RESUMO
The sequencing procedure has been used to determine the size of the CAG repeat expansion for the diagnosis of genetic disorders. Likewise, standard polymerase chain reaction (PCR) and gel electrophoresis techniques are applied for screening large number of patients. The trinucleotide repeats (TNR) region amplification by means of the PCR procedure was initially performed using 32-P end-labelled primers and currently carried out with fluorescently end-labelled primers. The goal to obtain reliable TNR quantification assays, at low cost and short assay times, represents a challenge for the molecular diagnosis aimed at massive screening of affected populations. In the current work, we obtained preliminary results of a new methodology for the detection and size estimation of CAG expanded alleles. The assay was based on an indirect enzyme linked immunosorbent assay (ELISA) for quantifying the amount of labelled cytidines in DNA molecules. The label, 6-(p-bromobenzamido)caproyl radical, was introduced by the transamination and acylation reactions. A group of model sequences containing different numbers of CAG repeats, as well as the ATXN3 (ataxin 3) gene (from subjects suffering type 3 spinocerebellar ataxia SCA3) were used for assay standardization. The assay is simple, inexpensive, and easy to perform and differentiates distinct degrees of CAG expansions.
Assuntos
Citidina/análise , Análise Mutacional de DNA/métodos , DNA/análise , Técnicas Genéticas , Ataxias Espinocerebelares/genética , Expansão das Repetições de Trinucleotídeos/genética , Ataxina-3 , Sequência de Bases/genética , Bioensaio/métodos , Citidina/química , Citidina/genética , DNA/química , DNA/genética , Ensaio de Imunoadsorção Enzimática/métodos , Testes Genéticos/métodos , Humanos , Biologia Molecular/métodos , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/análise , Proteínas Nucleares/genética , Proteínas Repressoras/análise , Proteínas Repressoras/genética , Ataxias Espinocerebelares/diagnósticoRESUMO
Four chimeric synthetic peptides (Q5, Q6, Q7(multiply sign in circle), and Q8(multiply sign in circle)), incorporating immunodominant epitopes of the core p19 (105-124 a.a.) and envelope gp46 proteins (175-205 a.a.), of HTLV-I were obtained. Also, two gp46 monomeric peptides M4 and M5(multiply sign in circle) (Ser at position 192) were synthesized. The analysis of the influence of the peptide lengths and the proline to serine substitution on the chimeric and monomeric peptides' antigenicity, with regard to the chimeric peptides Q1, Q2, Q3(multiply sign in circle), and Q4(multiply sign in circle), reported previously, for HTLV-I was carried out. The peptides' antigenicity was evaluated in an ultramicroenzyme-linked immunosorbent assay (UMELISA) using sera of HTLV-I/II. The peptides' antigenicity was affected appreciably by the change of the peptide length and amino acid substitutions into the immunodominant sequence of gp46 peptide.
Assuntos
Produtos do Gene env/imunologia , Produtos do Gene gag/imunologia , Antígenos HTLV-I/química , Epitopos Imunodominantes/química , Proteínas Oncogênicas de Retroviridae/imunologia , Substituição de Aminoácidos , Produtos do Gene env/química , Produtos do Gene gag/química , Antígenos HTLV-I/imunologia , Humanos , Epitopos Imunodominantes/imunologia , Peptídeos/síntese química , Peptídeos/química , Peptídeos/imunologia , Proteínas Recombinantes de Fusão/síntese química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/imunologia , Proteínas Oncogênicas de Retroviridae/química , Produtos do Gene gag do Vírus da Imunodeficiência HumanaRESUMO
The antigenicity of three chimeric synthetic peptides (Qm, Qm-16, and Qm-17) incorporating an immunodominant epitope of the gp41 transmembrane protein (587-617) and the different epitopes of the gp120 envelope protein (495-516), (301-335), (502-516) of human immunodeficiency virus (HIV-1), separated by two glycine residues, was evaluated by UltramicroEnzyme-linked immunosorbent assay (UMELISA) by using panels of anti-HIV-1 positive sera (n = 47). The specificity was evaluated with samples from healthy blood donors (n = 20) and anti-HIV-2 positive samples (n = 10). The results indicate that the chimeric peptide, Qm, was the most reactive one because it detected antibodies to virus efficiently. This may be related to peptide adsorption onto the solid surface, the C-terminal region of HIV-1 gp120 (495-516) combined with gp41 (587-617) in the chimera, and the epitope accessibility to the antibodies. This study showed the usefulness of the chimeric peptides as antigen to detect antibodies to HIV-1 virus.
Assuntos
Proteína gp120 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/química , Infecções por HIV/sangue , HIV-1/química , Peptídeos/química , Ensaio de Imunoadsorção Enzimática , Anticorpos Anti-HIV/sangue , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , Proteína gp41 do Envelope de HIV/imunologia , HIV-1/imunologia , Humanos , Peptídeos/imunologiaRESUMO
In the present work, a comparative study of 5-FdUrd, thy-, and metabolic in vivo labeling methods for plasmid and chromosomal DNA in E. coli DH5alpha cells was performed in order to achieve the best thymidine substitution method by 5-BrdUrd. According to the colorimetric immunoenzymatic results, we found that the minimal detectable labeled DNA (MDLD) was 312pg with the 5-FdUrd and thy- methods for 5-BrdUrd labeled plasmid DNA. 5-BrdUrd replaced about 96% of the total thymidine by 5-FdUrd methods; for the thy- and metabolic labeling methods, the MDLD value was 1,25 ng for denatured 5-BrdUrd chromosomal DNA. Pyrimidine nucleoside analogues were also evaluated as immunochemical markers for their in vivo introduction into DNA.
