Conclusion: Our novel strategy was proven effective both in vitro and in vivo. Significance: This approach will fill in a gap in the existing therapeutic strategy. These cytokines play crucial roles in the differentiation of the mature immune system and are involved in many human diseases. Furthermore, we provide evidence that our peptide, when conjugated to polyethylene glycol to gain stability in vivo , efficiently blocks the action of one of the target cytokines in animal models. This work was authored, in whole or in part, by National Institutes of Health staff. We also declare that these financial involvements of BIONIZ did not undermine the scientific objectivity and integrity of the presented work.
S1, Tables S1 and S2, and additional references. You'll be in good company. Journal of Lipid Research. View this article with LENS. Columns can be prepared where the antibodies are linked to a solid support. Antibodies raised against each IL-B30 will also be useful to raise anti-idiotypic antibodies.
These will be useful in detecting or diagnosing various immunological conditions related to expression of the respective antigens. The described peptide sequences and the related reagents are useful in detecting, isolating, or identifying a DNA clone encoding IL-B30, e. Typically, it will be useful in isolating a gene from mammal, and similar procedures will be applied to isolate genes from other species, e.
Cross hybridization will allow isolation of IL-B30 from the same, e. A number of different approaches will be available to successfully isolate a suitable nucleic acid clone. The purified protein or defined peptides are useful for generating antibodies by standard methods, as described above. Synthetic peptides or purified protein can be presented to an immune system to generate monoclonal or polyclonal antibodies. For example, the specific binding composition could be used for screening of an expression library made from a cell line which expresses an IL-B Screening of intracellular expression can be performed by various staining or immunofluorescence procedures.
Binding compositions could be used to affinity purify or sort out cells expressing a surface fusion protein. The peptide segments can also be used to predict appropriate oligonucleotides to screen a library. The genetic code can be used to select appropriate oligonucleotides useful as probes for screening.
In combination with polymerase chain reaction PCR techniques, synthetic oligonucleotides will be useful in selecting correct clones from a library. Complementary sequences will also be used as probes, primers, or antisense strands. Various fragments should be particularly useful, e. This invention contemplates use of isolated DNA or fragments to encode a biologically active corresponding IL-B30 polypeptide, particularly lacking the portion coding the untranslated 5' portion of the described sequence.
In addition, this invention covers isolated or recombinant DNA which encodes a biologically active protein or polypeptide and which is capable hybridizing under appropriate conditions with the DNA sequences described herein. Said biologically active protein or polypeptide can be an intact antigen having an amino acid sequence disclosed in, e. Further, this invention covers the use of isolated or recombinant DNA, or fragments thereof, which encode the proteins of the invention. The isolated DNA can have the respective regulatory sequences in the 5' and 3' flanks, e.
Alternatively, expression may be effected by operably linking a coding segment to a heterologous promoter, e. An "isolated" nucleic acid is a nucleic acid, e. The term embraces a nucleic acid sequence which has been removed from its naturally occurring environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogs or analogs biologically synthesized by heterologous systems. A substantially pure molecule includes isolated forms of the molecule.
Generally, the nucleic acid will be in a vector or fragment less than about 50 kb, usually less than about 30 kb, typically less than about 10 kb, and preferably less than about 6 kb. An isolated nucleic acid will generally be a homogeneous composition of molecules, but will, in some embodiments, contain minor heterogeneity. This heterogeneity is typically found at the polymer ends or portions not critical to a desired biological function or activity.
A "recombinant" nucleic acid is defined either by its method of production or its structure. In reference to its method of production, e. Alternatively, it can be a nucleic acid made by generating a sequence comprising fusion of two fragments which are not naturally contiguous to each other, but is meant to exclude products of nature, e. Thus, e. Such is often done to replace a codon with a redundant codon encoding the same or a conservative amino acid, while typically introducing or removing a sequence recognition site.
Alternatively, it is performed to join together nucleic acid segments of desired functions to generate a single genetic entity comprising a desired combination of functions not found in the commonly available natural forms. Restriction enzyme recognition sites are often the target of such artificial manipulations, but other site specific targets, e.
