Antibodies

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2D Gel Analysis

Biopharmaceutical products must be free of host cell proteins (HCPs) for approval by authorities such as the FDA and EMA. Commonly, ELISAs are used for HCP determination and regulatory authorities ask for a clear and manifest proof that the antibodies used in the HCP ELISA are fit for purpose. Their suitability needs to be shown, not only by the obtained ELISA data, but also by determination of the HCP coverage in percent.

The 2D Western blot method is currently the standard technique for detection of HCP coverage. In this method the proteins are separated in two dimensions according to their isoelectric points (IEF) and to their molecular weights (2D SDS-PAGE) with subsequent Western blotting and immunostaining. BioGenes is offering the fluorescence based 2D analysis but also colorimetric method.

2D DIGE Western Blot (fluorescence staining)

For the 2D DIGE Western Blot method the protein sample is labeled with Cy5 prior to running IEF/ 2D SDS-PAGE with subsequent Western blotting and immunostaining using Cy3-labeled antibodies. This results in one 2D DIGE Western Blot membrane on which both, the total HCPs and the anti-HCP antibodies can be detected.

As total HCP and anti-HCP antibodies are detected on the same membrane there is no need for inter-gel matching. Furthermore, the fluorescence staining allows for a complete electronic evaluation.

2D Western Blot (colorimetric staining)

For the commonly used colorimetric procedure, two gels are run. One gel is used for total protein staining by Coomassie or silver, and the other is used for Western blotting followed by immunostaining. Furthermore, for the detection of both abundant and scarce proteins two blots with low and high antibody concentrations are necessary. The coverage is determined by matching the stained protein spots on the gel with the immunodetected proteins on the membrane.

Fig.: Comparison of colorimetric (Coomassie BlueSilver, 600 μg HCP loaded) and fluorescence staining (Cy5, 50 μg HCP loaded).

BioGenes is also offering 2D DIGE analysis with electronic evaluation directly on the gel.

Please contact us for more information.