Development and application of monoclonal antibodies against gliadin and related prolamins

A family of monoclonal antibodies were obtained against gliadin but show binding abilities to other prolamins.
Full list of currently available anti-gliadin mAbs is shown in attached file
Most important mAbs are readily available from our research partner Zedira GmbH (www.zedira.com); the rest of the mAbs should be requested at XEMA

Hybridoma XGY7 producing mAbs against gliadin was used as a model in the experiments on binding of target antigens to hybridoma cells and immunomagnetic separation of hybridoma cells by target antigen-armed microspheres.

Epitope mapping of gliadin using XEMA anti-gliadin mAbs
We tried to develop gliadin epitope map using our mAb panel (see attached picture). We realize that the total number of epitope groups on gliadin is far from being covered by our mAbs; we continue to obtain more mAbs and this picture will be updated.

Immunoblotting studies of anti-gliadin mAbs
Most of anti-gliadin mAbs are reactive against gliadin in immunoblotting, sometimes showing selective binging to selected electrophoretic zone corresponding to alpha/omega classification (see pictures below, a figure below the stripped lane corresponds to XGY mAb series number).

ELISA kits for gliadins
Three ELISA kits were constructed from a.m. mAbs to detect ‘regular’ high MW gliadin; low MW gliadin which is characteristic for deeply processed foods and beverages; and an epitope on gliadin characteristic for Triticum aestivum, used for quality control of genuine (durum) pasta.

Kit performance data:
K380 Gliadin EIA
Test principle: sandwich, monoclonal antibodies
Total time of analysis: 30/30/15 min
Sensitivity: 5 ppm
Material tested: food

K380L LMW Gliadin EIA
Test principle: competition, monoclonal antibodies
Total time of analysis: 45/15 min
Sensitivity: appox. 1250 µg/g
Material tested: food

K381 Durum EIA
Test principle: single site, monoclonal antibodies
Total time of analysis: 30/30/15 min
Sensitivity: <1% w/w of T. aestivum
Material tested: flour, pasta

A pilot test kit (EIA) was also constructed to detect admixture of wheat or barley in RYE flour or bread – please inquire.

Prolamin fingerprinting by monoclonal antibodies – application for beer analysis
The antibodies initially produced against gliadin show binding to other prolamins. The routine method of analysis for prolamin-containing substances includes ethanol extraction and immobilization of prolamins on polystyrene plates. Direct binding with a number of anti-prolamin antibodies results in different immunoreactivity with prolamins extracted from different sources and exposed to different technological transformations, giving an unique ‘fingerprinting’ pattern for each prolamin-containing product, eg pasta, bread, beer raw materials (malt, wort, beer), different barley sorts

Application of this approach allows to monitor food production technology, maintaining of lot-to-lot and factory-to-factory reproducibility of branded foodstuffs, quality control and detection of fakes and adulterated products. The example of GOOD reproducibility within international beer brand manufactured in different cities and even countries is shown below.

Prolamin fingerprinting by monoclonal antibodies – application for barley sorts
4 samples of beer wort (from 3 different barley pure sorts) were fermented with 2 different ale-yeast strains during one fermentation procedure. Significant differences were observed between 2 yeast strains in prolamin epitopes transformation abilities (see figure below). Moreover two parallel samples of Xanadu sort obtaining wort show significant difference in immunoreactivity caused by microheterogeneity of yeast fermentation process. This fact shows that mAbs may distinguish the conformational epitopes on prolamin molecule, which are affected by processing or microenvironment.

Development of barley sorts homogeneity test
Determination of barley sort purity is a pivotal problem of beer industry. Immunoreactivity analysis of individual grains allows performing rapid test for barley lot crude sort-specified homogeneity. This simple model test cannot allow to determine sort purity because of low variability of mAb recognition between individual sorts. We hope that application of more mAbs may improve the performance of immunoassay for sort purity.

Parameter	Barley lot 1	Barley lot 2
Mean	1,144	1.669
SD	0.188	0.098
CV, %	16.4	5.8
Plates correlation	0.97	0.79

Antibodies* to prolamin of barley (Hordein) were also obtained.

Code	Specificity
Code	Hordein	Gliadin	Avenin	Secalin
XH2	++++	+	-	-
XH8	++++	-	-	-
XH15	++++	+++	-	-

* In the table are listed the main specificities, additionally there are at least 15 antibodies to different epitopes of hordein with different affinity.