Optical Density (OD)
Definition
The absorbance reading from a microplate reader that measures how much light is absorbed by the coloured product. Higher OD means more colour, which means more enzyme-labelled antibody captured, which means more antigen in the sample.
- Measured by
- Microplate reader
- Unit
- Absorbance, unitless (A)
- Relationship
- Higher OD correlates with more bound enzyme-antibody conjugate
- Used in
- ELISA-based screening assays
Common questions
Is a high optical density reading on its own proof of a positive result?+
No. OD is read against a calibration curve built from known standards and controls, and a cutoff threshold is set for the specific assay. A raw OD value has no meaning without comparison to that run's controls, since plate-to-plate variation in reagents and incubation affects absolute readings.
What can cause a falsely elevated optical density reading?+
Cross-reactive antibodies, residual wash buffer, bubbles in the well, or non-specific binding can all raise OD without true antigen presence. This is why immunoassay screens are treated as presumptive and confirmed with a more specific method before being reported as identification.
Related terms
- Cross-Reactivity Matrix
- A table documenting the assay's optical density response to blood from multiple non-target species, expressed as a percentage of the human blood...
- Horseradish Peroxidase (HRP)
- The most common enzyme label in immunoassays. When the substrate TMB (3,3'-5,5'-tetramethylbenzidine) is added, HRP converts it to a blue product that...
- Limit of Detection (LOD)
- The lowest concentration of analyte that produces a signal reliably distinguishable from the instrument noise, conventionally three times the standard deviation of...
- Sandwich ELISA
- An ELISA format in which a capture antibody immobilised on the plate binds one epitope of the target antigen, and a separate...
- Standard Curve
- A series of known antigen concentrations run on the same ELISA plate, used to convert the OD reading for an unknown sample...