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

Soo Shin In, Bo Shim Yun, Man Hong Choong, Hyun Chul Koh, Ho Lee Seok, Hwa Hong Seung

Research output: Contribution to journalArticle

3 Scopus citations

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°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%.

Original languageEnglish
Pages (from-to)505-510
Number of pages6
JournalArchives of Pharmacal Research
Volume25
Issue number4
StatePublished - 2002 Aug 31

Keywords

  • Hypotension
  • Kinetic Assay
  • Prekallikrein Activator

Fingerprint Dive into the research topics of 'An improved, reliable and practical kinetic assay for the detection of prekallikrein activator in blood products'. Together they form a unique fingerprint.

  • Cite this