Vol. 30, No. 8
JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 1992, p. 1907-1912
0095-1137/92/081907-06$02.00/0 Copyright © 1992, American Society for Microbiology
Serological Responses to Various Coccidioides Antigen Preparations in a New Enzyme Immunoassay WAYNE GADE,t* DAVID W. LEDMAN, RICHARD WETHINGTON, AND ARTHUR YI Research and Development, Meridian Diagnostics, Inc., Cincinnati, Ohio 45244 Received 23 September 1991/Accepted 24 March 1992
A new enzyme immunoassay (EIA) was used to investigate immunoglobulin M (IgM) and IgG responses to various coccidioidal antigen preparations. Direct binding and inhibition assays both revealed that the IgG responses of many serum specimens were directed largely to the tube precipitin (TP) antigen even when the microwells were coated with a combination of coccidioidal antigens (CCAs). From a panel of 28 serum specimens containing antibodies to CCAs, 4 serum specimens yielded high IgG signals (absorbance, >1.0) and were negative for IgM (absorbance, 2.0) with the CCA preparation and also bound directly to microwells coated only with TP antigens. The IgG signal (absorbance) of serum specimen 26 was reduced by 98% when it was preincubated with TP antigens prior to the assay. Significant IgG signals from several other serum specimens were observed when microwells were coated with TP antigen preparations, but they were absent when periodate-treated preparations were used. Two cerebrospinal fluid specimens yielded IgG signals with CCA-coated microwells, which were not inhibited by TP antigens, and yielded no signal with microwells coated only with TP antigens. The results are consistent with the concept that the typical serologic response to TP antigens occurs early in disease progression, but they suggest that TP antigens stimulate both IgM and IgG responses.
Coccidioides immitis is a soil fungus that is endemic to the southwestern United States, Mexico, and arid areas of Central and South America. Coccidioidomycosis is usually limited to a respiratory infection and is frequently asymptomatic (2, 14). However, together with immunosuppression and other risk factors, the infection can cause very serious disseminated disease (2, 14). The usual symptoms include influenzalike symptoms of fever, fatigue, headache, sore throat, and cough. The pulmonary symptoms associated with coccidioidomycosis are typically difficult to differentiate from those associated with other infections; thus, an accurate diagnosis usually involves a skin test, serological testing, or culture (2, 14). Classically, serologic testing involved the tube precipitin (TP) and complement fixation assays (2, 9-12, 14, 20). In the TP reaction, an antigen reacts with the early antibody (presumably immunoglobulin M [IgM]) (14, 15, 17). The complement fixation (CF) assay uses a heat-labile antigen (CF antigen) which reacts with antibody that appears later in infection, primarily IgG (2, 14, 15, 17). The immunodiffusion (ID) assays IDTP and IDCF typically use the same TP and CF antigen preparations as described above and are interpreted as demonstrating primarily IgM and IgG responses, respectively (2, 14, 19). While these tests and interpretations remain extremely valuable in the serological diagnosis and prognosis of coccidioidomycosis, recent enzyme immunoassay (EIA) results (from our laboratory and others [3, 4, 6, 7, 13, 18]) suggest that even purified antigen preparations may *
Corresponding author.
t Present address: Praxis Biologics, 300 East River Road, Roch-
ester, NY 14623-1214.
1907
react with both IgM and IgG. Cole et al. (3) reported that 13 serum specimens which were TP positive and CF negative were also positive for both IgM and IgG in an EIA by using
the 120-kDa TP antigen. While the antigen used in the IDTP-TP tests is reactive with IgM, EIAs demonstrate that such antigens are reactive with IgG. Specifically, IgG responses have been reported by using purified IDTP-TP preparations in other EIAs (3, 4, 7). Sawaki et al. (15) reported two serum specimens which were positive by the TP assay but negative for IgM and positive for IgG by radioimmunoelectrophoresis. We developed an EIA which uses a combination of coccidioidal antigens (CCAs) adsorbed to microwells and which differentially detects IgM and IgG. During the development of this assay system, we defined the response of IgG antibodies to purified TP antigen by several unique methods. The results demonstrate that it is not uncommon for the TP antigen(s) to react with IgG, as shown by EIA.
MATERIALS AND METHODS Serum specimens. Clinical specimens were obtained from serology laboratories where sera were submitted for coccidioidal antibody determinations. Specimens were obtained from the laboratories of Demosthenes Pappagianis, Davis, Calif., and James Johnson, Lexington, Ky. Additional information concerning follow-up specimens from serum specimen 26 was supplied by D. Pappagianis. Antigen purifications. Crude TP antigen was prepared from the alkali-soluble, water-soluble material described by Cox and Britt (5). Further treatment and purification of the antigen included the use of pronase (5 mg/ml for 30 min at 56°C and then boiling for 10 min) and dialysis against
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GADE ET AL.
