AI Article Synopsis
- A multiplex assay was developed to detect 8 breast cancer biomarkers on a single tissue slide using staining and imaging technologies from General Electric.
- An unexpected membranous staining of Ki-67 was found to be an artifact resulting from the binding of two antibodies in the multiplexing process.
- By blocking with rabbit serum, researchers established a successful method to multiplex the biomarkers without any antibody cross-reactivity.
Article Abstract
In the process of developing a multiplex of 8 common breast cancer biomarkers (Her2/neu, estrogen receptor, progesterone receptor, Ki-67, aldehyde dehydrogenase-1, NaK-ATPase, cytokeratin 8/18, and myosin smooth muscle) on a single formalin-fixed paraffin-embedded slide using a sequential staining, imaging, and dye bleaching technology developed by General Electric Company, membranous Ki-67 staining was observed and colocalized with Her2/neu staining. Using immunohistochemistry as gold standards, we discovered that membranous Ki-67 was an artifact caused by the binding of cyanine 5-conjugated rabbit polyclonal Ki-67 antibody to a secondary cyanine 3-conjugated donkey anti-rabbit antibody which was previously applied and bound to rabbit Her2/neu antibody in our multiplexing experiment. After blocking with rabbit serum, a successful protocol for 8 biomarker multiplexing without cross-reactivity of antibodies from the same species was developed.
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| http://dx.doi.org/10.1097/PAI.0000000000000202 | DOI Listing |
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