Version 4d Last updated 7 June 2023

ab108665
Cortisol ELISA Kit

A competitive immunoenzymatic assay for the quantitative

measurement of Cortisol in serum and plasma.

This product is for research use only and is not intended for diagnostic
use.

Copyright © 2023 Abcam. All rights reserved

Table of Contents

1. Overview 1
2. Protocol Summary 2
3. Precautions 3
4. Storage and Stability 3
5. Limitations 4
6. Materials Supplied 4
7. Materials Required, Not Supplied 5
8. Technical Hints 6
9. Reagent Preparation 7
10. Sample Preparation 8
11. Plate Preparation 10
12. Assay Procedure 11
13. Calculations 12
14. Typical Data 13
15. Typical Sample Values 14
16. Assay Specificity 15
17. Troubleshooting 16
18. Notes 18

Copyright © 2023 Abcam. All rights reserved

1. Overview

Abcam’s Cortisol in vitro competitive ELISA (Enzyme-Linked

Immunosorbent Assay) kit is designed for the accurate quantitative
measurement of Cortisol in serum and plasma.

A 96-well plate has been precoated with anti-Cortisol IgG. Samples

and the Cortisol-HRP conjugate are added to the wells, where any
Cortisol in the sample competes with the added Cortisol-HRP for
antibody binding. After incubation, the wells are washed to remove
unbound material and TMB substrate is then added which is catalyzed
by HRP to produce blue coloration. The reaction is terminated by
addition of Stop Solution which stops the color development and
produces a color change from blue to yellow. The intensity of signal is
inversely proportional to the amount of Cortisol in the sample and the
intensity is measured at 450 nm.

Cortisol is a steroid hormone released from the adrenal cortex in

response to the hormone ACTH (produced by the pituitary gland)
Cortisol is involved in the response to stress; it increases blood pressure,
blood sugar levels, suppresses the immune system and may cause
infertility in women. Cortisol acts through specific intracellular receptors
and has effects in numerous physiologic systems, including immune
function, glucose-counter regulation, vascular tone, substrate utilization
and bone metabolism. Cortisol is excreted primarily in urine in an
unbound (free) form. Cortisol is bound with high affinity in plasma to
corticosteroid-binding globulin (CBG, transcotin) and to albumin. Only
free Cortisol is available to most receptors.

The amount of Cortisol present in the serum undergoes diurnal

variation, with the highest levels present in the early morning, and lower
levels at night, several hours after the onset of sleep. Highest levels are
at about 6 – 8 a.m. and lowest levels are at about midnight. These
normal endogenous functions are basis for the physiological
consequences of chronic stress- prolonged Cortisol secretion causes
muscle wastage, hyperglycaemia, and suppresses immune/
inflammatory responses. The same consequences arise from long-term
use of glucocorticoid drugs.

ab108665 Cortisol ELISA Kit

2. Protocol Summary

Prepare all reagents, samples, controls and standards as instructed.

Add samples, standards and controls to wells used.

Add prepared labeled HRP-Conjugate to each well. Incubate at 37ºC.

After washing, add TMB substrate solution to each well. Incubate at

room temperature.

Add Stop Solution to each well. Read immediately.

ab108665 Cortisol ELISA Kit

3. Precautions

Please read these instructions carefully prior to beginning the assay.

 All kit components have been formulated and quality control
tested to function successfully as a kit.
 We understand that, occasionally, experimental protocols might
need to be modified to meet unique experimental circumstances.
However, we cannot guarantee the performance of the product
outside the conditions detailed in this protocol booklet.
 Reagents should be treated as possible mutagens and should be
handled with care and disposed of properly. Please review the
Safety Datasheet (SDS) provided with the product for information
on the specific components.
 Observe good laboratory practices. Gloves, lab coat, and
protective eyewear should always be worn. Never pipet by mouth.
Do not eat, drink or smoke in the laboratory areas.
 All biological materials should be treated as potentially hazardous
and handled as such. They should be disposed of in accordance
with established safety procedures.

