TABLE 2.
Strategies and methods for antibody/multiplex immunofluorescence panel validation.
| Strategy | Method | Advantage | Disadvantage |
|---|---|---|---|
| Genetic | In situ hybridization (ISH), CRISPR/CAS9 or siRNA/shRNA, Western blot | - Novel genes in spatial contest | - Limited co-expression |
| - The use of genome editing techniques is preferred | - Need functional knockdown reagents | ||
| - Provide a direct link between the gene, the target protein, and its detection by the antibody | - Cannot be used for human tissue samples and body fluids (plasma and serum) | ||
| - Useful for examining antibody specificity for proteins that come from related genes | - Time-consuming | ||
| Orthogonal | Fluorescent in situ hybridization (FISH), quantitative PCR, RNA-seq, Western blot | - Expression of the target protein is compared with an antibody-independent method | - Limited probes and parameters |
| - Co-expression in spatial context | - Need differential expression of target protein | ||
| Independent antibody | Immunofluorescence imaging, Immunohistochemistry, Western blot | - Co-expression can be in spatial context | - Limited parameters |
| - The data generated using several antibodies (different epitopes) in the same protein is compared | - Need antibodies with different epitopes | ||
| Tagged protein expression | Immunohistochemistry, Western blot | - Novel target in spatial context | - Limited co-expression |
| - Tagged proteins should be expressed at endogenous levels |
- Overexpression of the target protein might mask the detection of off-target binding events | ||
| - Limitations of this method are similar to those of the genetic approaches | |||
| - Avoid potential artifacts introduced by the tag itself | |||
| Immunocapture followed by mass spectrometry | Immunoprecipitation, chromatin immunoprecipitation | - Fast, easily co-expression | - Many proteins have similar size |
| -This is one of the best methods for identifying off-target protein binding | - Difficulty in distinguishing direct interactors with the antibody versus proteins that form relevant complexes with the target protein | ||
| - Some of the antibodies validated still do not perform in immunofluorescence assays |