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Detection and quantification of group 4 allergens in grass pollen extracts using monoclonal antibodies
Author(s) -
B Fahlbusch,
WolfDieter Müller,
O. Rudeschko,
Lothar Jäger,
Oliver Cromwell,
Helmut Fiebig
Publication year - 1998
Publication title -
clinical and experimental allergy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.462
H-Index - 154
eISSN - 1365-2222
pISSN - 0954-7894
DOI - 10.1046/j.1365-2222.1998.00297.x
Subject(s) - phleum , lolium perenne , allergen , pollen , lolium , epitope , biology , immunoglobulin e , monoclonal antibody , holcus lanatus , airborne allergen , dactylis glomerata , poa pratensis , alternaria , antibody , chemistry , immunology , botany , poaceae , allergy
Background Grass pollen extracts are complex mixtures consisting of different major allergenic and non‐allergenic components. Phl p 4 is an important allergen, because more than 75% of grass pollen allergic patients produce specific IgE antibodies against group 4 allergens. Objective This study was designed to investigate the specificity of monoclonal antibodies (MoAbs) produced against Phl p 4 and to verify the presence of group 4‐like proteins in different grass pollen. Furthermore the usefulness of MoAbs for quantification of group 4 allergens was studied. Methods Group 4 analogues were investigated by immunoblotting and ELISA inhibition using three MoAbs. The specificity of antibodies was studied using isolated group 1 and group 5 allergens. Quantification of group 4 allergen was achieved by a two‐site solid‐phase ELISA. Phl p 4 was purified from whole pollen extract by chromatographic or electrophoretic techniques and used as standard. Results The MoAbs studied bound strongly to proteins from timothy grass pollen extract at a mw of 55 kDa and a pI of 9.0–9.3. Phl p 4 homologes with similar mw were detected in Dactylis glomerata, Festuca pratensis, Holcus lanatus, Poa pratensis, Lolium perenne . Epitope mapping showed that all three MoAb recognized unrelated regions on Phl p 4. A two‐site binding ELISA using MoAbs was developed for determination of Phl p 4 in Phleum pratense extracts. The method was able to evaluate group 4 in mass units with a working range between 150 and 2000 ng/mL. The absolute amounts of group 4 in extracts of several grasses varied considerably but was always less than 1% of the total protein. Conclusion Group 4 homologes are present in the various grass extracts but to different extents. The group 4 ELISA could be very useful as a additional tool for providing information concerning the composition of grass pollen extracts.