The evaluation method of HCP antibody coverage generally adopts a combination of two-dimensional electrophoresis (2-DE) and protein immunoblotting (Western Blot) technology, namely 2D-Western Blot. Currently, the two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) in 2D-Western Blot is commonly used to detect and analyze HCP antibody coverage on a two-dimensional electrophoresis gel using different fluorescent labels and fluorescent color development methods. This has improved the accuracy of the evaluation to a certain extent, saving time and reagent costs. However, electrophoresis-based technology relies on high-quality electrophoresis results, requires clear stained protein spots, and has a heavy impact on human judgment during the result analysis, which can easily lead to unreliable results.
Many protocols at this stage use 2D-Western Blot (such as 2D-DIGE) methods for coverage detection and compare HCP antibody detection with total protein staining in the gel. This type of coverage analysis enables you to estimate the percentage of coverage and can guide you to use more specific ELISA or orthogonal analysis (such as mass spectrometry). Replacing the 2-DE method with a high-resolution mass spectrometry method can accurately identify the types and relative abundance of proteins contained in the HCP sample. Combined with the types of proteins that can be recognized by HCP polyclonal antibodies, matching calculations can obtain a more realistic coverage. Mass spectrometry can further qualitatively and quantitatively identify the HCP proteins recognized by HCP polyclonal antibodies.