Automated measurement of immunogalactosidase reactions with a fluorogenic substrate by the aperture defined microvolume measurement method and its potential application to Schistosoma mansoni immunodiagnosis

Abstract

For automated read-out of immunogalactosidase assays with a fluorogenic substrate, performed in microtitration trays, an inverted fluorescence microscope equipped with a photometer and a scanning stage and operated by a microprocessor has been used. Trays (60 and 96 wells) were scanned in 1 min and corrected measurements and a histogram of the results printed out in a few minutes. A lower level of 4 ng of the reaction product, 4-methylumbelliferone, was detected. By making the measuring system flexible we were also able to use it for fluorescence measurements of individual agarose beads or cells and for extinction measurements in ELISA reactions with a chromogenic substrate. On comparing FITC and galactosidase conjugates in a DASS-test, and peroxidase and galactosidase conjugates in an ELISA, with Schistosoma mansoni infection as test system, it appeared that the galactosidase modification of both assays was 30-50 times more sensitive than the FITC or peroxidase modification. For S. mansoni egg antigen, a lower detectable level of 0.1 ng antigen/ml was achieved.