Development of high‐sensitive enzyme immunoassays for gliadin quantification using the streptavidin‐biotin amplification system

Abstract

Optimization of three enzyme immunoassays of very high sensitivity using three antiprolamin monoclonal antibodies (MAbs) (13B4, 11C4 and 12A1) is presented here. These MAbs are specific for those prolamins toxic for coeliac patients, as determined by immunoblotting analysis. Biotinylated MAbs were used in two of the assays. In a competitive ELISA, the binding of each biotinylated MAb to a gliadin-coated solid phase was inhibited by gliadin in the fluid phase. The best result was obtained using the biotinylated MAbl3B4 (detection limit: 20 ng ml-1). With regard to capture ELISA, we tested the performance of the three MAbs. In this sandwich ELISA, the MAb used for antigenic capture was the same as that used as secondary biotinylated antibody. The MAbl2Al had the best performance (detection limit: 1 ng ml-1). The use of biotin-labelled gliadin in a quantitative immunoassay with a detection limit of 5 ng ml-1 is also reported. This assay involves an antigenic capture using the MAbl2Al followed by a competition between biotinylated and non-biotinylated gliadin. We have found the use of the streptavidin-biotin interaction as signal amplification system to be very useful. This technique, as far as we know, has not been previously reported for gliadin quantification.

Innovative approach to low-level gluten determination in foods using a novel sandwich enzyme-linked immunosorbent assay protocol

“…Furthermore, with the exception of one ELISA based on the use of three monoclonal antibodies [4], most methods are incapable of detecting wheat, rye and barley prolamins equally effectively [5][6][7]. Today's ELISAs based on monoclonal antibodies need to show a wider specificity towards prolamins toxic to coeliac patients, along with a higher degree of sensitivity, accuracy and reproducibility.…”

An innovative sandwich ELISA system based on an antibody cocktail for gluten analysis

“…MAbs were purified from ascites by affinity chromatography in a protein A‐Sepharose column (Pharmacia). One MAb (Rye 3), which recognized gliadins, secalins, hordeins and avenins in direct ELISA and immunoblotting (see below), was conjugated to HRP by using the periodate method [16].…”

Interference of denaturing and reducing agents on the antigen/antibody interaction. Impact on the performance of quantitative immunoassays in gliadin analysis

“…According to previous results, the eVect of denaturating agents appears to depend on the antibody and ELISA format employed. Therefore, we extended the analysis by assessing the combined eVect of 2-ME and GuHCl in competitive and capture ELISAs using diVerent antibody systems [38]. Altogether, these studies allow assessment of the eVects of 2-ME and GuHCl As reported for several proteins, 2-ME and GuHCl produce conformational changes leading to a decrease in the biological activity.…”

“…Capture ELISAs were developed using pairs of diVerent anti-gliadin monoclonal antibodies (mAbs) prepared by our group. 1B4E9 or 3B4H1 mAbs were used for antigen capture and biotinylated 2A1C4 or 1B4E9 mAbs were used as detection antibodies following the procedure described elsewhere [38]. BrieXy, plates were coated overnight at 4 °C with 1B4E9 or 3B4H1 mAb in PBS.…”