An improved, reliable and practical kinetic assay for the detection of prekallikrein activator in blood products

Abstract

An improved kinetic assay for prekallikrein activator (PKA), a potential vasodilator, has been developed to be used as an indicator for quality control during production of human albumin preparations. It consists of two reaction stages. In the first stage, PKA and prekallikrein are incubated at 37 degrees C for 45 min to allow the transformation into kallikrein. Kallikrein, a serine protease, catalyzes the splitting of p-nitroaniline (pNA) from its substrate H-D-Pro-Phe-Arg-pNA (S-2302). The rate at which pNA is released was measured spectrophotometrically at 405 nm. Prekallikrein, a substrate of PKA was purified by DEAE ion-exchange chromatography and the major potential variations in the assay were optimized; pH 8.0 and 150 mM sodium chloride were chosen to give a proper ionic strength. Reaction times in the range of 10 to 360 min provided linear dose-response curves. The concentration of prekallikrein was adjusted to fall between 1:1 and 1:3 dilutions to generate a linear standard calibration curve. Under the optimized conditions, reproducibility was checked. In a precision test, the coefficient of variation (CV) stayed within +/-4% and the dose-response curve showed a good correlation (r2=0.999). An accuracy test with an international standard of PKA afforded a mean recovery of 97.5%.

An international collaborative study to replace the 1st international standard for prekallikrein activator

“…The ratio of volume of test solution (PKA) to substrate solution (PKS) is recommended to be at least 1 : 9 in order to minimize matrix problems caused by difference in ionic strength and pH between PKA samples. It is known that determination of PKA activity is strongly influenced by buffer ionic strength [16] as a result of changes in the Michaelis constant ( K m ) of PKA for PKS [17]. Hence, differences in the ionic strength between albumin test samples and the PKA standard need to be minimized.…”