A similar concept is intended for a recombinant, e. Also disclosed are synthetic nucleic acids which, by genetic code redundancy, encode polypeptide similar to fragments of these antigens, and fusions of sequences from various different species or polymorphic variants. A significant "fragment" in a nucleic acid context is a contiguous segment of at least about 17 nucleotides, generally at least about 22 nucleotides, ordinarily at least about 29 nucleotides, more often at least about 35 nucleotides, typically at least about 41 nucleotides, usually at least about 47 nucleotides, preferably at least about 55 nucleotides, and particularly will be at least about 60 or more nucleotides, e.
There will be homologs in other species, including primates, rodents, canines, felines, and birds. Various IL-B30 proteins should be homologous and are encompassed herein. However, even proteins that have a mor distant evolutionary relationship to the antigen can readily be isolated under appropriate conditions using these sequences if they are sufficiently homologous. Primate IL-B30 proteins are of particular interest. Recombinant clones derived from the genomic sequences, e. Clinical Oncology Substantial homology, e.
Alternatively, substantial homology exists when the segments will hybridize under selective hybridization conditions, to a strand, or its complement, typically using a sequence of IL-B30, e. See, Kanehisa Nuc. Acids Res. The length of identity comparison, as described, may be over longer stretches, and may be over a stretch of at least about 17 nucleotides, usually at least about 28 nucleotides, typically at least about 40 nucleotides, and preferably at least about 75 to or more nucleotides.
Stringent conditions, in referring to homology in the hybridization context, will be stringent combined conditions of salt, temperature, organic solvents, and other parameters, typically those controlled in hybridization reactions. Stringent salt conditions will ordinarily be less than about mM, usually less than about mM, typically less than about mM, preferably less than about mM, including about , 50, or even 20 mM.
However, the combination of parameters is much more important than the measure of any single parameter. Hybridization under stringent conditions should give a background of at least 2-fold over background, preferably at least or more. For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
The sequence comparison algorithm then calculates the percent sequence identity for the test sequence s relative to the reference sequence, based on the designated program parameters. Optical alignment of sequences for comparison can be conducted, e. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments to show relationship and percent sequence identity.
It also plots a tree or dendrogram showing the clustering relationships used to create the alignment. The program can align up to sequences, each of a maximum length of 5, nucleotides or amino acids. The multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences.
This cluster is then aligned to the next most related sequence or cluster of aligned sequences. Two clusters of sequences are aligned by a simple extension of the pairwise alignment of two individual sequences. The final alignment is achieved by a series of progressive, pairwise alignments. The program is run by designating specific sequences and their amino acid or nucleotide coordinates for regions of sequence comparison and by designating the program parameters. For example, a reference sequence can be compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight 3.
Another example of algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described Altschul, et al. This algorithm involves first identifying high scoring sequence pairs HSPs by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence.
T is referred to as the neighborhood word score threshold Altschul, et al. These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences see, e. One measure of similarity provided by the BLAST algorithm is the smallest sum probability P N , which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.
A further indication that two nucleic acid sequences of polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions, as described below.
IL-B30 from other mammalian species can be cloned and isolated by cross-species hybridization of closely related species.
Biophysics UOS / Piehler Lab
Homology may be relatively low between distantly related species, and thus hybridization of relatively closely related species is advisable. Alternatively, preparation of an antibody preparation which exhibits less species specificity may be useful in expression cloning approaches. DNA which encodes the IL-B30 or fragments thereof can be obtained by chemical synthesis, screening cDNA libraries, or screening genomic libraries prepared from a wide variety of cell lines or tissue samples.
Biol, ; Gubler and Hoffman Gene ; and Glover ed. Alternatively, the sequences provided herein provide useful PCR primers or allow synthetic or other preparation of suitable genes encoding an IL-B30; including naturally occurring embodiments. This DNA can be expressed in a wide variety of host cells for the synthesis of a full-length IL-B30 or fragments which can in turn, e.
