Application of an enhanced luminol chemiluminescence reaction using 4-[4,5-di(2-pyridyl)-1H-imidazol-2-yl]phenylboronic acid to photographic detection of horseradish peroxidase on a membrane

Photographic detection of horseradish peroxidase (HRP) on a membrane by the luminol-hydrogen peroxide-HRP chemiluminescence reaction using 4-[4,5-di(2 -pyridyl)-1H-imidazol-2-yl]phenylboronic acid (DPPA) as an enhancer is desc ribed. The method is based on the long-lived chemiluminescence emission obt ained by using DPPA. Under the optimum conditions, as little as 0.10 ng (ca . 2.3 fmol) and 0.20 ng (ca. 4.6 fmol) per spot of HRP on a membrane were d etected as visible spots with exposure time of 60 and 10 min, respectively, by using an instant photographic film and a camera luminometer. The propos ed method was highly sensitive and was successfully applied to the detectio n of HRP conjugates as an alternative to the colorimetric method using a ch romogenic substrate in a commercially available assay kit of Western blotti ng. Copyright (C) 2001 John Wiley & Sons, Ltd.