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Summary for Patent: 4,446,240
|Title:||Pancreas specific protein systems|
|Abstract:||Pancreas specific protein systems, including Pan Ag purified antigen having molecular mass of about 2.25.times.10.sup.5 daltons and pancreas specific antibodies to such antigen, and methods for providing and utilizing such antigen and antibodies.|
|Inventor(s):||Nerenberg; Samuel T. (Huntington Beach, CA)|
|Patent Claims:||1. Substantially immunologically pure Pan Ag, an isotonic saline soluble pancreatic acinar cell cytoplasmic glycoprotein having a molecular weight of about
2.25.times.10.sup.5 daltons, a carbohydrate content of about 18 percent by weight, an antigenic stability in the range of from about pH 2.8 to about 8.5, an antigenic stability to incubation with deoxyribonuclease and ribonuclease, an antigenic
instability to incubation with trypsin and neuraminidase and which precipitates at between about 245 and about 340 grams per liter of ammonium sulfate saturation.
2. Pan Ag in accordance with claim 1 which is at least 99 percent by weight pure, exclusive of immunologically inert solvents, diluents or carriers.
3. An immunologically monospecific antibody to the Pan Ag of claim 1.
4. A purified antibody in accordance with claim 3 wherein said antibody is monospecific rabbit immunoglobin G antibody to Pan Ag.
5. A Pan Ag antibody in accordance with claim 3 wherein said antibody is radioactively labelled.
6. A Pan Ag antibody in accordance with claim 3 wherein said antibody is peroxidaselabelled.
7. A Pan Ag antibody in accordance with claim 3 wherein said antibody is labelled with a fluorescent dye.
8. A method for detecting the presence of Pan Ag in a biological sample, comprising the steps of contacting the sample with a monospecific antibody for Pan Ag, and detecting the Pan Ag-antibody reaction product.
9. A method in accordance with claim 8 wherein said reaction product is detected and quantified by radioimmunoassay, immunofluorescent or immunoperoxidase technique.
10. Pan Ag in accordance with claim 1, which is radioactively labelled.
11. Radioactively labelled Pan Ag in accordance with claim 10, which is complexed with an antibody for Pan Ag.
12. A method for detecting the presence of Pan Ag in a biological sample, comprising the steps of contacting the sample with a labelled complex of a Pan Ag as claimed in claim 1 and a monospecific antibody for said Pan Ag, and measuring the amount of labelled Pan Ag released by contact with said sample.
13. A method for providing a monospecific Pan Ag antiserum comprising the steps of percutaneously applying substantially immunologically pure Pan Ag antigen as claimed in claim 1 to a suitable mammal, collecting serum from said mammal after antibody development therein, and absorbing said animal serum or insolubilized normal human serum to provide a monospecific animal antiserum (antibody) to human pancreatic antigen.
14. A method for preparing and isolating Pan Ag, cytoplasmic pancreatic acinar cell protein, comprising the steps of
preparing an aqueous saline (9 grams per liter NaCl) extract of human pancreas,
dialyzing the saline extract against distilled water, and subsequently dialyzing against tris(hydroxy-methyl) methylamine(Tris)-glycine buffer (pH 9.1, 0.04 mol/liter) to provide a dialyzed extract,
centrifuging the dialyzed extract at 27,000.times.g for 20 minutes, collecting the supernate, fractionating the supernate by preparative polyacrylamide gel gradient electrophoresis to provide 14 principal protein bands, providing a mammal antiserum to the seventh protein band contacting the mammal antiserum with polymerized normal human serum, to produce an antiserum specific for pancreas and polymerizing the pancreasspecific antiserum with gluteraldehyde to form an insoluble immunoabsorbant,
preparing an aqueous saline (9 grams per liter NaCl) extract of human pancreas, heating the extract to 60.degree. C., immediately cooling the heated extract to 4.degree. C., centrifuging the cooled extract at 27,000.times.g for 20 minutes at 4.degree. C., dialyzing the centrifugation supernate against 0.05 mol/liter sodium phosphate buffer (pH 6.0), centrifuging the dialyzed supernate at 27,000.times.g, filtering the dialyzed centrifugation supernate, mixing the filtered dialyzed centrifugation supernate with carboxymethyl dextran polymer gel, washing the gel with 0.05 mol/liter sodium phosphate buffer (pH 6.0),
eluting adsorbed pancreatic proteins from the carboxymethyl dextran polymer gel to provide a protein solution, selectively absorbing components having a molecular weight of less than about 200,000 by subjecting said protein solution to gel filtration while passing through higher molecular weight components having molecular weight of more than about 200,000 in the gel filtration void volume, and collecting the gel filtration void volume solution,
subjecting the gel filtration void volume solution to preparative polyacrylamide gel electrophoresis in glycine buffer at pH of about 9.2 to separate the components into six principal proteins bands,
fractionating the third electrophoretically separated band (from the cathode) by immunoaffinity chromatography by contacting the protein with said insoluble immunoabsorbant to separate the pancreas-specific component from the non-absorbed components, eluting the pancreatic antigen (Pan Ag) from the insoluble immunoabsorbant, and subjecting the Pan Ag eluted from said insoluble immunoabsorbant to polyacrylamide disk gel electrophoresis to detect a single protein band, indicating a substantially pure pancreatic antigen containing about 18 percent by weight carbohydrate as determined by the phenolsulfuric acid method, which retains its antigenic activity in a pH range of from about 2.8 to about 8.5, but which is destroyed at pH 2.5, which is stable to incubation with deoxyribonuclease and ribonuclease, and which has a mass of about 2.25.times.10.sup.5 daltons as determined by polyacrylamide gel filtration versus IgE, follitropin, insulin, carcinoembryonic antigen and dextran reference markers.
15. The pancreatic antigen (Pan Ag) prepared by the method of claim 14.
16. A labelled immunologically monospecific antibody to the Pan Ag of claim 15.
17. A method in accordance with claim 8 for testing for an abnormal pancreatic condition in which the sample is a human blood sample and wherein the method further comprises determining whether the sample contains in excess of a predetermined quantity of Pan Ag in the range of from about 6 to about 10 micrograms per liter of Pan Ag.
18. A monospecific antibody in accordance with claim 8 which is immunologically unreactive toward the pancreatic proteins insulin, amylase, lipase, elastase, carboxypeptidase A, carboxypeptidase B, collagenase, leucine, aminopeptidase, phospholipase, carbonic anhydrase and gammaglutamyltransferase.
|Applicant||Tradename||Biologic Ingredient||Dosage Form||BLA||Number||Approval Date||Patent No.||Assignee||Estimated Patent Expiration||Status||Orphan||Source|
|Smith And Nephew||SANTYL||collagenase||OINTMENT;TOPICAL||101995||001||1965-06-04||⤷ Free Forever Trial||2039-03-29||RX||search|
|>Applicant||>Tradename||>Biologic Ingredient||>Dosage Form||>BLA||>Number||>Approval Date||>Patent No.||>Assignee||>Estimated Patent Expiration||>Status||>Orphan||>Source|
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