Development of high‐sensitive enzyme immunoassays for gliadin quantification using the streptavidin‐biotin amplification system (1)
Coeliac disease (CD) occurs in genetically susceptible individuals as a result of the ingestion of food containing prolamins of wheat, barley, rye and probably oat. Wheat proteins are widely used in the food industry to improve product quality because of their particular physicochemical properties. Wheat starch is also used as an excipient in medicines; consequently, gliadins can be found in pharmaceutical products. The tendency of prolamins to form macromolecular aggregates, their high homology with the resulting strong cross-reactivity and the difficulty to prepare isolated components are factors causing problems during the characterization of antibody reactivity as well as in the development of assays of high specificity and sensitivity.
Gliadin was dissolved in 70% aqueous ethanol at approximately 2 mg/ml. The solution was first filtered through filter paper of Whatman no. 1 and then through a 0.22-um Nucleopore membrane. A clear and stable gliadin solution was obtained using this procedure. The gliadin was dissolved in 0.001 M-NaOH and then filtered through a nitrocellulose membrane (0.45 um). This solution was dialyzed against 0.1 M-sodium borate buffer, pH 8.8, and was biotin conjugated.
The immunoblotting was obtained with the ethanol 70% soluble protein fractions of wheat, barley, rye, triticale, oat, soy, rice and maize. MAbs recognize alpha-, beta- and gamma-gliadins, and two intense bands from the w-gliadin region (50-55 kDa). In lane 2, (barley prolamins), two groups of bands are detected: one close to 50 kDa, corresponding to C-hordeins, and the other in the region of 40 kDa, corresponding to B-hordeins. A reactivity in the region of 50-40 kDa and 30 kDa is observed in rye proteins, corresponding to w-secalins and gamma-secalins. Both MAbs show reactivity against triticale proteins.
Although the three MAbs recognize alpha-, beta- and gamma-gliadins, significant differences can be observed in immunoblotting after A-PAGE. MAb13B4 (lane 1) reacts weakly with some w-gliadins. MAb11C4 (lane 2) recognizes alpha-, beta- and gamma-gliadins, but in a more restricted way. Mab12A1 (lane 3) reacts strongly with some gamma- and w-gliadins.
Assay using the MAb13B4 yielded the best results. This assay presents a broad range of linear response and a high sensitivity, parameters of the assay are as follows: 50% inhibition:120 ng/ml and a detection limit of 20 ng/ml.
In capture ELISA, mAb12A1 leads to higher OD405 values (approximately 1.5) than those obtained with MAb11C4. No positive response was observed when MAb13B4 was used. The sigmoid curve shows a working range of 0.3 to 50 ng/ml, and a detection limit of 1 ng gliadin/ml.
All MAbs efficiently captured the biotinylated gliadin. After determining the appropriate biotinylated gliadin concentration (1 ug/ml), a competitive ELISA was performed. the assay using the MAb12A1 presents the best performance with 50% inhibition at 40 ng gliadin/ml and a detection limit of 5 ng gliadin/ml. MAb13B4 presents a slightly lower detectability (50% inhibition = 110 ng gliadin/ml). The assay using the MAb11C4 shows only poor detectability (50% inhibition = 900 ng gliadin/ml).
Using the MAbl3B4, a high detectability quantitative assay has a detection limit of 20 ng gliadin/ml, corresponding to 2 mg gluten/100 g of dry product (at 1:50 sample dilution). A extra incubation step in competitive ELISA presents a detection limit of 1 ng gliadin/ml corresponding to 0.1 mg gliadin/100 g of dry product at 1/50 sample dilution. A capture ELISA presented a calibration curve with a broad range of lineal response (0.3-50 ng/ml) and a detection limit of 1 ng gliadin/ml (0.05 mg gluten/100 g at 1:50 of dry product sample dilution). A competitive ELISA using plates coated with MAb12A1 shows a 50% inhibition at 40 ng gliadin/ml and a detection limit of 5 ng gliadin/ml, corresponding to 0.5 mg gluten/100 g of dry product (at 1:50 sample dilution).