Successful Technique for the Selective Production of Monoclonal Antibodies Against a Major Allergenic Component in Timothy Pollen Extract

Abstract

Monoclonal antibodies (MAbs) were selectively raised against a major allergenic component of 38 kD in timothy grass pollen (Phleum pratense). We used a special prefractionating technique to isolate the 38 kD allergen, because immunizations with crude pollen extract had resulted in a wide variety of antibodies against other determinants, and cross-reactions between different-sized proteins occurred. Pollen extract was separated by Western blotting. Strips of the nitrocellulose membrane containing the allergen were excised, dispersed into allergen bearing particles, and used for immunization of BALB/c mice. Five MAbs were obtained that reacted with the 38 kD allergen. Four of these antibodies (BF 1, DC 9, EB 4, GE 2) exclusively detected the 38 kD protein, while one MAb (EB 6) additionally bound to a 32 kD allergen. Epitope mapping with the 3 IgG 1 antibodies (BF 1, EB 6, DC 9) was performed by ELISA inhibition tests using the purified 38 kD component. The antibodies did not interfere with each other. This confirms that they bind to different sites of the 38 kD molecule. Only a weak inhibitory effect of the MAbs on the binding of patient's IgE was determined, we suppose that the MAbs do not bind to IgE reactive epitopes. For standardization and further characterization of the 38 kD protein by peptide mapping the MAbs will be useful tools.