Crossed Radioimmunoelectrophoretic Studies of Distinct Allergens in Two Extracts of <i>Cladosporium herbaru</i><i>m</i>

Abstract

Cladosporium herbarum was supplied from two sources and extracted identically. Antisera against the extracts were produced in rabbits and two reference patterns were established using crossed immunoelectrophoresis (CIE). Both patterns showed more than 60 precipitates but less than 50% of the detectable antigens appeared to be identical in the two extracts. The allergens were identified by means of crossed radioimmunoelectrophoresis (CRIE) using sera from 35 individuals with proven or suspected allergy to C. herbarum. Four important and 10–20 less important allergens were demonstrated. Among the allergens present, there were none reacting with all patient sera. Only 1 out of 3 rabbits immunized with a suspension of broken cells of C. herbarum showed precipitating antibodies to the statistically most important allergen, while 9 rabbits immunized with aqueous extracts of the mold did not. The composition of the two extracts with respect to allergens differed. Allergens present in one extract were not always detectable in the other. The experiments also showed how CIE/CRIE with various combinations of antigens and antisera may be combined with CRIE inhibition, radioallergosorbent test (RAST) and RAST inhibition for comparing complex allergen extracts at the molecular level.

Allergenic components of isolates ofcladosporium herbarum

“…Electrophoresis in the first dimension was carried out at 10 V cm-' for 0.5 h and in the second at 2 V cm-' for.16 h. The plates were stained with Coomassie brilliant blue R-250. For CRIE, (Aukrust 1979) unstained CIE plates were incubated overnight with 400 pL of human atopic sera (pooled from six patients sensitive to C. he~barrrrtz) followed by anti-human IgE-1125 containing 400,000 cpm per plate for 16-18 h. The plates were subsequently exposed to.X-ray film for 21 days at room temperature. Controls were included in which the atopic serum was replaced by saline or a serum pool from six non-allergic individuals to check for non-specific absorption of the anti-human IgE-1125.…”

Allergenic components ofganoderma applanatum

“…Lchrer, New Orleans) and the solid phase disc was coupled with 200 11s of the extract. Crossed-immunoelectrophorcsis (CIE) and CRIE were pcrformed as described by Axelscn et al (1973) and Aukrust (1979) respectively. with slight modifications (Vijay et Protein components of the spore extract (1200 pg containing 96 pg of protein) were separated on SDS-PAGE by the method of Laemmli (1970) and were electrotransferred on to nitrocellulose (NC) membrane (Bio-Rad; 0.45 pm pore size) in a Bio-Rad Transblot cell as described by Towbin et al (1979).…”

Allergens ofAlternaria

“…Several allergens and antigens were common to Alternaria and Sternphyliitm extracts although the antisera used apparently did not detect the basic allergen in their CIE plates. Aukrust (1979) did demonstrate basic antigens with CIE of Cladosporiirni extracts, but did not demonstrate their allergenicity or their cross reactivity with other molds.…”

“…2. are minor and perhaps intermediate allergens as well. The definition of these categories has been based upon RAST or RAST inhibition, quantitative skin tests (Bjorkstkn et al 1984, Turkletaub et al 1982) quantitative immunoelectrophoresis (Aukrust 1979) or combinations of the above (Ingemann & Venov 1984). Alt-1 is a common and important allergen but it is by no means the only one (Lowenstein 1980, Bush et al 1983, Budd et al 1983a.…”