Isolation of a Kentucky Blue Grass Pollen Allergen Using a Murine Monoclonal Antibody Immunosorbent

Abstract

We have previously reported the production of murine monoclonal antibodies to the retentate fraction of Kentucky Blue Grass pollen (KBG-R). In the present study, one of these monoclonal antibodies (Mab 27) was used in a combination of methods employing Western and immunoblot analysis to establish its reactivity to various antigenic components present in KBG-R. Thus, Mab 27 reacted predominantly with an antigen having a MW of 30 kD and to a lesser extent with other antigenic components with MW of 35 and 17 kD. Fractions of KBG-R obtained by preparative isoelectrofocusing, revealed on analysis by ELISA, that Mab 27 reacted with several components differing in charge. From these observations it was concluded that KBG-R contained a group of related antigens detectable with Mab 27 and differing in size and charge. A reverse immunosorbent, prepared by coupling Mab 27 to CNBr-activated Sepharose 4B, was used to absorb KBG-R. Bound material was recovered by acid elution and designated as Ag 27. SDS-PAGE analysis revealed that Ag 27 consisted of at least two components with molecular weights of 30 and 17 kD. Analysis by RAST using sera from humans allergic to KBG indicated that Ag 27 was highly allergenic.