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To study the topology of Na+,K+‐ATPase monoclonal antibodies (MAbs) specific for membrane‐bound enzyme were produced. Using immunofluorescence staining of viable cells or smears of a pig kidney embryonic (PKE) cell line, two groups of MAbs were selected, namely those binding to extra‐ or intracellular portions of the α‐subunit. The extracellular location of peptide loop 804–841 linking the Vth and VIth intramembrane hydrophobic segments was proved using MAb VG2. Another MAb, IIC9, interacting with PKE cells only after membrane perforation (4% formaldehyde and 0.1% Tween‐20), was shown to bind to the hydrophilic loop 868–945. The antigenic determinants recognized by MAb IIC9 and VG2 are located in peptides 887–904 and 810–825, respectively. The C‐terminus of the α‐subunit molecule was positioned on the outer side of the cytoplasmic membrane utilizing affinity‐purified antibodies to the synthetic peptide corresponding to fragment 999–1008.

Using flow cytometry and fluorescence polarization analysis, specific muramyl peptide‐binding sites were shown to be located inside T‐lymphocytes, macrophages and neuroblastoma cells, but not inside B‐cells. No binding sites were found on the cell surface. The number of binding sites for each cell type was determined. Two types of binding sites were observed for myelomonocytic WEH1‐3 cells with K fd values of 21 and 540 nM. Inhibition analysis demonstrated that for effective binding, an intact glycopeptide molecule and D‐configuration of isoglutamine residue are important.

Mouse monoclonal antibodies against the most acutely toxic substances, botulinum neurotoxins (BoNTs) of types A, B, E, and F, was generated and characterized, that recognize their respective toxins in natural toxin complex. Based on these antibodies, we developed sandwich-ELISA for quantitative detection of these toxins. For each respective toxin the detection limit of the assay was: BoNT/A - 0.4 ng/ml, BoNT/B - 0.5 ng/ml; BoNT/E - 0.1 ng/ml; and for BoNT/F - 2.4 ng/ml. The developed assays permitted quantitative identification of the BoNTs in canned meat and vegetables. The BNTA-4.1 and BNTA-9.1 antibodies possessed neutralizing activity against natural complex of the botulinium toxin type A in vivo, both individually and in mixture, the mixture of the antibodies neutralized the higher dose of the toxin. The BNTA-4.1 antibody binds specifically the light chain (the chain with protease activity) of the toxin, whereas BNTA-9.1 interacts with the heavy chain. We believe that the BNTA-4.1 and BNTA-9.1 monoclonal antibodies are prospective candidates for development of humanized therapeutic antibodies for treatment of BoNT/A-caused botulism.