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222-F Antibody labeling reagents to rapidly screen for the binding and internalization of therapeutic antibodies
  1. Ryan Holly and
  2. Chris Langsdorf
  1. Thermo Fisher Scientific, Eugene, OR, USA


Background There is a growing need in the immunotherapy research field for tools to rapidly screen novel therapeutic antibodies, whether looking for cells expressing IgG, target binding, or tracking internalization. Thermo Fisher Scientific has developed the next generation of Invitrogen™ Zenon™ reagents to enable screening of potential therapeutic antibodies, working across a variety of sample types including primary B cells and hybridomas, while being compatible with a wide range of platforms including flow cytometry, high content imaging, and in-incubator imaging-based screening.

Methods The Invitrogen™ Zenon™ Alexa Fluor™ Plus IgG Labeling Reagents to screen for antibody binding provide a fast, versatile, and reliable method to evaluate antibody binding to cell surface antigens. Zenon screening reagents are labeled with Invitrogen™ Alexa Fluor™ Plus 488, Alexa Fluor™ Plus 594, or Alexa Fluor™ Plus 647 dyes (i.e., labeling reagent). The labeling reagent is directed against the Fc portion of an intact mouse or human IgG primary antibody. The formation of the zenon-antibody complex is rapid and requires no purification steps (figure 1).

The Invitrogen™ Zenon™ pHrodo™ Deep Red Labeling Reagents for screening antibody internalization provides a highly sensitive method for monitoring antibody internalization. Zenon pHrodo Deep Red utilizes the same screening reagent as Zenon Alexa Fluor Plus reagents, replacing Alexa fluor dyes with the pH-sensitive dye, pHrodo™ Deep Red. The pHrodo Deep Red sensors have minimal fluorescence at neutral pH and only upon entry into the late endosome and lysosome do they brightly fluoresce. Zenon pHrodo Deep Red Labeling Reagents are perfectly suited for screening the endocytosis and degradation of potentially therapeutic antibodies including antibody-drug conjugates.

Results Live cells stained with Zenon-labeled antibodies show remarkable signal to background in both flow imaging applications, while retaining their signal following fixation (figure 2). Therapeutic antibodies such as Trastuzumab labeled with the ZenonTM pHrodoTM Deep Red show as bright fluorescent puncta upon lysosomal degradation in the Alexa Fluor 647 channel and co-localize with lysosomal markers such as Invitrogen™ LysoTracker Red dye (figure 3). Both Zenon Alexa Fluor Plus (figure 4A) and Zenon phrodo Deep Red (figure 4B) are perfectly suited for screening antibody panels of up to 96 different antibodies.

Conclusions Thermo Fisher Scientific has developed sensitive and rapid Zenon reagents to allow for high throughput therapeutic antibody screening. These tools not only greatly improve the workflow from our existing Zenon reagents, but allow for increased sensitivity allowing the field to make decisive decisions when selecting candidate immunotherapies.

Abstract 222-F Figure 1

Zenon Alexa Fluor Plus lgG labeling reagent protocol

Abstract 222-F Figure 2

Zenon Alexa Fluor Plus IgG labeling reagents. (A) SKBR3 cells treated with Zenon reagent labeled mouse or human primary antibodies. (B) Signal to background calculated using Thermo Scientific™ CellInsights™ CX7. Background determined from Zenon reagent alone

Abstract 222-F Figure 3

Zenon Phrodo Deep Red labeling reagents. Internalization of Zenon pHrodo Deep Red labeled human or mouse primary antibodies showing co-localization with Lysotracker™ Red

Abstract 222-F Figure 4

Screening antigen positive cells with Zenon reagent labeled primary antibodies. High content imaging (A) and flow cytometry (B)

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