RESUMO
OBJECTIVE: To evaluate the oxidative state in patients with familial amyloidotic polyneuropathy type 1 (FAP1). DESIGN: From 3 unrelated families, patients with FAP1 carrying a transthyretin Met-30 mutation were studied. The diagnosis was confirmed by genetic analysis. Eleven of 21 patients carried the mutation; all were symptomatic and were clinically assessed using a clinical score. All of the patients were evaluated for copper-zinc superoxide dismutase type 1 activity in red blood cells using spectrophotometry. Plasma total reactive antioxidant potential was studied using a chemiluminescent method. The results were compared with those obtained from an age-matched control group. SETTING: A public and academic multidisciplinary research clinic. RESULTS: Six of the 11 FAP1-positive patients disclosed superoxide dismutase type 1 activity values greater than 55 U/mg of protein (upper control limit), whereas 9 of 10 patients in whom total reactive antioxidant potential was measured had values below the lower limit of the control group. No relationship was found between the levels of superoxide dismutase type 1 activity and the severity of the clinical involvement. CONCLUSIONS: Oxidative stress may be part of the mechanisms leading to tissue damage in patients with FAP1. The lack of correlation between the laboratory findings and the severity of clinical involvement may signal that oxidative processes are at work throughout the natural history of the disease.
Assuntos
Neuropatias Amiloides Familiares/sangue , Antioxidantes/metabolismo , Eritrócitos/enzimologia , Estresse Oxidativo , Espécies Reativas de Oxigênio/sangue , Superóxido Dismutase/sangue , Neuropatias Amiloides Familiares/diagnóstico , Neuropatias Amiloides Familiares/genética , Cobre/metabolismo , Brometo de Cianogênio/metabolismo , Genótipo , Humanos , Immunoblotting , Metionina/genética , Mutação , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Pré-Albumina/genética , Superóxido Dismutase-1 , Valina/genética , Zinco/metabolismoRESUMO
The widespread use of ciprofloxacin in human, animal and plant health has raised an environmental problem, paralleled by several other antibiotics. The aim of this work is the development of a rapid and sensitive ELISA assay for ciprofloxacin, which can constitute an alternative to time-consuming HPLC methods. For this purpose, we worked with antibody fragments, instead of whole antibodies, and used magnetic beads as solid support. Ciprofloxacin was successfully immobilized onto this support with a carbodiimide-mediated reaction. A library of phage particles that express human single-chain antibodies at their surface was then screened with an optimized protocol. Several positive fragments were isolated and identified as being V(L) fragments. These were then fully characterized. A reproducible competitive ELISA was developed using the magnetic beads - ciprofloxacin as support and the phages displaying the V(L) fragment as recognition entity. This assay showed limits of detection and quantification of 9.3 nM and 33 nM, respectively. Also, competitive ELISAs with ciprofloxacin homologues and other molecules showed cross-reactivities lower than 12%.