Assay Protocol
1. Use the plate layout sheet on
the back page to plan sample layout on plate and also aid in proper sample
and antibody identification after the assay. Each plate is laid out as
shown on the plate maps on the following pages, with each unique antibody
appearing in 6 positions on the plate. Rows A and H are not used in order
to minimize edge effects. We recommend that assays are carried out in
duplicate or (preferably) triplicate in order to minimize spurious
results. For example, we have shown the plate layout for an experiment in
triplicate, where the wells used for the control compound are highlighted
and the three rows underneath are used for the test compound. For an
experiment in duplicate, use rows B-C for the control and rows D-E and F-G
for two test compounds.
2. Dilute the 10xPBS with water
to 1x-strength, the 2 ml 10x PBS should be diluted to 20 ml with 18 ml
water.
3. Add 1 ml PBS to each of the
10 capturing antibody vials, mix well and coat each column of the 96-well
plate provided with one antibody solution. For example, column 1 will be
coated with Ab-1, column 2 coated with Ab-2 and so on. Only six rows will
be coated (B, C, D, E, F and G), each well is coated with 100 ¥ìl of
capturing antibody at 10 ¥ìg/ml. Incubate the plate at 4¡ÆC overnight with
the cover sheet provided.
4. The next day, dilute the
10xPBS-T and 5x Dilution buffer with water to 1x-strength. Check
concentrate bottles for precipitates before proceeding and if found warm
slightly in a water bath to dissolve before proceeding. The 50 ml of
10xPBS-T should be diluted to 500 ml with 450 ml water and the 5 ml of 5x
Dilution Buffer should be diluted to 25 ml with 20 ml water.
5. Wash the coated plate by
emptying contents and adding 250 ¥ìl of wash buffer to each well. Empty
wells again and tap the plate firmly upside down on a paper towel to fully
empty well. Repeat.
6. Dilute your sample and
Epoetin standard to a concentration of 2 ?g/ml in PBS-T; prepare at least
4 ml of each if samples are to be run in duplicate, 5 ml of each if run in
triplicate. Pipette 100 ¥ìL of 2 ¥ìg/ml sample or Epoetin standard into
each row of the plate. For replicates use multiple rows, i.e. Epoetin
standard in rows 2-3, sample 1 in rows 4-5 and sample 2 in rows 6-7. Cover
plates and incubate 1 hour at room temperature.
7. During the above incubation,
dilute the 1 mg/ml reporting antibody to 25 ¥ìg/ml by adding the entire
250 ¥ìL to 9.75 ml of PBS-T.
8. Wash plate by emptying
contents and adding 250 ¥ìl of wash buffer to each well. Empty wells again
and tap the plate firmly upside down on a paper towel to fully empty well.
Repeat.
9. Pipette 100 ¥ìL of 25 ¥ìg/ml
Reporting Antibody into each well. Cover plate and incubate plate 45 min
at room temperature.
10. During the above incubation,
dilute the 4 ?g/ml Streptavidin-HRP conjugate to 0.1 ¥ìg/ml by adding 375
¥ìL to 15 ml of Dilution Buffer.
11. Wash plate by emptying
contents and adding 250 ¥ìL of wash buffer to each well. Empty wells again
and tap the plate firmly upside down on a paper towel to fully empty well.
Repeat.
12. Pipette 100 ¥ìL of 0.1 ¥ìg/ml
Streptavidin-HRP conjugate into wells. Cover plate and incubate plate 30
min at room temperature.
13. Wash plate by emptying
contents and adding 250 ¥ìL of wash buffer to each well. Empty wells again
and tap the plate firmly upside down on a paper towel to fully empty well.
Repeat 2 more times
14. Add 100 ¥ìL of TMB substrate
to each well. Allow color development to proceed for exactly 12 minutes
and then stop reaction by adding 100 ¥ìL of Stop Solution to each well.
Upon addition of stop solution, developed color will change from blue to
yellow.
15. Read the optical density
generated from each well in a plate reader capable of reading at 450 nm,
Use wells A11, B11 and C11 as blank.
16. Export the plate reader data
into Excel and calculate an average and variance for each set of
replicates. If the variance is large inspect the raw data to determine the
problem. With data in triplicate, one outlier may be evident, but if data
is in duplicate, the higher value is generally suspect (it뭩 easier to
get a high value in error than a low value). Graph the data as a bar graph
so that for each array antibody the response can be compared between your
sample and Epoetin standard. Any differences between your sample and the
Epoetin standard should be apparent. |