ELISA Method for Estimation of Gliadin in Bread Samples

Immunology
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Table of Contents
Introduction 3
Aims 4
Results 4
Discussion 5
Conclusion 7
References 7
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Introduction:
The enzyme-linked immunosorbent assay (ELISA) is the most commonly used biochemical
assay technique. ELISA uses solid-phase enzyme immunoassay (EIA) for the detection of
ligand in a liquid sample through antibodies directed against protein. ELISA is being used in
medicine as a diagnostic tool and quality control tool in biochemical industries. In ELISA,
antigens are attached to solid surface like polystyrene microtiter plate followed by antibodies
added to bind to the antigens. This detects antibody form complex with the unknown
antigens. Detection antibody are usually attached to enzyme or it can be detected by
secondary antibody which is attached to enzyme through bioconjugation (Konstantinou,
2017). After completion of each step, ELISA plate needs to be washed with detergent to
remove unbound protein and enzymes. Final step comprises of addition of enzyme substrate.
End point in ELISA is colour change which is usually measured using UV-Visible detection.
Based on the sequence of bonding of analytes and antibodies, ELISA are categorised into
different types like Direct ELISA, Sandwich ELISA, Competitive ELISA and reverse ELISA.
ELISA is being used for the determination of serum antibody concentrations in HIV test, for
detection of food allergens and in serological blood test for coeliac disease (Hornbeck, 2015).
Gliadin is a class of prolamin protein available in wheat and other cereals from grass genus
Triticum. Gliadins are heterogenous mixture of single chain polypeptides which are soluble in
70 % alcohol and these contain intramolecular disulphide bonds. Gliadins are components of
gluten which is useful to rise bread properly during baking. Gliadin is water-insoluble and
glutenin is water soluble. Gliadin exists in three forms like α, β, γ, and ω (Urade et al., 2018).
α gliadins are with fastest mobility while ω gliadins are with slowest mobility. Body is
intolerant to gliadin in coeliac (or celiac) disease (CD). Gliadin has the ability to cross
intestinal epithelium. Gliadins are responsible for precipitating coeliac disease while
glutenins are nontoxic in nature. Peptides derived from the gliadins are immunostimulatory in
coeliac disease (Kelly et al., 2015). Both gliadin and glutenin play role in the formation of
gluten. Gliadins are responsible for food derived pathogenesis. ELISA assays are usually
sensitive, specific and relatively easy to perform for the estimation of gluten content in food
products including breads. However, it is necessary to use different extraction solvents for
different types of foods for the estimation of gliadin using ELISA. ELISA assay was
implemented for the estimation of α-gliadin and whole gliadin in the wheat using polyclonal
rabbit or mouse enzyme labelled antisera. Literature mentioned that minimum 0.4 ng quantity
of gliadin in food products can also be estimated using ELISA. ELISA assay is useful in
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