Plasma protein binding of luciferase substrates influences sensitivity and accuracy of bioluminescence imaging.
BACKGROUND
Potential confounding factors in bioluminescence imaging (BLI)-quantification need to be identified to improve its accuracy and repeatability.


OBJECTIVE
The aim of this study was to study the degree to which plasma protein binding of BLI substrates influences signal intensity and quantification, both in vitro and in vivo.


METHODS
Protein binding was assessed using ultrafiltration and spectrophotometry. Effects of increasing serum protein concentrations were studied in vitro, in intact cells expressing Firefly or Renilla luciferase, and in vivo, in a doxorubicin-induced hypoalbuminemia mouse model.


RESULTS
Increasing concentrations of serum proteins showed an important negative effect on BLI signal intensity, both for D-luciferin and coelenterazine-dependent reactions. This was due to a decrease in the free fraction of the substrate in the presence of serum proteins of up to 88%. In vivo, hypoalbuminemia was associated with higher BLI signal intensity, although this was only significant in animals with a major decrease in albumin (<2 g/dL). Important kinetic differences, including earlier peak luminescence and a more rapid decline in luminescence, were observed for coelenterazine-dependent BLI under hypoalbuminemic conditions. Changes in protein concentrations can significantly influence BLI quantification and its presence decreases sensitivity in vitro. In vivo, effects on signal intensity and kinetics are only noted at severe hypoalbuminemia and can be neglected in most experimental designs. These findings again emphasize the need for careful interpretation of BLI quantification data.
Keyaerts
Marleen
M
In Vivo Cellular and Molecular Imaging Laboratory, Vrije Universiteit Brussel, Brussels, Belgium. mkeyaerts@gmail.com
Heneweer
Carola
C
Gainkam
Lea O Tchouate
LO
Caveliers
Vicky
V
Beattie
Bradley J
BJ
Martens
Geert A
GA
Vanhove
Christian
C
Bossuyt
Axel
A
Blasberg
Ronald G
RG
Lahoutte
Tony
T
D000818:Animals; D001798:Blood Proteins / Q000378:metabolism*; D002460:Cell Line; D008156:Luciferases / Q000378:metabolism*; D049449:Luminescence; D008297:Male; D051379:Mice; D008819:Mice, Nude; D013379:Substrate Specificity
D001798:Blood Proteins; D008156:Luciferases
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