Hybridomas: Re-imagined

The central challenge for cloning antibodies by hybridomas is simple: how do you identify exactly which cell is making the mAb you want? Traditionally, this process is difficult and time-consuming, as every cell needs to be isolated and tested individually. Otherwise, there has been no way to accurately identify what cell is secreting the desired mAb.

With OCMS, we have re-imagined this process using fluorescent antigen binding and high-content imaging, so you can literally see what you are looking for.

OCMS stands for On-Cell mAb Screening: cells capture and display their mAbs so they can be screened for binding to fluorescent antigens. Multiplex screening is simplified, and cells expressing desired mAbs can be easily separated from non-expressors.

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The OCMS Screening Paradigm: See What You’ve Been Missing

This hybridoma expresses a human IgA specific for poliovirus.  Left panel: human IgA (maroon). Right panel: poliovirus (green).  Cell nuclei are stained blue.

Efficient Cell Processing

With conventional hybridoma cloning methods, there is no way to gather real-time information about which cells are making the desired mAbs. Thus, large numbers of cells need to be cultured and individually tested. This has been the primary barrier to Mega-Scale Hybridoma Screening.

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Conventional hybridomas all look the same (left panel), but OCMS reveals which cells are making mAbs (middle and right panels) so only expressing cells are cloned. As a result, you can screen more cells in less time, with dramatically reduced infrastructure and cost.

How It's Done

OCMS methods fit into existing hybridoma workflows and performs with mouse and human B cells. Below is an artistic rendering of the surface of an OCMS-enabled hybridoma.

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1. Steady State: Cells secrete mAbs (tan) and express an Anchor (purple) on their surface

2. Antibody Capture: The Linker (red) binds to the Anchor (purple) and captures the mAbs (tan)

3. mAb Screening: The labeled antigen (a virus with a green label) adheres to cells that make antigen specific mAbs

The key to OCMS is specificity: Cells bind only to the mAbs they make. Excess linker in the culture medium competes IgG from binding to non-expressing cells.

In this image, secreting and non-secreting cells have been mixed, and non-secreting cells are labeled green. Only non-labeled hybridomas capture IgG (maroon).

Specific Binding

Multiplex Screening

OCMS uses soluble antigens for mAb screening.  This requires significantly less antigen than traditional assays and preserves valuable binding epitopes. In addition, high-content optical screening technology simplifies multiplex screening.  For example, the Celigo Imaging Cytometer from Nexcelcom has bright field and four fluorescent channels, and it can read a 96 well plate in 15 minutes.

This plate shows Celigo image of a hybridoma secreting a human mAb, labeled in red.  Image Courtesy of Leo Chan, Nexcelom Bioscience, Lawrence, MA, www.nexcelcom.com