phosphate-buffered saline (PBS; pH 7.2). This material was applied to a concanavalin A-Sepharose column equilibrated with a 100 mM sodium acetate buffer containing 1 M NaCI and 1 mM concentrations of MgCI2, MnCl2, and CaCl2 (pH 6.3). Elution of the concanavalin A-bound (CAB) fraction was achieved with 100 mM mannose in the sodium acetate buffer. This pronase-treated CAB material (PCAB) was extensively dialyzed against PBS. The PCAB product has been shown to have precipitin activity in both the TP and IDTP assays. Two additional antigen preparations were used in this investigation. The purified 120-kDa protein, previously reported as the TP antigen (3), was obtained from Gary Cole, and a CAB preparation (7) was obtained from John Galgiani. Both antigens formed precipitin reactions in IDTP assays. PREMIER Coccidioides EIA. The PREMIER Coccidiodes EIA was produced by Meridian Diagnostics, Inc. (Cincinnati, Ohio), and the assays were performed as described in the package insert, except where noted. The PREMIER EIA microwells were Immulon II microwells (Dynatech, Chantilly, Va.) coated by a proprietary process with a CCA solution (which contained a combination of coccidioidal antigens active in both IDTP and IDCF assays). Briefly, the PREMIER EIA is an indirect EIA procedure which yields a result in approximately 70 min. Following dilution (1:400) of the specimen with a sample diluent provided in the kit, duplicate 100-1tl aliquots of each specimen were added to two microwells coated with CCA and incubated for 30 min at room temperature (RT). The incubation was terminated by inverting the microwells, tapping the contents onto an adsorbent pad, and filling the wells with the wash buffer provided with the kit. This washing procedure was repeated two more times. The second incubation involved the addition of 100 pul of either anti-human IgG or IgM conjugate contained in the kit to each duplicate well. This incubation was also for 30 min at RT. The washing procedure described above was repeated three more times, and 100 [lI of the substrate was incubated for 10 min at RT. The substrate incubation was terminated by the addition of 100 ,ul of stop solution. The plates were read after 2 min on a dual-wavelength EIA reader equipped with 450- and 630-nm filters. Reading of the microwells with the primary filter (450 nm) and the secondary filter (630 nm) automatically eliminated the light-scattering characteristics of the microwells and enabled a more accurate determination of the A450 of the converted reaction product. The cutoff value for positive specimens was 20.20, and that for negative specimens was .0.15. The cutoff for negative specimens was confirmed when the means and standard deviations for more than 1,000 negative specimens were calculated. The sum of the mean plus 2 standard deviations was rounded off to arrive at a convenient negative cutoff of 0.15. The range between these two values was considered indeterminate and would be retested. Antigen coating of microwells with TP antigens. Immulon II microwells were coated either with CCA preparation (as provided in the PREMIER EIA kit) or with the PCAB or the 120-kDa antigen overnight at RT. Microwells were then coated for 2 to 4 h with 1% bovine serum albumin to decrease nonspecific binding during the assay. Once the microwells coated with the PCAB and the 120-kDa antigens were produced, the assays were performed on these microwells as described above. Periodate treatment of TP antigens. Stock solutions with 450 ,ul of the PCAB or the 120-kDa antigen (each at approximately 6 ,ug/ml) in acetate buffer (pH 5.5) were incubated
J. CLIN. MICROBIOL.
with 50 ,ul of 100 mM NaTO4 solution (final concentration, 10 mM). The solution was placed at RT in the dark for 30 min, and then the reaction was stopped with 50 ,ul of ethylene glycol. This periodate-treated material was used to coat the microwells as described above. Separation of IgM and IgG fractions. The Quik-Sep System II (Isolab, Akron, Ohio) was used for the separation of IgM from IgG. Columns and buffers were purchased from Isolab. Sera were typically diluted 1:20 by using 100 RI of neat serum and adding 1.9 ml of Isolab wash buffer. In some cases, a lower volume of serum was available, so a 1:20 dilution of 50 ,I was used. The column was equilibrated with 30 ml of the wash buffer before the diluted sample was applied. The first (unbound) peak contained IgG. The bound IgM was eluted with the elution buffer and was collected in 0.5-ml fractions. Prior to assaying the column fractions with IgG and IgM conjugates, each fraction was diluted 1:20 with the sample diluent provided in the PREMIER EIA kit. TP antigen inhibition experiments. TP antigen preparations were used to inhibit various fractions from the column runs and diluted sera. Inhibition experiments were performed by preincubating (15 min, RT) specific column fractions or sera (in sample diluent) with a final concentration of 2.4 ,ug of each antigen preparation per ml, as described above. The percentage of inhibition was then determined by performing the assay procedure and comparing EIA results with and without antigen preincubation. Heterologous inhibition experiments. The