hymecromone and 2-nitrophenylgalactoside

The enzyme beta-galactosidase present in the Kupffer cells of the liver has potential as a marker of liver dysfunction prior to transplantation. Spectrophotometric methods have insufficient sensitivity.. Fluorimetric methods have the required sensitivity and we have optimised such a method in a microtitre plate format to improve its utility. beta-galactosidase acts on the substrate 4-methylumbelliferyl-galactoside (MUG) to produce 4-methylumbelliferone (4-MU), detected fluorimetrically with excitation wavelength 355 nm and emission wavelength 460 nm.. Reaction conditions in a citrate-phosphate buffer were optimised to give maximal enzyme activity: pH was optimal at 4.4 (range investigated 3.6-5.0) and substrate concentration at 3.33 mmol/l. A small specimen volume (10 microl) in 80 microl of substrate solution produced adequate fluorescent yield after an incubation period of 30 to 60 min at 37 degrees C. Reaction was terminated by addition of 200 microl of glycine-NaOH, pH 12.8. The assay is linear to 3,000 U/ml. The intra-assay coefficient of variation (CV%) at 50, 502, and 2,012 U/ml was 4.7, 3.1, and 3.4, respectively (n=10). Inter-assay CV% at 51, 496, and 1,986 U/ml was 7.0, 4.0, and 3.9, respectively (n=10).. The assay has greater practical utility and demonstrated significant differences in the perfusate beta-galactosidase between cold-stored and warm-perfused livers in a porcine model of transplantation.

Topics: Animals; beta-Galactosidase; Buffers; Calibration; Citrates; Fluorometry; Humans; Hydrogen-Ion Concentration; Hymecromone; Kinetics; Liver; Liver Diseases; Liver Transplantation; Models, Biological; Nitrophenylgalactosides; Phosphates; Reproducibility of Results; Sensitivity and Specificity; Swine

The use of 4-methylumbelliferyl phosphate (MUP) and ortho-nitrophenyl-beta-D-galactopyranoside (ONPG) for the identification of Clostridium perfringens was investigated. A liquid assay containing both MUP and ONPG was a highly specific alternative method for C. perfringens confirmation, reducing incubation time from 48 to only 4 h. The assay solution is easy to prepare, does not require anaerobic conditions for use, and has an extended shelf life.

Topics: Acid Phosphatase; Bacterial Typing Techniques; Chromogenic Compounds; Clostridium perfringens; Culture Media; Fluorescent Dyes; Hymecromone; Nitrophenylgalactosides; Sensitivity and Specificity; Time Factors

We have previously established a non-competitive solid-phase enzyme-linked immunosorbent assay (ELISA) specific for interleukin-1 alpha (IL-1 alpha) using a combination of polyclonal antibody as the immobilized antibody, biotinylated monoclonal antibody as the second antibody and avidin-peroxidase. The level of detection of that ELISA was 200-500 pg/ml. In order to improve its sensitivity, we have used streptavidin-beta-D-galactosidase and the fluorogenic substance 4-methylumbelliferyl-D-galactopyranoside as enzyme substrate. With this system IL-1 alpha could be detected at concentrations as low as 10-50 pg/ml, which was about 10-20 times more sensitive than conventional mouse thymocyte co-stimulator assays. Furthermore, the assay system was specific for IL-1 alpha in that neither IL-1 beta nor interleukin-2 (IL-2) interfered.

Topics: Antibodies, Monoclonal; Biological Assay; Biotin; Enzyme-Linked Immunosorbent Assay; Galactosides; Humans; Hymecromone; Interleukin-1; Nitrophenylgalactosides; Recombinant Proteins