RESUMO
Bacterial vaginosis is one of the most frequent vaginal infections. Its main etiological agent is Gardnerella vaginalis, which produces several virulence factors involved in vaginal infection and colonization, in particular, sialidase (SLD), a potential clinical biomarker that participates in immune response modulation and mucus degradation. The main objective of this work was the production and evaluation of a monoclonal antibody against G. vaginalis sialidase and its validation in immunoassays. For immunization of mice, a synthetic multiantigenic peptide was used, and hybridomas were generated. After fusion, hybridomas were evaluated for antibody production and cloned by limited dilution. One clone producing IgG1 was selected and characterized by indirect ELISA, dot blot, and Western blot, and we also tested clinical isolates and HeLa cells infected with G. vaginalis. The results showed that the anti-SLD antibody recognized a single protein of ~90 kDa that correlated with the estimated molecular weight of SLD. In addition, anti-SLD antibody recognized SLD from complete bacteria and from culture supernatants of infected Hela cells. In conclusion, our results showed that the anti-SLD antibody recognized SLD from different sources and could be considered a new tool for the diagnosis of bacterial vaginosis. KEY POINTS: ⢠Anti-sialidase mAb was generated using a synthetic peptide ⢠The mAb recognizes synthetic peptide and intact protein from multiple sources ⢠The antibody was characterized by several immunological methods.
Assuntos
Anticorpos Monoclonais/imunologia , Proteínas de Bactérias/imunologia , Gardnerella vaginalis/imunologia , Neuraminidase/imunologia , Peptídeos/imunologia , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/isolamento & purificação , Proteínas de Bactérias/química , Feminino , Gardnerella vaginalis/enzimologia , Gardnerella vaginalis/isolamento & purificação , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Neuraminidase/química , Peptídeos/síntese química , Vaginose Bacteriana/microbiologiaRESUMO
OBJECTIVE: To determine the prevalence of Gardnerella vaginalis, anaerobic bacteria and Mycoplasma hominis in vaginal specimens of women with and without bacterial vaginosis (BV) as well as to determine the sensitivity and specificity of the direct sialidase assay of vaginal fluid as a rapid test for diagnosing this syndrome. METHODS: Vaginal cultures were obtained from 109 nonpregnant women (mean age 33 +/- 7.1 years), 47 of them with clinical signs of BV (BV+) and 62 of them without BV (BV-). In addition, we determined the vaginal sialidase activity in both groups, which may serve as a feature of this syndrome. RESULTS: Anaerobic bacteria were isolated in 91% and 18% of the BV+ and BV- groups, respectively (p < 0.001). Peptostreptococcus spp., Prevotella bivia and Porphyromonas spp. were strongly associated with BV. P. bivia and Prevotella spp. represented 44% of all the anaerobes isolated in the BV+ group. All the isolated P. bivia strains presented sialidase activity. G. vaginalis and M. hominis were isolated in 76% and 42% of the BV+ and 1% and 0% of the BV- women, respectively (p < 0.001). Mobiluncus morphotypes were observed in 34% of the BV+ and 0% of BV- women. Sensitivity, specificity, positive predictive value and negative predictive value of sialidase activity were 81%, 94%, 90% and 86%, respectively. CONCLUSIONS: Our data demonstrate a strong association between G. vaginalis, M. hominis, and P. bivia and BV. Sialidase activity and Gram stain of vaginal fluid represent accurate methods for diagnosing BV.