A 1-step immunoenzymatic assay was used in this study. An automated
chemiluminescence enzyme immunoassay device, known as an
Access Immunoanalyzer (Beckman Coulter), and a special bone-type
ALP (BAP) kit (Access Ostase; Beckman Coulter) were used [19,22].
Beckman BAP Access Ostase operating instructions were followed. Samples
and reagents were mixed, and all operations were automatically
performed as long as the appropriate operating items were chosen.
Each 25 μl sample was mixed with 50 μl of a solid phase reagent (magnetic
particles coated with goat anti-mouse polyclonal antibodies) and
15 μl of an antibody reagent (mouse monoclonal anti-BAP antibodies),
and then washed after reacting at 37 °C for 14.4 min. Next, a 200 μl substrate
solution (lumigen PPD) was added and samples incubated at
37 °C for 5 min. After each reaction was complete, its luminous intensity
(λ max = 540 nm) was assayed by immunoanalyzer and the concentration
of BAP in the sample was automatically calculated using a standard
curve. Assays were repeated 3 times in reproducibility analysis, for
which intraday and interday CVs for BAP were 3.82–5.79% and
2.60–5.25% for a 10.39 μg/l concentration, 3.77–5.66% and 3.33–5.80%
for 28.07 μg/l.