Drostanolone Enanthate Made by: Specifically, aerosol includes a gas-borne suspension of droplets of an albumin fusion protein of the instant invention, as may be produced in a metered dose inhaler or nebulizer, or in a mist sprayer. The approach used by the BLAST program is to first consider similar segments between a query sequence and a database sequence, then to evaluate the statistical significance of all matches that are identified and finally to summarize only those matches which satisfy a preselected threshold of significance. Further, such polypeptides can be utilized for in vitro diagnostic procedures. Dragon Pharma Basal Chemical Name: Deca Basal Chemical Name:
Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non- glycosylated. Masteron Prime Substance: Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards. These conserved amino acids are likely important for protein- function. Such fragments may be of 10 or more amino acids in length or may include about 15, 20, 25, 30, 50, or more contiguous amino acids from the HA sequence or may include part or all of specific domains of HA. The authors further indicate which amino acid changes are likely to be permissive at certain amino acid positions in the protein.
In the latter case, viral propagation generally will occur only in complementing host cells. The polynucleotides encoding albumin fusion proteins of the invention may be joined to a vector containing a selectable marker for propagation in a host. Generally,, a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid.
If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells. The polynucleotide insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E.
Other suitable promoters will be known to the skilled artisan. The expression constructs will further contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the transcripts expressed by the constructs will preferably include a translation initiating codon at the beginning and a termination codon UAA, UGA or UAG appropriately positioned at the end of the polypeptide to be translated.
As indicated, the expression vectors will preferably include at least one selectable marker. Such markers include dihydrofolate reductase, G, glutamine synthase, or neomycin resistance for eukaryotic cell culture, and tetracycline, kanamycin or ampicillin resistance genes for culturing in E. Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. Appropriate culture mediums and conditions for the above- described host cells are known in the art.
Other suitable vectors will be readily apparent to the skilled artisan. For example, in E. Examples of signal sequences or proteins or fragments thereof to which the albumin fusion proteins of the invention may be fused in order to direct the expression of the polypeptide to the periplasmic space of bacteria include, but are not limited to, the pelB signal sequence, the maltose binding protein MBP signal sequence, MBP, the ompA signal sequence, the signal sequence of the periplasmic E.
Several vectors are commercially available for the construction of fusion proteins which will direct the localization of a protein, such as the pMAL series of vectors particularly the pMAL-p series available from New England Biolabs. In a specific embodiment, polynucleotides albumin fusion proteins of the invention may be fused to the pelB pectate lyase signal sequence to increase the efficiency of expression and purification of such polypeptides in Gram-negative bacteria.
Examples of signal peptides that may be fused to an albumin fusion protein of the invention in order to direct its secretion in mammalian cells include, but are not limited to, the MPIF-1 signal sequence e. A suitable signal sequence that may be used in conjunction with baculoviral expression systems is the gp67 signal sequence e. Vectors which use glutamine synthase GS or DHFR as the selectable markers can be amplified in the presence of the drugs methionine sulphoximine or methotrexate, respectively.
An advantage of glutamine synthase based vectors are the availabilty of cell lines e. Glutamine synthase expression systems can also function in. A glutamine synthase expression system and components thereof are detailed in PCT publications: Additionally, glutamine synthase expression vectors can be obtained from Lonza Biologies, Inc.
Expression and production of monoclonal antibodies using a GS expression system in murine myeloma cells is described in Bebbington et al. The present invention also relates to host cells containing the above-described vector constructs described herein, and additionally encompasses host cells containing nucleotide sequences of the invention that are operably associated with one or more heterologous control regions e.
The host cell can be a higher eukaryotic cell, such as a mammalian cell e. A host strain may be chosen which modulates the expression of the inserted gene sequences, or modifies and processes the gene product in the specific fashion desired. Expression from certain promoters can be elevated in the presence of certain inducers; thus expression of the genetically engineered polypeptide may be controlled.
Furthermore, different host cells have characteristics and specific mechanisms for the translational and post- translational processing and modification e. Appropriate cell lines can be chosen to ensure the desired modifications and processing of the foreign protein expressed. Introduction of the nucleic acids and nucleic acid constructs of the invention into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods.
Such methods are described in many standard laboratory manuals, such as Davis et al. It is specifically contemplated that the polypeptides of the present invention may in fact be expressed by a host cell lacking a recombinant vector. In addition to encompassing host cells containing the' vector constructs discussed herein, the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly mammalian origin, that have been engineered to delete or replace endogenous genetic material e.
