Charlotte D. Parker

Isolation and characterization of monoclonal antibodies to Bordetella pertussis

Monoclonal antibodies to Bordetella pertussis were produced by fusion of mouse myeloma cells and spleen cells of immunized mice. Cell lines were examined for specific antibody production against several crude antigen preparations and lipopolysaccharide. Cross reactivity of monoclonal antibodies was assessed by enzyme immunoassay using cell lysates prepared from Bordetella spp. and several other bacteria. Reactivity of monoclonal antibodies to cell surface components was determined by immunofluorescence microscopy. Monoclonal antibodies represent useful probes to study the antigenic profile and distribution of antigens among various species of Bordetella, as well as specific tools to study the structure and function of B. pertussis virulence factors.

Regulation of intestinal mucosa guanylate cyclase by hemin, heme and protoporphyrin IX

Mg2+-dependent activity of intestinal brush border guanylate cyclase was stimulated 4-5-fold by 50-100 microM hemin. Higher concentrations were inhibitory. In the presence of 25% dimethyl sulfoxide, which stimulated activity 9-times, 50 microM hemin further increased activity 1.7-fold. However, when activity was stimulated 32-fold by the Escherichia coli heat-stable enterotoxin, or 26-fold by Lubrol PX, hemin produced only concentration-dependent inhibition. The first type of activation was more sensitive to hemin than the second. Reduction of hemin by dithiothreitol eliminated stimulation of basal activity, while inhibition of Lubrol PX-stimulated activity remained. Protoporphyrin IX also had no effect on basal activity, however, it inhibited enterotoxin- and Lubrol PX-stimulated activities similarly, but only to half the extent of hemin. Substitution of Mn2+ for Mg2+ elevated basal activity 15-fold, and this Mn2+-dependent activity was inhibited by hemin. Mn2+-dependent activity was stimulated (43%) by enterotoxin, however, the stimulated activity was more sensitive to hemin inhibition than the basal Mn2+-dependent activity and both inhibition curves were congruent above 50 microM hemin. Hemin inhibition of Lubrol PX-stimulated activity was much less with Mn2+ than with Mg2+. These results were interpreted as suggesting two sites of hemin inhibition; on an inhibitory regulator and on the enzyme. We also found that the secretory effect of enterotoxin in the suckling mouse bioassay was reduced 56% by the oral administration of hemin.

Solubilization and reprecipitation from intestinal brush border membranes of a complex containing guanylate cyclase activatable by the heat-stable enterotoxin

Extraction of pig intestinal brush border membranes with the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (Chaps) in the presence of 0.5 M KCl yielded a solution which contained 60-70% of the receptor for the Escherichia coli heat-stable enterotoxin (STa) and of the Lubrol PX-activated guanylate cyclase activity present in the membrane. When the supernatant solution was diluted fivefold with 10 mM Hepes buffer (pH 7.4) and kept at 4 degrees C overnight, a precipitate formed. Centrifugation yielded a pellet (P2) which contained 25-30% of both the cyclase and the receptor in the original membranes, with a 2.5- to 3-fold enrichment of both. The process could be repeated for further enrichment (P4). The addition of MgCl2 to the diluted extract affected both basal and STa-stimulated activity of P2; 1 mM was optimal. P2 resembled membranes with respect to competitive inhibition of 125I-STa binding by STa, and the concentration-dependent activation of cyclase by STa. Guanylate cyclase in resolubilized P2 was also activated by STa. Most of the enzymes interfering with guanylate cyclase determinations were removed, as were the brush border marker enzymes sucrase and gamma-glutamyltransferase, and a GTP-binding protein that is a pertussis toxin substrate. Specific cross-linking of 125I-STa to receptors in the membrane was preserved in P2 and P4, the three proteins showing the strongest radioactivity having relative molecular masses of 55,000-60,000, 70,000-80,000, and 135,000-140,000. P2 and P4 appear to contain a complex of membrane proteins with certain functional properties intact.

Surface antigens of Bordetella pertussis

Bordetella pertussis and other Bordetella species cause respiratory infections in humans and in a variety of animals. Clinical isolates of B. pertussis have multiple virulence factors, several of which have been reported to induce protective immunity. Using cell surface iodination techniques and monoclonal antibody immunoblots we have identified several proteins which are exposed on the surface of B. pertussis cells, including the filamentous hemagglutinin and outer membrane proteins 91, 18, and 15. Protein 91 is unique to virulent B. pertussis strains. Antibodies to protein 18 are found in convalescent serum of both humans and mice recovering from infection with B. pertussis.

Monoclonal Antibodies to Bordetella pertussis

Publisher Summary Bordetella pertussis is a small gram-negative coccobacillus that causes whooping cough, undergoes frequent antigenic variations. Recently, monoclonal antibodies have been prepared that react with B. pertussis and its components. This chapter discusses the utility of such antibodies for investigation of relevant B. pertussis antigens. The major antigens for identifying and serotyping B. pertussis are six agglutinogens, so called because they induce agglutinating antibody. Bordetella pertussis also produces a variety of noxious substances involved in pathogenesis of the disease. Major virulence antigens include pertussis toxin (PT), adenylate cyclase, and filamentous hemagglutinin (FHA). Several monoclonal antibodies have been induced to B. pertussis and its products, such as FHA, PT, outer membrane proteins, lipopolysaccharide, and unidentified antigens. Antibodies to FHA showed that FHA is not fimbrial in nature but lies on the cell surface. Antibodies to PT have shown reactions with specific subunits of the toxin, neutralizing toxicity only if they react with subunit. Monoclonal antibodies have been used in immunoaffinity chromatography to purify both FHA and PT from culture fluids. Monoclonal antibodies are likely to play an important role in defining the antigens of B. pertussis, in providing diagnostic reagents, and in purifying cell components.