RESUMO
A simple method for the collection, preservation, shipment, and testing of minute amounts of dried monoclonal antibodies for typing Neisseria meningitidis B is described. The monoclonal antibodies collected on filter paper were extracted in PBS and evaluated by Dot-blot employing whole cells of N. meningitidis B as antigen. The dried filter paper with monoclonal antibodies could be stored at room temperature for as long as 30 days without detectable changes in antibody response when used for typing outer membrane antigens of N. meningitidis B.
Assuntos
Anticorpos Monoclonais , Immunoblotting/métodos , Neisseria meningitidis/classificação , Técnicas de Tipagem BacterianaRESUMO
A simple method for the collection, preservation, shipment, and testing of minute amounts of dried monoclonal antibodies for typing Neisseria meningitidis B is described. The monoclonal antibodies collected on filter paper were extracted in PBS and evaluated by Dot-blot employing whole cells of N. meningitidis B as antigen. The dried filter paper with monoclonal antibodies could be stored at room temperature for as long as 30 days without detectable changes in antibody response when used for typing outer membrane antigens of N. meningitidis B
Assuntos
Anticorpos Monoclonais , Immunoblotting , Técnicas In Vitro , Neisseria meningitidis/classificação , Técnicas de Tipagem BacterianaRESUMO
The predicted amino acid sequence was determined for the class-1 outer membrane protein, PorA, from a B:15:P1.7,3 strain of Neisseria meningitidis that is currently causing an epidemic of meningitis in Northern Chile. The P1.7,3 PorA showed a unique sequence in the exposed loop 4 of the putative porin structure that is different from all the reported PorA sequences. Based on the nucleotide (nt) sequence of the P1.7,3 porA, we designed two sets of PCR (polymerase chain reaction) primers that specifically amplified porA from any N. meningitidis strain, and a third set of primers that amplified porA only from the P1.7,3 strain. Using these primers, we developed a sensitive double hot-start nested PCR (HNPCR) strategy that could amplify porA and generate nt sequence from as low as a single colony-forming unit. This strategy consisted of three phases of PCR. The first two phases were designed to generate amplified target DNA that could be directly visualized by ethidium bromide staining starting from one to two molecules of Neisseria genome. The third phase was designed to generate a sequence of several hundred nt directly from the amplified DNA. A number of culture-negative cerebrospinal fluid samples from individuals suspected of meningitis during a vaccine trial were analyzed by this strategy to obtain more accurate information on the actual number of cases that occurred in the study and the non-study populations. The basic HNPCR strategy described here could be applied to amplify and sequence target DNAs from any low-copy-number biological sample.
Assuntos
Neisseria meningitidis/genética , Reação em Cadeia da Polimerase/métodos , Porinas/genética , Análise de Sequência de DNA/métodos , Sequência de Aminoácidos , Sequência de Bases , Chile/epidemiologia , Primers do DNA , DNA Bacteriano , Humanos , Infecções Meningocócicas/líquido cefalorraquidiano , Infecções Meningocócicas/epidemiologia , Infecções Meningocócicas/microbiologia , Dados de Sequência MolecularAssuntos
Proteínas da Membrana Bacteriana Externa/imunologia , Vacinas Bacterianas/uso terapêutico , Polissacarídeos Bacterianos/imunologia , Adolescente , Adulto , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Formação de Anticorpos , Cápsulas Bacterianas , Vacinas Bacterianas/efeitos adversos , Vacinas Bacterianas/imunologia , Criança , Pré-Escolar , Chile , Seguimentos , Humanos , Lactente , Vacinas MeningocócicasRESUMO
The genetic structure of populations of Neisseria meningitidis was examined by an analysis of electrophoretically demonstrable allelic variation at 15 structural genes encoding enzymes in 688 isolates. Variation among strains in serogroup and serotype has little relationship to the complex structure of populations revealed by enzyme electrophoresis, which involves 14 major lineages of clones diverging from one another at more than half their genetic loci. Clones of one of these lineages, the ET-5 complex, have been identified as the causative agent of recent outbreaks and epidemics of meningococcal disease in Europe, South Africa, Latin America, and the United States. There is evidence that organisms of the ET-5 complex reached Florida via human immigrants from Cuba.