RESUMO
The in vitro potency of botulinum neurotoxin (BoNT) serotypes is often measured by monitoring cleavage of their soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein substrates. A frequently used method is Western blot, whereby the full-length protein and cleaved form migrate at different molecular weights. Until now, it has been extremely difficult to detect the cleaved cellular form of the SNARE protein vesicle associated membrane protein 1, 2 or 3 (VAMP1, 2 or 3) by Western blot. These VAMP isoforms are the substrates of BoNT serotypes BoNT/B, D, F and G as well as tetanus neurotoxin. Using custom made anti-VAMP antibodies against epitopes either side of the cleavage sites for BoNT/B, BoNT/D and BoNT/F, we have successfully detected the cleaved C-terminal VAMP fragment in cortical neurons. These new antibodies enable quantitative assessment of the potency of VAMP-cleaving neurotoxins by a gain of signal Western blot assay.
Assuntos
Toxinas Botulínicas/toxicidade , Epitopos/efeitos dos fármacos , Neurotoxinas/toxicidade , Proteína 1 Associada à Membrana da Vesícula/imunologia , Proteína 2 Associada à Membrana da Vesícula/imunologia , Proteína 3 Associada à Membrana da Vesícula/imunologia , Sequência de Aminoácidos , Animais , Western Blotting , Córtex Cerebral/citologia , Córtex Cerebral/efeitos dos fármacos , Feminino , Neurônios/efeitos dos fármacos , Gravidez , Ratos , Proteínas SNARE/metabolismo , Toxina Tetânica/toxicidade , Proteína 1 Associada à Membrana da Vesícula/efeitos dos fármacos , Proteína 2 Associada à Membrana da Vesícula/efeitos dos fármacos , Proteína 3 Associada à Membrana da Vesícula/efeitos dos fármacosRESUMO
BACKGROUND: Here we evaluate auto-antibody response against two potential antigenic determinants of genes highly expressed in low Gleason Score prostate cancer (PC) tumor samples, namely FLJ23438 and VAMP3. METHODS: RT-PCR assays were used to analyze mRNA expression profiles of FLJ23438 and VAMP3 transcripts. The auto-antibody response against FLJ23438 and VAMP3 recombinant proteins was tested by immunoblot assays using PC, benign prostate hyperplasia (BPH), healthy donors (HD), and other human cancers plasma samples. RESULTS: Our data showed that 37% (10/27) and 7.4% (2/27) of PC plasma samples presented auto-antibodies against FLJ23438 and VAMP3, respectively. Only 8.3% (1/12) of BPH plasma samples were reactive for both auto-antibodies, while none (0/12) of HD plasma samples tested were reactive. CONCLUSIONS: The prevalence of 37% of positive PC plasma samples for anti-FLJ23438 antibodies suggests that humoral immune response against this antigenic determinant could be a potential serum marker for this cancer.
