Zenon antibody labeling system.
The Zenon antibody labeling system allows non-covalent coupling of fluorochromes to any unconjugated mouse IgG1 primary antibody. Unconjugated primary antibodies are briefly incubated with mouse Fc-specific Fab fragments coupled to a variety of fluorochromes (including most of the Alexa Fluor dyes, biotin, PE, APC, alkaline phosphatase and horseradish peroxidase), followed by a blocking step to block unbound andi-Fc sites. The resulting complex is relatively stable and can be used for both single and multicolor labeling as if it were a conventional direct conjugate. The Zenon system is particularly useful when direct antibody conjugates are unavailable or difficult to prepare, especially for polychromatic flow cytometry. Our preliminary evaluation is the system is below.
(Below). Zenon labeling mechanism. Unlabeled mouse IgG1 primary antibodies are non-covalently labeled for five minutes with anti-mouse Fc Fab fragments coupled to the required fluorochrome. The resulting complexes are then incubated with non-specific mouse IgG1, which blocks remaining anti-Fc binding sites. The complexes can then be used as is they were directly conjugated primary antibodies. Illustration courtesy of Molecular Probes, Inc.
(Below). Single color labeling with Zenon-coupled antibodies. PBMCs derived from apheresis flow-through were RBC-depleted over Ficoll-Hypaque and labeled with either anti-human CD16, CD19, CD45 or CD56 coupled with either Alexa Fluor 488, PE, APC or Alexa Fluor 647 using the Zenon system. All samples were analyzed on a BD FACSCalibur, with red diode excitation for APC and Alexa Fluor 647. The results are expressed as filled histograms (with percentage positive cells) below. Labeling was reasonably good overall, although phycobiliprotein labelings may have been subject to some steric hindrance.
Zenon conjugates worked well for multicolor labeling, both with each other and in combination with conventional direct conjugates.
(Below). Four-color labeling for human NK cells with Zenon APC conjugates. Human PBMCs were labeled with conventional PE-anti-CD3, PE-Cy5-anti-CD16 and Zenon-conjugated Alexa Fluor 488-anti-CD16 and APC-anti-CD45.
(Below). Four-color labeling for human NK cells with Zenon Alexa Fluor 647 conjugates. As above, but Zenon-conjugated Alexa Fluor 647 antibody substituted for APC.
(Below). Four-color labeling for human T and B cells with Zenon APC conjugates. Human PBMCs were labeled with conventional PE-anti-CD3, PE-Cy5-anti-CD4 and Zenon-conjugated Alexa Fluor 488-CD45 and APC-anti-CD19.
(Below). Four-color labeling for human T and B cells with Zenon Alexa Fluor 647 conjugates. As above, but Zenon-conjugated Alexa Fluor 647 antibody substituted for APC.
(Below). Four-color labeling for human myeloid, T and B cells with Zenon APC conjugates. Human PBMCs were labeled with conventional PE-anti-CD3, PerCP-Cy5.5-CD19 and Zenon-conjugated Alexa Fluor 488-CD16 and APC-anti-CD45.
(Below). Four-color labeling for human myeloid, T and B cells with Zenon Alexa Fluor 647 conjugates. As above, but Zenon-conjugated Alexa Fluor 647 antibody substituted for APC.
For more information on the Zenon system, go to the Molecular Probes Zenon website.