1. Practical methods BSS020X329A
ANALYSIS OF SALIVARY CORTISOL
Enzyme linked immuno-absorbance assay (ELISA) 1

2. measuring cortisol With ELISA
Anti-cortisol antibody coated 96-well plate
Assay buffer.
Conjugate: cortisol-HRP. (enzyme linked antigen):
Substrate: tetramethylbenzidine (TMB). –for detection.
Stop solution: 2M sulphuric acid.

3. measuring the adrenal response to ACTH.
With cortisol Assay
used to assess the proper functioning of the adrenal gland stress response
Two different tests can be used.
The ACTH stimulation test stimulate adrenal cortisol secretion.
dexamethasone suppression test: suppress endogenous production of cortisol in normal individuals.

4. material 96 well plate Waste beaker Universal container Conjugate
Sulphuric acid
Brown glass bottle
(TMB)
Micro centrifuge tubes

5. Competitive ELISA
The labelled antigen (HRP) competes for primary antibody binding sites with the sample antigen (unlabelled).
The more sample antigen in the sample, the less labelled antigen is retained in the well and the weaker the signal.

6. Reagents Anti-cortisol antibody coated 96-well plate Assay buffer.
Anti-cortisol antibody coated 96-well plate
Assay buffer.
Conjugate: cortisol-HRP. (enzyme linked antigen):
Wash solution.
Substrate: tetramethylbenzidine (TMB). –for detection CARCINOGENIC.
Stop solution: 2M sulphuric acid. CORROSIVE.
(add 0.1ml HRP to 25ml buffer)

7. Salivary cortisol measurement using ELISA
Add 25μl sample
in each well
Wash and Add TMB
in each well
Add Sulphuric acid
to stop reaction
Cortisol present
40 min
add
Sulphuric acid
Stop
Wash 4x
Then add 200uL TMB
Cortisol absent
Cortisol + HRP:
(conjugate)
Cortisol :
(samples)
Other antigen:

8. Samples (values on separate sheet)
Standards S1-S6
Quality controls – low, high QCL & QCH
Normal circadian rhythm samples R1-R6
ACTH stimulation test: Patient A & B
Dexamethasone suppression test: Patient C & D

9. Circadian rhythm Standards Quality Controls QC low 10 nmol/l
(One patient with a normal circadian rhythm)
Number Time Value
R nmol/l
R nmol/l
R nmol/l
R nmol/l
R nmol/l
R nmol/l
S nmol/l S2 1.2 nmol/l S nmol/l S4 9.2 nmol/l S nmol/l S nmol/l
Quality Controls
 QC low 10 nmol/l
QC high 30 nmol/l

10. s2 s1 s3 s4 s5 s6 R1 R2 R3 R4 R5 R6 A2 A1 A3 B1 B2 B3
QCL
QCH C1 C2 D2 D1

11. The success of this method depends on:
cleanliness of equipment – contamination with metals, azide and some organic molecules may be toxic to the enzyme label.
accuracy of pipetting – do this slowly and with care.
timing – each well should be incubated for the same time.

12. Method Add 25μl sample/standard per well
Add 25μl sample/standard per well
Add 200μl conjugate (cortisol:HRP) solution per well – **
Incubate and Agitate for 1 hour at room temperature
Wash wells 4 times with washer and buffer solution
Add 200μl TMB per well – **
Incubate and Agitate for 30 minutes at room temperature
Add 50μl 2M sulphuric acid per well
Read optical density at 450nm -

13. General Laboratory Safety
Personal
Protection
Equipment

14. When you are finished: Before you leave Clear your bench!!!!
Empty your trays into the red one at the back of the lab
Call me so that I can sign off your station sheets.

15. BUT before all take the next 5 min to…
Read the protocol (method) carefully before starting any procedure.
Some advice:
Do not deviate from those instructions / Do not rush. If you have doubt ask a member of staff in charge.
Remember that it is sensible to treat all materials as potentially hazardous and behave accordingly.

17. Write up
Choose from practical 1 or 2
words
Guideline will be posted on Moodle
Any question .

18. Patients
Patient A ACTH stimulation +ve test (ie adrenals produce cortisol) A1 0 minutes (basal level) nmol/l A2 30 minutes nmol/l A3 60 minutes nmol/l Patient B ACTH stimulation –ve test (ie adrenals fail to produce cortisol) B1 0 minutes (basal level) nmol/l B2 30 minutes nmol/l B3 60 minutes nmol/l
(ACTH administered after A1)
(ACTH administered after B1)

19. Patient C Dexamethasone suppression of ACTH (+ve test) C1 Day 0 9
Patient C Dexamethasone suppression of ACTH (+ve test) C1 Day am (basal level) nmol/l C2 Day am 0.5 nmol/l Patient D Dexamethasone suppression of ACTH (-ve test) D1 Day am (basal level) nmol/l D2 Day am 2 nmol/l

20. Reference for this practical
~p 943 Testing endocrine function
~p 983 The adrenal axis
(8th ed)