title: Production and characterization of monoclonal antibodies to serine proteinase allergens in Penicillium and Aspergillus species.
abstract: 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 monoclonal antibodies against these serine proteinase allergens.
  
  
  METHODS
  BALB/c mice were immunized individually with the Penicillium citrinum culture medium or the crude extract and culture medium preparations of Aspergillus fumigatus. Hybridoma cells that secrete monoclonal antibodies against serine proteinase allergens were selected by immunoblotting. Antigens in three different Penicillium (P. citrinum, P. notatum and P. oxalicum) and two different Aspergillus species (A. fumigatus, and A. flavus) recognized 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 antibodies against serine proteinase allergens were generated after fusion of NS-1 cells with spleen cells obtained from BALB/c mice immunized with antigens 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 proteinase in two Penicillium (P. citrinum, P. notatum) and two Aspergillus species (A. fumigatus, and A. flavus) tested. PCM16 reacted with the alkaline serine proteinase allergen in P. citrinum, A. fumigatus and A. flavus but 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. notatum) tested. Among these five monoclonal antibodies generated, only PCM39 and FUM20 can react with the vacuolar serine proteinase allergen in P. notatum, P. oxalicum and in A. fumigatus. The 35 kDa P. citrinum component that reacted with FUM20 has an N-terminal amino acid sequence of DSPSVEKNAP.
  
  
  CONCLUSIONS
  Five monoclonal antibodies against different epitopes of the serine proteinase major allergens in prevalent Penicillium and Aspergillus species were generated in the present study. Antibodies obtained may be useful in the characterization and standardization of serine proteinase allergens in crude fungal extracts.
authors.affiliation: Department of Medical Research, Veterans General Hospital, Taipei, Taiwan, Republic of China.
authors.forename: W L
authors.initials: WL
authors.lastname: Lin
authors.forename: H
authors.initials: H
authors.lastname: Chou
authors.forename: M F
authors.initials: MF
authors.lastname: Tam
authors.forename: M H
authors.initials: MH
authors.lastname: Huang
authors.forename: S H
authors.initials: SH
authors.lastname: Han
authors.forename: H D
authors.initials: HD
authors.lastname: Shen
mesh_terms: D000485:Allergens / Q000276:immunology; D000908:Antibodies, Fungal / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001230:Aspergillus / Q000276:immunology*; D003429:Cross Reactions; D005656:Fungal Proteins / Q000276:immunology*; D015151:Immunoblotting; D016056:Immunodominant Epitopes / Q000276:immunology; D007123:Immunoelectrophoresis, Two-Dimensional; D010407:Penicillium / Q000276:immunology*; D012697:Serine Endopeptidases / Q000276:immunology*
chemical_list: D000485:Allergens; D000908:Antibodies, Fungal; D000911:Antibodies, Monoclonal; D005656:Fungal Proteins; D016056:Immunodominant Epitopes; D012697:Serine Endopeptidases