ELISA
Two antibodies to different epitopes: one captures, one detects. High specificity and sensitivity (pg/mL) in complex samples like serum and supernatants.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
Sandwich
Two antibodies to different epitopes: one captures, one detects. High specificity and sensitivity (pg/mL) in complex samples like serum and supernatants.
How it differs from the standard method
- Needs a matched antibody pair.
- Biotin–streptavidin detection amplifies the signal; avoid milk as a blocker (it contains biotin).
1How an ELISA turns binding into colour
An enzyme-linked immunosorbent assay captures the molecule you want on the bottom of a plastic well and labels it with an enzyme, usually horseradish peroxidase (HRP). After each step, unbound material is washed away.
Coat
Capture antibody (or antigen) sticks to the polystyrene by passive adsorption.
Block
A protein such as BSA covers the remaining plastic so later reagents can't stick to it.
Bind and detect
Sample, then a labelled detection antibody, with washes between every step.
Develop and read
TMB colour develops in the dark; stop with acid and read OD 450.
2Four formats
- Direct
- Antigen on the plate, detected by one enzyme-labelled antibody. Quick, but less sensitive.
- Indirect
- Antigen on the plate, an unlabelled primary antibody, then an enzyme-labelled secondary. Used to measure antibodies in serum (titres).
- Sandwich
- Capture antibody on the plate binds the analyte; a second antibody to a different epitope detects it. The most specific and sensitive format for proteins in complex samples.
- Competitive
- Sample analyte competes with labelled analyte for limited antibody, so signal falls as concentration rises. Used for small molecules (hormones, drugs) that can't bind two antibodies.
3From OD to concentration
Every plate carries a serial dilution of a known standard. OD against log concentration gives an S-shaped curve, usually fitted with a four-parameter logistic (4PL).
Key terms
Practise these as flashcards →Signal for a dilution series of known concentrations, used to convert sample signal into concentration.
Four-parameter logistic: bottom, top, midpoint (EC50) and slope. The usual model for ELISA curves.
Wells with every reagent but no analyte. They measure background, which is subtracted.
Lower and upper limits of quantification: the concentration range where the curve gives accurate values.
Interference from other components of the sample (serum proteins, lipids) that changes the signal.
In one-step sandwich assays, extremely high analyte saturates both antibodies separately and paradoxically lowers the signal.
When to use it
- Measuring the concentration of one protein (cytokine, hormone, biomarker) in serum, plasma or culture supernatant.
- Screening many samples in parallel on 96- or 384-well plates.
- Measuring antibody titres after immunisation or infection (indirect format).
Compared with alternatives
Shows the protein's size and is semi-quantitative; ELISA gives concentrations for many samples faster.
Measures dozens of analytes in one small sample; ELISA is simpler, cheaper and well validated for a single analyte.
Bradford and BCA measure total protein; ELISA measures one specific protein.
Measures proteins on or in single cells rather than secreted into fluid.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
Two antibodies to different epitopes: one captures, one detects. High specificity and sensitivity (pg/mL) in complex samples like serum and supernatants.
- Needs a matched antibody pair.
- Biotin–streptavidin detection amplifies the signal; avoid milk as a blocker (it contains biotin).