The assay is an immunometric ('sandwich') EIA for the quantitative
measurement of AFP in human serum or plasma. Sample AFP is bound by 2
anti-AFP antibodies directed at different epitopes. One antibody is
attached to magnetic particles and the other is indirectly labelled with
alkaline phosphatase using the biotin/streptavidin system. The assay has
four main stages:
Magnetic anti-AFP and biotinylated anti-AFP are incubated
with sample (50 µL) for 12 - 24 hours at 4 - 8°C (overnight in a
refrigerator) followed by a magnetic wash step.
The magnetic particles are incubated with alkaline
phosphatase labelled streptavidin for 1 hour at 4 - 8°C followed
by 2 magnetic wash steps.
A coloured enzyme substrate is incubated with the particles for
30 minutes at 37°C. The presence of alkaline phosphatase causes a
colour change from yellow to pink.
The reaction is terminated by addition of Stop Buffer.
The tubes are then placed in the spectrophotometer or colorimeter.
The optical density (at
550nm or 492nm) of each tube can be measured and the results
calculated using a data processing program.