Characterization of Antigens from Aspergillus fumigatus III. Comparison of Antigenic Relationships of Clinically Important Aspergilli!
SANG J. KIM and S. D. CHAPARAS
SUMMARy __________________________________________________________ Antigenic relationships between strains of Aspergillus fumigatus, Aspergillus fumigatus var. ellipticus, Aspergillus phialiseptus, Aspergillus flavus, and Aspergillus niger were analyzed by fused rocket immunoelectrophoresis and by skin tests. Seventy-three to 89 % of the numbers of antigens detected between strains and species of the A. fumigatus series were shared. The degree of sharing between antigens of A. flavus, A. fumigatus series, and A. niger was much lower and ranged from 19 to 35 %. In reciprocal skin tests in sensitized guinea pigs, similar relationships were shown. Three fractions of A. fumigatus extract proved to be markedly specific for this species. Cross reactivity was associated primarily with carbohydrate and glycoprotein fractions.
Introduction In earlier studies (I, 2), the preparation, fractionation, and characterization of a large batch of mycelial extract of Aspergillus fumigatus were described that were intended to provide more sensitive and specific reagents for the control of diagnostic reagents for testing in vivo and in vitro. Because other species of Aspergillus can also produce infections and diseases (3-7) and serums from such patients react with antigens from A. fumigatus and other species, serologic data alone are insufficient to permit identification of the infecting organism. Cross reactivity has also been seen in skin testing (3, 7-14). In the present study, the reactivity and specificity of mycelial extracts and fractions of A. fumigatus were compared to those of mycelial extracts from clinical isolates of
(Received in original form June 18, 1979 and in revised form September 5,1979) 1 From the Mycobacterial and Fungal Antigens Branch, Division of Bacterial Products, Bureau of Biologics, Food and Drug Administration, 8800 Rockville Pike, Bethesda, Md. 20205.
other strains and species of Aspergillus by skin tests in homologously and heterologously sensitized guinea pigs, and by fused rocket immunoelectrophoresis (15). Methods Antigens. Mycelial extracts of A. fumigatus NIH 5233 and its fractions were prepared as described previously (1, 2). API fractions APIFA, APIFB, APIFC, and APIFD were precipitated with 75 % ammonium sulfate and eluted in order, respectively, from a Sephadex G-75 column. AS fractions ASI and ASH were soluble at 75 % ammonium sulfate. ASI was bound by a DE-52 ion exchange column, whereas ASH was not bound. Similar preparations of mycelial extracts have been prepared from clinical isolates of A. fumigatus (strain 004 and 005), A. fumigatus var. ellipticus (AFE) ATCC 16903, Aspergillus phialiseptus (APS) 010, Aspergillus flavus (AFV) ATCC 24133, Aspergillus niger (ANG) ATCC 1015, Aspergillus clavatus (ACT), and Aspergillus nidulans (AND). Isolates 004 and 005 of A. fumigatus were from cases of aspergilloma and had morphologic features similar to those of strain NIH 5233 except that both strains exhibited poor conidial production and retarded growth rate. Strain 004 also exhibited intercalarial or terminal round cells (10 to 30 /Lm). A. phialiseptus organisms were isolated from the sputum spe-
AMERICAN REVIEW OF RESPIRATORY DISEASE, VOLUME 120, 1979
1297
1298
KIM AND CHAPARAS
cimen of a patient with allergic bronchopulmonary aspergillosis. Immunization of animals. Antibodies were produced in rabbits using the mycelial homogenates, as described previously (I, 2). For each strain or species, 6 guinea pigs were sensitized subcutaneously in the footpads and intramuscularly in the nuchal area. Skin tests were performed 4 to 6 wk after sensitization by the injection of 0.1 ml of antigen in saline solution intracutaneously in the flank areas. All skin tests for fractions and extracts were per-
004
005
ME
AFE
005
APS ACT
formed at doses determined to be bioequivalent (13 to 15 mm) in dose-response titrations in homologously sensitized animals. Reaction diameters were recorded between IS and 24 h. Fused rocket immunoelectrophoresis. This procedure was performed according to a modification of the method of Svendsen (16) using 1 % agarose containing veronal buffer at pH S.6 and ionic strength 0.075, as described previously (1, 2, 15). The test was repeated when necessary, using different geometries and arrangements to produce lines of iden-
AND
005
AFV ANG
FA
FB
FC
ASI
Fig. 1. Fused rocket immunoelectrophoresis of extracts of species of Aspergillus and fractions of Aspergillus Jumigatus with anti-A. Jumigatus 005 serum. ACT = Aspergillus clavatus; AFE = A.Jumigatus var. ellipticus; AFV = Aspergillus flaws; AND = Aspergillus nidulans; ANG = Aspergillus niger; APS = Aspergillus phialiseptus; ME, 004, and 005 = A. Jumigatus NIH 5233, 004, and 005; ASI, FA, FB, and FC = fractions ASI, APIFA, APIFB, and APIFC derived from mycelial extract of A. Jumigatus NIH 5233.
