Other conjugated antibodies

In addition to HRP, PE, and Alexa Fluor® conjugates, a variety of other conjugated antibodies exist, including popular labels such as fluorescein isothiocyanate (FITC), allophycocyanin (APC), alkaline phosphatase (AP), and biotin.

How should I choose the right conjugate for my experiment?

Consider the detection systems available to you, the sensitivity required for your target, and the nature of your sample. For applications such as immunocytochemistry or flow cytometry, fluorescent labels such as FITC or APC may be preferred, whereas for quantitative studies like ELISA, enzyme-conjugated antibodies like AP or a Biotin conjugate may be more suitable.

FITC conjugated antibodies

FITC (fluorescein isothiocyanate) is a widely used green-fluorescent dye ideal for flow cytometry and immunocytochemistry.

APC conjugated antibodies

APC’s (Allophycocyanin) far-red emission (~660 nm) and exceptional brightness enable sensitive detection of low-expression proteins and rare cell types. APC conjugated antibodies are ideal for immunophenotyping in complex samples in flow cytometry.

AP conjugated antibodies

AP (Alkaline phosphatase) provides a great alternative to HRP-conjugated antibodies, especially when working at alkaline pH. AP is very stable, maintaining enzymatic activity during assay incubation and substrate development, allowing for reproducible results in ELISA, western blot and IHC.

Biotin conjugated antibodies

Biotin forms large complexes with avidin or streptavidin for signal amplification and can be used in two ways:

Avidin-biotin complex (ABC) method: large avidin-biotin complexes linked through reporter enzymes are incubated with biotinylated antibodies. The signal is amplified due to the high enzyme-to-antibody ratio.
Labeled streptavidin-biotin (LSAB) method: the signal is amplified through incubation of the biotinylated antibody with complexes made of streptavidin and reporter enzymes. Complexes in the LSAB method are smaller than in the ABC method.

LSAB methods have become more popular than ABC over the last few years, primarily due to the lower non-specific binding of streptavidin proteins. Benefits of LSAB methods are described below.

ABC

LSAB

Notes

Specificity

Lower

Higher

Avidin may show non-specific binding due to its carbohydrate moieties and its high isoelectric point (pI). In contrast, streptavidin lacks carbohydrate moieties and has a more neutral pI.

Sensitivity

High

Both methods show greater sensitivity than direct or indirect detection.

Tissue penetration

Lower

Higher

The complex size in LSAB methods is smaller, facilitating greater tissue penetration.

Sample processing

More complex

Simpler

Both methods require three incubation steps, but ABC methods require an additional incubation of avidin with the reporter enzyme.

Limitations of using biotinylated antibodies

Despite their wide adoption, these methods also come with their limitations. The presence of endogenous biotin in tissues can significantly increase background signal. Therefore, blocking endogenous biotin is particularly important in tissues with high expression of the molecule such as the liver and kidney. Note that non-biotin-based detection methods are recommended with frozen tissue sections due to their high amounts of endogenous biotin.