Chromogenic Substrate Methods For The Determination Of FVIIIc, Endotoxin And Plasminogen Activator

FVIII clotting activity in plasma and concentrates can be assayed with good reproducibility in the range 20-120% (C.V. approximately 3%) using purified FIXfl and FX in excess. By incubating the sample with above factors, Ca²⁺ and phospholipid, FX is activated in proportion to the amount of FVIIIc. Generated FX_a is measured with the substrate S-2222. FVIIIc in the range below 20% can be determined by modifying the reaction conditions.

Endotoxin in water solutions can be accurately determined down to 1 pg/ml by incubating the sample with an excess of Limulus lysate. The generated enzymatic activity which is linear in proportion to the amount of endotoxin, is then measured with the substrate S-2423 (Ac-Ile-Glu-Gly-Arg-pNA). Accurate endotoxin determinations in plasma and cerebrospinal fluid are also possible after destroying interfering inhibitors by heat treatment.

Plasminogen activator activities can be measured via plasminogen and the plasmin substrate S-2251 or directly by substrate S-2288 (H-D-Ile-Pro-Arg-pNA). Interfering activities can be minimized by strategic selection of various protease inhibitors.

In all the methods the assay conditions, reagents and procedure have been optimized.