ischemic event, and the nature and time of such event. The tests can, of course, be formatted for use with read-out facilities available in an ambulance, in an emergency room, in a doctor's office. The present monoclonal antibodies may be produced in a variety of cellular media, by known techniques described for producing hybridoma cells, utilizing, for example, fused mouse spleen lymphocytes and myeloma cells.

The general methodology for making monoclonal antibodies by hybridomas is well known. In the past, immortal, antibody-producing cell lines were also created by techniques other than fusion, such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus . See, e.g. , M. Schreier et al ., " Hybridoma Techniques" ( 1 980); Hammerling et al., "Monoclonal Antibodies And T-cell Hybridomas" ( 1 981 ); Kennett et al. , "Monoclonal Antibodies" ( 1980); see also U.S. Patent Nos. 4,341 ,761 ; 4,399, 1 21 ; 4,427,783; 4,444,887; 4,451 ,570; 4,466,91 7; 4,472,500; 4,491 ,632; 4,493,890.

Panels of monoclonal antibodies produced against a particular antigen can be screened for various properties; i.e., isotype, epitope, affinity, etc. Of particular interest are monoclonal antibodies that neutralize the activity of the antigen or its subunits. Such monoclonals can be readily identified in ELISA or radioimmunoassays. High affinity antibodies are also useful when immunoaffmity purification of native or recombinant antigen is possible.

It may be preferable for the antibody molecules produced herein be in the form of Fab, Fab' , F(ab')₂ or F(v) portions of whole antibody molecules

As suggested earlier, the diagnostic method of the present invention comprises examining a biological sample or medium by means of an assay including an effective amount of an antibody. In addition, it may be preferable for the antibody molecules produced and used herein to be in the form of Fab, Fab', F(ab')₂ or F(v) portions or whole antibody molecules Patients capable of benefiting from this method include those suffering from cancer, a pre-cancerous lesion, a viral infection or other like pathological derangement Methods for isolating the antibodies and for determining and optimizing the ability of antibodies to assist in the examination of a test sample are all well-known in the art

Methods for producing polyclonal antibodies are well-known in the art See U S Patent No 4,493, 795 to Nestor et al A monoclonal antibody, typically containing Fab and/or F(ab')₂ portions of useful antibody molecules, can be prepared using the hybridoma technology described in Antibodies - A Laboratory Manual, Harlow and Lane, eds , Cold Spring Harbor Laboratory, New York ( 1988) , which is incorporated herein by reference Briefly, to form the hybridoma from which the monoclonal antibody composition is produced, a myeloma or other self-perpetuating cell line (in the present invention, a stable chicken B-cell line) is fused with lymphocytes obtained from the spleen of a chicken hypeπmmunized with an antigen

Splenocytes are typically fused with a fusion partner using polyethylene glycol (PEG) 6000 Fused hybrids are typically selected by their sensitivity to HAT However, as noted above, the stable chicken B-cell line of the present invention does not exhibit sensitivity to HAT medium. Specifically, whereas a concentration of 100 mM 8- aminoguanidine is typically considered a concentration suitable for selecting for HAT- resistant cells, the present stable chicken B-cell line, and hybridomas produced therefrom, exhibit resistance to at least 300 mM 8-amιnoguanιdιne Hybridomas producing a monoclonal antibody useful in practicing this invention are identified by their ability to immunoreact with the particular antigen, in particular, by rosetting using the method schematically represented in Figure 7, and their ability to inhibit specified activity of the antigen in target cells or a biological sample. A monoclonal antibody useful in practicing the present invention can be produced by initiating a monoclonal hybridoma culture comprising a nutrient medium containing a hybridoma that secretes antibody molecules of the appropriate antigen specificity The culture is maintained under conditions and for a time period sufficient for the hybridoma to secrete the antibody molecules into the medium. The antibody-containing medium is then collected The antibody molecules can then be further isolated by well-known techniques

Media useful for the preparation of these compositions are both well-known in the art and commercially available and include synthetic culture media. An exemplary synthetic medium is Dulbecco's minimal essential medium (DMEM; Dulbecco et al., Virol 8 396 ( 1 959)) supplemented with 4 5 gm/l glucose, 20 mm glutamine, and 20% fetal calf serum