inhibition protocol described above was also performed by using antigen preparations from Histoplasma capsulatum and Blastomyces dermatitidis as well as 200-,ug/ml concentrations of galactose, mannose, and casein. A supernatant preparation from a Histoplasma culture was concentrated approximately fivefold (by using an Amicon Centri-Prep 30 concentrator) and was used as an inhibitor. Partially purified Blastomyces antigen was prepared by anion-exchange (DEAE-Sepharose) and S-300 (a high-molecular-weight sieve) chromatography (8). The concentration (2% [vol/vol]) of these fungal antigens used in the EIA inhibition studies was shown to inhibit known positive sera in the PREMIER ETAs for those organisms. Determination of the extent of IgG conjugate cross-reaction. Purified human IgM obtained from Jackson ImmunoResearch Laboratories, Inc. (West Grove, Pa.) was preincubated with both conjugates. The conjugates were then used to assay sera which previously yielded positive signals for both IgG and IgM. The extent of the IgG conjugate crossreaction was defined as the percentage of the IgG signal which was inhibited by an IgM concentration that caused >99% inhibition of the IgM signal. Protein determinations. The microwell protein assay was a modification of the procedure described by Bradford (1) in which a protein reagent (Bio-Rad, Richmond, Calif.) diluted with four volumes of deionized H20 was used. Aliquots (100 ,ul) of column fractions were then added to an equal volume of the diluted protein dye. Microwells were used so that the relative protein content of each fraction could be read with an EIA reader by using the 630-nm filter. The EIA reader was zeroed on a microwell containing 100 ,ul of H20 and 100 ,ul of diluted reagent. ID experiments. Antigens and control antisera used for the ID assays were from Meridian Diagnostics, Inc. These reagents consist of partially purified coccidioidal antigens and the standard positive control, which is a goat serum specimen against the appropriate coccidioidal antigen. Peak fractions of IgM and IgG from the Isolab separations were
NEW EIA FOR COCCIDIOIDES ANTIGENS
VOL. 30, 1992
TABLE 2. Direct binding of specimens to TP and periodate-treated TP
TABLE 1. Serological responses and TP inhibition of various serum specimens
Serum 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
CSF 1 2
IgG
IgM
1.719 0.844 0.839 0.470 0.875
0.523 0.863 2.060 1.819
IgG and TP
IgM and TP
IgG
IgM
0.794 0.086 0.131 0.075 0.279 0.049 0.085 0.010 0.158 0.145 0.108 0.534 0.178 0.119 0.276 0.038 0.615 0.259 0.087 0.605 0.008
54 90 84 84 68 95 84 99 92 50 90 72 88 63 88 98 >80 79 87 28 NA 86 88 90 53 97
94 80 73 85 NAa NA NA NA NA 95 NA >54 NA 90 76 NA 76 79 >38 >48 95 96 88 73 50 98
-11 -11
NA NA
1.235 2.724 0.654 >3.0 0.844 >3.0 0.011 0.382 0.303 2.501 2.413 2.260 2.850 0.265 0.511 0.561 2.098 1.905
0.042 0.265 0.283 0.240 0.063
0.031 0.170 0.547 0.264 0.008 0.020 0.013 0.011 0.045 0.087 0.006 1.631 0.008 0.192 0.305 0.006 0.515 0.574 1.868 1.556 0.018 0.112 0.299 0.074 0.280 0.043
0.008 0.045
0.805 0.704
0.009 0.008
1.001
0.007 0.012 0.013 0.143
0.526 0.695 1.926 0.115 1.647 0.292 1.083 0.124 1.899 >3.0 0.792 0.027 1.309 0.514 1.268 1.931 1.586 0.100 2.157 >3.0
0.722 0.633
Absorbance
% Inhibition
Absorbance
Specimen
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a NA, not applicable, because the control value was negative in the assay.
concentrated 5- to 10-fold with Centricon 30 devices (AmiDanvers, Mass.) prior to testing. TP experiments. The PCAB antigen preparation was shown to form a precipitin button with a known TP-positive serum specimen. The assay was performed by mixing 200 RIl of serum with 200 p.l of antigen (undiluted, 1:5, 1:50) and incubating the mixture for 24 h (14). Incubation was at RT, and the antigen reference standard was obtained from Ron Talbot, Bakersfield, Calif. con,
RESULTS Inhibition of IgG signal with TP antigens. The new PREMIER Coccidioides EIA, which uses microwells adsorbed with CCA, can detect both IgG and IgM antibodies. Table 1 illustrates the results when 26 serum specimens and 2 cerebrospinal fluid (CSF) specimens which contained antibodies to coccidioidal antigens were assayed for both IgM and IgG. The fraction of TP-specific antibody was estimated in each serum specimen by preincubating the diluted serum with 2.4 p.g of partially purified PCAB or 120-kDa antigen per ml and performing a competitive inhibition assay. In a majority of cases (19 of 28), greater than 75% of the IgG response was inhibited by TP antigens. A similar percentage of IgM responses (12 of 18) was inhibited (>75%) by the TP antigen. In four serum specimens (specimens 6, 9, 11, and 16), the IgM signal (1.0) was high, yet the IgG responses were inhibited by >90% by the addition of the TP antigen.
Specimen
TP only
% Loss
Periodate-treated TP
IgG
1gM
IgG
IgM
IgG
IgM
Serum 12 9 2 26
0.516 2.330 0.879 2.554
1.129 0.076 0.354 1.205
0.012 0.012 0.012 0.023
0.007 0.023 0.012 0.011
98 99 99 99
99 NAa 97 99
CSF 1 2
0.012 0.011
0.013 ND
NDb ND
ND ND
ND ND
ND ND
a
NA, not calculated, because the control value was negative in the assay. determined because of an insufficient volume of specimen.