4. Storage and Stability

Store kit at +4°C immediately upon receipt. Kit has a storage time of
1 year from receipt, providing components have not been
reconstituted.
Refer to list of materials supplied for storage conditions of individual
components. Observe the storage conditions for individual prepared
components in the Materials Supplied section.

ab108665 Cortisol ELISA Kit

5. Limitations

 Assay kit intended for research use only. Not for use in diagnostic
procedures.
 Do not mix or substitute reagents or materials from other kit lots or
vendors. Kits are QC tested as a set of components and
performance cannot be guaranteed if utilized separately or
substituted.

6. Materials Supplied

Storage
Item Quantity
Condition
Anti-Cortisol IgG Coated Microplate
96 wells 4°C
(12 x 8 wells)
Stop Solution 15 mL 4°C
Cortisol-HRP Conjugate 21 mL 4°C
TMB Substrate Solution 15 mL 4°C
10X Washing Solution 50 mL 4°C
Cortisol Control 1 mL 4°C
Cortisol Standard 0 – 0 ng/mL 1mL 4°C
Cortisol Standard 1 – 10 ng/mL 1 mL 4°C
Cortisol Standard 2 – 50 ng/mL 1 mL 4°C
Cortisol Standard 3 – 150 ng/mL 1 mL 4°C
Cortisol Standard 4 – 500 ng/mL 1 mL 4°C

ab108665 Cortisol ELISA Kit

7. Materials Required, Not Supplied

These materials are not included in the kit, but will be required to
successfully perform this assay:
­ Microplate reader capable of measuring absorbance at 450 nm or
620 nm
­ Incubator at 37°C
­ Multi- and single-channel pipettes to deliver volumes between 10
and 1,000 µL
­ HEPES buffer (0.1 M, pH 7.5) supplemented with 0.1% BSA
­ Optional: Automatic plate washer for rinsing wells.
­ Rotating mixer
­ Deionised or (freshly) distilled water.
­ Disposable tubes
­ Timer

ab108665 Cortisol ELISA Kit

8. Technical Hints

 Avoid foaming or bubbles when mixing or reconstituting

components
 Avoid cross contamination of samples or reagents by changing tips
between sample, standard and reagent additions
 Ensure plates are properly sealed or covered during incubation
steps
 Complete removal of all solutions and buffers during wash steps is
necessary for accurate measurement readings
 Addition of the TMB Substrate solution initiates a kinetic reaction,
which is terminated by the addition of the Stop Solution. Therefore,
the TMB Substrate and the Stop Solution should be added in the
same sequence to eliminate any time deviation during the reaction
 It is important that the time of reaction in each well is held constant
for reproducible results. Pipetting of samples should not extend
beyond ten minutes to avoid assay drift. If more than 10 minutes are
needed, follow the same order of dispensation. If more than one
plate is used, it is recommended to repeat the dose response curve
in each plate
 The incomplete or inaccurate liquid removal from the wells could
influence the assay precision and/or increase the background
 This kit is sold based on number of tests. A ‘test’ simply refers to a
single assay well. The number of wells that contain sample, control
or standard will vary by product. Review the protocol completely
to confirm this kit meets your requirements. Please contact our
Technical Support staff with any questions

ab108665 Cortisol ELISA Kit

9. Reagent Preparation

 Equilibrate all reagents to room temperature (18-25°C) prior to use.

The kit contains enough reagents for 96 wells.
 Prepare only as much reagent as is needed on the day of the
experiment.

9.1 1X Washing Solution

Prepare 1X Washing Solution by diluting 10X Washing Solution with
deionized water. To make 500 mL 1X Washing Solution combine
50 mL 10X Washing Solution with 450 mL deionized water. Mix
thoroughly and gently. Diluted solution is stable for 30 days at
4°C. In the concentrated solution it is possible to observe the
presence of crystals, in this case mix at room temperature until
complete dissolution of crystals.

­ All other solutions are supplied ready to use.

ab108665 Cortisol ELISA Kit

10. Sample Preparation

­ The determination of Cortisol can be performed in plasma as well

as in serum. This kit has been validated for use with Citrate and
Heparin Plasma, but not tested with EDTA plasma
­ For sample dilution, we suggest using a HEPES buffer (0.1 M, pH 7.5)
supplemented with 0.1% BSA.
­ Store the sample at -20°C if the determination is not performed on
the same day as the sample collection.
­ Treatment of the patient with corticosteroids, natural or synthetic
steroids can impair Cortisol determination.

