Production and characterization of monoclonal antibodies to serine proteinase allergens in Penicillium and Aspergillus species

Background Alkaline and/or vacuolar serine proteinases are major allergens in prevalent airborne Penicillium and Aspergillus species. Objective The object of this study is to generate and characterize monoclon al antibodies against these serine proteinase allergens. Methods BALB/c mice were immunized individually with the Penicillium citrin um culture medium or the crude extract and culture medium preparations of A spergillus fumigatus. Hybridoma cells that secrete monoclonal antibodies ag ainst serine proteinase allergens were selected by immunoblotting. Antigens in three different Penicillium (P. citrinum, P. notatum and P. oxalicum) a nd two different Aspergillus species (A. fumigatus, and A. flavus) recogniz ed by these monoclonal antibodies were analysed by sodium dodecyl sulphate and two-dimensional polyacrylamide gel electrophoresis immunoblotting and N -terminal amino acid sequence analysis. Results Four (PCM8, PCM10, PCM16 and PCM39) and one (FUM20) monoclonal anti bodies against serine proteinase allergens were generated after fusion of N S-1 cells with spleen cells obtained from BALB/c mice immunized with antige ns from P. citrinum and A. fumigatus, respectively. Immunoblotting results showed that PCM8 reacted with an alkaline serine proteinase allergen in P. citrinum and P. notatum. PCM10 and PCM39 reacted with the alkaline serine p roteinase in two Penicillium (P. citrinum, P. notatum) and two Aspergillus species (A. fumigatus, and A. flavus) tested. PCM16 reacted with the alkali ne serine proteinase allergen in P. citrinum, A. fumigatus and A. flavus bu t not with that in P. notatum. MoAb FUM20 reacted with the alkaline serine proteinase allergen in two Aspergillus species (A. fumigatus and A. flavus) but not with that in two different Penicillium species (P. citrinum, P. no tatum) tested. Among these five monoclonal antibodies generated, only PCM39 and FUM20 can react with the vacuolar serine proteinase allergen in P. not atum, P. oxalicum and in A. fumigatus. The 35 kDa P. citrinum component tha t reacted with FUM20 has an N-terminal amino acid sequence of DSPSVEKNAP. Conclusion Five monoclonal antibodies against different epitopes of the ser ine proteinase major allergens in prevalent Penicillium and Aspergillus spe cies were generated in the present study. Antibodies obtained may be useful in the characterization and standardization of serine proteinase allergens in crude fungal extracts.