title: The conjugation of an AQP1-directed immunotoxin in the study of site-directed therapy within the CNS.
abstract: OBJECTIVE
  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 cross-linker. The conjugate was purified by Superdex S-200 chromatography. Column fractions were analysed by SDS-PAGE 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 IC(50) of 1.81 μg/ml when exposed to the anti-AQP1 mAb-RCA conjugation reaction products. Selectivity of this reagent was demonstrated by the higher IC(50) exhibited by non-AQP1-expressing primary fibroblast cultures and cultures exposed to the reaction products from the non-specific IgG-RCA conjugation attempt.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Neurosurgery, The General Infirmary at Leeds, Great George Street, Leeds, LS1 3EX, UK. ssurash@mac.com
authors.forename: Surash
authors.initials: S
authors.lastname: Surash
authors.forename: Peter
authors.initials: P
authors.lastname: Nemeth
authors.forename: Aruna
authors.initials: A
authors.lastname: Chakrabarty
authors.forename: Paul
authors.initials: P
authors.lastname: Chumas
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000633:toxicity*; D051398:Aquaporin 1 / Q000276:immunology; D015153:Blotting, Western; D016923:Cell Death / Q000187:drug effects; D002478:Cells, Cultured; D002831:Choroid Plexus / Q000187:drug effects*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007150:Immunohistochemistry; D007167:Immunotherapy / Q000379:methods*; D000922:Immunotoxins / Q000633:toxicity*; D009474:Neurons / Q000187:drug effects; D051381:Rats; D012276:Ricin / Q000633:toxicity*
chemical_list: D000911:Antibodies, Monoclonal; D000922:Immunotoxins; D051398:Aquaporin 1; D012276:Ricin