Avoid repeated freezing and thawing.

ab108665 Cortisol ELISA Kit

 Refer to Dilution Guidelines for further instruction.
Guidelines for Dilutions of 100-fold or Greater
(for reference only)

100x 10000x
4 µl sample + 396 µl buffer (100X) A) 4 µl sample + 396 µl buffer (100X)
= 100-fold dilution B) 4 µl of A + 396 µl buffer (100X)
= 10000-fold dilution
Assuming the needed volume is less
than or equal to 400 µl Assuming the needed volume is less
than or equal to 400 µl

1000x 100000x
A) 4 µl sample + 396 µl buffer (100X) A) 4 µl sample + 396 µl buffer (100X)
B) 24 µl of A + 216 µl buffer (10X) B) 4 µl of A + 396 µl buffer (100X)
= 1000-fold dilution C) 24 µl of A + 216 µl buffer (10X)
= 100000-fold dilution
Assuming the needed volume is less
than or equal to 240 µl Assuming the needed volume is less
than or equal to 240 µl

ab108665 Cortisol ELISA Kit

11. Plate Preparation

­ The 96 well plate strips included with this kit are supplied ready to
use. It is not necessary to rinse the plate prior to adding reagents
­ Unused well strips should be returned to the plate packet and
stored at 4°C.
­ For each assay performed, a minimum of 1 well must be used as a
blank, omitting sample and conjugate from well addition.
­ For statistical reasons, we recommend each standard and sample
should be assayed with a minimum of two replicates (duplicates).

ab108665 Cortisol ELISA Kit

12. Assay Procedure

­ Equilibrate all materials and prepared reagents to room

temperature prior to use.
­ Please read the test protocol carefully before performing the assay.
Result reliability depends on strict adherence to the test protocol as
described.
­ If performing the test on ELISA automatic systems we recommend
increasing the washing steps from three to five and the volume of
washing solution from 300 µL to 350 µL to avoid washing effects.
­ Assay all standards, controls and samples in duplicate.

12.1 Prepare all reagents, working standards, and samples as directed

in the previous sections.
12.2 Remove excess microplate strips from the plate frame, return
them to the foil pouch containing the desiccant pack, reseal and
return to 4°C storage.
12.3 Add 20 µL standard, control or sample into their respective wells.
Add 200 µL Cortisol-HRP Conjugate to each well. Leave a blank
well for substrate blank.
12.4 Cover wells with the foil supplied in the kit.
12.5 Incubate for 1 hour at 37°C.
12.6 When incubation has been completed, remove the foil, aspirate
the content of the wells and wash each well three times with 300
µL diluted washing solution. Avoid overflows from the reaction
wells. The soak time between each wash cycle should be > 5
seconds. At the end carefully remove remaining fluid by tapping
strips on tissue paper prior to the next step.
Note: Washing is critical, insufficient washing results in poor precision
and falsely elevated absorbance values.
12.7 Add 100 µL TMB Substrate Solution into all wells.
12.8 Incubate for exactly 15 minutes at room temperature in the dark.
12.9 Add 100 µL Stop Solution into all wells in the same order and at
the same rate as for the TMB Substrate Solution. Shake the
microplate gently. Any blue color developed during the
incubation turns into yellow.
12.10 Measure the absorbance of the sample at 450 nm within 5
minutes of addition of the Stop Solution.

ab108665 Cortisol ELISA Kit

13. Calculations

Calculate the mean absorbance for each point of the standard curve
and each sample. Plot the mean value of absorbance of the
standards against concentration. Draw the best-fit curve through the
plotted points. (e. g.: Four-Parameter Logistic).

Interpolate the values of the samples on the standard curve to obtain

the corresponding values of the concentrations expressed in ng/mL.

ab108665 Cortisol ELISA Kit

14. Typical Data

Typical standard curve – data provided for demonstration purposes

only. A new standard curve must be generated for each assay
performed.