Vectors, as used herein, comprise plasmids, viruses, bacteriophage, integratable DNA fragments, and other vehicles which enable the integration of DNA fragments into the genome of the host. For purposes of this invention, DNA sequences are operably linked when they are functionally related to each other. For example, DNA for a presequence or secretory leader is operably linked to a polypeptide if it is expressed as a preprotein or participates in directing the polypeptide to the cell membrane or in secretion of the polypeptide.
A promoter is operably linked to a coding sequence if it controls the transcription of the polypeptide; a ribosome binding site is operably linked to a coding sequence if it is positioned to permit translation. Usually, operably linked means contiguous and in reading frame, however, certain genetic elements such as repressor genes are not contiguously linked but still bind to operator sequences that in turn control expression. Cell Biol. It will often be desired to express an IL-B30 polypeptide in a system which provides a specific or defined glycosylation pattern. The IL-B30, or a fragment thereof, may be engineered to be phosphatidyl inositol PI linked to a cell membrane, but can be removed from membranes by treatment with a phosphatidyl inositol cleaving enzyme, e.
This releases the antigen in a biologically active form, and allows purification by standard procedures of protein chemistry. Acta ; Tse, et al. Now that the IL-B30 has been characterized, fragments or derivatives thereof can be prepared by conventional processes for synthesizing peptides. The present invention provides reagents which will find use in diagnostic applications as described elsewhere herein, e. The gene may be useful in forensic sciences, e. The IL-B30 naturally occurring or recombinant , fragments thereof, and antibodies thereto, along with compounds identified as having binding affinity to IL-B30, should be useful as reagents for teaching techniques of molecular biology, immunology, or physiology.
Appropriate kits may be prepared with the reagents, e. The reagents will also be useful in the treatment of conditions associated with abnormal physiology or development, including inflammatory conditions. They may be useful in vitro tests for presence or absence of interacting components, which may correlate with success of particular treatment strategies.
In particular, modulation of physiology of various, e. For example, a disease or disorder associated with abnormal expression or abnormal signaling by an IL-B30 should be a likely target for an agonist or antagonist. The new cytokine should play a role in regulation or development of hematopoietic cells, e. Alternatively, it may affect vascular physiology or development, or neuronal effects.
In particular, the cytokine should mediate, in various contexts, cytokine synthesis by the cells, proliferation, etc. Antagonists of IL-B30, such as mutein variants of a naturally occurring form of IL-B30 or blocking antibodies, may provide a selective and powerful way to block immune responses, e. See also Samter, et al. Immunological Diseases vols. In addition, certain combination compositions would be useful, e.
See Berkow ed. Many other medical conditions and diseases involve activation by macrophages or monocytes, and many of these will be responsive to treatment by an agonist or antagonist provided herein. Immunological Diseases Little, Brown and Co. These problems should be susceptible to prevention or treatment using compositions provided herein.
The pancreatic islet localization suggests a possible relevance to diabetes. IL-B30, antagonists, antibodies, etc. These reagents can be combined for therapeutic use with additional active or inert ingredients, e. These combinations can be sterile filtered and placed into dosage forms as by lyophilization in dosage vials or storage in stabilized aqueous preparations. This invention also contemplates use of antibodies or binding fragments thereof, including forms which are not complement binding. Drug screening using IL-B30 or fragments thereof can be performed to identify compounds having binding affinity to or other relevant biological effects on IL-B30 functions, including isolation of associated components.
Subsequent biological assays can then be utilized to determine if the compound has intrinsic stimulating activity and is therefore a blocker or antagonist in that it blocks the activity of the cytokine. Likewise, a compound having intrinsic stimulating activity can activate the signal pathway and is thus an agonist in that it simulates the activity of IL-B This invention further contemplates the therapeutic use of blocking antibodies to IL-B30 as antagonists and of stimulatory antibodies as agonists.
This approach should be particularly useful with other IL-B30 species variants. The quantities of reagents necessary for effective therapy will depend upon many different factors, including means of administration, target site, physiological state of the patient, and other medicants administered. Thus, treatment dosages should be titrated to optimize safety and efficacy. Typically, dosages used in vitro may provide useful guidance in the amounts useful for in situ administration of these reagents.
