Method and device for determination of 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; 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; 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 5

A method according to any of claims 1 to 3, characterized in that the buffer is capable of maintaining a pH of 8.0 to 8.5.

Claim 6

A method according to any of claims 1 to 4, characterized in that the thrombin sensitive material is the fluorogenic substrate D - phe-pro-arg-AIE.

Claim 11

A device according to any of claims 6 to 8, characterized in that the buffer is capable of maintaining a pH of 8.0 to 8.5.

Claim 12

A device according to any of claims 6 to 9, characterized in that the thrombin sensitive material is the fluorogenic substrate D-phe-pro-arg-AIE.