Application of xanthine oxidase-catalyzed luminol chemiluminescence in a mouse interleukin-5 immunoassay.
A chemiluminescent substrate reagent for use in a sandwich immunoassay for the model antigen mouse interleukin-5 (IL-5) was developed using xanthine oxidase and luminol. Various parameters involved in this chemiluminescent reaction have been studied, including the substrate hypoxanthine, luminol and the Fe(II)-EDTA complex. Addition of the Fe(II)-EDTA complex enhances the chemiluminescence signal considerably. The xanthine oxidase-catalyzed chemiluminescent immunoassay was compared to horseradish peroxidase-linked immunoassays with luminol as chemiluminescent, and tetramethyl benzidine as colorimetric substrate. The detection limit of the xanthine oxidase-luminol assay was found to be about 0.6 pg/ml IL-5, whereas the peroxidase-catalyzed immunoassays have detection limits of about 1.3 (HRP-TMB) and 2.9 pg/ml (HRP-luminol) IL-5.
Rongen
H A
HA
Department of Pharmaceutical Analysis, Faculty of Pharmacy, Utrecht University, Netherlands. h.a.h.rongen@far.ruu.nl
van der Horst
H M
HM
van Oosterhout
A J
AJ
Bult
A
A
van Bennekom
W P
WP
D000818:Animals; D004492:Edetic Acid / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005296:Ferrous Compounds / Q000494:pharmacology; D006735:Horseradish Peroxidase; D015848:Interleukin-5 / Q000032:analysis*; D008163:Luminescent Measurements; D008165:Luminol; D051379:Mice; D014969:Xanthine Oxidase
D005296:Ferrous Compounds; D015848:Interleukin-5; D008165:Luminol; D004492:Edetic Acid; D006735:Horseradish Peroxidase; D014969:Xanthine Oxidase; C029451:ferrous chloride