1299
CHARACTERIZATION OF A. FUMIGATUS ANTIGENS
tity and optimal resolution of precipitin bands. Antigens were used at 20 mg/ml (dry weight). Fractions ASH and APIFD were excluded in most of the figures because they produced only a few, very poor precipitin bands that were more prominent in adjacent fractions. The PQS (percentage of qualitath'e sharing) values were determined as described elsewhere (15), using the formula: number of precipitin bands with heterologous preparation
PQS = - - - - - - - - - - - X 100. number of precipitin bands with homologous preparation In some anlayses, the reciprocal average PQS values
(RAP) for the comparison of any 2 species (e.g., A and B) were compared: PQS of A with B antiserum + PQS of B with A antiserum
RAP = - - - - - - - - - - - - - - 2 Results
Representative results and analyses with fused rocket immunoelectrophoresis are shown in figures I and 2 and in tables I and 2. In table 1, PQS values between 62 and 100 % suggest a close relationship between A. fumigatus strains, A. fumigatus var. ellipticus, and A. phialisep-
ANTI-A. phialiseptus
004 APS
005
AFE
APS
ME
ACT AND APS
AFV ANG
FA
FB
FC
ASI
Fig. 2. Fused rocket immunoelectrophoresis of extracts of species of Aspergillus and fractions of A. fumigatus with anti-Aspergillus phialiseptus serum. For definitions of abbreviations see figure 1.
1300
KIM AND CHAPARAS
TABLE 1 COMPARISON OF ANTIGENS FROM ASPERGILLUS SPP. WITH HOMOLOGOUS AND HETEROLOGOUS ANTISERA Antigens
Fractions of ME"
!g
.~
.9-
..
~
A. niger A. flavus A. phialiseptus A. fumigatus var ellipticus A. fumigatus 005
A. fumigatus NIH5233
~
21 .~
~ ~
.~
§
.
~
to
~
:I:
Z
~to
.~
.~
~
~
'
SANG J. KIM and S. D. CHAPARAS
SUMMARy __________________________________________________________ Antigenic relationships between strains of Aspergillus fumigatus, Aspergillus fumigatus var. ellipticus, Aspergillus phialiseptus, Aspergillus flavus, and Aspergillus niger were analyzed by fused rocket immunoelectrophoresis and by skin tests. Seventy-three to 89 % of the numbers of antigens detected between strains and species of the A. fumigatus series were shared. The degree of sharing between antigens of A. flavus, A. fumigatus series, and A. niger was much lower and ranged from 19 to 35 %. In reciprocal skin tests in sensitized guinea pigs, similar relationships were shown. Three fractions of A. fumigatus extract proved to be markedly specific for this species. Cross reactivity was associated primarily with carbohydrate and glycoprotein fractions.
Introduction In earlier studies (I, 2), the preparation, fractionation, and characterization of a large batch of mycelial extract of Aspergillus fumigatus were described that were intended to provide more sensitive and specific reagents for the control of diagnostic reagents for testing in vivo and in vitro. Because other species of Aspergillus can also produce infections and diseases (3-7) and serums from such patients react with antigens from A. fumigatus and other species, serologic data alone are insufficient to permit identification of the infecting organism. Cross reactivity has also been seen in skin testing (3, 7-14). In the present study, the reactivity and specificity of mycelial extracts and fractions of A. fumigatus were compared to those of mycelial extracts from clinical isolates of
(Received in original form June 18, 1979 and in revised form September 5,1979) 1 From the Mycobacterial and Fungal Antigens Branch, Division of Bacterial Products, Bureau of Biologics, Food and Drug Administration, 8800 Rockville Pike, Bethesda, Md. 20205.