b ND, not
The results in Table 1 were obtained by using the PCAB antigen preparation as a TP antigen. The purified 120-kDa protein obtained from G. Cole was used to confirm that the inhibition results in Table 1 reflect a true TP response rather than a contaminant in the PCAB preparation. When the CAB preparation obtained from J. Galgiani was used as a third antigen, the results were also very similar (data not shown). Direct binding of sera to TP-coated microwells. Since IgG inhibition by TP antigens was greater than expected, a direct binding procedure was used to confirm that the IgG signals were directed to the PCAB and the 120-kDa antigens. Both the 120-kDa protein and PCAB antigens were used to coat microwells, and the basic EIA protocol described above was repeated. A large IgG signal was observed when microwells coated with these antigens were tested with the four serum specimens (Table 2). Pretreatment of the TP antigen with periodate prior to coating eliminated greater than 99% of the IgG signal when serum specimens 2, 9, 12, and 26 were assayed. This result is consistent with reports that carbohydrates are the antigenic sites on the TP antigen (2-4, 14) and is consistent with the binding of IgG from these sera to the TP antigen. Interestingly, two CSF specimens (specimens 1 and 2) yielded IgG signals that were uninhibited by the PCAB antigen (Table 1) and that did not bind to PCAB-coated microwells (Table 2). The IgG signals obtained with CCAcoated microwells (0.722 and 0.633) were .50-fold higher than those obtained from assays with microwells coated with the PCAB or the 120-kDa antigen (0.012 and 0.011). Separation of IgG and IgM components. Several serum specimens were chromatographed to separate the IgG and IgM fractions by using the Isolab Quik-Sep II system. Figure 1 illustrates the elution profiles when three different serum specimens were chromatographed and assayed by the EIA procedure (by using the CCA coating on the microwells). Figure la also shows the typical protein profile for serum chromatographed in this way. Assays of the fractions with both conjugates confirmed that the first peak contained primarily IgG (i.e., it was not contaminated with IgM) and identified the second peak as containing the IgM fraction. Figure 2 illustrates the binding profiles of peak IgG and IgM fractions from serum specimen 26 when microwells coated with the PCAB or the 120-kDa antigen were compared with the CCA-coated microwells. Notice that substantial IgG binding occurred even when no CF antigen or other
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GADE ET AL.
J. CLIN. MICROBIOL.
.5.UUU-
.uuu- o- oIgG Profile/PCAU Ag 0- lgG Profile/i120 kDa Ag 2.500 - -A IgM Profile/PCAB Ag Ag v IgM Profile/120 kDa
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Il
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(a
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o
.:0
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FIG. 1. Elution profiles of three serum specimens fractionated on Quik-Sep columns and assayed by using CCA-coated microwells. (a) Data from serum specimen 26; (b) data from serum specimen 9; (c) data from serum specimen 20.
CCAs were present. The profile was essentially the same when either the PCAB or the more purified 120-kDa protein was adsorbed to the microwell. Cross-reaction of the IgM conjugate with the IgG conjugate. The possibility that IgG signals reflected a cross-reaction between the IgG conjugate and the IgM conjugate was also investigated. Purified human IgM (Jackson ImmunoResearch Laboratories) was added to the IgG conjugate prior to the assay of known positive serum specimens; the assay demonstrated that IgM inhibition results in modest decreases of less than 15% of the IgG signal. When the same ratio of IgM:conjugate was added to the IgM conjugate, the inhibition was greater than 99%.
A
5
10
15 20 25 Fraction Number
30
35
40
FIG. 2. Elution profiles of serum specimen 26 fractionated on Quik-Sep columns by using the direct TP-binding assay with either the 120-kDa protein or PCAB coated on the microwells. Ag, antigen.
IDTP and IDCF assays with the IgG fraction. ID assays used to confirm that IgG fractions are capable of precipitating both IDTP and IDCF antigens. A relatively sharp band of identity with IDTP-positive control standards was observed when concentrated IgG fractions from the sera were assayed with IDTP and IDCF reagents from the commerical kit. Concentrations of TP antigens required for inhibition. The antigen concentrations required to inhibit peak fractions of two different serum specimens (specimens 20 and 26) are illustrated in Fig. 3. The IgG peak from serum specimen 26 was totally (>99%) inhibited by both antigen preparations (PCAB and the 120-kDa antigen) at the highest concentration (19.2 ,ug/ml). Even at the intermediate antigen concentrations (1.9 ,ug/ml), the inhibition of serum specimen 26 was >95%. By contrast, the intermediate concentrations of antigen inhibited the IgG responses of serum specimen 20 by approximately 50%. The difference in inhibition between the two antigen preparations at lower concentrations could be accounted for by purity, molecular mass differences (the 120-kDa antigen is a purified glycoprotein [4], whereas the PCAB preparation primarily contains mannose-containing carbohydrates and no detectable protein), and quantitation differences (the 120-kDa antigen was quantified by a protein assay and the PCAB preparation was quantified by a carbohydrate assay [16]). The binding of the IgM peaks was also virtually eliminated by both antigen preparations at the high concentrations. Several other possible inhibitors were also added to the sample diluent and tested with sera known to contain antibodies to coccidioidal antigens. When 200-,.g/ml concentrations of mannose and casein were added to the sample diluent, inhibition was less than 5%. The same concentration of galactose caused inhibition of as high as 12%. When preparations of H. capsulatum and B. dermatitidis antigens were added to the sample diluent (2% [vol/voll), the inhibition was less than 20%. These concentrations were shown to inhibit sera which contained antibodies specific for those two pathogens by greater than 90%. When 10 ,l of a IDCF preparation (a 75% ammonium sulfate pellet from the IDCF reagent) per ml was added to the sample diluent, the inhibition was greater than 90%. Not all IgG responses were inhibited as much by TP antigens. The IgG responses from serum specimens 1, 10, 20, and 25 were inhibited by less than 60% by the addition of the PCAB antigen. The IgG peak of serum specimen 20 were
1.500
1.000-
0.5000-
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'0
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0
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U
0
0
VOL. 30, 1992 a 2.500 0 U
l
NEW EIA FOR COCCIDIOIDES ANTIGENS T
l signal/PCAB Ag IgG
i
=JIgG signal/i120 kDa Ag CM IgG control signal M IgM control signal =IgM signal/i 20 kDa Ag M IggM signal/PCAB Ag
i
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o~~~~~~~~~~~~~~~~~~~o on~~~~~~~~~~~~~~~~~~o
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a
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. 19.2
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FIG. 3. Inhibition of IgG and IgM signals from serum specimens 26 (a) and 20 (b) with various concentrations of two TP antigen (Ag)
preparations.
