Development and characterization of murine monoclonal antibodies specific for dissimilatoric copper nitrite reductase.

Abstract

Several hybridoma cell lines from mice were established, producing monoclonal antibodies (MAbs) directed against the dissimilatoric copper nitrite reductase (dNIR) to detect actual denitrifying bacteria at the single cell level under nondestructive conditions in the environment. The mice were immunized with native or recombinant enzyme gained from two different bacteria, Ochrobactrum anthropi and Alcaligenes faecalis. The antibodies obtained could be divided into two groups according to their different specificities for dNIRs of different bacteria: One group of MAbs had a broad specificity for dissimilatoric copper nitrite reductases from bacteria of different phylogenetic taxa; the other group gave only a clear signal with the corresponding immunogen. None of the raised MAbs showed a cross reactivity with the dissimilatoric heme nitrite reductase. One MAb from each group (MAb dNIR1a and MAb dNIR29) has been selected for further investigation. Data of enzyme-linked immunosorbent assay (ELISA), Western blotting, and immunofluorescence-microscopy are presented and compared with phylogenetic data. Furthermore, results of Western blotting experiments with cells, grown without nitrate under aerobic conditions, and cells cultivated with nitrate under anaerobiosis, are shown.