An improved, reliable and practical kinetic assay for the detection of prekallikrein activator in blood products.
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%.
Shin
In Soo
IS
Blood Product Division, Korea Food & Drug Administration, Seoul.
Shim
Yun Bo
YB
Hong
Choong Man
CM
Koh
Hyun Chul
HC
Lee
Seok Ho
SH
Hong
Soung Hwa
SH
D000581:Amidohydrolases / Q000032:analysis; D001774:Blood Chemical Analysis; D004305:Dose-Response Relationship, Drug; D015956:Factor XIIa / Q000032:analysis*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D007700:Kinetics; D012015:Reference Standards; D015203:Reproducibility of Results; D012965:Sodium Chloride
D007202:Indicators and Reagents; D012965:Sodium Chloride; D015956:Factor XIIa; D000581:Amidohydrolases; C019665:amidase