In addition, techniques known in the art may be -used to operably associate heterologous polynucleotides e.
Albumin fusion proteins of the invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, hydrophobic charge interaction chromatography and lectin chromatography. Most preferably, high performance liquid chromatography "HPLC" is employed for purification.
In specific embodiments the albumin fusion proteins of the invention are purified using Size Exclusion Chromatography including, but not limited to, sepharose S, S, S, superdex resin columns and their equivalents and comparabies. In specific embodiments the albumin fusion proteins of the invention are purified using Affinity Chromatography including, but not limited to, Mimetic Dye affinity, peptide affinity and antibody affinity columns that are selective for either the HSA or the "fusion target" molecules.
In preferred embodiments albumin fusion proteins of the invention are purified using one or more Chromatography methods listed above. Additionally, albumin fusion proteins of the invention may be purified using the process described in International Publication No. One of skill in the art could easily modify the process described therein for use in the purification of albumin fusion proteins of the invention.
Albumin fusion proteins of the present invention may be recovered from: Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non- glycosylated.
In addition, albumin fusion proteins of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes. Thus, it is well known in the art that the N-terminal methionine encoded by the translation initiation codon generally is removed with high efficiency from any protein after translation in all eukaryotic cells.
While the N-terminal methionine on most proteins also is efficiently removed in most prokaryotes, for some proteins, this prokaryotic removal process is inefficient, depending on the nature of the amino acid to which the N-terminal methionine is covalently linked. In one embodiment, the yeast Pichia pastoris is used to express albumin fusion proteins of the invention in a eukaryotic system. Pichia pastoris is a methylotrophic yeast which can metabolize methanol as its sole carbon source.
A main step in the methanol metabolization pathway is the oxidation of methanol to formaldehyde using O 2. This reaction is catalyzed by the enzyme alcohol oxidase. In order to metabolize methanol as its sole carbon source, Pichia pastoris must generate high levels of alcohol oxidase due, in part, to the relatively low affinity of alcohol oxidase for O 2. Consequently, in a growth medium depending on methanol as a main carbon source, the promoter region of one of the two alcohol oxidase genes AOX1 is highly active.
J, et al, Yeast 5: Thus, a heterologous coding sequence, such as, for example, a polynucleotide of the present invention, under the transcriptional regulation of all or part of the AOX1 regulatory sequence is expressed at exceptionally high levels in Pichia yeast grown in the presence of methanol.
In one example, the plasmid vector pPIC9K is used to express DNA encoding an albumin fusion protein of the invention, as set forth herein, in a Pichea yeast system essentially as described in "Pichia Protocols: Methods in Molecular Biology," D. R- Higgins and J. This expression vector allows expression and secretion of a polypeptide of the invention by virtue of the strong AOX1 promoter linked to the Pichia pastoris alkaline phosphatase PHO secretory signal peptide i.
In another embodiment, high-level expression of a heterologous coding sequence, such as, for example, a polynucleotide encoding an albumin fusion protein of the present invention, may be achieved by cloning the heterologous polynucleotide of the invention into an expression vector such as, for example, pGAPZ or pGAPZalpha, and growing the yeast culture in the absence of methanol.
In addition, albumin fusion proteins of the invention can be chemically synthesized using techniques known in the art e. Structures and Molecular Principles, W. For example, a polypeptide corresponding to a fragment of a polypeptide can be synthesized by use of a peptide synthesizer. Furthermore, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the polypeptide sequence.
Furthermore, the amino acid can be D dextrorotary or L levorotary. Thelnvention encompasses albumin fusion proteins of the present invention which are differentially modified during or after translation, e. Any of numerous chemical modifications may be carried out by known techniques, including but not limited, to specific chemical cleavage by cyanogen bromide,, trypsin, chymotrypsin, papain, V8 protease, NaBH 4 ; acetylation, formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin; etc.
Additional post-translational modifications encompassed by the invention include, for example, e. The albumin fusion proteins may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the protein.
In specific embodiments, albumin fusion proteins of the present invention or fragments or variants thereof are attached to macrocyclic chelators that associate with radiometal ions, including but not limited to, 7 Lu, 90 Y, Ho, and Sm, to polypeptides. In a preferred embodiment, the radiometal ion associated with the macrocyclic chelators is 11 'In.
In another preferred embodiment, the radiometal ion associated with the macrocyclic chelator is 0 Y. In other specific embodiments, DOTA is attached to an antibody of the invention or fragment thereof via linker molecule. Examples of linker molecules useful for conjugating DOTA to a polypeptide are commonly known in the art - see, for example, DeNardo et al. As mentioned, the albumin fusion proteins of the invention may be modified by either natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art.
