Serotyping HIV-1 with V3 peptides: detection of high avidity antibodies presenting clade-specific reactivity

Abstract

The main objective of the present study was to assess the specificity and sensitivity of a modified assay using short synthetic peptides of the V3 region of HIV-1 gp120, which is the main target for neutralizing antibodies. Results from an enzyme immunoassay (EIA) employing a panel of synthetic peptides of HIV-1 subtypes and using urea washes to detect high avidity antibodies (AAV3) were compared with those obtained by the heteroduplex mobility assay and DNA sequencing. The EIA correctly typed 100% of subtype B (sensitivity = 1.0; specificity = 0.95), 100% of HIV-1 E samples (sensitivity = 1.0; specificity = 1.0), and 95% of subtype C specimens (sensitivity = 0.95; specificity = 0.94). In contrast, only 50% of subtype A (sensitivity = 0.5; specificity = 0.95), 60% of subtype D (sensitivity = 0.6; specificity = 1.0), and 28% of subtype F samples (sensitivity = 0.28; specificity = 0.95) were correctly identified. This approach was also able to discriminate in a few samples antibodies from patients infected with B variants circulating in Brazil and Thailand that reacted specifically. The assays described in this study are relatively rapid and simple to perform compared to molecular approaches and can be used to screen large numbers of serum or plasma samples. Moreover, the classification in subtypes (genotypes) may overestimate HIV-1 diversity and a classification into serotypes, based on antigenic V3 diversity or another principal neutralization domain, may be more helpful for vaccine development and identification of variants.

Anti-human immunodeficiency virus type 1 humoral immune response and highly active antiretroviral treatment

“…Duplicates of sequentially diluted (1:100 at a first step, followed by serial dilutions using factor 2) heat inactivated plasma were incubated (1h/37 o C followed by an overnight incubation at 4 o C) with different biotinylated synthetic peptides (5 mg/well) bound to multiwell plates (Maxisorb, Nunc, Roskilde, Denmark) precoated with streptavidin (10 mg/well). Specific reactivity was assessed (including two 8M urea washes after antibody incubation according to Casseb et al 2002) by peroxidase-conjugated anti-human-IgG binding and peroxide/ tetrametilbenzidine revelation. Reactivity was assessed as dilution titer reaching at least twice the mean binding of the duplicate control plasma pools (obtained from local HIV negative blood donors) included in each assay plate.…”

Valuable antibody detection method for classifying hepatitis E virus genotypes

“…The serial samples from JE13, JE53, and JE54, infected with genotype 4, showed strong reactivity to the ORF3 peptide from genotype 4, whereas, the antibodies against the genotype 1 and 3 ORF3 peptides showed very weak reactivity (Figure C‐E). Although the serum sample from JE54 reacted weakly with the genotype 3 ORF3 peptide, with an S/CO value of 2.79 at 6.5 weeks, the sample was negative for the genotype 3 ORF3 peptide on the competitive EIA …”

Inibição da resposta de hipersensibilidade do Tipo I da prole de camundongos mediada pela imunização materna com o ácaro Dermatophagoides pteronyssinus

“…Aos 45 dias de idade, período em que a prole já adquiriu competência imunológica para produzir anticorpos antígenos T dependentes, foi detectada importante inibição da produção de anticorpos IgG1 e também de IgE anti-Dp. Jarrett et al 4,5 descreveram que ratos fêmeas imunizadas com ovalbumina (OVA) transmitem anticorpos que exercem efeito inibitório na resposta de IgE, e aumento da produção de IgG na prole imune. Posteriormente, os autores evidenciaram que a supressão de ambas as respostas IgE e IgG, depende do protocolo de imunização materna, ou seja, da dose do antígeno, uso de adjuvante e via utilizada 6 .…”

“…A avidez dos anticorpos anti-Dp nos soros dos camundongos fêmeas imunizadas a termo e no soro transplacentário transmitido a prole foi analisada por ELISA, utilizando a uréia 8M como descrito por Casseb et al 4 com algumas modificações. A reação de ELISA foi procedida similarmente ao descrito no item 2.5, com exceção de que as amostras dos soros foram incubadas em duplicatas e uma das replicatas foi lavada com PBS 0,1% Tween e outra com PBS 0,1% Tween uréia 8M.…”