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Stereoselective method for high-yield synthesis of 4-trifluoromethylumbelliferyl-~-D-galactopyranoside -fluorigenic substrate for 0-galactosidase e--li, as enzyme label for ELISA, has been developed. The substrate is readily available in pure and stable form, nonfluorescent under the detection conditions and has no significant nonenzymatic degradation to fluorescent products. Compared to the well known 4-methylumbelliferone ( Aex = 360 nm, Aem = 450 nm), 4-trifluoromethylumbelliferone has a slightly large Stokes shift (Aex = 400 nm,hem = 500 nm), a longer excitation wavelength and emits green fluorescence through the enzymatic reaction. In the present ELISA we used 4-trifluoromethylumbelliferyl-~-D-galactopyranoside as sensitive enzyme substrate for rapid and visual screening of desired hybridoma clones producing monoclonal antibodies. With this immunoassay we succesfully selected antiperoxidase monoclonal antibodies which inhibited peroxidase activity.

Monoclonal antibodies (mcAbs) specific to alkaline isoenzymes of horseradish peroxidase were used to characterize the antigenic properties of horseradish peroxidase. The results of a competitive binding assay indicated that monoclonal antibodies can be divided into three groups directed against distinct parts of the protein. The interaction of monoclonal antibodies with native and modified horseradish peroxidase showed also three different patterns of reactivity. Antibodies from groups I and II are directed against epitopes which are conformational and formed by tertiary structure elements. Epitopes recognized by these antibodies are sensitive to heme removal or partial denaturation of peroxidase. Antibodies from group III bind specifically with epitopes consisting of primary or secondary structure elements. The antigenic determinants recognized by antibodies from group III PO(1) and 36F(9) were shown to be linear (continuous) and formed by amino acid residues 261-267 and 271-277, respectively, as determined by the peptide scanning method (PEPSCAN). The location of revealed linear antigenic determinants in the molecular structure of peroxidase is analyzed.