Methods for producing monoclonal antibodies are also well-known in the art See

Niman et al , Proc Natl Acad Sci USA, 80 4949-4953 ( 1983) Typically, the antigen or a peptide analog is used either alone or conjugated to an immunogenic carrier, as the immunogen in the before described procedure for producing antigen-specific monoclonal antibodies The hybridomas are screened for the ability to produce an antibody that immunoreacts with the antigen

The present invention further contemplates therapeutic compositions useful in practicing the therapeutic methods of this invention A subject therapeutic composition includes, in admixture, a pharmaceutically acceptable excipient (carrier) and one or more of the monoclonal antibodies produced by the method of the present invention or fragment thereof as described herein as an active ingredient

The preparation of therapeutic compositions which contain antibodies, analogs or active fragments as active ingredients is well understood in the art Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions, however, solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared The preparation can also be emulsified The active therapeutic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents which enhance the effectiveness of the active ingredient.

An antibody, analog or active fragment can be formulated into the therapeutic composition as neutralized pharmaceutically acceptable salt forms. Pharmaceutically acceptable salts include the acid addition salts (formed with the free ammo groups of the polypeptide or antibody molecule) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium , calcium, or ferric hydroxides, and such organic bases as isopropylamme, tnmethylamine, 2-ethylamιno ethanol, histidine, procame, and the like.

The therapeutic antibody-, analog- or active fragment-containing compositions are conventionally administered intravenously, as by injection of a unit dose, for example. The term "unit dose" when used in reference to a therapeutic composition of the present invention refers to physically discrete units suitable as unitary dosage for humans, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier, or vehicle.

The compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount. The quantity to be administered depends on the subject to be treated, capacity of the subject's immune system to utilize the active ingredient, and degree of inhibition or neutralization of antigen binding capacity desired Precise amounts of active ingredient required to be administered depend on the _judgment of the practitioner and are peculiar to each individual. However, suitable dosages may range from about 0 1 to 20, preferably about 0.5 to about 10, and more preferably one to several, milligrams of active ingredient per kilogram body weight of individual per day and depend on the route of administration. Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed by repeated doses at one or more hour intervals by a subsequent injection or other administration. Alternatively, continuous intravenous infusion sufficient to maintain concentrations of ten nanomolar to ten micromolar in the blood are contemplated. The therapeutic compositions may further include an effective amount of the antibody, analog or fragment thereof, and one or more of the following active ingredients: an antibiotic, a steroid. Exemplary formulations are given below:

Formulations

Intravenous Formulation I

Ingredient mg/ml cefotaxime 250.0 antibody 10.0 dextrose USP 45.0 sodium bisulfite USP 3.2 edetate disodium USP 0.1 water for injection q.s.a.d. 1 .0 ml

Intravenous Formulation II

Ingredient mg/ml ampicillin 250.0 antibody 10.0 sodium bisulfite USP 3.2 disodium edetate USP 0.1 water for injection q.s.a.d. 1 .0 ml

Intravenous Formulation III Ingredient mg/ml gentamicin (charged as sulfate) 40.0 antibody 1 0.0 sodium bisulfite USP 3.2 disodium edetate USP 0.1 water for injection q.s.a.d. 1 .0 ml

Intravenous Formulation IV

Ingredient mg/ml antibody 10.0 dextrose USP 45.0 sodium bisulfite USP 3.2 edetate disodium USP 0.1 water for injection q.s.a.d. 1 .0 ml

As used herein, "pg" means picogram, "ng" means nanogram, "ug" or "μg" mean microgram, " mg" means milligram, "ul" or "μ\" mean microliter, "ml" means milliliter, "I " means liter.

The present invention also relates to a variety of diagnostic applications, including methods for detecting the presence of a particular antigen, by reference to its ability to bind to the present antibodies. As mentioned earlier, the antigen can be used to produce antibodies to itself by a variety of known techniques, and such antibodies could then be isolated and utilized as in tests for the presence of particular antigen, or its activity in suspect target cells.

As described in detail above, antibody(ies) to the antigen can be produced and isolated by standard methods including the well known hybridoma techniques. For convenience, the antιbody(ιes) to the antigen will be referred to herein as Ab, and antιbody(ιes) raised in another species as Ab₂.

