Reagent strip test for antithrombin-III

Claim 1

A method for the quantitative determination of AT-III in mammalian blood plasma which comprises: (a) contacting the plasma with excess heparin and a 3 layered reagent strip comprising: i. a first layer of a carrier matrix containing excess thrombin and a buffer; ii. a second lower layer adjacent to and in liquid communication with the first layer said second layer comprising a carrier matrix containing a thrombin sensitive fluorogenic or chromogenic substrate and a buffer, said substrate being capable of interacting with thrombin in such a manner that a time related chemical change detectable by fluorometric or spectrophotometric means takes place when thrombin and the substrate are contacted in a suitable liquid environment, wherein the material of the first layer is capable of adsorbing the plasma and allowing a portion of it to flow through into the lower layer which lower layer is of a material which allows the plasma fluid to solubilize the substrate and permits it to diffuse from the lower layer to the first layer after the AT-III in the plasma and the thrombin in the first layer have formed a thrombin AT-III complex; and iii. a layer of water impermeable material beneath the lower layer; (b) monitoring any chemical change in the substrate by reflectance spectrofluorometric or reflectance spectrophotometric means repeatedly over a period of time to obtain at least 2 fluorometric or spectrophotometric values as a function of time; and (c) comparing the relationship between the values obtained in step (b) with values obtained in a like manner using plasma samples containing known amounts of antithrombin-III and using such comparison to determine the concentration of antithrombin-III in the plasma sample being tested.

Claim 2

A method for the quantitative determination of AT-III in mammalian blood plasma which comprises: (a) contacting the plasma with excess heparin and a 3 layered reagent strip comprising: i. a first layer of a carrier matrix containing excess thrombin and a buffer; ii. a second lower layer adjacent to and in liquid communication with the first layer said second layer comprising a carrier matrix containing a thrombin sensitive fluorogenic or chromogenic substrate and a buffer, said substrate being capable of interacting with thrombin in such a manner that a time related chemical change detectable by fluorometric or spectrophotometric means takes place when thrombin and the substrate are contacted in a suitable liquid environment, wherein the material of the first layer is capable of adsorbing the plasma and allowing a portion of it to flow through into the lower layer which lower layer is of a material which allows the plasma fluid to solubilize the substrate and permits it to diffuse from the lower layer to the first layer after the AT-III in the plasma and the thrombin in the first layer have formed a thrombin AT-III complex; and iii. a layer of water impermeable material beneath the lower layer; (b) monitoring any chemical change in the substrate by reflectance spectrofluorometric or reflectance spectrophotometric means repeatedly over a period of time to obtain at least 2 fluorometric or spectrophotometric values as a function of time; and (c) comparing the relationship between the values obtained in step (b) with values obtained in a like manner using plasma samples containing known amounts of antithrombin-III and using such comparison to determine the concentration of antithrombin-III in the plasma sample being tested.