For enzymatic and biotin detection, e.g. in WB or ELISA, we suggest a secondary antibody conjugated to horseradish peroxidase (HRP), alkaline phosphatase (AP) or biotin. Both avidin and streptavidin bind very strongly to biotin and enable signal amplification, regardless of the host species of the antibody.
If a laser light is involved, e.g. in Flow Cytometry, ICC/IF or IHC, we suggest fluorescent detection with a secondary antibody conjugated to a fluorochrome.
View more details about secondary detection methods:
Detection with biotin
For amplification of signal, a two-step biotin-avidin-enzyme may be used (commonly abbreviated as "ABC" reagent). A biotinylated secondary antibody is applied first, then avidin or streptavidin conjugated to an enzyme or fluorochrome binds to multiple sites on the biotinylated secondary antibody.
Learn more about biotin binding proteins.
Enzymatic detection
HRP, popular for use in chemiluminescent detection systems, is less expensive, rapid and a more stable enzyme than AP. However, AP is considered more sensitive particularly when colorimetric detection is used.
Table: Guide to enzymatic detections
| Enzyme | Substrate | Applications | Advantages | Drawbacks |
|---|---|---|---|---|
| HRP | Chromogenic, soluble (TMB, ABTS, OPD...) | ELISA | Easy to use | Light sensititive coloration |
| Chromogenic, precipitating (CN, AEC, DAB,...) | WB, SB, IHC | Easy to use | Background in blood samples and some other tissues Staining stability lower than AP | |
| Fluorogenic (ADHP/resorufin) Chemiluminescent | ELISA | High sensitivity | Need a fluorescent equipment | |
|
| Luminol | WB, SB, IHC | High sensitivity | Need radiographic equipment or light scanner |
| AP | Chromogenic, soluble (pNPP) | ELISA | Linear kinetic Often more sensitive than HRP | Unstable |
| Chromogenic, precipitating(BCIP/NBT,...) | WB, SB, IHC | Staining stability higher than HRP | Interfere with nuclear counterstain | |
|
| Fluorogenic (4-MUP) | ELISA, IHC | Sensitivity | Need fluorescence equipment |
WB: Western Blot, SB: Southern Blot, IHC: Immunohistochemistry, ELISA: Enzyme linkied immunosorbent assay
Fluorescent detection
Due to their novel electronic configurations, fluorochromes have a unique and characteristic spectra for absorption (excitation) and emission.
A single dye is excited at a particular wavelength and emits a photon at another wavelength.
A tandem dye consists of a donor and acceptor fluorochrome molecule, placed in close proximity, allowing for energy transfer between the two. The tandem dye is excited at the excitation wavelength of the acceptor molecule and emits a photon at the emission wavelength of the donor molecule.
Download the guide to fluorochromes
Learn more about fluorochromes and how fluorescent dyes work.
The guide is also available in our 'Understanding secondary antibodies' card. Download the entire card (8 MB) or email us to request your free copy today!