The presence of the antigen in cells or samples can be ascertained by the usual immunological procedures applicable to such determinations. A number of useful procedures are known. Three such procedures which are especially useful utilize either the antigen labeled with a detectable label, antibody Ab, labeled with a detectable label, or antibody Ab₂ labeled with a detectable label.

The procedures and their application are all familiar to those skilled in the art and accordingly may be utilized within the scope of the present invention. The "competitive" procedure is described in U.S. Patent Nos. 3,654,090 and 3,850,752. Still other procedures are known such as the "double antibody" , or "DASP" procedure.

In each instance, the antigen forms complexes with one or more antιbody(ιes) or binding partners and one member of the complex is labeled with a detectable label. The fact that a complex has formed and, if desired, the amount thereof, can be determined by known methods applicable to the detection of labels.

It will be seen from the above, that a characteristic property of Ab₂ is that it will react with Ab, . This is because Ab, raised in one species has been used in another species as an antigen to raise the antibody Ab₂. For example, Ab₂ may be raised in a mammal using the present chicken antibodies as antigens. Ab₂ therefore would be anti-chicken antibody raised in a mammal. For purposes of this description and claims, Ab, will be referred to as a primary or anti-antigen antibody, and Ab₂ will be referred to as a secondary or anti-Ab, antibody

The labels most commonly employed for these studies are radioactive elements, enzymes chemicals which fluoresce when exposed to ultraviolet light, and others

A number of fluorescent materials are known and can be utilized as labels These include for example, fluorescein, rhodamine, auramine, Texas Red, AMCA blue and Lucifer Yellow A particular detecting material is anti-rabbit antibody prepared in goats and conjugated with fluorescein through an isothiocyanate

The antibody or its fragments or binding partner(s) can also be '^«,beled with a radioactive element or with an enzyme The radioactive label can be detected by any of the currently available counting procedures The preferred isotope ma ' be selected from ³H,

¹⁴C, ³²P, ³⁵S, ³⁶CI, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁹⁰Y, ¹²⁵l, ^13, l, and ¹⁸⁶Re.

Enzyme labels are likewise useful, and can be detected by any of he presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, ampe ometric or gasometric techniques The enzyme is conjugated to the selected particle by reaction with bridging molecules such as carbodnmides, diisocyanates, glutaraldehyde and the like Many enzymes which can be used in these procedures are known and c an be utilized The preferred are peroxidase, β glucuronidase, β-D-glucosidase, β-D-g -lactosidase, urease, glucose oxidase plus peroxidase and alkaline phosphatase U S I 'atent Nos

3,654,090 3, 850,752, and 4,01 6,043 are referred to by way o1 example for their disclosure of alternate labeling material and methods

Alternatively, the monoclonal antibodies of the present invention may be conjugated to a label which can be visualized by electron microscopy, such as metal particles, preferably gold

Accordingly, a purified quantity of the antibody may be radiolabe ed and/or combined, for example, with antibodies or other binding partners thereto, af er which binding studies would be carried out Solutions would then be prepared that contain various quantities of labeled and unlabeled uncombmed antibody, and samples would then be inoculated and thereafter incubated The resulting samples are then washed, solubilized if necessary, and then counted in a gamma counter for a length of time sufficient to yield a standard error of < 5% . These data are then subjected to Scatchard analysis after which observations and conclusions regarding material activity can be drawn. While the foregoing is exemplary, it illustrates the manner in which an assay for binding of the antibody to antigen may be performed and utilized, in the instance where the cellular binding ability of the assayed material may serve as a distinguishing characteristic

In a further embodiment of this invention, commercial test kits suitable for use by a medical specialist may be prepared to determine the presence or absence of antigen in a sample suspected to contain such an antigen. In accordance with the testing techniques discussed above, one class of such kits will contain at least the labeled antibody or its binding partner, for instance an antibody specific thereto, and directions, of course, depending upon the method selected, e.g , "competitive", "sandwich", " DASP" and the like The kits may also contain peripheral reagents such as buffers, stabilizers, etc

Accordingly, a test kit may be prepared for the demonstration of the presence or absence of an antigen or antigen activity, comprising- (a) a predetermined amount of at least one labeled immunochemically reactive component obtained by the direct or indirect attachment of the present antibody or a specific binding partner thereto, to a detectable label,

