Salimetrics Salivary Cortisol ELISA
5/8/02 AMP
Specimen Collection
Use non-citric acid cotton
Freeze all saliva samples prior to assay in order to precipitate mucins
Thaw completely and centrifuge at 1500g( @3000rpm ) for 15 minutes
Preparing for the Assay:
1.
Make sure you have enough autoclaved microcentrifuge tubes to prepare standards and
unknowns. You will need 48 tubes total ( 8(standards), 37(unknowns), 3(NSB, Zero, 1.8).
2.
Gather all supplies that will need before beginning preparation (pipetters, disposable tube
to hold 24mL, serological pipette, etc.)
Procedure
Step 1:
1.
Bring all reagents to room temperature except concentrated enzyme conjugate (2-8C)
before use.
2.
Plate Layout
1
A Standard
1
B Standard
2
C Standard
3
D Standard
4
E Standard
5
F Standard
6
G Standard
7
H
NSB
2
3
Standard
1
Standard
2
Standard
3
Standard
4
Standard
5
Standard
6
Standard
7
NSB
Standard
8
Standard
9
Zero
4
5
6
7
8
9
10
Standard Sample 6 Sample 6 Sample 14 Sample 14 Sample 22 Sample 22
8
Standard Sample 7 Sample 7 Sample 15 Sample 15 Sample 23 Sample 23
9
Zero
Sample 8 Sample 8 Sample 16 Sample 16 Sample 24 Sample 24
Sample 1 Sample 1 Sample 9 Sample 9 Sample 17 Sample 17 Sample 25 Sample 25
Sample 2 Sample 2
Sample Sample 10 Sample 18 Sample 18 Sample 26 Sample 26
10
Sample 3 Sample 3 Sample Sample 11 Sample 19 Sample 19 Sample 27 Sample 27
11
Sample 4 Sample 4 Sample12 Sample12 Sample 20 Sample 20 Sample 28 Sample 28
Sample 5 Sample 5
Sample
13
Sample 13 Sample 21 Sample 21 Sample 29 Sample 29
11
12
Sample
30
Sample
31
Sample
32
Sample
33
Sample
34
Sample
35
Sample
36
Sample
37
Sample
30
Sample
31
Sample
32
Sample
33
Sample
34
Sample
35
Sample
36
Sample
37
Step 2:
 Label 8 microcentrifuge tubes ( 1.5, 1.2, 0.9, 0.6, 0.2, 0.67, 0.022, 0.007)
 Extra standards added that have special preparations (See next page).
 Label 37 microcentrifuge tubes for unknowns and put aside for later ( only if running a full
plate)
 Pipette 100ul of assay diluent in tubes 0.6, 0.2, 0.67, 0.022, 0.007.
 Serially dilute the standard by adding 50ul of the 1.8 ug/dL standard (Standard 1) to tube 0.6
(Standard 5). Mix well.
 After changing pipette tips, remove 50ul from tube 0.6 (Standard 5) to tube 0.2 (Standard 6).
Mix well.
 After changing pipette tips, remove 50ul from tube 0.2 (Standard 6) to tube 0.067 (Standard
7). Mix well. Continue for tubes 0.022 and 0.007.
 To make tube 1.5 (Standard 2), tube 1.2 (Standard 3), tube 0.9 (Standard 4):
 Tube 1.5: 50ul of 1.8ug,dL Cortisol Standard with 10ul of assay diluent
 Tube 1.2: 40ul of 1.8ug/dL Cortisol Standard with 20ul of assay diluent.
 Tube 0.9: 25ul of 1.8ug/dL Cortisol Standard with 25ul of assay diluent.
 Label 3 microcentrifuge tubes NSB, Zero, and 1.8.
 Pipette 50ul of assay diluent in tubes NSB and Zero (Remember done in duplicate)
 Pipette 50ul of Cortisol Standard in tube 1.8.
 Prepare unknowns with appropriate dilutions ( See separate sheet for correction dilution
factor)
NOTE: Whether you prefer to prepare your samples before your standards’ is
your preference.
 Pipette 24mLs of assay diluent into a disposable tube. Set aside for Step 4.
 NOTE: Each microcentrifuge tube should have enough sample to fill two wells. Remember
everything (standards and unknowns) is done in duplicates!
Step 3:
 Pipette 25ul of standards and unknowns into appropriate wells. Standards and samples
should be assayed in duplicate.
 Pipette 25ul of NSB, Zero, and 1.8 into appropriate wells. Assayed in duplicate.
Step 4:
 Add 15ul of the enzyme conjugate to the 24mL of assay diluent prepared in Step 2.
 Mix well and pipette 200ul into each well using a multichannel pipette.
Step 5:
 Mix plate on rotator for 5 minutes at 500 rpm ( or tap to mix) and incubate at room
temperature for 55 minutes.
 10 minutes before time is up take out TMB solution from 4C and let sit before use and
prepare wash buffer solution for Step 6. Preparations in Step 6.
Step 6:
 Wash plate 4 times with 1X wash buffer.
 Washing may be done by gently squirting wash buffer into each well with a squirt bottle or
pipetting 300ul of wash buffer prepared in each well, and then discarding the liquid by
inverting the plate over a sink.
 To prepare buffer: Dilute the wash buffer concentrate 10 fold with room temperature
de-ionized water(100ml of 10x wash buffer to 900ml of de-ionized water)
 After each wash, the plate should be thoroughly blotted on paper towels before being
turned upright.
Step 7:
 Add 200ul of TMB solution to each well with a multichannel pipette.
 Mix on plate rotator for 5 minutes at 500rpm ( or tap to mix) and incubate the plate in the
dark at room temperature for 25 minutes.
Step 8: VERY IMPORTANT!!!
 10 minutes before time is up, take out stop solution from 4C and set up template on
computer. Have everything ready before you add the stop solution. (See Absorbance MeterDeltaSoft instructions)
Step 9:
 Add 50ul of stop solution to each well with a mulitchannel pipette.
 Mix on a plate rotator for 3 minutes at 500rpm (or tap to mix). Caution: DO NOT mix too
vigorously. Wells are very full!
 Wash off bottom of plate with a Kim-wipe.
 Read in a plate reader at 450 nm. Read plate within 10 minutes of adding stop solution.