Effect of monoclonal antibody to pertussis toxin on toxin activity

Abstract

Two distinct monoclonal antibodies, one to pertussis toxin subunit S2, called 9G8, and another to subunits S2 and S3, called 11E6, were generated from the hybridomas of myeloma SP2/0 and spleen cells of BALB/c mice immunized mainly with the subunit S234 complex. Binding ability of 9G8 and 11E6 to the subunits was confirmed by the enzyme-linked immunosorbent assay and immunoblotting analysis. Generation of 11E6 bound to both S2 and S3 might mean that there is common antigenicity between S2 and S3. Neutralizing activities of 9G8 and 11E6 on various biological activities of pertussis toxin, including ADP-ribosyltransferase and leukocytosis-promoting, islet-activating, permeability-increasing. Chinese hamster ovary (CHO) cell-clustering, and hemagglutinating activities, were compared with those of anti-S1 monoclonal antibodies 1B7 and 3F10, which were isolated and characterized in a previous study (H. Sato, A. Ito, J. Chiba, and Y. Sato, Infect. Immun. 46:422-428, 1984). 1B7 and 3F10 neutralized ADP-ribosyltransferase activity of pertussis toxin or S1, but 9G8 and 11E6 did not. 1B7 showed very potent neutralization against leukocytosis-promoting, islet-activating, permeability-increasing, and CHO cell-clustering activities of pertussis toxin, but 3F10 did not, although anti-ADP-ribosyltransferase activities of both antibodies were identical. 11E6 neutralized leukocytosis-promoting, islet-activating, CHO cell-clustering, and hemagglutinating activities but not permeability-increasing activity. 9G8 showed slight neutralization of leukocytosis-promoting and CHO cell-clustering activities. Specific activities of 1B7 and 11E6 in each neutralization test were higher than or almost comparable to those of polyclonal antibodies to pertussis toxin. The neutralizing mechanism of 1B7 and 11E6 in leukocytosis-promoting activity was compared. 11E6 seemed to interfere with the binding of pertussis toxin to receptors on mouse spleen cells.

Stable expression of pertussis toxin in Bordetella bronchiseptica under the control of a tightly regulated promoter

“…The biological activity of the purified PT was determined by the Chinese hamster ovary (CHO) cell-clustering test as described by Hewlett et al (14). Neutralization of CHO cell-clustering by antibody E19 (38) was carried out as described by Sato et al (29). The relative CHO cell-clustering activity was defined as the ratio between the apparent concentration of recombinant PT determined by the CHO cell-clustering assay and the absolute concentration determined by dot-immunoblotting experiments, expressed as a percentage.…”

Humanised monoclonal antibodies neutralise pertussis toxin by receptor blockade and reduced retrograde trafficking

“…These are formed by two or more antibodies binding simultaneously to one toxin molecule and are rapidly cleared via the increased avidity of multimerized Fc for Fcγ receptors (Montero-Julian et al, 1995). Hu11E6 binds an epitope present in each of the homologous S2 and S3 subunits, which means two high-affinity epitopes are present per toxin molecule (Sato et al, 1987). Hu1B7 binds a single epitope on the S1 subunit with weak binding to the S4 subunit on the B-oligomer (Sutherland & Maynard, 2009).…”

“…Antibody hu11E6 binds an epitope in the S2 and S3 subunits of the B-oligomer, which also contains the receptor-binding epitope. This antibody has been proposed to interfere with PTx binding to receptors (Sato et al, 1987).…”

“…Prior work defined the hu1B7 epitope with amino acid-level resolution (Kowalsky et al, 2015;Sutherland & Maynard, 2009), revealing an epitope that is dominated by S1 but appearing to bridge the subunit interface to weakly engage the hetero-pentameric PTx B subunit (Sutherland & Maynard, 2009). The m11E6 epitope has been determined to include the S2/3 subunits near the receptor-binding site (Sato, Sato, Ito, & Ohishi, 1987). Neutralising antibodies can potentially interfere with any of the steps involved in PTx secretion, receptor binding, and intracellular trafficking (Wagner & Maynard, 2018).…”

Characterization of murine monoclonal antibodies that recognize defined epitopes of pertussis toxin and neutralize its toxic effect on Chinese hamster ovary cells

“…The myeloma used in the fusion experiment was X63Ag8 (10). Hybridoma supernatant fluids were screened for PT subunit recognition by Western blot analysis carried out essentially as described by Burnette (3) and by inhibition of CHO cell clustering (7, 8) as described by Sato et al (22). PT was electrophoresed through a polyacrylamide gel by the method of Laemmli (13) by using a 3.85% acrylamide stacking gel and a 15% acrylamide separating gel.…”