(b) other reagents, and

(c) directions for use of said kit

More specifically, the diagnostic test kit may comprise

(a) a known amount of the antibody as described above (or a binding partner) generally bound to a solid phase to form an immunosorbent, or in the alternative, bound to a suitable tag, or plural such end products, etc (or their binding partners) one of each,

(b) if necessary, other reagents; and

(c) directions for use of said test kit

In a further variation, the test kit may be prepared and used for the purposes stated above, which operates according to a predetermined protocol (e.g. "competitive" , "sandwich" , "double antibody" , etc.) , and comprises: (a) a labeled component which has been obtained by coupling the antibody to a detectable label,

(b) one or more additional immunochemical reagents of which at least one reagent is a ligand or an immobilized ligand, which ligand is selected from the group consisting of

(i) a ligand capable of binding with the labeled component (a); (n) a ligand capable of binding with a binding partner of the labeled component (a) ,

(in) a ligand capable of binding with at least one of the component(s) to be determined, and

(iv) a ligand capable of binding with at least one of the binding partners of at least one of the component(s) to be determined, and

(c) directions for the performance of a protocol for the detection and/or determination of one or more components of an immunochemical reaction between the antibody and a specific binding partner thereto

The invention also extends to devices in which the chicken monoclonal antibodies of the present invention are utilized The materials comprise the monoclonal antibody or antibodies which are binding partners specific to the antigen to be detected In this embodiment, one antibody of a pair of antibodies, which are specific for a particular antigen, is irreversibly immobilized onto a solid support, this antibody is alternately referred to hereinafter as a capture antibody The other antibody specific for the same antigen is labeled, and is capable of moving with a sample to the location on the solid support of the capture antibody This antibody is sometimes referred to herein as the detection antibody

According to one aspect of this embodiment of the invention there is provided a device comprising a housing means containing a membrane unit or section, with a detector section and a capture section, preferably with a filter section The detector section contains the antibody or antibodies specific to an epitope on an antigen or antigens to be detected in a sample, preferably a sample of blood, serum or plasma. The capture section contains capture antibodies specific to another epitope of each of the antigens to be detected The capture section is positioned distal to the position of the detector section, wherein the capture antibodies are irreversibly immobilized in the capture section, the detector antibodies are reversibly immobilized in the detector section and migrate with the sample into the capture section, when the device is in use. The detector antibodies may be suitably labeled to give a measurable reaction when the marker is present an is bound in accordance with the process of this invention. Binding of the binding partner or antibody to its cognate antigen, the marker, in a sample can be detected by other detection means, such as optical detection, biosensors, homogenous immunoassay formats, and the like.

In accordance with the above, an assay system for screening potential drugs effective to modulate the presence or activity of the antigen may be prepared. The antibody may be introduced into a test system, and the prospective drug may also be introduced into the resulting test system, and the test system thereafter examined to observe any changes in the presence or activity of the antigen in the sample, due either to the addition of the prospective drug alone, or due to the effect of added quantities of the known antibody.

The following examples are presented in order to more fully illustrate the preferred embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention.

EXAMPLE 1

Isolation of stable chicken B-cell line

A chicken which had previously been immunized with a branched peptide, B40, for generating antibodies to myosin light chaιn- 1 (MLC-1 ), was used as a source of splenocytes after not generating a satisfactory anti-MLC-1 titer. 1 mg B40 had been administered to the chicken one week prior to sacrifice. Splenocytes were isolated as described below in Example 2. Adherent cells were removed after 1 hour at 39 C in T- 1 50 flasks. Cell clumps and debris were removed by filtering the cell suspension through 4 layers of sterile gauze pad using CELLECTOR. A total of 1 .1 25 x 1 0⁹ cells were obtained, with a viability of 52 %.

The cells (4.7 x 10⁷/ml) were panned using a 100 mm dish coated with 1 Y-263. A total of 3.65 x 1 0⁷ cells were obtained, with a viability of 92 %. A portion of the panned cells (6.5 x 10⁶) were seeded onto a 24-well plate at 5 x 10⁵ cells/well. Cultures were fed with fresh GM (RPMI-1640 supplemented with 10% FCS, 5 % chicken serum, 1 mM sodium pyruvate, 0.1 mM non-essential ammo acids, 50 μM 2-ME and 50 μg/ml gentamycin) . A portion of the spent medium was removed and replaced with fresh GM every 3 days. 39 days post-isolation, rapidly growing lymphocyte cultures were observed in 5 original wells. These were designated 80-2, 80-5, 80-6, 80-12 and 80-13. One of these cell cultures, cB-6, had a doubling time of approximately 1 2.6 hours (Figure 5) . Cell line cB- 6 has been deposited with the American Type Culture Collection, 1 2301 Parklawn Drive, Rockville, Maryland 20852, USA, on under accession number

