Introductory Lecture (Part 2): Wildlife Endocrinology [ELISA]

Question 1

ELISA stands for?

Answer 1

Enzyme-Linked Immunosorbent Assay.

Question 2

ELISA?

Answer 2

= a biochemical method used to detect & measure specific hormones (eg., cortisol, testosterone, progesterone) in a biological sample.

Question 3

Why use ELISA in Wildlife endocrinology? (4)

Answer 3

Hormones are often present in very small amounts, so ELISA is:

• Sensitive enough to detect low hormone levels.
• Specific to the hormone of interest.
• Relatively fast & cost-effective.
• Usable with many matrices (faeces, urine, hair, saliva, blood, etc).

Question 4

How does ELISA work (process)? (5)

Answer 4

• Coat plate.
• Add sample.
• Washing.
• Add substrate.
• Read results.

Question 5

ELISA Step 1: Coat the plate?

Answer 5

= a special antibody that binds to cortisol is attached to the bottom of a microplate (usually 96 wells).

Question 6

Microplate/96-well plate?

Answer 6

= a flat plastic plate with tiny test tubes (resembles a mini ice cube tray).

Question 7

ELISA Step 2: Add the sample? (2)

Answer 7

• Add the extracted hormone sample (eg., from faeces dissolved in ethanol & buffer) to the wells.
• The cortisol in your sample competes with a known, enzyme-linked version of cortisol for binding sites on the antibody.

Question 8

ELISA Step 3: Washing?

Answer 8

= unbound substances are washed away, leaving only the antibody-bound components.

Question 9

ELISA Step 4: Add substrate? (2)

Answer 9

• A colour-changing substrate is added.
• If the enzyme-linked cortisol is present (i.e., if your sample had low cortisol, leaving more enzyme-labelled cortisol to bind), a colour reaction occurs.

Question 10

ELISA Step 5: Read the results? (3)

Answer 10

• A spectrophotometer measures the intensity of the colour.
• The darker the colour, the less hormone was in the original sample (because more enzyme-labelled hormone got to bind).
• Results are compared to a standard curve created using known hormone concentrations.

Question 11

NB about ELISA?

Answer 11

The more hormone in the sample, the less colour you get in the end.

Question 12

ELISA Outcome? (3)

Answer 12

The data are then used to:

• Plot hormone concentrations across individuals or timepoints.
• Compare conditions (eg., before vs after a stressor).
• Correlate hormone levels with behaviour, season, reproduction, etc.

Question 13

ELISA Process: Well designation? (4) [BUCS]

Answer 13

Specific wells are designated for:

• Blanks.
• Unknown samples.
• Controls.
• Standards.

Question 14

ELISA wells: Blanks?

Answer 14

= wells containing all reagents except the sample & are used to measure background signal.

Question 15

ELISA wells: Unknown samples?

Answer 15

= the actual test samples whose hormone concentrations are to be determined.

Question 16

ELISA wells: Controls?

Answer 16

= samples with known high or low hormone levels to ensure assay accuracy.

Question 17

ELISA wells: Standards?

Answer 17

= known concentrations of the target hormone to generate a standard curve.

Question 18

Assay?

Answer 18

= a test used to measure the presence, amount, or activity of a substance (eg., hormone, pathogen, drug, enzyme) in a sample.

Question 19

Therefore, whenever you see “run an assay” or “assay hormone levels”?

Answer 19

= test a sample to see how much of a specific hormone is in it.