Animal testing of effective doses for treatment of particular disorders will provide further predictive indication of human dosage. Various considerations are described, e. Methods for administration are discussed therein and below, e. Pharmaceutically acceptable carriers will include water, saline, buffers, and other compounds described, e. Slow release formulations, or a slow release apparatus will often be utilized for continuous or long term administration. IL-B30, fragments thereof, and antibodies to it or its fragments, antagonists, and agonists, may be administered directly to the host to be treated or, depending on the size of the compounds, it may be desirable to conjugate them to carrier proteins such as ovalbumin or serum albumin prior to their administration.
Therapeutic formulations may be administered in many conventional dosage formulations. While it is possible for the active ingredient to be administered alone, it is preferable to present it as a pharmaceutical formulation. Formulations typically comprise at least one active ingredient, as defined above, together with one or more acceptable carriers thereof.
Each carrier should be both pharmaceutically and physiologically acceptable in the sense of being compatible with the other ingredients and not injurious to the patient. Formulations include those suitable for oral, rectal, nasal, topical, or parenteral including subcutaneous, intramuscular, intravenous and intradermal administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Both naturally occurring and recombinant forms of the IL-B30s of this invention are particularly useful in kits and assay methods which are capable of screening compounds for binding activity to the proteins.
Several methods of automating assays have been developed in recent years so as to permit screening of tens of thousands of compounds in a short period. The development of suitable assays can be greatly facilitated by the availability of large amounts of purified, soluble IL-B30 as provided by this invention. Mutational analysis can be performed, e. Helices A and D are most important in receptor interaction, with the D helix being the more important region.
Helix A would run in the human from about pro 7 to his 27 , while helix D would run from about trp to gly Surface exposed residues would affect receptor binding, while embedded residues would affect general structure. Predicted residues of particular importance would likely correspond to arg , ser , gln , ala , ala , val , ala , arg , ala , and his For example, antagonists can normally be found once the antigen has been structurally defined, e.
Testing of potential interacting analogs is now possible upon the development of highly automated assay methods using a purified IL-B In particular, new agonists and antagonists will be discovered by using screening techniques described herein. Of particular importance are compounds found to have a combined binding affinity for a spectrum of IL-B30 molecules, e. One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant DNA molecules expressing an IL-B Cells may be isolated which express an IL-B30 in isolation from other molecules.
Such cells, either in viable or fixed form, can be used for standard binding partner binding assays. See also, Parce, et al. USA , which describe sensitive methods to detect cellular responses. First, large numbers of different small peptide test compounds are synthesized on a solid substrate, e. Then all the pins are reacted with solubilized, unpurified or solubilized, purified IL-B30, and washed. The next step involves detecting bound IL-B Rational drug design may also be based upon structural studies of the molecular shapes of the IL-B30 and other effectors or analogs.
Effectors may be other proteins which mediate other functions in response to binding, or other proteins which normally interact with IL-B30, e. One means for determining which sites interact with specific other proteins is a physical structure determination, e. These will provide guidance as to which amino acid residues form molecular contact regions, as modeled, e. For a detailed description of protein structural determination, see, e. This invention also contemplates use of IL-B30 proteins, peptides, and their fusion products in a variety of diagnostic kits and methods for detecting the presence of another IL-B30 or binding partner.
Typically the kit will have a compartment containing either a defined IL-B30 peptide or gene segment or a reagent which recognizes one or the other, e. A kit for determining the binding affinity of a test compound to an IL-B30 would typically comprise a test compound; a labeled compound, for example a binding partner or antibody having known binding affinity for IL-B30; a source of IL-B30 naturally occurring or recombinant ; and a means for separating bound from free labeled compound, such as a solid phase for immobilizing the molecule.
Once compounds are screened, those having suitable binding affinity to the antigen can be evaluated in suitable biological assays, as are well khown in the art, to determine whether they act as agonists or antagonists to the IL-B30 signaling pathway. The availability of recombinant IL-B30 polypeptides also provide well defined standards for calibrating such assays.