Polypeptides of the invention may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Albumin fusion proteins of the invention and antibodies that bind a Therapeutic protein or fragments or variants thereof can be fused to marker sequences, such as a peptide to facilitate purification. Avenue, Chatsworth, CA, , among others, many of which are commercially available.
As described in Gentz et al. Other peptide tags useful for purification include, but are not limited to, the "HA" tag, which corresponds to an epitope derived from the influenza hemagglutinin protein Wilson et al. Further, an albumin fusion protein of the invention may be conjugated to a therapeutic moiety such as a cytotoxin, e.
A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and. Therapeutic agents include, but are not limited to, antimetabolites e.
The conjugates of the invention can be used for modifying a given biological response, the therapeutic agent or drug moiety is not to be construed as limited to classical chemical therapeutic agents.
For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Techniques for conjugating such therapeutic moiety to proteins e. Albumin fusion proteins may also be attached to solid supports, which are particularly useful for immunoassays or purification of polypeptides that are bound by, that bind to, or associate with albumin fusion proteins of the invention.
Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. Also provided by the invention are chemically modified derivatives of the albumin fusion proteins of the- invention which may provide additional advantages such as increased solubility, stability and circulating time of the polypeptide, or decreased immunogenicity see U. The albumin fusion proteins may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.
The polymer may be of any molecular weight, and may be branched or unbranched. For polyethylene glycol, the preferred molecular weight is between about 1 kDa and about kDa the term "about" indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight for ease in handling and manufacturing.
Other sizes may be used, depending on the desired therapeutic profile e. For example, the polyethylene glycol may have an average molecular weight of about , , , , ,. As noted above, the polyethylene glycol may have a branched structure. Branched polyethylene glycols are described, for example, in U. The polyethylene glycol molecules or other chemical moieties should be attached to ' the protein with consideration of effects on functional or antigenic domains of the protein.
There are a number of attachment methods available to those skilled in the art, such as, for example, the method disclosed in EP 0 coupling PEG to G-CSF , herein incorporated by reference; see also Malik et al. For example, polyethylene glycol may be covalently bound through amino acid residues via reactive group; such as a free amino or carboxyl group. Reactive groups are those to which an activated polyethylene glycol molecule may be bound.
Sulfhydryl groups may also be used as a reactive group for attaching the polyethylene glycol molecules. Preferred for therapeutic purposes is attachment at an amino 5 group, such as attachment at the N-terminus or lysine group. As suggested above, polyethylene glycol may be attached to proteins via linkage to any of a number of amino acid residues.
For example, polyethylene glycol can be linked to proteins via covalent bonds to lysine, histidine, aspartic acid, glutamic acid, or cysteine. One or more reaction chemistries may be employed to attach polyethylene glycol to 10 specific amino acid residues e. One may specifically desire proteins chemically modified at the N-terminus. Using polyethylene glycol as an illustration of the present composition, one may select from a.
The method of obtaining the N-terminally pegylated preparation i. Selective proteins chemically modified at the N-terminus modification may be accomplished by reductive alkylation which exploits differential reactivity of different types of primary amino groups lysine versus the N-terminal available for derivatization in a particular protein.
Under the appropriate reaction conditions, substantially selective. As indicated above, pegylation of the albumin fusion proteins of the invention may be accomplished by any number of means. For example, polyethylene glycol may be attached to the albumin fusion protein either directly or by an intervening linker. Linkerless systems for 30 attaching polyethylene " glycol to proteins are described in Delgado et al.
Upon reaction of protein with tresylated MPEG, polyethylene glycol is directly attached to amine groups of the protein. Thus, the invention includes protein- polyethylene glycol conjugates produced by reacting proteins of the invention with a polyethylene glycol molecule having a 2,2,2-trifluoreothane sulphonyl group.
Polyethylene glycol can also be attached to proteins using a number of different intervening linkers. Protein-polyethylene glycol conjugates wherein the polyethylene glycol is attached to the protein by a linker can also be produced by reaction of proteins with compounds such as MPEG-succinimidylsuccinate, MPEG activated with l,r-carbonyldiimidazole, MPEG-2,4,5-trichloropenylcarbonate, MPEG-p- nitrophenolcarbonate, and various MPEG-succinate derivatives. A number of additional polyethylene glycol derivatives and reaction chemistries for attaching polyethylene glycol to proteins are described in International Publication No.
