The conjugation of an AQP1-directed immunotoxin in the study of site-directed therapy within the CNS
- نوع فایل : کتاب
- زبان : انگلیسی
- مؤلف : Surash Surash & Peter Nemeth & Aruna Chakrabarty & Paul Chumas
- چاپ و سال / کشور: 2010
Description
Purpose The water channel, aquaporin (AQP)1, is highly specific to the choroid plexus (CP) epithelium within the brain. It is therefore a potential target through which therapeutic agents could be selectively directed to the CP. Here we describe the conjugation of a monoclonal antibody (mAb), raised against an extra-cellular domain of AQP1, to the A chain of ricin (RCA). This reagent should allow study of a highly specific chemical lesion of the CP. Methods A published method was used to couple the anti- AQP1 mAb to the A chain of ricin using an SPDP crosslinker. The conjugate was purified by Superdex S-200 chromatography. Column fractions were analysed by SDSPAGE and Western blotting. Fractions containing conjugate were assayed for cytotoxicity on rat CP cells in culture, using a WST-1 viability assay to assess cell death. Results SDS-PAGE and Western blotting demonstrated separation of conjugated anti-AQP1 mAb from unconjugated ricin by the Superdex column. Cultured rat CP cells were killed with an IC50 of 1.81 ىg/ml when exposed to the anti-AQP1 mAb-RCA conjugation reaction products. Selectivity of this reagent was demonstrated by the higher IC50 exhibited by non-AQP1-expressing primary fibroblast cultures and cultures exposed to the reaction products from the non-specific IgG-RCA conjugation attempt. Conclusion We have been able to demonstrate selective cytotoxicity in vitro of an anti-AQP1 mAb-RCA conjugate. We hope that this work will generate further interest in the use of this site-specific conjugate to target the CP in conditions such as hydrocephalus, tumours that arise from here, and other AQP1-expressing tumours
Childs Nerv Syst (2011) 27:811–818 DOI 10.1007/s00381-010-1336-2 Received: 9 February 2010 / Accepted: 9 November 2010 / Published online: 23 November 2010
