Antibody Optimization for Automated Immunohistochemistry (IHC) Improves Data Quality

IHC is routinely automated using instruments such as the Leica Bond, Ventana BenchMark or Tissue-Tek Genie. These instruments are offered by their manufacturers together with a range of Ready-to-Use (RTU) antibodies and predefined protocols for clinical applications. Clinically approved antibody lists are not comprehensive and often don’t offer the flexibility required for research applications. Instead, Research Use Only (RUO) antibodies can be selected and optimized for use on automated immunostaining instruments. Scientists at Reveal Biosciences are available to offer guidance in selecting the best antibody for your study requirements as needed.

Before starting any new IHC assay, it is critical to optimize the antibody staining conditions to ensure a strong positive signal and low background. Careful experimental design using positive and negative controls is also necessary to assess antibody specificity. Commercial antibodies or those developed in-house may be used, and antibodies can be optimized and tested on tissue from a wide variety of species. Reveal Biosciences combines the power of Leica Bond immunostainers with the experience of our team to generate high quality IHC data. We offer the following levels of antibody optimization depending on your needs and research goals:

BRONZE

Single antibody

This optimization is useful for research applications. A single antibody, selected by the client or our technical team, is tested on positive control tissue at four different concentrations, each with two different antigen retrieval conditions plus appropriate negative controls. An experienced scientist or pathologist will assess the slides and recommend the optimal staining conditions for future experiments. In some cases, issues such as antibody specificity or staining intensity may arise that could require additional optimization or the selection of an alternative antibody.

SILVER

Multiple antibodies

This optimization is also useful for research applications. A number of antibodies (usually 3 – 4) are selected from different sources that target the same biomarker. These antibodies are each tested and compared to determine which gives the best results for the tissue(s) of interest. Antibodies are tested on positive control tissue at four different concentrations, each with two different antigen retrieval conditions plus appropriate negative controls. An experienced scientist or pathologist will assess the slides and recommend the optimal antibody and staining conditions for future experiments.

Optional: Antibodies can also be tested by Western blot to assess specificity.

GOLD

Single or multiple antibodies

This level of optimization is most suited for antibodies that will be used in clinical applications such as companion diagnostics. Antibodies are tested and optimized as described above, with the addition of a full clinical validation following guidelines from the College of American Pathologists (CAP).

For each level of optimization the following tissue or cell pellet controls may be used:

Tissue controls – tissue samples known to be positive or negative for the protein of interest. Helpful information about protein expression in a variety of tissue types can be found at: www.proteinatlas.com

Cell pellet controls – if available, cells with known positive and/or negative expression of the protein of interest can be made into cell pellets, fixed, and processed into paraffin wax (FFPE) and used as controls for IHC assay development.

Assay Negative controls – an isotype control or no primary antibody is used in place of the protein-specific primary antibody for the assay. This will demonstrate any non-specific staining that may be caused by the secondary reagents alone.

Please contact us for more information about antibody optimization or to discuss your study with an experienced member of our team: [email protected]

Leave a Comment

Your email address will not be published. Required fields are marked *