Patent ID: 9140710
Filing Date: 2015-09-22
Classification: C07K,G01N

Abstract:
1. A volume-independent assay method for the determination of a ratio of holo transcobalamin to total transcobalamin content in a transcobalamin-containing liquid sample, said method comprising: (i) providing said liquid sample, in which the quantity of said sample is not precisely determined; contacting said sample with a porous substrate having immobilized thereon in a first region a first transcobalamin-immobilizing antibody, being capable of specific binding to holo transcobalamin, so as to generate a first transcobalamin bound region having holo transcobalamin bound thereto, and in a second region a second transcobalamin-immobilizing antibody being capable of immobilizing apo or apo and holo transcobalamin, so as to generate a second transcobalamin bound region having apo or apo and holo transcobalamin bound thereto; (ii) before, during or after step (i) above, contacting transcobalamin of said sample with a reporter-labeled binding partner for transcobalamin, so as to immobilize said reporter-labeled binding partner by binding to said first and second transcobalamin bound regions; and (iii) detecting signals from the reporter-labeled binding partner immobilized on the first and second regions and determining the relative levels of holo transcobalamin and total transcobalamin in the sample based on the detected signals; wherein the relative level of holo transcobalamin in the sample is determined as the signal from the first region; and wherein the relative level of total transcobalamin is determined as the signal from the second region when the second antibody is capable of immobilizing apo and holo transcobalamin; or as the sum of the signals from the first and second regions when the second antibody is capable of immobilizing apo transcobalamin; (iv) calculating the ratio of the relative holo transcobalamin level to the relative total transcobalamin level; wherein the method is performed without determining absolute amounts of holo transcobalamin and total transcobalamin.