A Murine Monoclonal Antibody Based Enzyme-Linked Immunosorbent Assay for Almond (Prunus dulcis L.) Detection

Abstract

A sandwich enzyme-linked immunosorbent assay (ELISA) using anti-almond soluble protein rabbit polyclonal antibodies as capture antibodies and murine monoclonal antibody 4C10 as the detection antibodies was developed. The assay is specific and sensitive (3-200 ng almond protein/mL) for almond detection. The standardized assay is accurate (<15% CV) and reproducible (intra- and inter assay variability <15% CV). The assay did not register any cross-reactivity with the tested food matrices, suggesting the assay to be almond amandin specific. The assay could detect the presence of declared almond in the tested matched commercial samples. Further, the assay reliably detected the presence of almonds in the laboratory prepared food samples spiked with almond flour.

Immunoreactivity of Biochemically Purified Amandin from Thermally Processed Almonds (Prunus dulcis L.)

“…The mAb 4C10 detected 61 and 63 kDa bands (Figure B) and mAb 4F10 recognized approximately 38, 50, 55, 61, and 63 kDa polypeptides (indicated by arrows in the right margin, Figure C). These observations are consistent with earlier reports (Su et al., ). Isolated amandin, regardless of processing treatment, retained immunoreactivity.…”

“…Nonpareil whole almonds were a gift from the Almond Board of California (Modesto, CA, U.S.A.). Sources of chemicals have been reported earlier (Su et al., ). Animal Care and Use Committee at Florida State Univ.…”

“…Prior to analysis, to compensate for processing-induced protein solubility reduction, each amandin extract was normalized to 8000 ng/mL. A murine mAb-based indirect sandwich ELISA was used as descibed by Su et al (2013). Ninety-six-well microtiter plates were coated with a rabbit antiwhole almond polyclonal capture antibody 99R20 (604 ng in 50 μL/well) in a citric acid coating buffer (48.5 mM citric acid, 103 mM sodium phosphate, pH 5.0) and incubated at 37°C for 2 hr.…”

Almond Allergy: An Overview on Prevalence, Thresholds, Regulations and Allergen Detection

“…The ELISA immunoreactivity variations were up to 8-fold among genotypes/hybrids and 2.5 fold amongst the almond marketing varieties. Another study has reported the development of a sandwich ELISA using anti-almond soluble protein rabbit polyclonal antibodies as capture antibodies and murine-mAb as detection antibodies [ 53 ]. The assay was specific and sensitive (3–200 ng almond protein/mL) for almond detection.…”

Identification and characterisation of the proteins bound by specific phage‐displayed recombinant antibodies (scFv) obtained against Brazil nut and almond extracts

“…The enzyme‐linked immunosorbent assay (ELISA) takes advantage of antibodies that specifically recognise and bind a particular antigen, which can be either a marker protein or the allergen itself . Traditionally, those specific antibodies have been raised in animals . However, there is a tendency towards the replacement of antibodies raised in animals with antibodies synthesised in vitro , products of synthetic libraries .…”