A rapid, quantitative assay for measuring alkaline phosphatase activity in osteoblastic cells in vitro

https://doi.org/10.1016/S0169-6009(08)80187-0Get rights and content

Abstract

Alkaline phosphatase (ALP) is the most widely recognized biochemical marker for osteoblast activity. Although its precise function is poorly understood, it is believed to play a role in skeletal mineralization. The aim of this study was to develop an assay suitable for measuring the activity of this enzyme in microtiter plate format. Using the well-characterized osteoblast-like cell line Saos-2, this paper describes an optimized biochemical assay suitable for measuring ALP activity in tissue culture samples. We have determined that a p-nitrophenyl phosphate substrate concentration of 9 mM provides highest enzyme activities. We have found that cell concentration, and hence enzyme concentration, affects both the kinetics and precision of the assay. We also tested several methods of enzyme solubilization and found that freeze-thawing the membrane fractions twice at −70°C/37°C or freeze-thawing once with sonication yielded highest enzyme activities. The activity of the enzyme decreased by 10% after 7 days storage. This assay provides a sensitive and reproducible method that is ideally suited for measuring ALP activity in isolated osteoblastic cells, although sample preparation and storage can influence results.

References (33)

  • GA Rodan et al.

    Expression of the osteoblast phenotype

  • M Skojdt et al.

    Bone cell biology and the regulation of bone turnover

  • H Harris

    The human alkaline phosphatase, what we know and what we don't know

    Clin Chim Acta

    (1989)
  • DE Cole et al.

    Mutations affecting bone-forming cells

  • SB Rosalki et al.

    Two new methods for separating and quantifying bone and liver alkaline phosphatase isoenzymes in plasma

    Clin Chem

    (1984)
  • CS Hill et al.

    The preparation of monoclonal antibodies which react preferentially with human bone alkaline phosphatase and not liver alkaline phosphatase

    Clin Chim Acta

    (1989)
  • Cited by (0)

    View full text