RESUMO
This report describes a novel mu chain capture ELISA for the detection of IgM antibodies against a Plasmodium falciparum antigen. A fragment of the 220 kDa P. falciparum glutamate rich protein containing amino acid residues 489-1271 was expressed in E. coli as a recombinant chimeric beta-galactosidase fusion protein and used as antigen after purification and biotinylation. Specific IgM antibodies were found in 51% (39/77) of sera from adult Liberians immune to malaria. The binding of IgM antibodies was specific for the malaria portion of the fusion protein and no cross-reactivity was found in sera from patients with IgM antibodies due to other diseases. Inhibition studies with a fusion protein containing amino acid residues 816-1134 (GLURP816-1134) representing the carboxy-terminal repeat region suggested a different use of epitopes for IgM antibodies in different individuals.
Assuntos
Anticorpos Antiprotozoários/análise , Antígenos de Protozoários/imunologia , Imunoglobulina M/análise , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Animais , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Malária Falciparum/imunologiaRESUMO
We have examined the number of genes coding for a group of high-molecular-mass rhoptry protein(s) in the malaria parasite Plasmodium yoelii, and studied variation in the gene family within the parasite's genome. A region of the genes was amplified using oligonucleotides based on conserved DNA sequences and the products cloned. The sequences could be divided into 7 groups by restriction-fragment-length polymorphism. Further variation was detected by sequence analysis; 11 different sequences were detected in the 16 clones analyzed. The genes in the family were distributed on 6 chromosomes probably at 9 or more loci.
Assuntos
Genes de Protozoários , Plasmodium yoelii/genética , Proteínas de Protozoários/genética , Animais , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , Variação Genética , Camundongos , Dados de Sequência Molecular , Plasmodium yoelii/imunologia , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Proteínas de Protozoários/imunologia , Alinhamento de Sequência , Homologia de Sequência do Ácido NucleicoRESUMO
A gene coding for a 220-kDa glutamate rich protein (GLURP), an exoantigen of Plasmodium falciparum, was isolated and its nucleotide sequence was determined. The deduced amino acid sequence contains 2 repeat regions. The sequence of one of these was shown to be conserved among geographically dispersed isolates, and a fusion protein containing that sequence was able to stimulate B- and T-cells. Antibodies against GLURP stained erythrocytic stages of the parasite as well as the hepatic stage as detected by electron microscopy.
Assuntos
Antígenos de Protozoários/genética , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Mapeamento Cromossômico , DNA de Protozoário/genética , Eritrócitos/parasitologia , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologiaRESUMO
A method for purification of a recombinant Plasmodium falciparum protein produced in E. coli and its use in an enzyme-linked immunosorbent assay (ELISA) is described. The cloned gene fragment encodes GLURP,489-1271 the carboxy-terminal 783 amino acid residue portion of a 1271 amino acid residue P. falciparum glutamate rich protein (GLURP), with a molecular weight of 220 kilodalton. The protein is associated with all parasite stages in the human host. Examination of sera from 105 adult Liberians living in a malaria endemic area revealed anti-GLURP IgG antibodies in 98% of the sera. The recombinant GLURP489-1271 was expressed as a chimeric protein, fused with E. coli beta-galactosidase. However, antibodies in sera were directed only against the malaria part of the fusion protein and not against beta-galactosidase. Antigen from in vitro P. falciparum cultures of isolates from Tanzania (F32), Papua New Guinea (MAD20) and Honduras (HB3) completely absorbed specific antibodies, indicating the presence of conserved epitopes produced by all isolates of P. falciparum. Recombinant GLURP489-1271 ELISA is sensitive and rapid, and therefore well-suited for sero-epidemiological studies, and for control of the immunogenicity of a possible future P. falciparum vaccine utilizing epitopes from GLURP.
