Comparison of Six Commercial ELISA Kits for Their Specificity and Sensitivity in Detecting Different Major Peanut Allergens(1)
Several methods are being used to detect and quantify peanut residues including DNA-based methods, such as polymerase chain reaction (PCR), and protein-based methods such as enzyme linked immunosorbent assay (ELISA). The recovery and detection of peanut proteins with the use of different extraction buffers, food matrices, and processing methods showed a wide variation in the recovery of peanut proteins exists among different kits.
Ara h 1 is a 7S vicilin-type protein, while Ara h 3 is an 11S legumin-type protein. Ara h 2 and Ara h 6 are both 2S albumins that show close homology to each other. Ara h1 and Ara h3 are the more abundant peanut proteins, several studies have proven that Ara h 2 and Ara h 6 bind more strongly with IgE from peanut allergic patients and release mediators more efficiently from basophils, which confirm that these are also more potent allergens in in vitro systems and in vivo.
The range of quantification of each of the test kit standards in μg peanut protein/ml were calculated. By applying different dilutions, as specified in the kit, this ELISA also reached a sensitivity range (relative to the unextracted food products) similar to that of the other kits, that is, 4−60 ppm.
With the Veratox and Romer kits, a higher percentage of peanut was recovered when the sample was prepared as an extract rather than a suspension. However, recovery was comparable with both methods of sample preparation with the BioKits, R- Biopharm, and ELISA Systems kits.
The gel profile and molecular sizes of the allergens Ara h 1 (63 kDa), Ara h 2 (17−20 kDa doublet), Ara h 3 (a series of polypeptides ranging from 14−45 kDa), and Ara h 6 (15 kDa) are published. Ara h 3 is the most abundant protein and that the other three allergens are also present but in lower amounts.
Five of the kits were most sensitive in the recognition of Ara h 3, while their reactivity for the other allergens was substantially lower. The Morinaga kit stands out from the other five kits in being the most sensitive in detecting Ara h 2 and Ara h 6, the two allergens that were least recognized by the other kits.
The Veratox, Romer, and Morinaga kit manufacturers do not indicate that their peanut ELISAs are targeted against specific peanut proteins/allergens. For the BioKits ELISA, it is stated that this kit specifically detects the peanut allergen, Ara h 1. Both the R-Biopharm and the ELISA Systems kits specify that their antibodies target peanut proteins including the allergens Ara h 1 and Ara h 2. Antibodies raised against raw peanuts would have a lower affinity to heat-treated antigens. The Morinaga kit differs significantly from the rest in containing the reducing agent, β-mercaptoethanol (2%), in its extraction buffer. This same manufacturer has developed a similar ELISA to detect egg albumin where they use an extraction buffer containing both a surfactant (SDS) and a reducing agent (β-mercaptoethanol), and the antibodies are also raised against denatured proteins. Of the total protein content of peanut kernels, Ara h 3 is the most abundant and Ara h 1 content is about 12−16%, while both Ara h 2 and Ara h 6 are present in approximately the same concentration, 6−9%. Heating/roasting of peanuts results in a decrease in Ara h 1 levels presumably owing to a loss of solubility.