Pegylated protein products produced using the reaction chemistries set out herein are included within the scope of the invention. The number of polyethylene glycol moieties attached to each albumin fusion protein of the invention i.
For example, the pegylated proteins of the invention may be linked, on average, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, or more polyethylene glycol molecules. Similarly, the average degree of substitution within ranges such as , , , , , , , , , , , , , , , , , or polyethylene glycol moieties per protein molecule. Methods for determining the degree of substitution are discussed, for example, in Delgado et al.
The polypeptides of the invention can be recovered and purified from chemical synthesis and recombinant cell cultures by standard methods which include, but are not limited to, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography.
The presence and quantity of albumin fusion proteins of the invention may be determined using ELISA,. In an alternate version of this protocol, the ELISA plate might be coated with the anti-Therapeutic protein specific antibody and the labeled secondary reagent might be the anti-human albumin specific antibody.
Each of the polynucleotides identified herein can be used in numerous ways as reagents. The following description should be considered exemplary and utilizes known techniques.
The polynucleotides of the present invention are useful to produce the albumin fusion proteins of the invention. As described in more detail below, polynucleotides of the invention. Polynucleotides of the present invention are also useful in gene therapy.
One goal of gene therapy is to insert a normal gene into an organism having a defective gene, in an effort to correct the genetic defect. The polynucleotides disclosed in the present invention offer a means of targeting such genetic defects in a highly accurate manner. Another goal is to insert a new gene that was not present in the host genome, thereby producing a new trait in the host cell. Additional non-limiting examples of gene therapy methods encompassed by the present invention are more thoroughly described elsewhere herein see, e.
Each of the polypeptides identified herein can be used in numerous ways. Albumin fusion proteins of the invention are useful to provide immunological probes for differential identification of the tissue s e. Albumin fusion proteins can be used to assay levels of polypeptides in a biological sample using classical immunohistological methods known to those of skill in the art e.
Other methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay ELISA and the radioimmunoassay RIA. Ga, 67 Ga ,. Albumin fusion proteins of the invention can also be detected in vivo by imaging. Labels or markers for in vivo imaging of protein include those detectable by X-radiography, nuclear magnetic resonance NMR or electron spin relaxtion ESR.
For X-radiography, suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject. Suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the albumin fusion protein by labeling of nutrients given to a cell line expressing the albumin fusion protein of the invention.
An albumin fusion protein which has been labeled with an appropriate detectable imaging moiety, such. It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images.
In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99m Tc. The labeled albumin fusion protein will then preferentially accumulate at locations in the body e.
Alternatively, in the case where the albumin fusion protein comprises at least a fragment or variant of a Therapeutic antibody, the labeled albumin fusion protein will then preferentially accumulate at the locations in the body e. In vivo tumor imaging is described in S. The Radiochemical Detection of Cancer, S. The protocols described therein could easily be modified by one of skill in the art for use with the albumin fusion proteins of the invention.
In one embodiment, the invention provides a method for. In one example, the invention provides a method for delivering a Therapeutic protein into the targeted cell. In another example, ' the invention provides a method for delivering a single stranded nucleic acid e. In another embodiment, the invention provides a method for the specific destruction of cells e.
By "toxin" is meant one or more compounds that bind and activate endogenous cytotoxic effector systems, radioisotopes, holotoxins, modified toxins, catalytic subunits of toxins, or any molecules or enzymes not normally, present in or on the surface of a cell that under defined conditions cause the cell's death.
Toxins that may be used according to the methods of the invention include, but are not limited to, radioisotopes known in the art, compounds such as, for example, antibodies or complement fixing containing portions thereof that bind an inherent or induced endogenous cytotoxic effector system, thymidine kinase, endonuclease, RNAse, alpha toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheria toxin, saporin, momordin, gelonin, pokeweed antiviral protein, alpha-sarcin and cholera toxin.
In a specific embodiment, the invention provides a method for the specific destruction of cells e. In another specific embodiment, the invention provides a method for the specific destruction of cells e. In a further specific embodiment, the invention provides a method for the specific destruction of cells e.
Techniques known in the art may be applied to label , polypeptides of the invention. Such techniques include, but are not limited to, the use of bifunctional conjugating agents see e.
Such disorders include, but are not limited to, those described herein under the section heading "Biological Activities," below. Thus, the invention provides a diagnostic method of a disorder, which involves a assaying the expression level of a certain polypeptide in cells or body fluid of an individual using an albumin fusion protein of the invention; and b comparing the assayed polypeptide expression level with a standard polypeptide expression level, whereby an increase or decrease in the assayed polypeptide.
