title: Optimising the radiolabelling properties of technetium tricarbonyl and His-tagged proteins.
abstract: BACKGROUND
  To date, the majority of protein-based radiopharmaceuticals have been radiolabelled using non-site-specific conjugation methods, with little or no control to ensure retained protein function post-labelling. The incorporation of a hexahistidine sequence (His-tag) in a recombinant protein can be used to site-specifically radiolabel with 99mTc-tricarbonyl ([99mTc(CO)3]+). This chemistry has been made accessible via a technetium tricarbonyl kit; however, reports of radiolabelling efficiencies and specific activities have varied greatly from one protein to another. Here, we aim to optimise the technetium tricarbonyl radiolabelling method to produce consistently >95% radiolabelling efficiencies with high specific activities suitable for in vivo imaging.
  
  
  METHODS
  Four different recombinant His-tagged proteins (recombinant complement receptor 2 (rCR2) and three single chain antibodies, α-CD33 scFv, α-VCAM-1 scFv and α-PSMA scFv), were used to study the effect of kit volume, ionic strength, pH and temperature on radiolabelling of four proteins.
  
  
  RESULTS
  We used 260 and 350 μL [99mTc(CO)3]+ kits enabling us to radiolabel at higher [99mTc(CO)3]+ and protein concentrations in a smaller volume and thus increase the rate at which maximum labelling efficiency and specific activity were reached. We also demonstrated that increasing the ionic strength of the reaction medium by increasing [Na+] from 0.25 to 0.63 M significantly increases the rate at which all four proteins reach a >95% labelling efficiency by at least fourfold, as compared to the conventional IsoLink® kit (Covidien, Petten, The Netherlands) and 0.25 M [Na+].
  
  
  CONCLUSIONS
  We have found optimised kit and protein radiolabelling conditions suitable for the reproducible, fast, efficient radiolabelling of proteins without the need for post-labelling purification.
authors.forename: Adam
authors.initials: A
authors.lastname: Badar
authors.forename: Jennifer
authors.initials: J
authors.lastname: Williams
authors.forename: Rafael Tm
authors.initials: RT
authors.lastname: de Rosales
authors.forename: Richard
authors.initials: R
authors.lastname: Tavaré
authors.forename: Florian
authors.initials: F
authors.lastname: Kampmeier
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Blower
authors.affiliation: Division of Imaging Sciences and Biomedical Engineering, King's College London, 4th Floor, Lambeth Wing, St Thomas' Hospital, London SE1 7EH, UK. greg.mullen@kcl.ac.uk.
authors.forename: Gregory Ed
authors.initials: GE
authors.lastname: Mullen