EXAMPLE 2

Preparation of Lymphocytes

Immunization of chickens. Antigen was prepared by emulsification in FCA at 1 : 1 . Female chickens at approximately 8 weeks old were given primary immunizations of antigen in amounts of 1 -3 mg/chicken in a total volume of less than 1 ml. Immunizations were administered i.m. and/or s.c. at approximately 3 sites. After a six week period, secondary immunizations were given as described except that antigen was prepared using FIA during emulsification. After ten weeks tertiary immunizations were administered . Antigen was prepared using a sterile buffer such as PBS in a total volume of approximately 1 ml. Injections were administered i.v. using < 0.1 ml of sample and i .m using the remaining sample. After 4-5 days chickens were sacrificed and spleens removed using sterile techniques known in the art.

Isolation of single cell suspension from immunized chicken spleen. The isolation of splenocytes from chicken is performed as described previously for mice. Once removed , chicken spleen was placed in a 100 mm sterile petri dish and fat and other adhering tissue was removed . Spleen was next minced into 0.5 mm³ sized sections using sterile forceps and scissors. Using circular movements, minced spleen tissue was pressed against the screen of a stainless steel strainer using a glass syringe plunger until only fibrous tissue remained on top of the strainer screen. The tissue and lymphocytes that were forced through the strainer was collected into a sterile petri dish. Isolated splenocytes were then suspended in a total volume of 100 ml RPMI-1640 with 100 μg/ml Gentamycin (Rinse medium) . Any clumps were dispersed by a 10-ml pipette drawing tip and expelling several times. The splenocyte suspension was then transferred into two 50 ml conical centrifuge tubes and left to stand for 4 minutes at room temperature. Without disturbing settled large debris, the top 95 % of the cell suspension was harvested. The splenocytes were next pelleted by centrifugation for 10 minutes at 1000 r.p.m. (Beckman, CS-6K) . Supernatant (SN) was aspirated and the cell pellet resuspended in a total volume of 100 ml chicken growth medium (cGM) (cGM = RPMI-1640 (GIBCO) supplemented with 10% FBS, 2.5 % chicken serum, 4 mM L- glutamine, 1 mM sodium pyruvate, 0.1 mM non-essential ammo acids, 50 μg/ml gentamycin) Viable cell count was performed using trypan blue dye exclusion on the 100 ml cell suspension Cell density was adjusted to approximately 5 x 10⁷ cells/ml, and pipetted into five 1 50 cm² T-flasks at approximately 25 mg cell suspension per flask The splenocytes were next incubated for 2 hours at 38°C in 5% C0₂ -in-air with 98% relative humidity in a C0₂ incubator After incubation, non-adherent cells were harvested and pooled followed by filtration through 6 layers of pre-wetted (in PBS or saline) sterile gauze pads Cells were pelleted as described and resuspended in 50 ml rinse medium

Removal of erythrocytes and nonviable cells from chicken splenocytes suspension Removal of abundant large nucleated erythrocytes and nonviable cells, dead or damaged, from cell suspensions of chicken spleen is necessary for efficient somatic cell fusions mediated by polyethylene glycol (PEG) Selective removal of erythrocytes and nonviable cells from splenocytes suspension via buoyant density centrifugation at 400 x g on a Ficoll-Paque (FP) (Pharmacia Biotech, Uppsala, Sweden) cushion was performed Each 100 ml FP contains 5 7 g Ficoll 400, and 9 0 g diatπzoate sodium Five ml of splenocytes suspensions containing 1 - 3 x 10⁸ cells in culture medium were added to ten 1 5 ml centrifuge tubes and underlayered by 6 ml FP solution per tube Tubes were centrifuged at approximately 400 x g for 20 minutes at room temperature Viable lymphocytes were collected at the medium-FP interface and nonviable cells and erythrocytes were collected from the pellets After washing, a cell count was performed using trypan blue dye exclusion method, and viable cells were used for somatic cell fusion or immediately frozen in liquid nitrogen for future use