With respect to cancer, the presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.
For example, patients can be administered a polypeptide of the present invention in an effort to replace absent or decreased levels of the polypeptide e. In particular, albumin fusion proteins comprising of at least a fragment or variant of a Therapeutic antibody can also be used to treat disease as described supra, and elsewhere herein. Similarly, administration of an albumin fusion protein comprising of at least a fragment or variant of a Therapeutic antibody can activate the polypeptide to which the Therapeutic antibody used to make the.
At the very least, the albumin fusion proteins of the invention of the present invention. Can be used as molecular weight markers on SDS-PAGE gels or on molecular sieve gel filtration columns using methods well known to those of skill in the art. Moreover, the albumin fusion proteins of the present invention can be used to test the biological activities described herein. Such disorders include, but are not limited to, those described for each Therapeutic protein in the corresponding row of Table 1 and herein under the section headings "Immune Activity," "Blood Related Disorders,".
For a number of disorders, substantially altered increased or decreased levels of gene expression can be detected in tissues, cells or bodily fluids e. Thus, the invention provides a diagnostic method useful during diagnosis of a disorder, which involves measuring the expression level of the gene encoding a polypeptide in tissues, cells or body fluid from an individual and comparing the measured gene expression level with a standard gene expression level, whereby an increase or decrease in the gene expression level s compared to the standard is indicative of a disorder.
These diagnostic assays may be performed in vivo or in vitro, such as, for example, on blood samples, biopsy tissue or autopsy tissue. The present invention is also useful as a prognostic indicator, whereby patients exhibiting enhanced or depressed gene expression will experience a worse clinical outcome By "assaying the expression level of the gene encoding a polypeptide" is intended qualitatively or quantitatively measuring or estimating the level of a particular polypeptide e.
Preferably, the polypeptide expression level or mRNA level in the first biological sample is measured or estimated and compared to a standard polypeptide level or mRNA level, the standard being taken from a second biological sample obtained from an individual not having the disorder or being determined by averaging levels from a population of individuals not having the disorder.
As will be appreciated in the art, once a standard polypeptide level or mRNA level is known, it can be used repeatedly as a standard for comparison. By "biological sample" is intended any biological sample obtained from an individual, cell line, tissue culture, or other source containing polypeptides of the invention including portions thereof or mRNA. As indicated, biological samples include body fluids such as sera, plasma, urine, synovial fluid and spinal fluid and tissue sources found to express the full length or fragments thereof of a polypeptide or mRNA.
Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art. Where the biological sample is to include mRNA, a tissue biopsy is the preferred source. Total cellular RNA can be isolated from a biological sample using any suitable technique such as the single-step guanidinium-thiocyanate-phenol-chloroform method described in Chomczynski and Sacchi, Anal.
Levels of mRNA encoding the polypeptides of the invention are then assayed using any appropriate method. The present invention also relates to diagnostic assays such as quantitative and diagnostic assays for detecting levels of polypeptides that bind to, are bound by, or associate with albumin fusion proteins of the invention, in a biological sample e. Thus, for instance, a diagnostic assay in accordance with the invention for detecting abnormal expression of polypeptides that bind to, are bound by, or associate with albumin fusion proteins compared to normal control tissue samples may be used to detect the presence of tumors.
Assay techniques that can be used to determine levels of a polypeptide that bind to, are bound by, or associate with albumin fusion proteins of the present invention in a sample derived from a host are well-known to those of skill in the art. Assaying polypeptide levels in a biological sample can occur using any art-known method. Assaying polypeptide levels in a biological sample can occur using a variety of techniques. Other methods useful for detecting polypeptide gene expression include immunoassays, such as the enzyme linked immunosorbent assay ELISA and the radioimmunoassay RIA.
The tissue or cell type to be analyzed will generally include those- which are known, or suspected, to express the gene of interest such as, for example, cancer. The protein isolation methods employed herein may, for example, be such as those described in Harlow and Lane Harlow, E. The isolated cells can be derived from cell culture or from a patient. The analysis of cells taken from culture may be a necessary step in the assessment of cells that could be used as part of a cell-based gene therapy technique or, alternatively, to test the effect of compounds on the expression of the gene.
