Detection and quantification of group 4 allergens in grass pollen extracts using monoclonal antibodies.
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.


CONCLUSIONS
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.
Department of Clinical Immunology, Faculty of Medicine, University of Jena, Germany.
B
B
Fahlbusch
W D
WD
Müller
O
O
Rudeschko
L
L
Jäger
O
O
Cromwell
H
H
Fiebig
D000485:Allergens; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D003429:Cross Reactions; D004587:Electrophoresis, Agar Gel; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D015151:Immunoblotting; D051379:Mice; D010936:Plant Extracts / Q000737:chemistry* / Q000276:immunology*; D010940:Plant Proteins / Q000032:analysis* / Q000276:immunology / Q000302:isolation & purification*; D006109:Poaceae / Q000276:immunology*; D011058:Pollen / Q000276:immunology*
D000485:Allergens; D000911:Antibodies, Monoclonal; C100980:Phl p 4 protein, Phleum pratense; D010936:Plant Extracts; D010940:Plant Proteins