Results

Lymphocyte yield after FP-puπfication of splenocytes was 1 - 3 x 10⁹ viable cells The viability of cells before FP purification was 80-85 % while after purification viability was 93-96 % For somatic cell fusion, 2 x 10^B purified splenocytes were used per fusion Remaining cells were frozen in liquid nitrogen in vials containing approximately 3 x 10⁸ cells/ml (FBS + 10% DMSO) EXAMPLE 3

Chicken cell fusion. PEG mediated somatic cell fusion was performed as described for the murine system (Fuller et al ( 1987) Preparation of monoclonal antibodies, Ausubel et al, eds., Publishing Associates) using the cB-6 cell line as the fusion partner. Purified spleen cells isolated from immunized chickens were fused with cB-6 cells (splenocytes:cB-6, 3: 1 - 5: 1 ) using 42% (w/v) polyethylene glycol (PEG 4000) at room temperature. Each fusion used 2 x 10⁸ splenocytes. Fused cells in cGM containing 5% of chicken serum (i.e. , with an additional 2.5 % chicken serum) were plated into 400 wells of 96-well microtiter plates at cell seeding density of 5 - 7 x 10⁵ cells per well. After 5 to 7 days of culturing, supernatants from growing hybrid cell cultures were screened for the presence of specific antibody by enzyme-linked immunosorbent assay (ELISA) .

Results

ELISA screening of chicken hybridomas

P/N Ratio^' ELISA OD Number Number post/total culture hybridomas post for screened specific Ab (% post)

> 20 > 0.940 3 3/400 0.75 %

> 1 5 > 0 705 1 2 1 2/400 3%

> 1 0 > 0.470 144 1 44/400 36%

* P/N ratio = (mean NC OD - sample OD) x 100

NC (negative control) = chicken hybridoma growth medium (RPMI- 1640 supplemented with HAT, 10% FBS, 5 % chicken serum)

A comparison of cell fusion between cB-3 and cB-6 and splenocytes was performed. 2 x 10^s splenocytes were fused to 4 x 10⁷ cB cells. Results were as follows: Fusion partner cells Panning with mouse IgG-coated dish

Adsorbed cells Non-adsorbed cells cB-3 2.6 x I O⁵ cells (0.6% 4.4 x 10⁷ cells (99.4% viability) viability) cB-6 1 .9 x 10⁶ cells (7.3% 2.4 x 10⁷ cells (92.7% viability) viability)

EXAMPLE 4

Selection of chicken B-cells or chicken hybridomas using antigen-coated magnetic beads Specific antibody producing B-cells from populations of splenocytes or specific antibody-producing chicken hybridomas from mixed population, non-specific antibody producing chicken hybridoma cultures or hybridoma cultures that do not produce antibody were separated using antigen-coated magnetic beads . Cultures were incubated with beads for 10 - 30 minutes at room temperature using bead-to-cell ratios of 3: 1 to 6: 1 . Only antibody producing cells specific to antigen on beads form rosettes and may be isolated with a magnet. After isolating selected hybridoma cell populations that are 100% positive for specific antibody, cloning by a soft-agar method or other known cloning method in the art was performed.

Preparation of immunomagnetic beads. Dynabeads M-450 tosyl-activated , 4.3 μm diameter, 30 mg/ml (Dynal Inc.. NY, U.S.A.) were coated with antigen-specific purified antibody, monoclonal (MAb) or polyclonal antibody (PAb) . The covalent coupling of antibody to the beads was performed according to the manufacturers instructions Antibody was diluted in 0.5 M borate solution, pH 9.5 to 1 50 μg/ml. One ml of the antibody solution was next mixed with 1 ml Dynabeads M-450 (4 x 10⁸ beads) . The mixture was incubated for 24 hours at 22 °C on a rotator at slow speed. The immunomagnetic beads were washed 3 times, 10 minutes per wash, once for 30 minutes, and overnight at 4 °C using a PBS/BSA solution (5 ml of 10 mM PBS, pH 7.4, containing 0.1 % bovine serum albumin) . The beads were collected by a magnetic particle concentrator (MPC) (Dynal Inc.), and resuspended in 2 ml PBS/BSA solution, i.e., 2 x I O⁸ beads/ml and stored in a glass vial at 4°C.

