Are Physicochemical Properties Shaping the Allergenic Potency of Plant Allergens?

This review searched for published evidence that could explain how different physicochemical properties impact on the allergenicity of food proteins and if their effects would follow specific patterns among distinct protein families. Owing to the amount and complexity of the collected information, this literature overview was divided in two articles, the current one dedicated to protein families of plant allergens and a second one focused on animal allergens. Our extensive analysis of the available literature revealed that physicochemical characteristics had consistent effects on protein allergenicity for allergens belonging to the same protein family. For example, protein aggregation contributes to increased allergenicity of 2S albumins, while for legumins and cereal prolamins, the same phenomenon leads to a reduction. Molecular stability, related to structural resistance to heat and proteolysis, was identified as the most common feature promoting plant protein allergenicity, although it fails to explain the potency of some unstable allergens (e.g. pollen-related food allergens). Furthermore, data on physicochemical characteristics translating into clinical effects are limited, mainly because most studies are focused on in vitro IgE binding. Clinical data assessing how these parameters affect the development and clinical manifestation of allergies is minimal, with only few reports evaluating the sensitising capacity of modified proteins (addressing different physicochemical properties) in murine allergy models. In vivo testing of modified pure proteins by SPT or DBPCFC is scarce. At this stage, a systematic approach to link the physicochemical properties with clinical plant allergenicity in real-life scenarios is still missing.

Standardisation of allergen products: 4. Validation of a candidate European Pharmacopoeia standard method for quantification of major grass pollen allergen Phl p 5.

BACKGROUND: The aim of the BSP090 project is the establishment of European Pharmacopoeia Chemical Reference Substances (CRSs) in combination with corresponding standard ELISA methods for quantification of major allergens in allergen products. Here, we present data of a Phl p 5-specific sandwich ELISA that proved suitable for the quantification of Phl p 5, one of the major Timothy grass (Phleum pratense) pollen allergens. METHODS: A Phl p 5-specific ELISA system was assessed with respect to accuracy, precision, inter-assay (within laboratory) and inter-laboratory variations, in a ring trial including 14 laboratories in Europe and the USA. Model samples containing recombinant Phl p 5a CRS as well as native grass pollen extracts were analysed. Each participant was instructed to perform at least one preliminary assay to familiarise with the protocol, followed by three independent assays. RESULTS: The candidate standard ELISA proved suitable to quantify recombinant and native Phl p 5 with satisfactory precision (93% of results within ±30% acceptance range). Inter-assay variation (max. GCV 24%) and especially inter-laboratory variation (max. GCV 13%) showed conclusive results. When assessing accuracy by means of recovery of recombinant spikes from a grass pollen extract matrix, similarly satisfactory spike recovery results were observed for the two spikes with higher concentrations (all within ±30% acceptance range), whereas recovery of the lowest concentration spike was slightly poorer with mean results of six laboratories exceeding acceptance range. CONCLUSIONS: Based on the collaborative study results, the assessed Phl p 5-specific immunoassay is appropriate to be proposed as European Pharmacopoeia standard method.

Development and in-house validation of an allergen-specific ELISA for quantification of Bet v 4 in diagnostic and therapeutic birch allergen products.

Birch (Betula) pollen is a major cause of allergy in northern and central Europe. The allergenic potency of products for diagnosis and therapy of birch pollen allergy is adjusted nearly exclusively to the major birch pollen allergen Bet v 1. Although every fifth patient is additionally sensitized to Bet v 4, both content and variability of this minor allergen in birch allergen products remain unclear due to a lack of simple and cost-effective quantitative methods. This study aimed to develop and in-house validate the first Bet v 4-specific sandwich enzyme-linked immunosorbent assay (ELISA). Based on a murine monoclonal antibody in combination with a polyclonal rabbit antiserum, the ELISA proved to be highly sensitive, with a lower limit of quantification of 30 pg/ml Bet v 4. After confirmation of satisfactory accuracy, reproducibility, and robustness, the ELISA was utilized to quantify Bet v 4 in 30 authorized birch allergen products. The allergen was detected in all samples tested, ranging from 0.2 to 4.4 µg/ml. No significant correlation of Bet v 4 was found with the respective amount of Bet v 1. In contrast to Bet v 1, also no correlation of Bet v 4 with total protein content or total allergenic activity could be observed. Thus, it seems presently unfeasible to base birch allergen product standardization additionally on Bet v 4. In light of these results, the continuous monitoring of Bet v 4 in birch allergen products with the presented ELISA will provide a basis for the understanding of the clinical relevance of minor allergens.

Detectability of lupine seeds by ELISA and PCR may be strongly influenced by potential differences between cultivars.

Accurate methods for allergen detection are needed for the verification of allergen labeling and the avoidance of hidden allergens. But systematic data on the influence of different cultivars of allergenic crop species on their detectability in enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) are lacking. As one example, seeds of 14 different cultivars of lupine (Lupinus albus, Lupinus angustifolius, Lupinus luteus) were investigated for total protein according to a Kjeldahl method, and for their relative quantitative detectability in three commercial lupine-specific ELISA tests and four lupine-specific PCR methods. Total Kjeldahl nitrogen allowed an accurate quantification of total protein. Relative differences in quantitative response between cultivars of 390-5050% and 480-13,600% were observed between ELISA kits and PCR methods, respectively. Hence, quantitative results of selected ELISA and PCR methods may be strongly influenced by the examined lupine cultivar.

Development and in-house validation of allergen-specific ELISA tests for the quantification of Dau c 1.01, Dau c 1.02 and Dau c 4 in carrot extracts (Daucus carota).

Even though carrot allergy is common in Europe, the amount of different allergens in carrots is still unknown due to a lack of methods for quantitative allergen measurements. The current study aimed at the development of quantitative ELISA tests for the known carrot allergens, namely Dau c 1.01, Dau c 1.02, and Dau c 4 in pure carrot extracts. Monoclonal antibodies targeting the major carrot allergen isoforms Dau c 1.01 and Dau c 1.02 were generated and combined in sandwich ELISA with rabbit antisera against Api g 1, the celery homologue of Dau c 1. A competitive ELISA for the carrot profilin Dau c 4 was based on a polyclonal rabbit antiserum. The three ELISA tests were allergen-specific and displayed detection limits between 0.4 and 6 ng allergen/ml of carrot extract. The mean coefficient of variation (CV) as a means of intraassay variability of the Dau c 1.01, Dau c 1.02 and Dau c 4 ELISA tests was 8.1%, 6.9%, and 11.9%, and the mean interassay CV 13.3%, 37.1% and 15.6%, respectively. Target recovery ranged between 93 and 113%. In conclusion, the specific, accurate and reproducible quantification of three important carrot allergens may help to identify less allergenic carrot varieties, as well as to standardize the amount of allergens in extracts used for carrot allergy diagnosis.