following fractionation was inhibited only 47% by 2.4-,ug/ml concentrations of the PCAB or the 120-kDa antigen preparations (Fig. 3). It is interesting that neither CSF specimen included in this investigation was inhibitable by the PCAB or the 120-kDa antigen preparations and neither CSF specimen bound to microwells coated with either PCAB or the 120kDa antigen (Table 2). However, IgG signals from the four serum specimens listed in Table 2 were reduced by 98 to 99% when the PCAB or the 120-kDa antigen preparations were treated with periodate prior to coating the microwells. DISCUSSION The EIA data presented in this report indicate that the IgG fractions from several positive serum specimens contained large amounts of antibodies specific for the TP antigen. The IgM fractions also contained anti-TP activity, as expected. However, the percentage of IgG signal which could be inhibited in the EIA by the TP antigens or which directly bound to TP antigens was significantly larger than expected. This observation, which was determined by using the PREMIER Coccidioides EIA, is similar to the findings of other investigators (3, 4, 7) who have used EIA technology to measure the IgG and IgM responses to different antigen preparations, including the 120-kDa preparation. Indeed, the traditional precipitation reactions may primarily be IgM; however, a significant number of specimens appear to contain IgG antibodies which react with the TP-IDTP antigens in the EIA format.
1911
The PREMIER EIA kit provides microwells which are coated with a combination of antigens (CCAs). When these CCA-coated microwells were used to assay serum specimens 3 and 26, which were IDTP positive and IDCF negative, there was a significant IgG signal (Table 1). Inhibition experiments demonstrated that a surprisingly high percentage of the IgG signal in most serum specimens was inhibited by preincubation with the PCAB antigen. This occurred even when highly purified TP antigen (120-kDa protein) was used as the inhibitor. In this investigation, chromatographic methods were used to physically separate the IgG and IgM fractions prior to many experiments. The fractions were analyzed for protein content and by the EIA to detect antibody binding to various Coccidioides antigens. When the column fractions were assayed by using TP-coated microwells (either the PCAB or the 120-kDa antigen), the profile was similar to those generated with the microwells coated with CCA preparations (compare Fig. 1 and 2). Again, a substantial amount of IgG was bound to microwells which were coated with the PCAB or the 120-kDa antigen. The classical assays for TP antigen (or IDTP) are frequently interpreted as indicating an early coccidioides infection and the IgM response (14, 15, 17). The recent observation of significant IgG responses to purified TP antigen relates to the technology shift from precipitin reactions to the more sensitive EIA technology, which can detect both responses equally. In the present investigation, we demonstrated a substantial IgG response to PCAB and the 120-kDa antigen and verified that isolated IgG antibodies could yield a band of identity in an IDTP assay system. It is interesting that serum specimen 26 was IDCF negative and IDTP positive and that the IgG signal was essentially eliminated following preincubation with the TP antigen. This was the earliest specimen obtained from the patient. Assays of subsequent specimens described the standard transition to IDCF positive and IDTP positive and then IDCF positive and IDTP negative. Unfortunately, these subsequent specimens were not available for this EIA investigation. This case history is consistent with the observations that antibodies directed to the TP antigen are typically the early or the transient response and that the' antibodies specific for other antigens (including CF) are the later responses. The diagnosis of the early or acute phase of disease on the basis of TP or IDTP detection of IgM remains valid, although there is also an IgG response to the same antigen, as shown by EIA. REFERENCES 1. Bradford, M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254. 2. Bronnimann, D. A., and J. N. Galgiani. 1989. Coccidioidomycosis. Eur. J. Clin. Microbiol. Infect. Dis. 8:466-473. 3. Cole, G. T., D. Kruse, and K. R. Seshan. 1991. Antigen complex of Coccidioides immitis which elicits a precipitin antibody response in patients. Infect. Immun. 59:2434-2446. 4. Cole, G. T., D. Kruse, S. Zhu, K. R. Seshan, and R. W. Wheat. 1990. Composition, serologic reactivity, and immunolocalization of a 120-kilodalton tube precipitin antigen of Coccidioides immitis. Infect. Immun. 58:179-188. 5. Cox, R. A., and L. A. Britt. 1986. Isolation of a coccidioidin component that reacts with immunoglobulin M precipitin antibody. Infect. Immun. 53:449-453. 6. Galgiani, J. N., K. 0. Dugger, J. I. Ito, and M. A. Wieden. 1984. Antigenemia in primary coccidioidomycosis. J. Trop. Med. Hyg. 33:645-649. 7. Galgiani, J. N., J. I. Ito, an,d K. 0. Dugger. 1985. Enzyme-linked
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immunosorbent assay detection of coccidioidal antigens in human serum. Coccidioidomycosis, p. 239-249. In H. Einstein and A. Catanzaro (ed.), Coccidioidomycosis, Proceedings of the 4th Internatational Conference. National Foundation for Infectious Diseases, Washington, D.C. 8. Green, J. H., W. K. Harrell, J. E. Johnson, and R. Benson. 1980.
9. 10.
11. 12.
13.
