RESUMEN
A panel of monoclonal antibodies specific for the Clostridium perfringens alpha-toxin was produced by the fusion of X63.Ag8-653 cells with splenocytes from mice immunized either intrasplenically or intraperitoneally with an alpha-toxoid. The toxin-binding activity of each monoclonal antibody was evaluated. The monoclonal antibodies were also screened for their toxin-neutralizing potential in vitro, as determined by the inhibition of phospholipase C and hemolytic activities. In vivo inhibition of toxicity was assessed by the survival of mice challenged with preincubated alpha-toxin-antibody mixtures. Only one monoclonal antibody (3A4D10) was protective in vivo and neutralizing in both in vitro assays. Since 3A4D10 could inhibit both activities, the evidence suggests that these are colocated in the same area of the toxin molecule. This paper identifies a significant continuous linear binding region for 3A4D10 at positions 193 to 198 in the primary amino acid sequence of alpha-toxin.
Asunto(s)
Toxinas Bacterianas/inmunología , Proteínas de Unión al Calcio , Clostridium perfringens/metabolismo , Epítopos/análisis , Fosfolipasas de Tipo C , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Clostridium perfringens/inmunología , Femenino , Inmunización , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Pruebas de NeutralizaciónRESUMEN
A small-scale method for the extraction of the K88 major fimbrial subunit from enterotoxigenic Escherichia coli (ETEC) based on heat extraction has been developed. Variation in the buffer composition, time and temperature of extraction had negligible effect on the subsequent SDS-PAGE profile. There was, however, a correlation between the pH of the extraction buffer and the ensuing amount of K88 released. Reduction of the pH from 7 to 4 reduced by six-fold the amount of K88 released from the cells. We suggest that the relative stability of the K88 fimbriae at acid pH may influence the site of infection by ETEC in the intestine.
Asunto(s)
Antígenos Bacterianos/aislamiento & purificación , Antígenos de Superficie/aislamiento & purificación , Proteínas de Escherichia coli , Escherichia coli/inmunología , Proteínas Fimbrias , Tampones (Química) , Electroforesis en Gel de Poliacrilamida , Concentración de Iones de Hidrógeno , Métodos , Temperatura , Factores de TiempoRESUMEN
A monoclonal antibody (BALB/c mouse) with specificity for a neutralizing epitope on the epsilon-toxin produced by Clostridium perfringens type D was used to raise anti-idiotypic antibodies (anti-Id) in different strains of mice and rabbits. These were purified and used in cross-immunization studies to induce anti-(anti-idiotype). All strains of mice and rabbits immunized with BALB/c-derived anti-Id showed a high-titer antibody response directed towards the active site of the toxin. This protected the animals against toxin challenge and against an oral dose of the vegetative organisms. Animals immunized with other anti-Id preparations showed no specific antibody response and were not protected. Guinea pig peritoneal macrophages have a cell surface receptor for the toxin, and incubation of these cells with BALB/c anti-Id allowed them to survive toxin challenge, indicating that occupation of the receptors by the anti-Id prevented binding by the toxin. In conclusion, we have shown that an internal-image anti-Id preparation will induce protective immunity in syngeneic and xenogeneic animals and furthermore that immunity to a single epitope on the exotoxin is sufficient to protect against the toxin and clinical sequelae evoked by the disease-causing organism itself.
Asunto(s)
Anticuerpos Antiidiotipos/administración & dosificación , Infecciones por Clostridium/prevención & control , Clostridium perfringens/inmunología , Animales , Anticuerpos Antiidiotipos/biosíntesis , Anticuerpos Antiidiotipos/inmunología , Anticuerpos Antibacterianos/biosíntesis , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/inmunología , Toxinas Bacterianas/administración & dosificación , Toxinas Bacterianas/inmunología , Infecciones por Clostridium/inmunología , Infecciones por Clostridium/mortalidad , Epítopos/inmunología , Femenino , Cobayas , Inmunización , Masculino , Ratones , Ratones Endogámicos , ConejosRESUMEN
A fragment of DNA containing the gene coding for the phospholipase C (alpha-toxin) of Clostridium perfringens was cloned into Escherichia coli. The cloned DNA appeared to code only for the alpha-toxin and contained both the coding region and its associated gene promoter. The nucleotide sequence of the cloned DNA was determined, and an open reading frame was identified which encoded a protein with a molecular weight of 42,528. By comparison of the gene sequence with the N-terminal amino acid sequence of the protein, a 28-amino-acid signal sequence was identified. The gene promoter showed considerable homology with the E. coli sigma 55 consensus promoter sequences, and this may explain why the gene was expressed by E. coli. The cloned gene product appeared to be virtually identical to the native protein. A 77-amino-acid stretch that was close to the N terminus of the alpha-toxin showed considerable homology with similarly located regions of the Bacillus cereus phosphatidylcholine, preferring phospholipase C and weaker homology with the phospholipase C from Pseudomonas aeruginosa.