Development of a Monoclonal Antibody-Based Enzyme-Linked Immunosorbent Assay To Quantify Soluble β-Glucans in Oats and Barley

Abstract

A set of 31 murine monoclonal antibodies was produced against (1-->3,1-->4)beta-d-glucan from oats (Avena sativa L.) chemically cross-linked to keyhole limpet hemocyanin. Monoclonal antibodies were tested for their cross-reactivity to related and unrelated polysaccharides. The antibodies reacted strongly to unmodified beta-glucan from oats and barley (Hordeum vulgare L.) and to lichenan from Icelandic moss, a polysaccharide with a structure similar to that of beta-glucan but which is not encountered in cereals. Cross-reaction to other polysaccharides tested was minimal at physiological levels. An enzyme-linked immunosorbent assay (ELISA) that could routinely detect and quantify nanogram levels of soluble beta-glucan extracted from the flour of oats or barley was designed with one of these monoclonal antibodies. The beta-glucan extraction procedure from ground oat and barley samples and the ELISA were both optimized for reproducibility, accuracy, and throughput, and results were compared to values obtained from an established, commercially available enzyme-based assay. Correlations between the two assays were consistently high (r (2) > 0.9), indicating that the ELISA presented in this paper is a valuable alternative for assaying beta-glucan levels in cereals and cereal products, both routinely and in preparations in which beta-glucans are present in nanogram amounts. Development of the extraction procedure for ELISA is discussed.

Beta Glucan: Health Benefits in Obesity and Metabolic Syndrome

“…The AOAC method 995.16 is used to analyze β -glucan content in flours from whole grains, milling fractions, and unsweetened cereal products [47]. In addition to AOAC methods, there are other methods including enzyme-linked immunosorbent assay (ELISA) [51], near-infrared spectroscopy [52], and fluorescence assay of complex formed between β -glucan and calcofluor [53], which are all specifically designed to measure β -glucan.…”

Current potential and limitations of immunolabeling in cereal grain research

“…Polyclonal and monoclonal antibodies against different motifs encountered in xylans and AX have now been designed McCartney, Marcus, & Knox, 2005;Ordaz-Ortiz, et al, 2004). Monoclonal antibodies against (13),(14)--D-glucans (Rampitsch, Ames, Storsley, & Marien, 2003), xyloglucans (Marcus, et al, 2008), pectic homogalacturonan (Clausen, Willats, & Knox, 2003;Verhertbruggen, Marcus, Haeger, Ordaz-Ortiz, & Knox, 2009), xylogalacturonan ) and p-coumaric acid (Tranquet, et al, 2009), and polyclonal antibodies against ferulic acid (Philippe, Tranquet, Utille, Saulnier, & Guillon, 2007) have been generated and characterized. Pattathil, et al (2010) described a comprehensive toolkit of approximately 180 plant cell wall-directed monoclonal antibodies, of which approximately 130 were newly generated.…”

Sampling for indoor fungi☆

“…Two candidate proxies for total fungal biomass under investigation include ergosterol and Beta-1,3-D glucan. 40 Currently, each substance is difficult to measure, 41,42 and few studies have been done comparing their concentrations with actual fungal exposure. 43,44 Beta-1,3-D glucans are straight or branched chain glucose polymers that are present in most fungi either bound to chitin or on the cell wall as free polymers.…”

“…As new methods for measurement of ergosterol and glucans are developed, use of these fungal biomarkers might be more widely available. 42,49 Mycotoxins. Many genera of fungi have evolved the ability to produce toxic metabolites.…”