Patent EA-003185-B1 (EA)
Title: Rapid method of cancer diagnosis
Inventor: PUSCAS IOAN
IPC: C12Q1/527
Filed: 1998-03-13
Abstract: 1. A method for cancer diagnosis comprising (a) preparing a first reaction mixture by combining a diluted blood serum sample of the subject to be tested and a solution of purified carbonic anhydrase II, wherein the concentration of carbonic anhydrase II ranges from 0.01 nM to 1.0 mM; (b) determining they activity of the carbonic anhydrase II in the first reaction mixture; (c) determinig the activity of carbonic anhydrase II in a second reaction mixture containing all constituents of the first reaction mixture except the blood serum; and (d) assessing the degree of activation of carbonic anhydrase II in step (b) relative to step (c). 2. The method of claim 1, wherein the diluted blood serum sample contains from 0.1 to 50 vol.%, preferably from 1 to 10 vol.% of blood serum. 3. The method of claim 1 or 2, wherein the diluent of the diluted blood serum sample is selected from water and aqueous organic solvents, which diluent may optionally contain a buffer. 4. The method claim 1, wherein the concentration of carbonic anhydrase II in the solution of purified carbonic anhydrase II ranges from 0.01 nM to 1.0 mM, preferably from 1.0 nM to 1.0 NM, wherein the solvent of said solution is selected from water and aqueous organic solvents, and wherein said solvent may further contain a buffer and/or indicator. 5. The method of claim 1, wherein the diluted blood serum sample and the solution of purified carbonic anyhdrase are combined at a ratio of 100:1 to 1:100, preferably at a ratio of 10:1 to 1:10, and more preferably at a ratio of 1:1. 6. The method of claim 1, wherein the first reaction mixture contains from 0.05 to 25 vol.%, preferably from 0.1 to 10 vol.% and more preferably from 0.5 to 5 vol.% of blood serum. 7. The method of claim 6, wherein an activation of 100 % or higher in the first reaction mixture, which contains 25 vol.% or less, preferably 5 vol.% or less serum, relative to the carbonic anhydrase II activity of the second reaction mixture indicates the presence of a carcinogenic process in the subject. 8. The method of claim 1, wherein the diluted blood serum sample is an aqueous solution containing 10 vol.% serum, the solution of purified anhydrase II is an aqueous buffer solution containing from 1.0 to 10 nM carbonic anhydrase II and the first reaction mixture is prepared by combining the diluted blood serum sample and the solution of purified carbonic anhydrase at a ratio of 1:1. 9. The method of claim 1 or 8, wherein the determination of the carbonic anhydrase II is performed by a stop-flow kinetic measuring the CO2 hydration of said enzyme. 10. The method of claim 9, wherein the initial pH of the first and second reaction mixture is 7.5, and wherein the determination of the activity of the enzyme comprises combining the first or second reaction mixture with a substrate solution being an aqueous solution containing from 5 to 50 mM CO2, preferably 10 to 25 mM CO2 and more preferably 15 mM CO2, and measuring the time till the final pH of 6.5 is reached. 11. The method of claim 1 which is suitable to detect a cancer process in the subject irrespective of the stage of the cancer process, in particular in the asymptomatic phase or in early stage. 12. The method of claim 1 which is suitable for a screening test of cancer diagnosis in different stages and localizations, including pharyngeal cancer, gastric cancer, esophageal cancer, colorectal cancer, hepatocellular cancer, pancreatic, pulmonary, brest, ovarian, uterine cancer, testicular, prostatie, vesica urinary cancer, thyroidian, lips cancer, leukemia, malignant melanomas and Hodgkin lymphoma and any other form of cancer. 13. A kit for performing the method for cancer diagnosis as defined in claim 1 or 12, which comprises a composition containing carbonic anhydrase II, and at least one tumor marker, said tumor marker activating carbonic anhydrase II at concentrations of 1 muM or below. 14. The kit of claim 15 wherein the tumor marker is selected from tumor necrose factor alpha and beta, carcinoembryonic antigen, sialic acid, alpha1-antichymothrypsine and sp 185 Her2 oncoprotein.