Eden Padayachee and Stefan Barth
Health Science University of Cape Town, South Africa
Posters & Accepted Abstracts: J Neurol Neurophysiol
Objective: To design an IL4-specific diagnostic probe to image glioblastoma as part of a multiplex immunofluorescence imaging
Method: The DNA sequences for the single chain variable fragments (scFV) for IL4 was inserted into the SfiI and NotI-digested site of eukaryotic SNAP expression vector and restriction enzyme digestion and ligation was performed to engineer anti-IL4-SNAP. These fusion proteins were transfected into HEK293T cells using XtremeGene HP transfection reagent. Plasmid uptake was estimated by the microscopic viewing of GFP expression in live cells. Zeocin selection (100 ng/ml) was applied if transfection efficiency appeared low. Transfected cells will be grown in enriched media and supernatant containing secreted protein will be collected every 3-4 days for a period of ±3 weeks. Supernatants will be pooled and centrifuged to remove cells and purification was performed using immobilized metal affinity chromatography.
Results: Anti-IL4-SNAP was purified after GFP-expression in HEK293T mammalian cells, purified and conjugated to Alexa 488 fluorophore in a 1:1 stoichiometry and confocal microscopy and flow cytometry were used to confirm internalization and binding. After which the fusion protein was labeled with multiple fluorescent tags and applied to patient biopsies to achieve multiplex labeling to detect the type of a tumor important for further therapeutic treatment.
Conclusion: Glioblastoma is associated with a poor prognosis due to its aggressive progression, the absence of specific targeted therapy, resistance to conventional chemotherapeutic methods. Anti-IL4 SNAP is a diagnostic probe for specific diagnosis of glioblastoma and can be used with Alexa-488 in combination with cyanine dyes cy2, cy3 and cy5 to identify many biomarkers simultaneously in one sample to achieve complex SNAP-tag fusion protein multiplex labeling and allow for further therapeutic measures.