Quantification of Peach Fruit Allergen Lipid Transfer Protein by a Double Monoclonal Antibody-based Sandwich ELISA

Abstract

Fruit is one of the most commonly reported food allergy sources in China, and peach lipid transfer protein (Pru p 3) has been identified as the major allergen inducing systematic symptoms. Crude allergen extracts and single component allergens have been used in food allergy diagnosis and immunotherapy. Reliable and sensitive analytical methods to quantify Pru p 3 content in fruit will help to identify lowallergenic cultivars among the abundant peach genetic resources in China. In this study, we developed a sensitive sandwich ELISA method to measure Pru p 3 in peach fruit based on two monoclonal antibodies with high sensitivity and linearity. Significant variability was observed in peach cultivars, with a much higher Pru p 3 level in peach peel than in pulp. This method will be very useful to select peach varieties with low Pru p 3 content and as a diagnostic product to detect hidden allergens in processed food.

Development of a H2O2‐sensitive quantum dots‐based fluorescent sandwich ELISA for sensitive detection of bovine β‐lactoglobulin by monoclonal antibody

“…To date, a number of analytical methods have been developed to detect food allergens, including reversed-phase high-performance liquid chromatography (RP-HPLC), 7 mass spectrometry (MS), 8 LC-MS, 9 real-time polymerase chain reaction, 10 surface plasmon resonance, 11 electrochemical immunosensor, 12 and enzyme-linked immunosorbent assay (ELISA). 13,14 Among them, ELISA is the most commonly used method for detection of food allergens with advantages of speed, specificity, high sensitivity, and easy application. Moreover, antibody is an important element in ELISA for testing of food allergens, because epitopes play a key role in allergic reaction.…”

Localization of Four Allergens in Artemisia Pollen by Immunofluorescent Antibodies

“…Antibody-producing hybridoma cells were screened by ELISA and later by Western blot with immunized pollen extracts, recombinant (r)Art v 1.0101 [2, 20] and Art v 3 allergen rArt v 3.0201 [21], to select specific monoclonal antibodies (mAbs). mAbs were purified by protein G column and isotype determination as described previously [22]. One of the specific mAbs for Art an 1, Art ar 2, and Art an 3 was used to purify the target protein on a CNBr-activated Sepharose TM 4 fast flow column (71–5000–15AF preactivated media, spin column, 10 mL, C006727, GE Healthcare, USA).…”

“…One of the specific mAbs for Art an 1, Art ar 2, and Art an 3 was used to purify the target protein on a CNBr-activated Sepharose TM 4 fast flow column (71–5000–15AF preactivated media, spin column, 10 mL, C006727, GE Healthcare, USA). The purified protein was first tested with A. argyi -allergic sera pool by ELISA as described previously [22] to confirm it is an allergen, and then analyzed by LC-MS (Applied Biosystem MALDI-TOF/TOF 5800) or LC-MS/MS (Thermo Scientific Q Exactive) for its identity-matching to known Artemisia allergens.…”

“…These methods follow a previously published protocol [22]. Crude pollen extracts were analyzed by Tricine-SDS-PAGE and visualized by staining with Coomassie brilliant blue R-250.…”

“…Pollen of A. annua without antibody or antifade treatment was observed for autofluorescence. Chenopodium alba pollen, mAb A7–1 specific to peach allergen Pru p 3 [22], and α-SMA mAb to human actin were used as negative controls.…”