Conc.
O.D
(ng/mL)

0 2.95
10 2.11
50 1.16
150 0.61
500 0.25

Figure 1. Example of Cortisol standard curve.

ab108665 Cortisol ELISA Kit

15. Typical Sample Values

REFERENCE VALUES –
Human serum or plasma Cortisol reference values:

Between 8-10 am 60 - 230 ng/mL

Normal Patient
At 4 pm 30 – 150 ng/mL
Patient treated with ACTH 280 - 600 ng/mL
Patient treated with dexamethasone 0 – 50 ng/mL

Each laboratory should consider the range given by the manufacturer

as a general indication and produce their own range of expected
values based on the indigenous population where the laboratory
works.

SENSITIVITY –
The lowest detectable concentration of Cortisol that can be
calculated from the zero standard is 2.44 ng/mL at the 95% confidence
limit.

PRECISION –

Intra-assay Precision Inter-Assay Precision

n= 60 30
CV (%) < 9.0 < 9.8

RECOVERY –
The recovery of 12.5 – 25 – 50 – 100 ng/mL of Cortisol added to samples
gave an average value (±SD) of 103.56% ± 8.17% with reference to the
original concentrations.

ab108665 Cortisol ELISA Kit

16. Assay Specificity

The cross reaction of the antibody calculated at 50% is:

Cortisol 100 %
Prednisolone 46.2 %
11-Deoxycortisol 4%
Cortisone 3.69 %
Prednisone 3.10 %
11OH Progesterone 1%
Progesterone < 0.1 %
Aldosterone < 0.1 %
Pregnenolone < 0.1 %
17 beta Estradiol < 0.1 %
Estrone 3-sulfate < 0.1 %
Estriol < 0.1 %
Testosterone < 0.1 %
Spironolactone < 0.1 %
DHEA < 0.1 %
DHEA-S < 0.1 %
Androstenedione < 0.1 %
Androsterone < 0.1 %
DHT < 0.1 %
Danazol < 0.1 %
Cholesterol < 0.1 %
Dexamethasone < 0.1 %

Please contact our Technical Support team for more information.

ab108665 Cortisol ELISA Kit

17. Troubleshooting

Problem Cause Solution

Incubation time to Try overnight incubation at 4
short °C
Precipitate can
form in wells upon
substrate addition Increase dilution factor of
when sample
concentration of
Low
target is too high
signal
Using
incompatible Detection may be reduced
sample type (e.g. or absent in untested
serum vs. cell sample types
extract)
Sample prepared Ensure proper sample
incorrectly preparation/dilution
Ensure no bubbles present
Bubbles in wells
prior to reading plate
Check that all ports of plate
All wells not
washer are
washed
unobstructed/wash wells as
equally/thoroughly
recommended
Incomplete Ensure all reagents/master
Large CV reagent mixing mixes are mixed thoroughly

Inconsistent Use calibrated pipettes &

pipetting ensure accurate pipetting
Ensure consistent sample
Inconsistent
preparation and optimal
sample
sample storage conditions
preparation or
(e.g. minimize freeze/thaws
storage
cycles)

ab108665 Cortisol ELISA Kit

 Problem Cause Solution
Wells are
Wash wells as per protocol
insufficiently
recommendations
washed

Contaminated
High Make fresh wash buffer
wash buffer
background

Waiting too long

to read plate Read plate immediately
after adding stop after adding stop solution
solution
Store all reagents as
Improper storage recommended. Please
of note all reagents may not
ELISA kit have identical storage
Low requirements.
sensitivity Using
Detection may be
incompatible
reduced
sample type (e.g.
or absent in untested
Serum vs. cell
sample types
extract)

ab108665 Cortisol ELISA Kit

18. Notes

ab108665 Cortisol ELISA Kit

ab108665 Cortisol ELISA Kit
ab108665 Cortisol ELISA Kit
ab108665 Cortisol ELISA Kit
Technical Support
Copyright © 2023 Abcam. All Rights Reserved. The Abcam logo is a registered
trademark. All information / detail is correct at time of going to print.

For all technical or commercial enquiries please go to:

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www.abcam.co.jp/contactus (Japan)

Copyright © 2023 Abcam. All rights reserved