Characterizations and validations of novel antibodies toward translational research.
OBJECTIVE
There is significant need for well-characterized antibodies to the spectrum of human proteins encoded by the genome. Advances in tissue-based proteomic profiling have led to the discovery of many candidate molecular biomarkers and therapeutic targets for which development of clinical assays is depending on high quality antibodies. We developed an antibody validation approach for screening of new mAbs.


METHODS
We utilized a multi-stage approach of protein array and immunohistochemistry. In the first phase, we screened the NCI60 panel of cell lines by means of protein array and select antibodies based on concordance of mRNA expression to protein array signal. Results of this assay are used to predict antibody titer for immunohistochemistry on the NCI60 cell lines, presented as a tissue microarray. In the final stage, we created a tissue-based protein expression map by performing immunohistochemistry on a multi-tumor tissue microarray.


RESULTS
The success rate of this systematic antibody-screening tool was approximately 93% as measured by the results from the protein array. Data from the NCI60 protein array could be used to predict antibody titer for immunohistochemistry, improving the success rate of immunohistochemical assay development.


CONCLUSIONS
The presented strategy of antibody validation and characterization can be provided a new tool for exploration of human proteome.
Tissue Array Research Program, Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.
Reginald
R
Williams
Joon-Yong
JY
Chung
Kris
K
Ylaya
Gordon
G
Whiteley
Stephen M
SM
Hewitt
D000911:Antibodies, Monoclonal; D015415:Biomarkers / Q000032:analysis*; D045744:Cell Line, Tumor; D015894:Genome, Human / Q000276:immunology*; D006801:Humans; D007150:Immunohistochemistry; D040081:Protein Array Analysis / Q000379:methods; D020543:Proteome / Q000276:immunology*; D057170:Translational Research, Biomedical
D000911:Antibodies, Monoclonal; D015415:Biomarkers; D020543:Proteome