Isolation of an antigen from Blastomyces dermatitidis that is specific for the diagnosis of blastomycosis. Curr. Microbiol. 4:293-296. Huppert, M., and J. W. Bailey. 1965. The use of immunodiffusion tests in coccidioidomycosis. Am. J. Clin. Pathol. 44:364368. Huppert, M., and J. W. Bailey. 1965. The use of immunodiffusion tests in coccidioidomycosis. Am. J. Clin. Pathol. 44:369373. Kaufman, L. 1973. Value of immunodiffusion tests in the diagnosis of systemic mycotic diseases. Ann. Clin. Lab. Sci. 3:141-146. Kaufman, L., P. G. Standard, M. Huppert, and D. Pappagianis. 1985. Comparison and diagnostic value of the coccidioidin heat-stable (HS and tube precipitin) antigens in immunodiffusion. J. Clin. Microbiol. 22:515-518. Leonard, J., and R. Talbot. 1987. Indirect enzyme immunosorbent assay of coccidioidal 1gM antibody in serum. Abstr. 31st
J. CLIN. MICROBIOL. Ann. Coccidioides Study Group Meeting, p. 11. 14. Pappagianis, D., and B. L. Zimmer. 1990. Serology of coccidioidomycosis. Clin. Microbiol. Rev. 3:247-268. 15. Sawaki, Y., M. Iluppert, J. W. Bailey, and Y. Yagi. 1966. Patterns of human antibody reactions in coccidioidomycosis. J. Bacteriol. 91:422-427. 16. Scott, T. A., and E. H. Melvin. 1953. Determination of dextran with anthrone. Anal. Chem. 25:1656-1661. 17. Smith, C. E., M. T. Saito, and S. A. Simons. 1956. Pattern of 39,500 serologic tests in coccidioidomycosis. JAMA 160:546552. 18. Talbot, R. C., R. C. Johnson, and J. Leonard. 1986. Enzyme linked immunosorbent assay of coccidioidal antibody in CSF specimens. Abstr. 13th Ann. Coccidioides Study Group Meeting, p. 1. 19. Wieden, M. A., J. N. Galgiani, and D. Pappagianis. 1983. Comparison of immunodiffusion techniques with standard complement fixation assay for quantitation of coccidioidal antibodies. J. Clin. Microbiol. 18:529-534. 20. Zimmer, B. L., and D. Pappagianis. 1988. Characterization of a soluble protein of Coccidioides immitis with activity as an immunodiffusion-complement fixation antigen. J. Clin. Microbiol. 26:2250-2256.
JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 1992, p. 1907-1912
0095-1137/92/081907-06$02.00/0 Copyright © 1992, American Society for Microbiology
Serological Responses to Various Coccidioides Antigen Preparations in a New Enzyme Immunoassay WAYNE GADE,t* DAVID W. LEDMAN, RICHARD WETHINGTON, AND ARTHUR YI Research and Development, Meridian Diagnostics, Inc., Cincinnati, Ohio 45244 Received 23 September 1991/Accepted 24 March 1992
A new enzyme immunoassay (EIA) was used to investigate immunoglobulin M (IgM) and IgG responses to various coccidioidal antigen preparations. Direct binding and inhibition assays both revealed that the IgG responses of many serum specimens were directed largely to the tube precipitin (TP) antigen even when the microwells were coated with a combination of coccidioidal antigens (CCAs). From a panel of 28 serum specimens containing antibodies to CCAs, 4 serum specimens yielded high IgG signals (absorbance, >1.0) and were negative for IgM (absorbance, 2.0) with the CCA preparation and also bound directly to microwells coated only with TP antigens. The IgG signal (absorbance) of serum specimen 26 was reduced by 98% when it was preincubated with TP antigens prior to the assay. Significant IgG signals from several other serum specimens were observed when microwells were coated with TP antigen preparations, but they were absent when periodate-treated preparations were used. Two cerebrospinal fluid specimens yielded IgG signals with CCA-coated microwells, which were not inhibited by TP antigens, and yielded no signal with microwells coated only with TP antigens. The results are consistent with the concept that the typical serologic response to TP antigens occurs early in disease progression, but they suggest that TP antigens stimulate both IgM and IgG responses.
Coccidioides immitis is a soil fungus that is endemic to the southwestern United States, Mexico, and arid areas of Central and South America. Coccidioidomycosis is usually limited to a respiratory infection and is frequently asymptomatic (2, 14). However, together with immunosuppression and other risk factors, the infection can cause very serious disseminated disease (2, 14). The usual symptoms include influenzalike symptoms of fever, fatigue, headache, sore throat, and cough. The pulmonary symptoms associated with coccidioidomycosis are typically difficult to differentiate from those associated with other infections; thus, an accurate diagnosis usually involves a skin test, serological testing, or culture (2, 14). Classically, serologic testing involved the tube precipitin (TP) and complement fixation assays (2, 9-12, 14, 20). In the TP reaction, an antigen reacts with the early antibody (presumably immunoglobulin M [IgM]) (14, 15, 17). The complement fixation (CF) assay uses a heat-labile antigen (CF antigen) which reacts with antibody that appears later in infection, primarily IgG (2, 14, 15, 17). The immunodiffusion (ID) assays IDTP and IDCF typically use the same TP and CF antigen preparations as described above and are interpreted as demonstrating primarily IgM and IgG responses, respectively (2, 14, 19). While these tests and interpretations remain extremely valuable in the serological diagnosis and prognosis of coccidioidomycosis, recent enzyme immunoassay (EIA) results (from our laboratory and others [3, 4, 6, 7, 13, 18]) suggest that even purified antigen preparations may *
Corresponding author.
t Present address: Praxis Biologics, 300 East River Road, Roch-
ester, NY 14623-1214.
