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How well are your HCP ELISAs covered?
any change that could affect HCP composition.
Standard coverage assays and their challenges
A coverage assay can be quite straightforward, often
just 2D gel electrophoresis/Western blot (Figure 1A).
Comparing the colorimetric anti-HCP Western blot to a
total protein stain, and identifying the matching spots,
provides an estimate of antibody-to-HCP coverage.
This approach provides good resolution of individual
HCPs as a 2D map, but it has several challenges for
something that's so crucial to get right (USP 1132).
These include:

* Denaturation of HCP antigens, which might not
represent native antigen in ELISA.

* Variability in electrophoresis and transfer
efficiency between gels.

* Reliability of matching spots between blot and gel.
* Consistency between users in judging coverage.

* Time required for experimental preparation and
data analysis.

So, what can we do about this?
The U.S. Pharmacopeia proposes using an immunoaffinity approach, followed by differential in gel electrophoresis (DIGE) for checking coverage. DIGE is an
established multiplexing variation of 2D-gel electrophoresis/Western blot (Figure 1B) that enables you to
separate and image up to three samples simultaneously, simplifying workflows and improving the reliability of spot analysis.
In this case, the method labels the column chromatography fractions-input (all HCPs), eluate (HCPs covered
by anti-HCP antibodies), and flowthrough (HCPs missed
by antibodies)-with different fluorophores. These
fractions can run on a single 2D-gel, benefiting from
multiplexed fluorescence imaging and objective
analysis by software.

If your lab currently uses 2D-gel electrophoresis/
Western Blot for coverage assays, is there a simple way
of gaining some or all the benefits of DIGE without
changing approach?
Improving speed and accuracy with 2D DIBE
If we take the immunodetection method used in
Western blotting and combine it with the fluorescent
tagging and imaging of DIGE, the result is 2D DIBE:
differential in blot electrophoresis (Figure 2).
This assay format retains the high sensitivity of a
primary/secondary antibody system and the ability to
multiplex using fluorescent dyes. When switching from
a colorimetric Western blot, taking this approach allows
you to run one gel rather than two.
Reducing the number of gels saves time, conserves
reagents, and removes any variation from imperfect
duplicate gels and transfers. Using a single sample of

Figure 1B. Generic
differential gel
electrophoresis (DIGE)
workflow comparing
three samples.

15

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https://www.ncbi.nlm.nih.gov/pubmed/9420172 https://www.ncbi.nlm.nih.gov/pubmed/9420172 https://www.ncbi.nlm.nih.gov/pubmed/9420172 https://www.cytivalifesciences.com/shop/molecular-biology/nucleic-acid-electrophoresis--blotting--and-detection/molecular-imaging-for-nucleic-acids/amersham-typhoon-gel-and-blot-imaging-systems-p-00192?extcmp=CY20363-GL-GCR-PD-HCPprelaunchEbookGEN-exteml-exteml-ebook https://www.cytivalifesciences.com/shop/protein-analysis/molecular-imaging-for-proteins/imaging-software/melanie-9-coverage-software-p-09525?extcmp=CY20363-GL-GCR-PD-HCPprelaunchEbookGEN-exteml-exteml-ebook https://www.cytivalifesciences.com/shop/protein-analysis/molecular-imaging-for-proteins/imaging-software/melanie-9-coverage-software-p-09525?extcmp=CY20363-GL-GCR-PD-HCPprelaunchEbookGEN-exteml-exteml-ebook http://www.GENengnews.com

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Contents
Cytiva eBook - 1
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