Sensitivity limitations encountered in enhanced horseradish peroxidase catalysed chemiluminescence

Journal of Bioluminescence and Chemiluminescence
M A Motsenbocker

Abstract

Conditions for the enhanced horseradish peroxidase (HRP) catalysed reaction between luminol and hydrogen peroxide were optimized to determine detection limits for HRP conjugated to antibody fragment (HRP-Fab) in solution phase. Light output was linear with respect to HRP-Fab concentration but became nonlinear at low HRP-Fab concentrations when an accelerator (enhancer) of the reaction was used. para-Phenylphenol was a more effective enhancer than p-iodophenol at HRP-Fab concentrations below 20 pmol/l. The detection limit for HRP-Fab was 1.2 femtomoles in the absence of p-phenylphenol and 0.08 femtomole in the presence of p-phenylphenol. The acceleration of peroxidase activity at the lowest HRP-Fab concentrations occurred after an incubation time period of up to five minutes. This lag time limited the sensitivity and the mechanism for it was sought. Preincubation experiment results indicated that the lag time phenomenon may involve a reversible alteration in HRP catalytic activity and that enhancer, peroxide, luminol and HRP-Fab had to be incubated together some time before maximum activation could occur.

Citations

Jun 30, 1994·Journal of Biotechnology·C Kessler
Nov 19, 2010·Bioanalysis·Christophe A Marquette, Loïc J Blum
Feb 1, 1994·Scandinavian Journal of Clinical and Laboratory Investigation·D NelsonJ Börjeson
Aug 28, 2007·Electrophoresis·Edita AksamitieneAnatoly Kiyatkin
Jul 1, 1989·Journal of Bioluminescence and Chemiluminescence·F McCapraJ Branson
Jan 1, 1994·Journal of Bioluminescence and Chemiluminescence·M A Motsenbocker, K Kondo
Sep 16, 1992·Biochemical and Biophysical Research Communications·J M BinstockA L Southren

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