Coupling antigen to beads. Antigen in PBS, pH 7.4, was mixed with immunomagnetic beads in PBS/BSA to a total volume of approximately 0.5 ml (approximately 100 μg antigen : 1 x 10⁸ immunomagnetic beads) . The antigen/bead mixture was incubated for 30 minutes at room temperature on a rotator at slow speed. Free antigen was removed by repeated washing as described above and resuspended in 1 ml PBS/BSA (i.e., 2 x 10⁸ immunomagnetic beads coupled to antigen).

Rosetttng Rosetting was accomplished in 5 ml sterile plastic tubes (Falcon) with continuous gentle mixing of the suspension on a rotator for 30 minutes at room temperature 1 x 10⁶ cells were incubated with 3 - 5 x 10⁶ immunomagnetic beads in 1 ml RPMI- 1 640 containing 5 % fetal bovine serum. After incubation, the cell suspension was diluted to 4 ml with ice-chilled RPMI-1 640 medium Rosetted cells were separated from non-rosetted cells by holding the tube in the MPC for 30 seco nds. Rosetted cells were washed with 3 ml chilled medium by resuspension of the cells followed by further magnetic separation. Washing steps were repeated 3 - 5 times. P( rcentages of rosetted cells were determined by cell count on a hemacytometer

The isolation of antigen-specific antibody-producing cells can be achieved using various cell populations such as splenocytes isolated from immunized chickun, bulk cultures of hybridoma shortly after cell fusion, and ELISA positive primary hybr doma cultures

Results

After cell fusion, hybrid cells were incubated in 75 cm² flasks. Cultures were subjected to rosettmg with immunomagnetic beads to separate populations of hybrid cells producing specific antibody. Isolation was performed on day 1 , day 4 and day 8 post-fusion.

Approximately 1 8% of the fused cell population was producing IgG, however hybridomas producing specific antibody were only 2-3% of the total cell population. The low percentage of cells producing specific antibody on day 8 separation is likely due to non-producing hybridomas over-growing the culture.

EXAMPLE 5

Analysis of RNA from chicken cB-6^* cells

Polymerase Chain Reaction Total RNA was purified from cultured chicken CB-6 ^* cells with RNeasy kit from QIAGEN (Chatsworth, CA. USA) . Control total RNA was purified from B cells isolated from chicken spleen. Primers flanking chicken immunoglobulin heavy and light chain variable regions were designed using OLIGO 4.06 program from National Biosciences Inc. (Plymouth, MN, USA) based on published cDNA or genomic sequences

RT-PCR was performed with the thermostable rTth reverse transcriptase RNA PCR kit from Perkin-Elmer (Norwalk, CT, USA) . For reverse transcription, 2 μl 10X RT buffer ( 100 mM Tns-HCl, 900 mM KCI) , 1 mM MnCI₂, 200 μM each dATP, dCTP, dGTP and dTTP, 1 μM downstream primer, 1 μg of total RNA and 5 U of rTth DNA polymerase were used. Water was added to bring the final volume to 20 μl. Reverse transcription was accomplished at 57 °C for 5 minutes followed by 70°C for 5 minutes. For amplification of cDNA, 80 μl of a master mixture containing 80 μl of chelating buffer, 2 mM MgCI₂ and 0.25 μM upstream primers were added to the RT reaction. After denaturation at 94°C for 90 seconds, 40 cycles of 94°C for 30 seconds, 59°C for 45 seconds and 72 °C for 45 seconds were performed in a GeneAmp F CR System 2400 (Perkin-Elmer) . Final extension was performed for 7 minutes.

Results In order to determine whether chicken cB-6 cells produce immunoglobulin molecules, RNA PCR was performed to estimate the levels of immunoglobulin transcripts. Both immunoglobulin heavy chain and light chain variable regions were amplified from control RNA (see Figure 6, lanes 3 and 5) but not in RNA isolated from cB-6 cells (see Figure 6, lanes 2 and 4) , thus indicating that CB-6 ⁺ cells do not produce immunoglobulin molecules.

