Application of an enhanced luminol chemiluminescence reaction using 4‐[4,5‐di(2‐pyridyl)‐1 H ‐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)‐1 H ‐imidazol‐2‐yl]phenylboronic acid (DPPA) as an enhancer is described. The method is based on the long‐lived chemiluminescence emission obtained 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 detected as visible spots with exposure time of 60 and 10 min, respectively, by using an instant photographic film and a camera luminometer. The proposed method was highly sensitive and was successfully applied to the detection of HRP conjugates as an alternative to the colorimetric method using a chromogenic substrate in a commercially available assay kit of Western blotting. Copyright © 2001 John Wiley & Sons, Ltd.