Detection of crossreactive determinants in grass pollen extracts using monoclonal antibodies against group IV and group V allergens

Abstract

Monoclonal antibodies (MoAb) to defined allergens of P. pratense were raised. Five of them were selected for detailed studies by means of immunoblotting after SDS-PAGE and IEF of extracts from P. pratense and L. perenne. Three antibodies (1D11, 3B2, 2D1) recognize structures with mol. wt of 29 and 34 kD and pI of 4.8-7.6, corresponding to group V allergens. Two other MoAbs (2D8, 3C4) are directed against strong basic structures with a mol. wt of 50 kD and pI of 7.8-9.5 according to group IV allergens. The specificity of antibodies was supported by direct ELISA with purified group V and IV allergens. The isolated allergens were characterized before by SDS-PAGE and CIE and that allergenicity was detected with sera of patients with allergic rhinitis. Using our selected MoAbs crossreactive epitopes on group V and IV allergens have been excluded. Our antibodies have been used to detect crossreactivity in 14 grass pollen extracts. The evaluation of the pollen extracts has been performed by enzyme immunoassay (EIA) inhibition. One MoAb (3C4) is able to recognize group IV allergens in all grass species analysed whereas the MoAb 2D8 seems to identify group IV structures in selected grasses only. Binding to conserved structures of group V has been proved for MoAb 1D11. Other group V specific MoAbs (2D1, 3B2) identify similar, however incomplete, spectra. These results have been confirmed also by the dot immunobinding assay.