ELISA, short for enzyme-linked immunosorbent assay, is a medical laboratory test used to detect specific proteins in a liquid sample. It is widely used to test for hormones, antibodies, disease markers, allergens, and proteins linked to infection. The method matters because it can identify very small amounts of a target molecule with high specificity.
In a medical device such as a microplate reader, many samples can be tested at the same time in a 96-well plate.
Key Facts
- ELISA stands for enzyme-linked immunosorbent assay.
- Antibodies bind specific target proteins because their binding sites match the target antigen.
- Signal strength increases when more enzyme-labeled antibody is bound to the target protein.
- Absorbance is measured by a microplate reader using light passed through each well.
- Beer-Lambert law: A = εlc, where absorbance increases with concentration for many solutions.
- A standard curve relates known protein concentrations to absorbance values so unknown concentrations can be estimated.
Vocabulary
- Antibody
- A Y-shaped immune protein that binds to a specific molecule called an antigen.
- Antigen
- A molecule or part of a molecule that is recognized and bound by an antibody.
- Enzyme-linked antibody
- An antibody attached to an enzyme that produces a color change when the correct substrate is added.
- Substrate
- A chemical that reacts with an enzyme to make a detectable product such as a colored solution.
- Absorbance
- A measure of how much light a sample absorbs, often used to estimate the concentration of a colored product.
Common Mistakes to Avoid
- Skipping wash steps, which leaves unbound antibodies or enzymes in the well and creates a falsely high color signal.
- Assuming darker color always means a positive result, which is wrong because the color must be compared with controls and a standard curve.
- Mixing up antigen and antibody roles, which is wrong because the antigen is the target being detected while the antibody is the binding tool.
- Reading unknown samples outside the standard curve range, which is wrong because concentration estimates are unreliable beyond the calibrated values.
Practice Questions
- 1 A standard curve gives the relationship A = 0.40c + 0.05, where A is absorbance and c is protein concentration in ng/mL. If an unknown sample has A = 0.85, what is its protein concentration?
- 2 In a 96-well ELISA plate, 12 wells are used for standards and 8 wells are used for controls. How many wells remain for patient samples?
- 3 A well turns deep blue after substrate is added, while a negative control remains nearly clear. Explain what this suggests about the target protein and why controls are needed.