A preferred kit for determining the concentration of, e. Compartments containing reagents, and instructions, will normally be provided. Such diagnostic assays can employ lysates, live cells, fixed cells, immunofluorescence, cell cultures, body fluids, and further can involve the detection of antigens related to the antigen in serum, or the like.
Diagnostic assays may be homogeneous without a separation step between free reagent and antigen-binding partner complex or heterogeneous with a separation step. Anti-idiotypic antibodies may have similar use to diagnose presence of antibodies against an IL-B30, as such may be diagnostic of various abnormal states. For example, overproduction of IL-B30 may result in production of various immunological reactions which may be diagnostic of abnormal physiological states, particularly in proliferative cell conditions such as cancer or abnormal activation or differentiation.
Moreover, the distribution pattern available provides information that the cytokine is expressed in pancreatic islets, suggesting the possibility that the cytokine may be involved in function of that organ, e. Frequently, the reagents for diagnostic assays are supplied in kits, so as to optimize the sensitivity of the assay.
For the subject invention, depending upon the nature of the assay, the protocol, and the label, either labeled or unlabeled antibody or binding partner, or labeled IL-B30 is provided. This is usually in conjunction with other additives, such as buffers, stabilizers, materials necessary for signal production such as substrates for enzymes, and the like. Preferably, the kit will also contain instructions for proper use and disposal of the contents after use. Typically the kit has compartments for each useful reagent. Desirably, the reagents are provided as a dry lyophilized powder, where the reagents may be reconstituted in an aqueous medium providing appropriate concentrations of reagents for performing the assay.
Many of the aforementioned constituents of the drug screening and the diagnostic assays may be used without modification or may be modified in a variety of ways. For example, labeling may be achieved by covalently or non-covalently joining a moiety which directly or indirectly provides a detectable signal. In any of these assays, the binding partner, test compound, IL-B30, or antibodies thereto can be labeled either directly or indirectly. Possibilities for direct labeling include label groups: radiolabels such as I, enzymes U.
Possibilities for indirect labeling include biotinylation of one constituent followed by binding to avidin coupled to one of the above label groups. There are also numerous methods of separating the bound from the free IL-B30, or alternatively the bound from the free test compound. The IL-B30 can be immobilized on various matrixes followed by washing.
Other suitable separation techniques include, without limitation, the fluorescein antibody magnetizable particle method described in Rattle, et al. Methods for linking proteins or their fragments to the various labels have been extensively reported in the literature and do not require detailed discussion here. Many of the techniques involve the use of activated carboxyl groups either through the use of carbodiimide or active esters to form peptide bonds, the formation of thioethers by reaction of a mercapto group with an activated halogen such as chloroacetyl, or an activated olefin such as maleimide, for linkage, or the like.
Fusion proteins will also find use in these applications. Oligonucleotide or polynucleotide sequences may be taken from the sequence of an IL-B These sequences can be used as probes for detecting levels of the IL-B30 message in samples from patients suspected of having an abnormal condition, e. Since the cytokine may be a marker or mediator for activation, it may be use useful to determine the numbers of activated cells to determine, e.
The preparation of both RNA and DNA nucleotide sequences, the labeling of the sequences, and the preferred size of the sequences has received ample description and discussion in the literature. See e. Diagnostic kits which also test for the qualitative or quantitative expression of other molecules are also contemplated. Diagnosis or prognosis may depend on the combination of multiple indications used as markers.
Thus, kits may test for combinations of markers. Other kits may be used to evaluate other cell subsets. Having isolated a ligand of a specific ligand-receptor interaction, methods exist for isolating the receptor. See, Gearing, et al. For example, means to label the IL-B30 cytokine without interfering with the binding to its receptor can be determined. For example, an affinity label can be fused to either the amino- or carboxyl-terminus of the ligand.
Such label may be a FLAG epitope tag, or, e. An expression library can be screened for specific binding of the cytokine, e. Sci USA ; and Liu, et al. Alternatively, a panning method may be used. Protein cross-linking techniques with label can be applied to isolate binding partners of the IL-B30 cytokine. This would a allow identification of proteins which specifically interact with the cytokine, e.