Figure 6 shows a comparison between immunoglobulin heavy chain and light chain mRNA transcript levels from chicken cB-6 cells and chicken B cells. Lane 1 : DNA marker comprising a 100 base pair ladder; Lanes 3 and 5: DNA amplified from control RNA; Lanes 2 and 4: DNA amplified from RNA isolated from cB-6; Lanes 1 and 2: light chain variable region primers were used to amplify a 430 base pair DNA fragment; Lanes 3 and 4: heavy chain variable region primers used to amplify a 41 0 base pair DNA fragment. Fifteen μl of PCR product was analyzed using 1 .2 % TBE agarose gel electrophoresis.

Claims

WHAT IS CLAIMED IS:

1 A method for preparing chicken hybridoma cells which secrete a monoclonal antibody specific for a particular antigen comprising the steps of: a isolating a splenocyte suspension from a chicken immunized with the particular antigen; b isolating splenocytes from the splenocyte suspension by removing large nucleated erythrocytes and nonviable cells from the splenocyte suspension via buoyant density centrifugation; c fusing the isolated splenocytes with a stable chicken B-cell line; d incubating the cells fused in step (c) with antigen-coated immunomagnetic beads which are coupled to the particular antigen, such that the hybridoma cells which secrete the monoclonal antibody specific for the particular antigen form rosettes around the immunomagnetic beads; and e isolating the fused cells which have formed rosettes.

2 The method of Claim 1 , wherein the stable chicken B-cell line is not transformed with a virus

3 The method of Claim 1 , wherein the stable chicken B-cell line does not secrete antibody

4 The method of Claim 1 , wherein the stable chicken B-cell line is cB-6

5 The method of Claim 4, wherein the cB-6 cell line is ATCC accession number CRL 1 1 984

6 The method of Claim 1 , wherein the stable chicken B-cell line has a doubling time of less than 1 3 hours

7 The method of Claim 1 , wherein the cells from the stable chicken B-cell line and the chicken splenocytes are fused with polyethylene glycol.

8. The method of Claim 1 , further comprising the step of confirming the specificity of the isolated hybridoma by analyzing antibody secreted from the cell by ELISA.

9. The method of Claim 1 , wherein the immunomagnetic beads comprise polyclonal antibody.

10. The method of Claim 1 , wherein the immunomagnetic beads comprise monoclonal antibody.

1 1 . A method for preparing chicken monoclonal antibody specific for a particular antigen comprising the steps of: a. isolating a splenocyte suspension from a chicken immunized with the particular antigen; b. isolating splenocytes from the splenocyte suspension by removing large nucleated erythrocytes and nonviable cells from the splenocyte suspension via buoyant density centrifugation; c. fusing the isolated splenocytes with a stable chicken B-cell line; d . incubating the cells fused in step (c) with antigen-coated immunomagnetic beads which are coupled to the particular antigen, such that the hybridoma cells which secrete the monoclonal antibody specific for the particular antigen form rosettes around the immunomagnetic beads; e . isolating the fused cells which have formed rosettes; f . culturing the fused cells in a suitable medium; and g . isolating monoclonal antibody secreted by the cultured fused cells.

1 2. The method of Claim 1 1 , further comprising the step of confirming the specificity of the monoclonal antibody by ELISA.

1 3. A stable chicken B-cell line suitable as a fusion partner for the production of monoclonal antibody-secreting hybridoma cells, which stable chicken B-cell line is not transformed with a virus.

14. The stable chicken B-cell line of Claim 1 3 which has a doubling time of less than 1 3 hours.

15. The stable chicken B-cell line of Claim 1 3 which is cell line cB-6.

1 6. The stable chicken B-cell line of Claim 1 3 which has ATCC accession number

1 7. A hybridoma cell which secretes monoclonal antibodies, which is produced by fusion of the stable chicken B-cell line of Claim 1 3 to an antibody-secreting cell.

1 8. A hybridoma cell which secretes monoclonal antibodies, which is produced by fusion of the stable chicken B-cell line of Claim 14 to an antibody-secreting cell.

1 9. A hybridoma cell which secretes monoclonal antibodies, which is produced by fusion of the stable chicken B-cell line of Claim 1 5 to an antibody-secreting cell.

20. A hybridoma cell which secretes monoclonal antibodies, which is produced by fusion of the stable chicken B-cell line of Claim 16 to an antibody-secreting cell.

21 . The hybridoma cell of Claim 1 7, wherein the antibody-secreting cell is a chicken splenocyte.

22. The hybridoma cell of Claim 1 8, wherein the antibody-secreting cell is a chicken splenocyte