1907
react with both IgM and IgG. Cole et al. (3) reported that 13 serum specimens which were TP positive and CF negative were also positive for both IgM and IgG in an EIA by using
the 120-kDa TP antigen. While the antigen used in the IDTP-TP tests is reactive with IgM, EIAs demonstrate that such antigens are reactive with IgG. Specifically, IgG responses have been reported by using purified IDTP-TP preparations in other EIAs (3, 4, 7). Sawaki et al. (15) reported two serum specimens which were positive by the TP assay but negative for IgM and positive for IgG by radioimmunoelectrophoresis. We developed an EIA which uses a combination of coccidioidal antigens (CCAs) adsorbed to microwells and which differentially detects IgM and IgG. During the development of this assay system, we defined the response of IgG antibodies to purified TP antigen by several unique methods. The results demonstrate that it is not uncommon for the TP antigen(s) to react with IgG, as shown by EIA.
MATERIALS AND METHODS Serum specimens. Clinical specimens were obtained from serology laboratories where sera were submitted for coccidioidal antibody determinations. Specimens were obtained from the laboratories of Demosthenes Pappagianis, Davis, Calif., and James Johnson, Lexington, Ky. Additional information concerning follow-up specimens from serum specimen 26 was supplied by D. Pappagianis. Antigen purifications. Crude TP antigen was prepared from the alkali-soluble, water-soluble material described by Cox and Britt (5). Further treatment and purification of the antigen included the use of pronase (5 mg/ml for 30 min at 56°C and then boiling for 10 min) and dialysis against
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GADE ET AL.
phosphate-buffered saline (PBS; pH 7.2). This material was applied to a concanavalin A-Sepharose column equilibrated with a 100 mM sodium acetate buffer containing 1 M NaCI and 1 mM concentrations of MgCI2, MnCl2, and CaCl2 (pH 6.3). Elution of the concanavalin A-bound (CAB) fraction was achieved with 100 mM mannose in the sodium acetate buffer. This pronase-treated CAB material (PCAB) was extensively dialyzed against PBS. The PCAB product has been shown to have precipitin activity in both the TP and IDTP assays. Two additional antigen preparations were used in this investigation. The purified 120-kDa protein, previously reported as the TP antigen (3), was obtained from Gary Cole, and a CAB preparation (7) was obtained from John Galgiani. Both antigens formed precipitin reactions in IDTP assays. PREMIER Coccidioides EIA. The PREMIER Coccidiodes EIA was produced by Meridian Diagnostics, Inc. (Cincinnati, Ohio), and the assays were performed as described in the package insert, except where noted. The PREMIER EIA microwells were Immulon II microwells (Dynatech, Chantilly, Va.) coated by a proprietary process with a CCA solution (which contained a combination of coccidioidal antigens active in both IDTP and IDCF assays). Briefly, the PREMIER EIA is an indirect EIA procedure which yields a result in approximately 70 min. Following dilution (1:400) of the specimen with a sample diluent provided in the kit, duplicate 100-1tl aliquots of each specimen were added to two microwells coated with CCA and incubated for 30 min at room temperature (RT). The incubation was terminated by inverting the microwells, tapping the contents onto an adsorbent pad, and filling the wells with the wash buffer provided with the kit. This washing procedure was repeated two more times. The second incubation involved the addition of 100 pul of either anti-human IgG or IgM conjugate contained in the kit to each duplicate well. This incubation was also for 30 min at RT. The washing procedure described above was repeated three more times, and 100 [lI of the substrate was incubated for 10 min at RT. The substrate incubation was terminated by the addition of 100 ,ul of stop solution. The plates were read after 2 min on a dual-wavelength EIA reader equipped with 450- and 630-nm filters. Reading of the microwells with the primary filter (450 nm) and the secondary filter (630 nm) automatically eliminated the light-scattering characteristics of the microwells and enabled a more accurate determination of the A450 of the converted reaction product. The cutoff value for positive specimens was 20.20, and that for negative specimens was .0.15. The cutoff for negative specimens was confirmed when the means and standard deviations for more than 1,000 negative specimens were calculated. The sum of the mean plus 2 standard deviations was rounded off to arrive at a convenient negative cutoff of 0.15. The range between these two values was considered indeterminate and would be retested. Antigen coating of microwells with TP antigens. Immulon II microwells were coated either with CCA preparation (as provided in the PREMIER EIA kit) or with the PCAB or the 120-kDa antigen overnight at RT. Microwells were then coated for 2 to 4 h with 1% bovine serum albumin to decrease nonspecific binding during the assay. Once the microwells coated with the PCAB and the 120-kDa antigens were produced, the assays were performed on these microwells as described above. Periodate treatment of TP antigens. Stock solutions with 450 ,ul of the PCAB or the 120-kDa antigen (each at approximately 6 ,ug/ml) in acetate buffer (pH 5.5) were incubated
