Flow Cytometry (FCM) /FACS | Non-Conjugated Antibody

Indirect staining

In indirect staining, the primary antibody is not fluorochrome-labeled, but is detected by a fluorochrome-labeled secondary antibody. This second reagent may be an antibody with specificity for the first antibody. Alternatively, the avidin-biotin system can be used, whereby an antibody is conjugated to biotin and detected with fluorochrome-labeled avidin.

With the wide range of conjugated antibodies now available, this method means that unconjugated primary antibodies raised against many different targets can be used in conjunction with a labeled secondary antibody for FACS analysis. This widens the choice of target proteins for the researcher.

When using secondary antibodies - All the primary antibodies have been raised in different species to ensure cross labeling via the secondary antibodies is not encountered. The secondary antibodies recognize one of the species exclusively.

The advantage of indirect immunoassay

• High sensitivity: More than one labeled antibody is bound per antigen molecule; The signal intensity could be amplified on a single epitope.
• Flexible: Different primary antibodies can be used with a single labeled secondary antibody; The reservoir of primary antibodies suitable for flow cytometry would be broadened for no limitation of fluorochrome labeling.
• Cost-saving: Fewer labeled antibodies are required.