Development of a Specific, Sensitive, and Robust Enzyme-Linked Immunosorbent Assay (ELISA) to Detect Trace Amounts of Almond (Prunus Dulcis L.)

Some 30000 people in the U. S. go to emergency rooms each year due to allergic reactions to food; more alarming, there are approximately 150-200 deaths every year, usually adolescents and young adults, in comparison to 50 deaths caused each year by E. coli O157:H7. Families with a food-allergic child experienced more stress and scored lower than the general population on scales of general health, emotional health and family activities. Although almonds are nutritionally and economically important, almond ranked third accounting for 15% of total allergic reactions triggered by an individual tree nut. Strict avoidance is currently the only way to prevent serious health consequences. Allergic reactions from accidental exposure are still relatively common even if patients attempt to avoid known allergens. The objectives of this study were to: (1). Identify and characterize murine monoclonal antibodies (mAbs) demonstrating high sensitivity and specificity to almond protein(s) but devoid of crossreactivity with other protein(s) for almond detection; (2). Construct, optimize, and validate a sensitive, specific, and reliable sandwich ELISA using mAbs for almond trace detection in routine food analyses; (3). Assess the applicability of the assay for robustness in terms of sample source, conditions; and the stability of almond protein(s) towards the selected mAb(s). Using protein G affinity chromatography, seven pre-selected murine mAbs were purified. Five mAbs that demonstrated high affinity and sensitivity in Western blotting as the optimized Ab titer were 58.02, 5.88, 4.78, 30.29, and 78.87 ng/ml for 4C10, 4F10, 2A3, 4G2, and 3B4 respectively that can detect as low as 30, 50, 100, 500, and 8000 ng of almond protein respectively. No cross-reactive protein was found in the tested foods or food ingredients. All five mAbs can sensitively detect both native and denatured almond protein(s) suggesting a stable interaction between the mAb and the corresponding recognized epitope on the almond protein. ELISA additivity test and amandin preparation using column chromatography revealed all five mAbs were recognizing shared or overlapping epitope (s) on amandin, the major storage protein that accounts for ~65% of the total seed proteins. Amandin is the major allergen in almond seeds. Molecular Weight of 4C10 reactive polypeptide was determined to be ~69 kDa by SDS PAGE using Fisher BioReagents* EZ-Run* Prestained Rec Protein Ladder 3602 and 3603 as well as ChemiDocTM XRS+ System with Image LabTM Software. 4C10 and 4F10 reactive polypeptides were purified using Cyanogen Bromide (CNBr) Affinity Chromatography. N-terminal sequencing of the affinity purified polypeptides further confirmed amandin to be recognized by mAb 4C10 and 4F10. Therefore, mAbs 4C10 and 4F10 are both excellent candidates for almond assay development. Using rabbit anti whole almond polyclonal antibodies (pAb) as capture Ab, mAb 4C10 as detection Ab, a sandwich ELISA was constructed and optimized resulting in the following protocol: Corning® Costar 2797 plates were coated with 50 µl rabbit anti-whole almond protein extract pAbs in citrate buffer (48.5 mM citric acid, 103 mM Na2HPO4, pH 5.0, 604 ng/well) for 2 hrs at 37ºC. Coated plates were then blocked with 200 µl 5% (w/v) non-fat dry milk (NFDM) in Tris buffered saline (TBS-T, 10 mM Tris, 0.9% w/v NaCl, 0.05% v/v Tween 20, pH 7.6). Plates were incubated sequentially with almond protein or amandin (8000 ng/ml, 4), mAb 4C10 (4 ng/well), and alkaline phosphatase-labeled goat anti-mouse pAb (24 ng/well) in 1% NFDM in TBS-T. The plates were washed 3× with TBS-T after each incubation step (37ºC for 1 hrs). Color was developed using p-nitrophenyl phosphate substrate [1 tablet/ 5 ml substrate buffer (0.0049% w/v MgCl2, 0.096% v/v diethanolamine, pH 9.8)] at 37 ºC for 30 mins and stopped by adding 50 µl/well 3 M NaOH. Plates were read at 405 nm in an ELISA reader (model EL 307, Bio-Tek Instruments Inc., Riverton, NJ) and KC4 software were used to analyze data and generate four parameter curve for quantification. The constructed sandwich ELISA was validated to have LOD of 7 ng/ml (0.007 ppm), protein concentration at 50% maximum signal of 39.46 ± 0.54 ng/ml (n=510), True Positive Rate (aka. sensitivity) of 99.96%, and True Negative Rate (aka. specificity) of 99.05% without cross-reactive to a majority of tested food matrices except for a weak reactivity to corn, corn flakes, and red potato at % immuno-reactivity of 0.08 ± 0.01% (n=2), 0.14 ± 0.00 % (n=2), 0.37 ± 0.03 % (n=4) respectively. This sandwich ELISA is reproducible and accurate as all the CVs tested were less than 15% and the average recoveries were within 15% of the actual value. The general applicability of the constructed 4C10 sandwich ELISA was examined. This assay was able to detect and quantify amandin in all tested samples regardless of gene origin and growing condition as well as purified native and recombinant (Pru du 6.01) amandins. The assay can detect amandin under a variety of tested conditions including: presence/absence of lipids; several food processing methods; pH (1-13) exposure; and long-term storage. The assay can detect 2.11 ng/well of amandin in all the tested food matrices spiked with 50 ng/ml of almond protein (final concentration). Food matrices affect amandin recovery causing over- or under-estimation (275-24%). Overall, the constructed mAb 4C10-dependent sandwich ELISA is sensitive, specific, and robust as amandin is a stable allergen.