other strains and species of Aspergillus by skin tests in homologously and heterologously sensitized guinea pigs, and by fused rocket immunoelectrophoresis (15). Methods Antigens. Mycelial extracts of A. fumigatus NIH 5233 and its fractions were prepared as described previously (1, 2). API fractions APIFA, APIFB, APIFC, and APIFD were precipitated with 75 % ammonium sulfate and eluted in order, respectively, from a Sephadex G-75 column. AS fractions ASI and ASH were soluble at 75 % ammonium sulfate. ASI was bound by a DE-52 ion exchange column, whereas ASH was not bound. Similar preparations of mycelial extracts have been prepared from clinical isolates of A. fumigatus (strain 004 and 005), A. fumigatus var. ellipticus (AFE) ATCC 16903, Aspergillus phialiseptus (APS) 010, Aspergillus flavus (AFV) ATCC 24133, Aspergillus niger (ANG) ATCC 1015, Aspergillus clavatus (ACT), and Aspergillus nidulans (AND). Isolates 004 and 005 of A. fumigatus were from cases of aspergilloma and had morphologic features similar to those of strain NIH 5233 except that both strains exhibited poor conidial production and retarded growth rate. Strain 004 also exhibited intercalarial or terminal round cells (10 to 30 /Lm). A. phialiseptus organisms were isolated from the sputum spe-
AMERICAN REVIEW OF RESPIRATORY DISEASE, VOLUME 120, 1979
1297
1298
KIM AND CHAPARAS
cimen of a patient with allergic bronchopulmonary aspergillosis. Immunization of animals. Antibodies were produced in rabbits using the mycelial homogenates, as described previously (I, 2). For each strain or species, 6 guinea pigs were sensitized subcutaneously in the footpads and intramuscularly in the nuchal area. Skin tests were performed 4 to 6 wk after sensitization by the injection of 0.1 ml of antigen in saline solution intracutaneously in the flank areas. All skin tests for fractions and extracts were per-
004
005
ME
AFE
005
APS ACT
formed at doses determined to be bioequivalent (13 to 15 mm) in dose-response titrations in homologously sensitized animals. Reaction diameters were recorded between IS and 24 h. Fused rocket immunoelectrophoresis. This procedure was performed according to a modification of the method of Svendsen (16) using 1 % agarose containing veronal buffer at pH S.6 and ionic strength 0.075, as described previously (1, 2, 15). The test was repeated when necessary, using different geometries and arrangements to produce lines of iden-
AND
005
AFV ANG
FA
FB
FC
ASI
Fig. 1. Fused rocket immunoelectrophoresis of extracts of species of Aspergillus and fractions of Aspergillus Jumigatus with anti-A. Jumigatus 005 serum. ACT = Aspergillus clavatus; AFE = A.Jumigatus var. ellipticus; AFV = Aspergillus flaws; AND = Aspergillus nidulans; ANG = Aspergillus niger; APS = Aspergillus phialiseptus; ME, 004, and 005 = A. Jumigatus NIH 5233, 004, and 005; ASI, FA, FB, and FC = fractions ASI, APIFA, APIFB, and APIFC derived from mycelial extract of A. Jumigatus NIH 5233.
1299
CHARACTERIZATION OF A. FUMIGATUS ANTIGENS
tity and optimal resolution of precipitin bands. Antigens were used at 20 mg/ml (dry weight). Fractions ASH and APIFD were excluded in most of the figures because they produced only a few, very poor precipitin bands that were more prominent in adjacent fractions. The PQS (percentage of qualitath'e sharing) values were determined as described elsewhere (15), using the formula: number of precipitin bands with heterologous preparation
PQS = - - - - - - - - - - - X 100. number of precipitin bands with homologous preparation In some anlayses, the reciprocal average PQS values
(RAP) for the comparison of any 2 species (e.g., A and B) were compared: PQS of A with B antiserum + PQS of B with A antiserum
RAP = - - - - - - - - - - - - - - 2 Results
Representative results and analyses with fused rocket immunoelectrophoresis are shown in figures I and 2 and in tables I and 2. In table 1, PQS values between 62 and 100 % suggest a close relationship between A. fumigatus strains, A. fumigatus var. ellipticus, and A. phialisep-
ANTI-A. phialiseptus
004 APS
005
AFE
APS
ME
ACT AND APS
AFV ANG
FA
FB
FC
ASI
Fig. 2. Fused rocket immunoelectrophoresis of extracts of species of Aspergillus and fractions of A. fumigatus with anti-Aspergillus phialiseptus serum. For definitions of abbreviations see figure 1.
1300
KIM AND CHAPARAS
TABLE 1 COMPARISON OF ANTIGENS FROM ASPERGILLUS SPP. WITH HOMOLOGOUS AND HETEROLOGOUS ANTISERA Antigens
Fractions of ME"
!g
.~
.9-
..
~
A. niger A. flavus A. phialiseptus A. fumigatus var ellipticus A. fumigatus 005
A. fumigatus NIH5233
~
21 .~
~ ~
.~
§
.
~
to
~
:I:
Z
~to
.~
.~
~
~
'
