Specificity characteristics of monoclonal antibodies to wheat grain storage proteins

(Received 10 June 1986)
Key words: Wheat; Gluten protein; Monoclonal antibody; Structural homology
A variety of monoclonal antibodies was prepared to wheat (Triticum aestivum L.) gluten proteins, and their antigenic specificities were assessed using non-denaturing polyacrylamide gradient and SDS-polyacrylamide gel electrophoresis and immunoblotting techniques. While most anti-gliadin monoclonal antibodies bound to all gliadin protein bands separated by one-dimensional electrophoresis, several antibodies binding to small groups of gliadin proteins were identified. At high concentrations, these 'specific' antibodies bound to an increasing number of gliadins; this is likely due to the very high sequence homologies between groups of gliadin proteins. Several monoclonal antibodies with specificities for glutenin proteins were produced. These antibodies bound to all major ghitenin subunits, although at low antibody concentrations some bound selectively to a single subunit. Other anti-glutenin antibodies bound to minor glutenin subunits and a variety of gliadin proteins. Several bound to gamma gliadins and high-molecular-weight glutenins, suggesting that these groups of proteins bear considerable homology. The results are reviewed in relation to known information on gluten protein structure, derived from DNA-sequencing studies.
Bio~imica et Biophvsica Acta 874 (1986) 245-254
245
Elsevier BBA 32682
Specificity characteristics of m o n o c l o n a l antibodies to wheat grain storage proteins
J o h n H. S k e r r i t t a a n d P. A n n e U n d e r w o o d b
" C S I R O Wheat Research Unit and h Dit#sion of Molecular Biology, North Rvde, N.S.W. (Australia)
Introduction
The major storage proteins of the wheat-grain endosperm, termed gliadins and glutenins, constitute over 80% of the total grain protein and are responsible for the unique theological properties of wheaten flour doughs [1-4]. The gliadins are monomeric proteins (M r 30000-80000) with unusually high glutamine and proline contents and low content of charged groups. They are poorly soluble in neutral buffers but dissolve readily in aqueous alcoholic, alkaline or acidic solutions. On the basis of electrophoretic mobility at acid pH they have been classified into a, fl, 3' and ¢0 classes; as a result of gene duplication and diDr. J.H. Skerritt, CSIRO Wheat Research Unit, PO Box 7, North Ryde, NSW 2113, Australia.
gence, over 50 discrete gliadin components exist in each variety [1-5]. Glutenins exist as polymeric moieties (subunit M r 11500-150000) associated by hydrophobic and hydrogen bonding and intermolecular sulfhydryl bonds [1-5]. In wheat, three homoeologous chromosomes are thought to bear structural genes for gluenins, giving rise to several dozen discrete polypeptides. The complexity of gluten, with its large number of components, all rather low in aqueous solubility, and bearing high degrees of amino acid sequence homology to one another, has complicated analysis of the structures and functions of the various protein components. Recent advances in understanding have come from application of DNA cloning and sequencing methods to genes for cereal seed storage proteins (see Refs. 6 and 7 for reviews). Immunochemical methods have also provided valuable information on homologies be-
Materials and Methods
Preparation of endosperm protein fractions Bread wheat (Triticum aestioum, cv. Timgalen)
was milled (Buhler mill, straight run) to produce white flour (75.5% extraction rate, 13.7% protein). Salt-soluble proteins were extracted by two 1 h extractions with 20 vol. 10% (w/v) NaC1 at 20°C and crude gliadin was prepared by extracting the saline-soluble residue twice with 70% (v/v) ethanol (20 vol., 2 x 1 h, 20°C). A glutenin fraction was extracted by treatment of this residue with 50% propanol/2% mercaptoethanol/1% acetic acid (10 vol., 16 h, 20°C). This glutenin fraction was chosen over SDS-extracted glutenin for screening of antibodies in dot-immunoassays since the detergent has been shown to markedly reduce glutenin antigenicity [14,15]. These extracts were dialyzed against distilled water (or 50 mM acetic acid, in the case of glutenin) then freeze-dried. Albumins and globulins were prepared from the dialyzed and lyophilized 10% (w/v) NaC1 extract as follows. Contaminating gliadin was removed by 1 h shaking in 0.5 ml/mg 70% (v/v) ethanol; the undissolved material was dissolved in 10% (w/v) NaCI and dialyzed against water. The precipitate forming upon dialysis is considered to contain globulins and the supernatant, albumins. Gliadin was repurified by dissolving the lyophilized ethanol extract in 0.5 ml/mg 70% ethanol, and precipitating gliadin by addition of 2 vol.