Use of polyclonal antibodies for flow cytometry staining of membrane and cytoplasmic antigens.

Differently from monoclonal antibodies, polyclonal antibodies are rarely used for flow cytometry because

they give a high background due to Fc receptor binding (very high with rabbitt Abs).
they are not homogeneous and conjugation is unpredictable and variable. Titer vary from batch to batch.
primary sera may have unpredictable crossreactivty and affinity purification is necessary.

Staining live or permeabilized cells in indirect immunostaining with rabbit or goat polyclonal antibodies is therefore not recommended.

The method detailed here exploits in vitro conjugation of the primary antibody with F(ab) fragments of the fluorochrome-conjugated secondary antibody ^Ref, as for the Mouse-on-Mouse technique. Alternatively, direct biotin conjugation of the purified antiserum should be tested.

In the experiment shown below an affinity purified rabbit anti Hen Egg Lysozyme antibody (anti HEL; Chemicon International, AB1223) at 1 µg/ml final dilution has been used to stain spleen cells from a wild type and an MD4 mouse transgenic for an anti-HEL surface immunoglobulin ^Ref. Both mice were injected I.P one hour before with 50 µg purified HEL protein ^Ref. A purified rabbit antibody non-reactive with the antigen was used as a control, at the same concentration.

Cells were stained with one of the following combinations:

Rb anti-HEL (or control) + Goat anti Rabbit-biotin F(ab) 10 µg/ml (reacted for 20 min) + 100µg/ml rabbit purified Ig, followed by Avidin-FITC.

Rb anti-HEL (or control) + Goat anti Rabbit-FITC F(ab) 10 µg/ml (reacted for 20 min) + 100µg/ml rabbit purified Ig

Rb anti-HEL (or control) + Goat anti Rabbit-Cy5 F(ab) 10 µg/ml (reacted for 20 min) + 100µg/ml rabbit purified Ig

Rb anti-HEL (or control) followed by Goat anti Rabbit-FITC 1:200.

The seconday antibodies for this experiment were purchased from Jackson ImmunoResearch Laboratories. More recently, a full range of fluorochrome- and enzyme-conjugated F(ab) antibodies has been made available by Molecular Probes, (see the special website Zenon Technology).

The shaded area is the negative control antibody, the dark line is the anti-HEL antibody. click on the image to enlarge

Gt anti rabbit FITC and Cy5 F(ab) preparations gave clean staining on both animals.

The primary and secondary antibodies should be allow to react first for 20 min., then the blocking rabbit Ig should be added. Prepare and use the mixture immediately before staining and discard the leftover.

This technology can be successfully applied also to monoclonal antibodies. See an extensive discussion at the Zenon Technology website.

We strongly suggest to perform a similar experiment as described above to verify possible crossreactivities or unwanted reactivities, particularly when using a sensitive technique such as flow cytometry.