Group 5 determination in Pooideae grass pollen extracts by monoclonal antibody‐based ELISA. Correlation with biologic activity

Abstract

A solid-phase, monoclonal antibody-based ELISA was set up to quantitate group 5 allergens in pollen extracts of wild and cultivated Pooideae grasses. The method was able to evaluate group 5 concentration in mass units with a sensitivity in the ng/ml range and a practical working range of 1-100 ng/ml. The group 5 ELISA was compared with rocket immunoelectrophoresis for determination of allergen levels in several Phleum pratense extracts, and a very good quantitative correlation was found (r = 0.98; P < 0.0001). A highly significant correlation (r > 0.8) was also obtained in comparing allergenic potency determined by RAST inhibition to group 5 content in several wild and cultivated grass species. The results proved the usefulness of the method in the standardization of Pooideae pollen extracts employed in diagnosis and treatment.

Antigenic Similarity among Group 1 Allergens from Grasses and Quantitation ELISA Using Monoclonal Antibodies to Phl p 1

“…Group 1 is more prevalent but group 5 usually induces higher IgE titers than group 1. As a consequence, when a serum pool is used, there is a good correlation between group 5 content and biological activity determined by RAST inhibition [13]. We have not seen this clearly in the case of group 1 (data not shown).…”

“…However, correction factors have to be established to compensate for the observed shift along the x-axis, which is most likely due to the fact that the affinity of the antibodies is not exactly the same for all allergens. This ELISA, along with the previously described ELISA method to quantify group 5 allergens [13], will allow us to standardize allergen products on the basis of the content of the two major grass pollen allergens. The exact ratio between these two major allergens must be defined after careful evaluation of the natural variability.…”

“…Groups 1 and 5 are the predominant allergens with an IgE prevalence >80% [8,9,10]. Group 5 is exclusive of the Pooideae subfamily [11,12,13], whereas group 1 has also been identified in members from other subfamilies [14,15,16,17]. Group 1 allergens are 30- to 37-kDa glycoproteins [14,15,16,17,18,19,20,21,22,23,24] which make up a subgroup of the β-expansin family of cell wall-loosening proteins in plants and may play a role in pollen germination on the stigma [25,26,27], through a biochemical mechanism that has not yet been fully elucidated [27,28,29,30].…”

Variation of the group 5 grass pollen allergen content of airborne pollen in relation to geographic location and time in season

“…15 Different species of the Poaceae family express homologues of Phl p 5 16,17 that show similar immunologic responses. 18 It is not possible to distinguish grass pollen grains from different grass species by using light microscopy, but the identity of allergens and the amount of allergen released from pollen varies between species. 8,17 Natural variation of allergen from biological sources was reported for birch pollen, 19 olive pollen, 20 horses, 21 and cats.…”

Evaluation of allergen vaccine potency

“…An optimal combination frequently consists of a monoclonal capture antibody and a polyclonal second antibody to confer maximum specificity and sensitivity. A large number of double-bind ELISAs have been developed for a variety of important allergens [27][28][29][30][31][32][33][34][35][36][37], and because of the increased sensitivity over the traditional RID, this assay format is currently the most widely used for specific allergen measurements in allergen preparations.…”