Ole e 3, a Candidate for in vivo Diagnosis of Polcalcin Sensitization

Abstract

<b><i>Introduction:</i></b> Polcalcins belong to the family of calcium-binding proteins. They are ubiquitous in the plant kingdom and highly conserved, which leads to these panallergens showing a high degree of inter-cross-reactivity. They are responsible for allergic polysensitization, and therefore, their diagnosis is necessary for correct selection of immunotherapy. The objectives were to develop a method to purify native polcalcin with intact allergenic properties and to validate its use for diagnosis of polcalcin sensitization. <b><i>Methods:</i></b> Ole e 3 was purified by immunoaffinity chromatography using anti-rChe a 3 polyclonal antibodies and identified by mass spectrometry. Calcium-binding assays were performed in immunoblot and ELISA assays. Diagnostic capacity of Ole e 3 was analyzed by ELISA and compared to ImmunoCAP with sera from a pollen-sensitized population. Cross-reactivity with other polcalcins was investigated by ImmunoCAP inhibition. <b><i>Results:</i></b> Immunogenicity of purified Ole e 3 was not affected by the addition of calcium. However, the presence of a calcium chelator agent completely inhibited IgG binding by immunoblot and produced a 32.3% reduction in IgE binding by ELISA. Ole e 3 enabled diagnosis of polcalcin-sensitized patients, and a good correlation was revealed with ImmunoCAP. A 50% inhibition in IgE binding was obtained with 2.8 ng of Ole e 3 for rBet v 4 and 3.9 ng for rPhl p 7. <b><i>Discussion/Conclusion:</i></b> Native Ole e 3 was purified by maintaining its allergenic properties. This innovative method enables obtaining this active native allergen to be used for in vivo diagnosis of polcalcin sensitization.

EAACIMolecular Allergology User's Guide 2.0

“…Polcalcins are only expressed in pollen, thus are not related to food allergy, contrary to other pan‐allergens such as profilin or nsLTPs. It is considered as a pan‐allergen and therefore could be a confounding factor in the diagnosis of polysensitized pollen‐allergic patients and may lead to a diagnosis of “allergy mirages” 1397,1517 (Figure 166). Through IgE inhibition assays, Asero et al found important differences in IgE reactivity between rBet v 4 and rPhl p 7, since only grass pollen extract was able to markedly inhibit rPhl p 7.…”

“…However, in some research articles, an extract derived from palm pollen has been used and prepared by ALK (Madrid, Spain) 1412,1421 . Recently a method to purify the olive polcalcin, Ole e 3, has been described 1517,1518 . For specific IgE determinations, there are only three polcalcins available; Phl p 7, Bet v 4 and Aln g 4.…”

“…It is considered as a pan-allergen and therefore could be a confounding factor in the diagnosis of polysensitized pollen-allergic patients and may lead to a diagnosis of "allergy mirages" 1397,1517 (Figure 166). That suggests that in some areas the primary source of sensitization for polcalcin is grass pollen.…”

“…1412,1421 Recently a method to purify the olive polcalcin, Ole e 3, has been described. 1517,1518 For specific IgE deter-…”