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

• Published in: Clinical and experimental allergy Vol. 28; no. 7; pp. 799 - 807
• Main Authors: FAHLBUSCH, B, MÜLLER, W.-D, RUDESCHKO, O, JÄGER, L, CROMWELL, O, FIEBIG, H
• Format: Journal Article
• Language: English
• Published: Oxford BSL Blackwell Science Ltd 01.07.1998; Blackwell
• Subjects: Allergens; Animals; Antibodies, Monoclonal - immunology; Antibody Specificity; Antigens, allergens; Biological and medical sciences; Cross Reactions; cross-reactivity; Electrophoresis, Agar Gel; Enzyme-Linked Immunosorbent Assay; Epitope Mapping; Fundamental and applied biological sciences. Psychology; Fundamental immunology; grass pollen extracts; Immediate hypersensitivity. Allergy. Anaphylaxis, etc; Immunobiology; Immunoblotting; Mice; monoclonal antibodies; Phl p 4; Plant Extracts - chemistry; Plant Extracts - immunology; Plant Proteins - analysis; Plant Proteins - immunology; Plant Proteins - isolation & purification; Poaceae - immunology; Pollen - immunology; purification; two-site binding ELISA; Monocotyledones; Purification; Quantization; Extract; Monoclonal antibody; Enzyme immunoassay; Gramineae; Angiospermae; Cross reaction; Spermatophyta; Pollen; Aeroallergen; ELISA assay; Allergen
• ISSN: 0954-7894; 1365-2222
• DOI: 10.1046/j.1365-2222.1998.00297.x

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.