It is also quite possible that these functional receptor complexes may share many or all components with an IL-B30 receptor complex, either a specific receptor subunit or an accessory receptor subunit. Many of the standard methods below are described or referenced, e. Methods for protein purification include such methods as ammonium sulfate precipitation, column chromatography, electrophoresis, centrifugation, crystallization, and others. Matsudaira ed. Combination with recombinant techniques allow fusion to appropriate segments epitope tags , e.
Genetic Engineering. Standard immunological techniques are described, e. Cytokine assays are described, e. Assays for vascular biological activities are well knows in the art. They will cover angiogenic and angiostatic activities in tumor, or other tissues, e. Assays for neural cell biological activities are described, e.
Methodology of developmental systems is described, e. FACS analyses are described in Melamed, et al. The sequence of the gene is provided in Table 1. The sequence is derived from a cDNA library made from melanocyte, fetal heart, and pregnant uterus.
Cytokines: A Practical Approach
It is also found from a cDNA library sequence derived from a pancreatic islet. These sequences allow preparation of PCR primers, or probes, to determine cellular distribution of the gene. The sequences allow isolation of genomic DNA which encode the message.
Using the probe or PCR primers, various tissues or cell types are probed to determine cellular distribution. PCR products are cloned using, e. The resulting cDNA plasmids are sequenced from both termini on an automated sequencer Applied Biosystems. Typically, the probe is labeled, e. The expression is probably in the cell types described, and perhaps also in pancreatic islets. No signal was detected in the other samples. In summary, the distribution shows IL-B30 elevated in activated macrophages, suggesting a role in inflammation; activated Th1 cells, suggesting a regulation or effector role in T helper subsets, particularly Th1 immune responses; and activated dendritic cells, suggesting a role in antigen presentation or germinal center T or B cell interactions with DC.
High signal was detected in the monocytes cell line RAW Other samples showed no signal. The expression in the RAW Chromosome mapping is a standard technique. Circumstantial evidence suggests that the mouse IL-B30 gene maps to chromosome Multiple transfected cell lines are screened for one which expresses the cytokine at a high level compared with other cells. Various cell lines are screened and selected for their favorable properties in handling. Natural IL-B30 can be isolated from natural sources, or by expression from a transformed cell using an appropriate expression vector.
Purification of the expressed protein is achieved by standard procedures, or may be combined with engineered means for effective purification at high efficiency from cell lysates or supernatants. FLAG or His 6 segments can be used for such purification features. Alternatively, affinity chromatography may be used with specific antibodies, see below. Protein is produced in coli, insect cell, or mammalian expression systems, as desired.
The IL-B30 cDNA, or other species counterpart sequence, can be used as a hybridization probe to screen a library from a desired source, e. Many different species can be screened both for stringency necessary for easy hybridization, and for presence using a probe. Appropriate hybridization conditions will be used to select for clones exhibiting specificity of cross hybridization.
Screening by hybridization using degenerate probes based upon the peptide sequences will also allow isolation of appropriate clones. Alternatively, use of appropriate primers for PCR screening will yield enrichment of appropriate nucleic acid clones. Similar methods are applicable to isolate either species, polymorphic, or allelic variants. Species variants are isolated using cross-species hybridization techniques based upon isolation of a full length isolate or fragment from one species as a probe.
Alternatively, antibodies raised against human IL-B30 will be used to screen for cells which express cross-reactive proteins from an appropriate, e. Synthetic peptides or purified protein are presented to an immune system to generate monoclonal or polyclonal antibodies. The resulting antibodies are used for screening, purification, or diagnosis, as described. Polyclonal serum, or hybridomas may be prepared. In appropriate situations, the binding reagent is either labeled as described above, e.
Immunoselection and related techniques are available to prepare selective reagents, as desired. The effect on proliferation of various cell types are evaluated with various concentrations of cytokine. Monocytes are purified by negative selection from peripheral blood mononuclear cells of normal healthy donors. Briefly, 3 x 10 8 ficoll banded mononuclear cells are incubated on ice with a cocktail of monoclonal antibodies Becton-Dickinson; Mountain View, CA consisting, e. Antibody bound cells are washed and then incubated with sheep anti-mouse IgG coupled magnetic beads Dynal, Oslo, Norway at a bead to cell ratio of Antibody bound cells are separated from monocytes by application of a magnetic field.
