A simple and sensitive immunoassay method for the detection of low-molecular-weight proteins and neuropeptides

BACKGROUND: Low-molecular-weight proteins and peptides perform numerous regulatory roles in biological systems and could be useful as biomarkers. Hence, qualitative and quantitative measurement of peptide levels has become increasingly important. AIM: This present study was aimed at developing a simple and sensitive dot immunoblot assay (DIA) by modifying existing protocols. MATERIALS AND METHODS: Using polyclonal antibody generated against FMRFamide peptide, at a dilution ratio of 1:10,000 in buffer, as a primary antibody, alkaline phosphatase-conjugated goat anti-rabbit antibody as secondary antibody and nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate development system, peptides were dotted on 0.2μm nitrocellulose membrane in volumes of 1μl, 0.5μl and 0.2μl. RESULTS: Results show immunoreactivity of FMRFamide antibody to small quantities of dotted neuropeptide as low as 0.2ng and 33.4pM. CONCLUSION: This indicates that this protocol is sensitive with the additional advantage of simplicity, speed, small sample volume, low cost, and production of nonhazardous waste. This DIA protocol could be useful in resource-poor settings and laboratories with low budgets.