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

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.

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.

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

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.

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)

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:

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

Circadian rhythm Standards Quality Controls QC low 10 nmol/l   (One patient with a normal circadian rhythm) Number Time Value R1 06.00 31.25 nmol/l R2 09.00 71.50 nmol/l R3 13.00 31.25 nmol/l R4 16.00 20.80 nmol/l R5 20.00 8.06 nmol/l R6 24.00 1.04 nmol/l S1 0.33 nmol/l S2 1.2 nmol/l S3 3.06 nmol/l S4 9.2 nmol/l S5 27.6 nmol/l S6 82.8 nmol/l Quality Controls  QC low 10 nmol/l QC high 30 nmol/l

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

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.

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 -

General Laboratory Safety Personal Protection Equipment

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.

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.

Write up Choose from practical 1 or 2 1200 - 1500 words Guideline will be posted on Moodle Any question . Email: laurent.lacroix@Roehampton.ac.uk

Patients Patient A ACTH stimulation +ve test (ie adrenals produce cortisol) A1 0 minutes (basal level) 10.40 nmol/l A2 30 minutes 71.50 nmol/l A3 60 minutes 31.25 nmol/l Patient B ACTH stimulation –ve test (ie adrenals fail to produce cortisol) B1 0 minutes (basal level) 31.25 nmol/l B2 30 minutes 20.80 nmol/l B3 60 minutes 31.25 nmol/l (ACTH administered after A1) (ACTH administered after B1)

Patient C Dexamethasone suppression of ACTH (+ve test) C1 Day 0 9 Patient C Dexamethasone suppression of ACTH (+ve test) C1 Day 0 9.00am (basal level) 62.50 nmol/l C2 Day 2 9.00am 0.5 nmol/l Patient D Dexamethasone suppression of ACTH (-ve test) D1 Day 0 9.00am (basal level) 71.50 nmol/l D2 Day 2 9.00am 2 nmol/l

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