Antibody Validation for Research

Increase Confidence in the Detection of Specific Binding

Our IHC service offers a stepwise and flexible approach. For each antibody/antigen combination, IHC assay conditions are initially optimized and validated for key parameters and endpoints including consideration of:

  • Formalin-fixed, paraffin-embedded (FFPE) or frozen tissue sections
  • Colorimetric or fluorescent endpoint
  • Positive and negative control cells and/or tissues
  • 2 antigen retrieval methodologies (FFPE) or 2 fixation techniques (frozen)
  • 3 to 5 primary antibody concentrations with matching non-immune IgG isotype and ‘no primary antibody’ controls
  • Appropriate system for detection of primary antibody binding, e.g. Dako EnVision™ FLEX System or Vector® ImmPRESS™ Excel Amplified HRP Polymer Staining System
  • Positive control antibody included with every assay to validate detection reagents
  • Expert analysis to confirm specificity and correct cellular location

Our 7-step process methodically optimizes and validates IHC protocol prior to experimental investigation.

7-step-antibody-validation

 

Fully optimized and validated IHC assays are then used to investigate expression of the target protein(s) in a selected tissue panel


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An example of Asterand’s standard optimization of IHC assay parameters, using 2 antigen retrieval techniques and three antibody concentrations. FFPE sections of human cerebellum were incubated with three concentrations of Abcam’s anti-PKC gamma antibody (ab71558) or with an equivalent concentration of non-immune IgG. The red box indicates the recommended assay conditions for optimal immunostaining of PKC gamma in Purkinje cells.

 

Peptide blocking experiments may be performed for further validation of antibody specificity

antibody2Development of an IHC assay for B7H3 in breast tumors for a major pharmaceutical company. In addition to our standard optimization and validation, the client wished to further validate the specificity of the anti-B7H3 antibody by peptide blocking. Specific, membrane-associated B7H3-immunoreactivity was observed in the breast tumor stained with the anti-B7H3 antibody. Pre-incubation of the antibody with recombinant B7H3 peptide before application to the sections virtually eliminated immunostaining of the tissues, thereby confirming specificity of the antibody.

 

 

In addition to our standard optimization and validation, the client wished to further validate the specificity of the anti-B7H3 antibody by peptide blocking. Specific, membrane-associated B7H3-immunoreactivity was observed in the breast tumor stained with the anti-B7H3 antibody. Pre-incubation of the antibody with recombinant B7H3 peptide before application to the sections virtually eliminated immunostaining of the tissues, thereby confirming specificity of the antibody.

Further antibody validation may include western blotting analysis or gene knock-down in suitable cell lines using RNAi or shRNA techniques.

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For further information, contact us at advantage@asterandbio.com.