Coeliac disease (CD) is a gastrointestinal afliction triggered by the ingestion of prolamins from wheat, barley, rye and possibly oats. The only treatment for CD is a strict diet, free of the toxic components. Immunochemical methods are usually applied to certify foods aimed at coeliac patients. The characterization of a panel of four anti-prolamin monoclonal antibodies (MAbs) to be used to certify gluten-free products is described here. To this aim, purified gliadin, secalin and hordein fractions were obtained by preparative electrophoresis at acid pH. This procedure provides purified fractions not exposed to denaturing conditions. The specificity of the MAbs was tested by ELISA against purified fractions and ethanol extracts of wheat, barley, rye, oats, rice, maize, buckwheat, sorghum and soy. The four MAbs recognized only coeliac-toxic cereals. Each MAb reacted strongly with gliadins and showed differential reactivity against the different prolamin purified fractions. Some MAbs showed a broad pattern of recognition whereas others presented a more restricted one. The reactivity observed corresponded to structural homologies among gliadin, secalin and hordein fractions. It is remarkable that some fractions obtained by electrophoresis in the presence of sodium dodecylsulphate were not recognized by some MAbs, whereas the same components obtained by preparative A-PAGE showed high reactivity. This reinforces the suitability of the purification method employed in this study to isolate prolamin fractions. Using these purified prolamins, characterization of anti-prolamin MAb reactivity was achieved.