A method for the quantification of a colored or fluorescent signal in enzyme immunoassays by photodensitometry

A colored or fluorescent signal is generally evaluated with the naked eye, or by means of different more or less sophisticated and costly instruments. Photodensitometry is an additional technique which is both inexpensive and simple to perform. This technique can satisfactorily quantify a signal without the use of either a spectrophotometer or a fluorometer. In this study we compared readings obtained by spectrophotometry, fluorometry and photodensitometry in 96-well ELISA plates and in Terasaki plates. In ELISA plates, it is possible to detect 1220-300,000 femtograms (fg) of peroxidase by spectrophotometry and 4800-125,000 fg by photodensitometry. In Terasaki plates, we were able to measure between 3.8 and 8000 fg of beta-galactosidase per sample by spectrofluorometry, and from 30 to 8000 fg by photodensitometry. Using a sandwich procedure in Terasaki plates we were able to measure between 100 and 10,000 fg of IgE per sample by spectrofluorometry and from 500 to 10,000 fg by photodensitometry. Photodensitometry is the least expensive technique for the reliable detection of enzyme or enzymatic marker in small sample volumes treated with a fluorogenic substrate.