Monoclonal Antibody-Based ELISA to Quantify the Major Allergen of <i>Cynodon dactylon</i> (Bermuda Grass) Pollen, Cyn d 1

Abstract

Background: Pollen of Bermuda grass (Cynodon dactylon) is an important cause of pollinosis in many areas of the world. Most patients show sensitivity to the major allergen Cyn d 1, a glycoprotein composed of a number of isoforms with a molecular mass of 31–32 kDa. The aim of this work was to develop a monoclonal antibody (mAb)-based ELISA to quantify Cyn d 1, and to assess the correlation of the allergen content with the biological activity of C. dactylon pollen extracts. Methods:After fusion of myeloma cells with spleen cells from a BALB/c mouse immunized with C. dactylon pollen extract, Cyn d 1-specific mAbs secreting hybridomas were selected, and the antibodies characterized. One of them (4.4.1) was used as the capture antibody in an ELISA method for Cyn d 1 quantitation. An anti-Cyn d 1 rabbit serum was used as the second antibody. Cyn d 1 was purified by immunoaffinity chromatography with mAb 4.4.1, characterized, and used as the standard in the assay. Results: The identity, purity and isoallergen composition of affinity-purified Cyn d 1 was confirmed by N-terminal amino acid sequencing, SDS-PAGE, Western blot and 2D electrophoresis. The Cyn d 1 ELISA is highly specific and sensitive, with a detection limit of 0.24 ng/ml and a linear range of 1.1–9.2 ng/ml. An excellent correlation was found when the content of Cyn d 1, measured in 16 different extracts, was compared with the allergenic activity of the same extracts determined by RAST inhibition. Conclusions: The results prove the usefulness of the Cyn d 1 ELISA for the standardization of C. dactylon-allergen products on the basis of major allergen content.

Differences among Pollen-Allergic Patients with and without Plant Food Allergy

“…In this study we have analyzed 2 well-defined pollen-allergic patient groups and tested a wide and well-documented panel of purified allergens [10,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39]. Clinical data were compared with both the results obtained with the specific IgE responses to this panel and with a battery of commercially available pollen extracts used for skin tests.…”

“…The following natural or recombinant allergens, isolated by previously described methods, were included in the diagnostic panel: Phleum pratense: nPhl p 1 [19] and nPhl p 5 [20]; Cynodon dactilon: nCyn d 1 [21]; A. vulgaris: nArt v 1 [22]; O. europaea: nOle e 1 [23] and Ole e 9, as a mix of rN-terminal domain and rC-terminal domain from Ole e 9 [24,25]; P. lanceolata: nPla l 1 [26]; P. judaica: nPar j 1 [27]; C. sempervirens: nCup s 1 [10]; S. kali: nSal k 1 [28]; Chenopodium album: rChe a 1 [29], rChe a 2 (profilin) [30] and rChe a 3 (polcalcin) [31]; P. acerifolia :rPla a 1 and nPla a 2 (BIAL-Aristegui, Bilbao, Spain). As pan-allergens, we used polcalcin: rChe a 3 from C. album pollen [31]; nonspecific LTP from peach: rPru p 3 [32]; a mixture of 3 profilins: rCuc m 2 from muskmelon [33], rChe a 2 from C. album pollen [30] and rMal d 4 from apple [10].…”

Relevance of Allergenic Sensitization to Cynodon dactylon and Phragmites communis: Cross-reactivity With Pooideae Grasses

“…P communis sIgE values showed the highest correlation with Phl p 1 sIgE (R 2 =0.8), although the correlation was also high with Cyn d 1 (R 2 =0.7). Duffort el al [23] developed a monoclonal antibody to quantify Cyn d 1 that also recognized P communis. Both results suggest that Phr a 1 shares epitopes with Cyn d 1 and with Phl p 1.…”

Standardization of allergen extracts for immunotherapy: where do we stand?

“…The working range of the highly reproducible assays was in the low (Pla a 1 and Par j 1/2) to intermediate (Cup a 1 and Cup s 1) ng range and a good correlation with the IgE reactivity of the relevant pollen extracts was reported. Another study [36] focused on the group I allergen from Bermuda grass pollen, in which Cyn d 1-specific monoclonal antibodies were selected from spleen cells from BALB/c mice immunized with Bermuda grass pollen extract. One mAb was combined to a sandwich with a rabbit anti-Cyn d 1 antiserum, and natural Cyn d 1 was used as the reference compound.…”