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Complementing IHC with real‐time interaction analysis on tissue sections Hanna Björkelund Ridgeview Instruments AB ridgeview instruments ab Ridgeview Instruments • Uppsala‐based company • LigandTracer – Study protein‐cell interactions in real‐time – Existing products – Radioactivity, soon fluorescence • TissueTracer – Study the binding of ligands (mainly antibodies) to tissue sections in real‐time – Possible result: A way of stratifiying cancer patients using this knowledge ridgeview instruments ab Tissue analysis today Immunohistochemistry (IHC) • Compare expressions in tumor and normal tissue Problems • Requires experience – operator dependent • Call for standardization • End‐point measurement: Risk for blind spots ridgeview instruments ab http://www.wjso.com/content/3/1/38 TissueTracer strategy • Instead of just looking at the amount of receptors on a tissue: try looking at the dynamic behavior of the interaction • The way (how fast, how strong) an antibody binds potentially reveals more than”how much” • Complement IHC analysis with binding traces Signal Time ridgeview instruments ab Affinity is only half of the truth Compare simulated binding traces for three antibodies with 1nM affinity and different interaction dynamics. Look at 2 hours of incubation at 10 nM concentration, then 7 hours of retention. 100 % bound target 0 0 2 4 6 8 h ridgeview instruments ab Link to IHC 100 % bound target Optimize wash Optimize incubation 0 0 2 4 6 8 h ridgeview instruments ab Binding traces on tissue... • Solution? Study the binding as it happens using LigandTracer – Operator independent – Real‐time measurements • LigandTracer today: – Protein‐cell interactions – Protein‐protein interactions • In this project: – Tissue sections ridgeview instruments ab LigandTracer technology Target cells with bound protein Detector Buffer with labeled protein Cell‐dish Motor ridgeview instruments ab Radiation intensity Peak height LigandTracer technology Time (min) ridgeview instruments ab Results: Anti‐HER2 + Anti‐IgG‐FITC binding to SKOV3 tissue A specific signal for anti‐IgG‐FITC is observed using SKOV3 tissue incubated with Anti‐HER2 ridgeview instruments ab Breast cancer High binding level High staining Tonsill Medium binding level Medium staining Placenta No binding Low staining ridgeview instruments ab Novacastra Summary • We are developing a tool for studying molecules binding to tissue in real‐time • A complement to IHC • A modification of LigandTracer, does not require much alterations • Promising results with detectable signals from specific binding ridgeview instruments ab Thank you for listening! • Acknowledgments – – – – – Ass. Prof Lars Gedda Stiftelsen för strategisk forskning Ass. Prof Kenneth Wester HPR group, Uppsala Biomedical radiation sciences, Uppsala University www.ligandtracer.com ridgeview instruments ab