For example, albumin fusion proteins may be used to quantitatively or qualitatively detect the presence of polypeptides that bind to, are bound by, or associate with albumin fusion proteins of the present invention. This can be accomplished, for example, by immunofluorescence techniques employing a fluorescently labeled albumin fusion protein coupled with light microscopic, flow cytometric, or fluorimetric detection.
In a preferred embodiment, albumin fusion proteins comprising at least a fragment or variant of an antibody that immunospecifically binds at least a Therapeutic protein disclosed herein e. This can be accomplished, for example, by immunofluorescence techniques employing a fluorescently labeled antibody coupled with light microscopic, flow cytometric, or fluorimetric detection.
The albumin fusion proteins of the present invention may, additionally, be employed histologically, as in immunofluorescence, immunoelectron microscopy or non-immunological assays, for in situ detection of polypeptides that bind to, are bound by, or associate with an albumin fusion protein of the present invention. In situ detection may be accomplished by removing a histological specimen from a patient, and applying thereto a labeled antibody or polypeptide of the present invention.
The albumin fusion proteins are preferably applied by overlaying the labeled albumin fusion proteins onto a biological sample. Through the use of such, a procedure, it is possible to determine not only the presence of the polypeptides that bind to, are bound by, or associate with albumin fusion proteins, but also its distribution in the examined tissue. Using the present invention, those of ordinary skill will readily perceive that any of a wide variety of histological methods such as staining procedures can be modified in order to achieve such in situ detection.
Immunoassays and non-iminunoassays that detect polypeptides that bind to, are bound by, or associate with albumin fusion proteins will typically comprise incubating a sample, such as a biological fluid, a tissue extract, freshly harvested cells, or lysates of cells which have been incubated in cell culture,.
The biological sample may be brought in contact with and immobilized onto a solid phase support or carrier such. The support may then be washed with suitable buffers followed by treatment with the detectably labeled albumin fusion protein of the invention.
The solid phase support may then be washed with the buffer a second time to remove unbound antibody or polypeptide. Optionally the antibody is subsequently labeled. The amount of bound label on solid support may then be detected by conventional means. By "solid phase support or carrier" is intended any support capable of binding a polypeptide e. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite.
The nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention. The support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to a polypeptide. Thus, the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod.
Alternatively, the surface may be flat such as a sheet, test strip, etc. Preferred supports include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation.
The binding activity of a given lot of albumin fusion protein may be determined according to well known methods. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation. In addition to assaying polypeptide levels in a biological sample obtained from an individual, polypeptide can.
For example, in one embodiment of the invention, albumin fusion proteins of the invention are used to image diseased or neoplastic cells. Suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the albumin fusion protein by labeling of nutrients of a cell line or bacterial or yeast strain engineered. Additionally, albumin fusion proteins of the invention whose presence can be detected, can be administered.
Chemical Data, Structures and Bibliographies. Hall 6 December Concise Dictionary of Pharmacological Agents: Larry Jameson; Leslie J.
De Groot 25 February Adult and Pediatric E-Book. Int J Sports Med. Androstanolone stanolone, dihydrotestosterone, DHT Androstanolone esters Bolazine capronate Drostanolone propionate dromostanolone propionate Epitiostanol Mepitiostane Mesterolone Metenolone acetate methenolone acetate Metenolone enanthate methenolone enanthate Stenbolone acetate Nortestosterone derivatives: Bolandiol dipropionate Nandrolone esters e.
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The specific primers may contain restriction endonuclease recognition sites which can be used for cloning into expression vectors using methods known in the art. This can be accomplished, for example, by immunofluorescence techniques employing a fluorescently labeled albumin fusion protein coupled with light microscopic, flow cytometric, or fluorimetric detection. Furthermore, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the polypeptide sequence.
Enantat Produced by: Moreover, the albumin fusion proteins of the present invention can be used to test the biological activities described herein.
Each of the polynucleotides identified genzyme methenolone enanthate 100 can be used in numerous ways as reagents. Injectable Anabolic Steroid Basal Ingredient: A polypeptide-specific antibody or antibody fragment which has been labeled with an appropriate detectable imaging moiety, such as a radioisotope for example, 13I I, In, 99m Tca radio-opaque substance, or a material detectable by nuclear magnetic resonance, is introduced for example, parenterally, subcutaneously or intraperitoneally into the mammal to be examined for a disorder. The genzyme methenolone enanthate 100 of the chemiluminescent-tagged albumin fusion protein is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Other suitable vectors meditech stanozolol 10mg review be readily apparent to the skilled artisan. Dianabol Basic Chemical Name:
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