Analyses of the expression of cell surface molecules can be performed by direct immunofluorescence. Cells are pelleted at x g. Exemplary mAbs are used, e. In addition, monocytes are stimulated with LPS E. Subsequently cells are washed, resuspended in permeabilization buffer 0. Cells 2 x 10 5 are centrifuged and resuspended in 20 ml directly conjugated anti-cytokine mAbs diluted in permeabilization buffer for 20 minutes at RT.
Total PBMC are isolated from buffy coats of normal healthy donors by centrifugation through ficoll-hypaque as described Boyum, et al. The native, recombinant, and fusion proteins would be tested for agonist and antagonist activity in many other biological assay systems, e. USA 90, ; Ho, et al.
IL-B30 will also be evaluated for effects on NK cell stimulation. Assays may be based, e. B cell growth and differentiation effects will be analyzed, e. Transgenic mice can be generated by standard methods. Such animals are useful to determine the effects of deletion of the gene, in specific tissues, or completely throughout the organism.
Such may provide interesting insight into development of the animal or particular tissues in various stages. Moreover, the effect on various responses to biological stress can be evaluated. Cold Spring Harbor Laboratory Press. A transgenic mouse has been generated, and while the animal seems to survive birth, it fails to thrive, and typically dies within a few weeks. The construct is based upon an actin promoter with a CMV enhancer, which should lead to broad and high expression. The mice, like IL-6 transgenic mice, are runted. Moreover, they exhibit a bloated abdomen, inflammation of the stomach and intestines, infiltration of cells into the liver, and typically die before day These mice do not breed.
A second subset of the transgenic mice have a less severe phenotype, and attemps to breed them are taking place. The genomic structure for the mouse IL-B30 has been determined. A strategy for the production of IL-B30 knock-out mice has been developed, and constructs have been started. An expression vector comprising the polynucleotide of Claim 2. A host cell containing the expression vector of Claim 5.
The host cell of Claim 6, wherein the host cell is a eukaryotic cell. A method of making a polypeptide comprising expressing a recombinant polynucleotide of Claim 2. An antibody or antigen binding fragment thereof that specifically binds to a polypeptide with an amino acid sequence consisting of the sequence of SEQ ID NO: 2, 4 or 5. The antibody or antigen binding fragment thereof of Claim 9, wherein said antibody or antigen binding fragment thereof is a monoclonal antibody, Fab, or F ab 2.
The antibody or antigen binding fragment thereof of Claim 9, wherein said antibody or antigen binding fragment thereof is detectably labeled. A method for using the antibody or antigen binding fragment thereof of Clam 9, comprising contacting said antibody or antigen binding fragment thereof with a biological sample comprising an antigen, wherein said contacting results in formation of an antibody: antigen complex or an antigen binding fragments: antigen complex.
The method of Claim 12, wherein said biological sample is from a human. A kit comprising the antibody or antigen binding fragment thereof of Claim 9, and: a instructional material for the use of said antibody or antigen binding fragment thereof for said detection; or. The polypeptide of Claim 15, wherein the polypeptide is detectably labeled. An antibody or antigen binding fragment thereof that specifically binds to a polypeptide with an amino acid sequence consisting of the mature amino acid sequence of SEQ ID NO: 2 or 4. A pharmaceutical formulation comprising the antibody or antigen binding fragment of Claim 9 and a pharmaceutically acceptable carrier.
A pharmaceutical formulation comprising the polypeptide of Claim 17 and a pharmaceutically acceptable carrier. A pharmaceutical formulation comprising the antibody or antigen binding fragment of Claim 18 and a pharmaceutically acceptable carrier. USA true EPB1 en. JPB2 en. KRB1 en. CNA en. ARA1 en. ATT en. AUB2 en. BRA en. CAC en. COA1 en. CZB6 en. DED1 en. DKT3 en.