A Novel Universal Detection Agent for Time-Gated Luminescence Bioimaging.
Luminescent lanthanide chelates have been used to label antibodies in time-gated luminescence (TGL) bioimaging. However, it is a challenging task to label directly an antibody with lanthanide-binding ligands and achieve control of the target ligand/protein ratios whilst ensuring that affinity and avidity of the antibody remain uncompromised. We report the development of a new indirect detection reagent to label antibodies with detectable luminescence that circumvents this problem by labelling available lysine residues in the linker portion of the recombinant fusion protein Linker-Protein G (LPG). Succinimide-activated lanthanide chelating ligands were attached to lysine residues in LPG and Protein G (without Linker) and the resulting Luminescence-Activating (LA-) conjugates were compared for total incorporation and conjugation efficiency. A higher and more efficient incorporation of ligands at three different molar ratios was observed for LPG and this effect was attributed to the presence of eight readily available lysine residues in the linker region of LPG. These Luminescence-Activating (LA-) complexes were subsequently shown to impart luminescence (upon formation of europium(III) complexes) to cell-specific antibodies within seconds and without the need for any complicated bioconjugation procedures. The potential of this technology was demonstrated by direct labelling of Giardia cysts and Cryptosporidium oocysts in TGL bioimaging.
Macquarie University, Department of Chemistry and Biomolecular Sciences, Sydney, NSW 2109, Australia.|Macquarie University, ARC Centre of Excellence for Nanoscale BioPhotonics, Sydney, NSW 2109, Australia.
Nima
N
Sayyadi
Macquarie University, Department of Chemistry and Biomolecular Sciences, Sydney, NSW 2109, Australia.|Macquarie University, ARC Centre of Excellence for Nanoscale BioPhotonics, Sydney, NSW 2109, Australia.
Andrew
A
Care
Macquarie University, Department of Physics and Astronomy, Sydney, NSW 2109, Australia.
Russell E
RE
Connally
Macquarie University, Department of Chemistry and Biomolecular Sciences, Sydney, NSW 2109, Australia.
Andrew C
AC
Try
Macquarie University, Department of Chemistry and Biomolecular Sciences, Sydney, NSW 2109, Australia.|University of Auckland, Department of Molecular Medicine and Pathology, Auckland, 92019, New Zealand.
Peter L
PL
Bergquist
Macquarie University, Department of Chemistry and Biomolecular Sciences, Sydney, NSW 2109, Australia.
Anwar
A
Sunna
D002614:Chelating Agents / Q000737:chemistry* / Q000494:pharmacology; D003458:Cryptosporidium / Q000302:isolation & purification / Q000472:pathogenicity; D005063:Europium / Q000737:chemistry; D005872:Giardia / Q000302:isolation & purification / Q000472:pathogenicity; D028581:Lanthanoid Series Elements / Q000737:chemistry* / Q000494:pharmacology; D008024:Ligands; D049449:Luminescence; D008163:Luminescent Measurements / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000494:pharmacology
D002614:Chelating Agents; D028581:Lanthanoid Series Elements; D008024:Ligands; D011993:Recombinant Fusion Proteins; D005063:Europium