Antigenic relationships among aspergilli by ELISA inhibition

21 BASIDIOSPORE SENSITIVITY AND ASTHMA. John Santilli, Jr., H.D., David G. Marsh, Ph.D., Ralph P. Collins, Ph.D., Joseph F. Alexander Jr., M.D. and Phillip S. Horman, M.D., Dridgeport, Connecticut, Storrs, Connecticut and Baltimore, Maryland The role of Basidiospores in the allergic diathesis is poorly understood. Dialyzed extracts of Agaricus compestris, Coprinus micaeus, Fuligo ~eptica, Lycoperdon perlatum, Scleroderma lycoperdoides, Ustilago maydis, =nd sooty mold were preparec from fresh spores. The study group consisted of 62 patients with asthma and 75 patients with allergic rhinitis serving as controls. These 137 patients had symptoms occurring during October and November. All were intradermally skin tested with the 7 extracts at l Opg/ml. Skin tests were recorded at 15 min. (pos = 5mm wheal with lOmm erythema or greater) at 6 and 24 hrs. (pos = 6mm or greater of induration & erythema). A total of 251 immediate reactions were observed in the asthmatic group (4.05/pt) compared to I04 in the control group (l.38/pt) P<.OOl (t test). However, the delayed reactions were just the opposite. The asthmatic group had 139 delayed reactions or 2.24/pt compared to 332 or 4.42/pt in the control group, Pr It appears that immediate skin test sensitivity to Basidiospores may, be significant of asthma occurring during October and November.

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22 CROSSED RADIOIMMUNOELECTROPHORETIC (CRIE) ANALYSIS OF SERUM IgG AND IgE RESPONSES TO ASPERGILLUS IN PATIENTS WITH CYSTIC FIBROSIS (CF) AND ALLERGIC BRONCHOPULMONARY ASPERGILLOSIS (ABPA). R.K. Bush, M.D., and M.J. Voss, M.D. Madison, Wisconsin Serum from patients with ABPA have both IgG and IgE antibodies toward Aspergillus (ASP) antigens. In this study we used CRIE to determine whether the IgE and IgG antibodies in ABPA serum reacted to the same ASP antigens. First dimension electrophoretic separation of a commercial Aspergillus antigen preparation was performed in a 1% agarose-Tris-barbital-calcium lactate buffer, pH 8.4 at i0 v/cm for 25 minutes. The second dimension gel contained 20 ~i/ cm 2 of each patient's own serum which had been passed over an anti-IgE-Sepharose column. The anti-IgE sorbent column removed 89.6%, 52.4%, and 48% of the total serum IgE activity respectively, as determined by ELISA. Second dimension immunoelectrophoresis was carried out for 18 hrs at 2 v/cm. The plates were then incubated with 400 ~i of each patient's own untreated serum or saline control for 4 hrs. The gels were next incubated with 125I-labeled anti-human IgE, 200,000 cpm/plate, overnight. The gels were washed, dried, and exposed for 18 days to x-ray film which was then processed. Coomassie Blue stain was used to identify the gel immunoprecipitins. The Coomassie Blue stained plates and the autoradiographs demonstrated 2-7 precipitating antigens recognized by both IgG and IgE antibodies. Thus CRIE simultaneously detected both IgE and IgG ASP responses in sera of patients with ABPA and CF.

MEASUREMENTOF ASPERGILLUS SPECIES-SPECIFIC IgG AND IgE ANTIBODIES BY ELISA. B.E.Birkbx, M.D.~ H.A.Bur~e, Ph.D., M.L.Muilenber9, M.S., W.R. Solomon~ M.D., Ann Arbor, MI. The enzyme linked immunosorbent assay (ELISA) measures protein (P) or non-protein (NP) specific IgG & IgE with exquisite s e n s i t i v i t y . We used whole, P, & NP culture f i l t r a t e fractions of Aspergillus fumigatus (Af) (4 strains), A.niger TAn), A.wentii (Aw), A.sydowi___i_i(As), & A.versicolor (Av) prepared in our laboratory and a commercial lyophilized Af preparation in an indirect microtiter plate ELISA for specific IgG & IgE. We assayed sera from 50 normals and 83 sera submitted from patients suspected of harboring aspergilli for Af precipitins (PN). 19 of these were Pn +. In a l l of these plus l negative sera, ELISA detected Af P & NP specific IgG & IgE. I t appears Af P, but not NP specific IgG in aspergilloma is at least twice that in ABPA. 59 sera were negative against Af by PN and ELISA, but significant a c t i v i t y was detected against P antigens of An, Aw, & Av in l and As in 38 sera. Of 9 Af + sera tested, there was a c t i v i t y against An in 5, Aw in 4, Av in 4, and As in 9. The results of this rapid non-radioactive assay were reproducible with clear differentiation of elevated values (0. D.405, mean normal = 0.I07 § 0.016; positive = 1.004 ~ 0.148). Each strain ~ f A f detected a l l PN + sera, even at dilutions up to 1:50,000. ELISA offers advantages of simplicity and s e n s i t i v i t y over PN for AsperBillus associated disease and is adaptable to species other than Af of potential pathogenic significance. We therefore believe i t merits wider c l i n i c a l application.

24 ANTIGENIC RELATIONSHIPSAMONGASPERGILLI BY ELISA INHIBITION. H. Burge PhD, B. Birkbu MD, M. Muilenber9 MS, W. Solomon MD, Ann Arbor, MI. Although illness due to other aspergilli is increasingly noted, in v i t r o diagnostic tests remain confined largely to A. fumigatus (Af) antigens. We evaluated Af e~tracts for antigens possibly common to aspergilli and examined cross-reactivity among familiar form species. Aliquots of an Af precipitin positive serum pool (diluted 1:5000) were used in microtiter plate IgG ELISA against culture f i l t r a t e protein fractions of A. wentii(Aw), A. versicolor(Av), A. s~dowii(AsT, ~ n~er(An)-and Af. Direct reactions and t i t e r s after preincubating serum with the same antigens and Alternaria alternata in concentrations of l , lO, and lO0 ~g/ml of diluted serum were compared. Direct % binding values (using Af t i t e r s as lO0) were: for Av 76%; As 63%; Aw 39%; and An 21%. Antigen concentrations (~g/ml) producing 50% i n h i b i t i o n of ELISA on preincubation were: Aw As Af Av An Alt(Inhib) Aw l O 0 >lO0 As 2 l 14 48 > l O 0 >lO0 Af 7 >lOO lOO > l O 0 >lO0 Av 2 l O 0 >lO0 An l 20 4 >lO0 18 65 Although IgG binding to all species exceeded that for normal sera, specific differences were evident by direct and inhibition assays. An resembled other species least closely, but Af cross-reactivity also was limited. These findings suggest that species-specific reagents w i l l be essential in serological studies of non-Af aspergilli--especially An and other distantly related forms.

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