DETECTION AND QUANTIFICATION OF GROUP-4 ALLERGENS IN GRASS-POLLEN EXTRACTS USING MONOCLONAL-ANTIBODIES

Background Grass pollen extracts are complex mixtures consisting of di fferent major allergenic and non-allergenic components. Ph1 p 4 is an important allergen, because more than 75% of grass pollen allergic pat ients produce specific IgE antibodies against group 4 allergens. Objec tive This study was designed to investigate the specificity of monoclo nal antibodies (MoAbs) produced against Phl p 4 and to verify the pres ence of group 4-like proteins in different grass pollen. Furthermore t he usefulness of MoAbs for quantification of group 4 allergens was stu died. Methods Group 4 analogues were investigated by immunoblotting an d ELISA inhibition using three MoAbs, The specificity of antibodies wa s 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 elec trophoretic 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 usi ng 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 gr oup 4 in extracts of several grasses varied considerably but was alway s less than 1% of the total protein. Conclusion Group 4 homologes are present in the various grass extracts but to different extents. The gr oup 4 ELISA could be very useful as a additional tool for providing in formation concerning the composition of grass pollen extracts.