Step 1: Label each strip on its end tab to ensure identity
should the strips become detached from the plate frame during the
Designate one well on the plate and leave empty. This well will
serve as a substrate blank.
Step 3: Pipette 200
µl of standards #1-6 into duplicate wells.
Step 4: Pipette 200
µl of each specimen into duplicate wells.
Step 5: Cover
the microplate with a plate sealer and incubate the plate for 30
minutes at 37° C.
Step 6: Aspirate the
contents of each well and wash the wells 4 times with 1X Plate Wash
Buffer. To wash, fill the wells with 300 µl of 1X plate wash buffer
and aspirate. Perform 4 fill/aspirate cycles. After the final wash
cycle, thoroughly blot the plate by carefully striking the plate on
a pad of absorbent paper towels. Continue until no visible droplets
of Plate Wash Buffer are observed.
Pipette 100 µl of Detector Antibody into each standard and specimen
Do not add Detector Antibody to the substrate blank well.
Step 8: Cover the
plate with a plate sealer and incubate for 30 minutes at 37° C.
Step 9: Wash the
plate 4 times with Plate Wash Buffer as described in Step 6.
Step 10: Pipette 100
µl of Substrate into each well including the substrate blank
Step 11: Incubate
the plate for 30 minutes at room temperature. A blue color will
develop in wells containing human IgG.
Step 12: Pipette 100
µl of Stop Solution into each well. A color change from blue to
yellow will occur.
Step 13: Within 15
minutes, read the optical density of each well at 450 nm
using a microtiter plate reader.