Assuntos
Anticorpos Antiprotozoários/sangue , Ensaio de Imunoadsorção Enzimática , Plasmodium falciparum/imunologia , Proteínas de Protozoários/isolamento & purificação , Animais , Especificidade de Anticorpos , Cromatografia em Gel , Humanos , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
A primer-set was designed for specific detection of genes that encode for 16S rRNA of Helicobacter pylori, using direct polymerase chain reaction (PCR). The primers were selected in the hypervariable regions, derived from a complete small subunit 16S rRNA sequence of the reference strain H. pylori CCUG 17874. The primer-set amplified a 537 base pair (bp) sequence specifically from chromosomal H. pylori DNA. Amplification of purified chromosomal H. pylori DNA was achieved at concentrations as low as 1 femto gram (fg), equivalent to 5 bacteria. Furthermore, as few as 1 lysed H. pylori cell was detected by this PCR technique. The specificity of the primers was 100%, since purified chromosomal DNA was detected from all 32 various H. pylori isolates, whereas no other bacteria species were detected, whether related to Helicobacter or not. The 16S rDNA primers successfully detected H. pylori in antral biopsy specimens collected from infected patients.
Assuntos
DNA Ribossômico/genética , Infecções por Helicobacter/diagnóstico , Helicobacter pylori/isolamento & purificação , Reação em Cadeia da Polimerase , RNA Ribossômico 16S/genética , Adulto , Idoso , DNA Bacteriano/análise , Feminino , Helicobacter pylori/genética , Humanos , Masculino , Pessoa de Meia-Idade , Antro Pilórico/microbiologiaRESUMO
BACKGROUND: Individuals carrying the factor V Leiden mutation have been shown to have an increased risk of developing venous thromboembolism. Our aim was to develop an ELISA-like assay to detect the mutation in PCR-amplified genomic DNA using novel, high-affinity DNA analogs, termed locked nucleic acids (LNAs). METHODS: LNA octamer probes complementary to the factor V wild-type or mutated sequence were covalently attached to individual wells of a microtiter plate. Biotinylated factor V amplicons were added, and hybridization to the immobilized LNA probes was scored colorimetrically using a horseradish peroxidase-anti-biotin Fab conjugate and tetramethylbenzidine substrate. RESULTS: In a prospective study of 53 patients, the assay reproducibly scored both factor V homozygotes and heterozygotes with excellent sensitivity and specificity. All results were in complete agreement with the results obtained with the conventional PCR-restriction fragment length polymorphism technique. CONCLUSIONS: The simplicity of the assay and its procedural relatedness to the widely used ELISA format should make it useful for routine factor V testing in the clinical laboratory.
Assuntos
Fator V/genética , Sondas de Oligonucleotídeos , Alelos , Antraquinonas , Ensaio de Imunoadsorção Enzimática , Humanos , Mutação , Sondas de Oligonucleotídeos/síntese química , Reação em Cadeia da Polimerase/métodos , Estudos Prospectivos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Raios UltravioletaRESUMO
We have characterized a family of repetitive DNA elements with homology to the MgPa cellular adhesion operon of Mycoplasma genitalium, a bacterium that has the smallest known genome of any free-living organism. One element, 2272 bp in length and flanked by DNA with no homology to MgPa, was completely sequenced. At least four others were partially sequenced. The complete element is a composite of six regions. Five of these regions show sequence similarity with nonadjacent segments of genes of the MgPa operon. The sixth region, located near the center of the element, is an A+T-rich sequence that has only been found in this repeat family. Open reading frames are present within the five individual regions showing sequence homology to MgPa and the adjacent open reading frame 3 (ORF3) gene. However, termination codons are found between adjacent regions of homology to the MgPa operon and in the A+T-rich sequence. Thus, these repetitive elements do not appear to be directly expressible protein coding sequences. The sequence of one region from five different repetitive elements was compared with the homologous region of the MgPa gene from the type strain G37 and four newly isolated M. genitalium strains. Recombination between repetitive elements of strain G37 and the MgPa operon can explain the majority of polymorphisms within our partial sequences of the MgPa genes of the new isolates. Therefore, we propose that the repetitive elements of M. genitalium provide a reservoir of sequence that contributes to antigenic variation in proteins of the MgPa cellular adhesion operon.