J. CLIN. MICROBIOL.
with 50 ,ul of 100 mM NaTO4 solution (final concentration, 10 mM). The solution was placed at RT in the dark for 30 min, and then the reaction was stopped with 50 ,ul of ethylene glycol. This periodate-treated material was used to coat the microwells as described above. Separation of IgM and IgG fractions. The Quik-Sep System II (Isolab, Akron, Ohio) was used for the separation of IgM from IgG. Columns and buffers were purchased from Isolab. Sera were typically diluted 1:20 by using 100 RI of neat serum and adding 1.9 ml of Isolab wash buffer. In some cases, a lower volume of serum was available, so a 1:20 dilution of 50 ,I was used. The column was equilibrated with 30 ml of the wash buffer before the diluted sample was applied. The first (unbound) peak contained IgG. The bound IgM was eluted with the elution buffer and was collected in 0.5-ml fractions. Prior to assaying the column fractions with IgG and IgM conjugates, each fraction was diluted 1:20 with the sample diluent provided in the PREMIER EIA kit. TP antigen inhibition experiments. TP antigen preparations were used to inhibit various fractions from the column runs and diluted sera. Inhibition experiments were performed by preincubating (15 min, RT) specific column fractions or sera (in sample diluent) with a final concentration of 2.4 ,ug of each antigen preparation per ml, as described above. The percentage of inhibition was then determined by performing the assay procedure and comparing EIA results with and without antigen preincubation. Heterologous inhibition experiments. The inhibition protocol described above was also performed by using antigen preparations from Histoplasma capsulatum and Blastomyces dermatitidis as well as 200-,ug/ml concentrations of galactose, mannose, and casein. A supernatant preparation from a Histoplasma culture was concentrated approximately fivefold (by using an Amicon Centri-Prep 30 concentrator) and was used as an inhibitor. Partially purified Blastomyces antigen was prepared by anion-exchange (DEAE-Sepharose) and S-300 (a high-molecular-weight sieve) chromatography (8). The concentration (2% [vol/vol]) of these fungal antigens used in the EIA inhibition studies was shown to inhibit known positive sera in the PREMIER ETAs for those organisms. Determination of the extent of IgG conjugate cross-reaction. Purified human IgM obtained from Jackson ImmunoResearch Laboratories, Inc. (West Grove, Pa.) was preincubated with both conjugates. The conjugates were then used to assay sera which previously yielded positive signals for both IgG and IgM. The extent of the IgG conjugate crossreaction was defined as the percentage of the IgG signal which was inhibited by an IgM concentration that caused >99% inhibition of the IgM signal. Protein determinations. The microwell protein assay was a modification of the procedure described by Bradford (1) in which a protein reagent (Bio-Rad, Richmond, Calif.) diluted with four volumes of deionized H20 was used. Aliquots (100 ,ul) of column fractions were then added to an equal volume of the diluted protein dye. Microwells were used so that the relative protein content of each fraction could be read with an EIA reader by using the 630-nm filter. The EIA reader was zeroed on a microwell containing 100 ,ul of H20 and 100 ,ul of diluted reagent. ID experiments. Antigens and control antisera used for the ID assays were from Meridian Diagnostics, Inc. These reagents consist of partially purified coccidioidal antigens and the standard positive control, which is a goat serum specimen against the appropriate coccidioidal antigen. Peak fractions of IgM and IgG from the Isolab separations were
NEW EIA FOR COCCIDIOIDES ANTIGENS
VOL. 30, 1992
TABLE 2. Direct binding of specimens to TP and periodate-treated TP
TABLE 1. Serological responses and TP inhibition of various serum specimens
Serum 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
CSF 1 2
IgG
IgM
1.719 0.844 0.839 0.470 0.875
0.523 0.863 2.060 1.819
IgG and TP
IgM and TP
IgG
IgM
0.794 0.086 0.131 0.075 0.279 0.049 0.085 0.010 0.158 0.145 0.108 0.534 0.178 0.119 0.276 0.038 0.615 0.259 0.087 0.605 0.008
54 90 84 84 68 95 84 99 92 50 90 72 88 63 88 98 >80 79 87 28 NA 86 88 90 53 97
94 80 73 85 NAa NA NA NA NA 95 NA >54 NA 90 76 NA 76 79 >38 >48 95 96 88 73 50 98
-11 -11
NA NA
1.235 2.724 0.654 >3.0 0.844 >3.0 0.011 0.382 0.303 2.501 2.413 2.260 2.850 0.265 0.511 0.561 2.098 1.905
0.042 0.265 0.283 0.240 0.063
0.031 0.170 0.547 0.264 0.008 0.020 0.013 0.011 0.045 0.087 0.006 1.631 0.008 0.192 0.305 0.006 0.515 0.574 1.868 1.556 0.018 0.112 0.299 0.074 0.280 0.043
0.008 0.045
0.805 0.704
0.009 0.008
1.001
0.007 0.012 0.013 0.143
0.526 0.695 1.926 0.115 1.647 0.292 1.083 0.124 1.899 >3.0 0.792 0.027 1.309 0.514 1.268 1.931 1.586 0.100 2.157 >3.0
0.722 0.633
Absorbance
% Inhibition
Absorbance
Specimen
1909
a NA, not applicable, because the control value was negative in the assay.
concentrated 5- to 10-fold with Centricon 30 devices (AmiDanvers, Mass.) prior to testing. TP experiments. The PCAB antigen preparation was shown to form a precipitin button with a known TP-positive serum specimen. The assay was performed by mixing 200 RIl of serum with 200 p.l of antigen (undiluted, 1:5, 1:50) and incubating the mixture for 24 h (14). Incubation was at RT, and the antigen reference standard was obtained from Ron Talbot, Bakersfield, Calif. con,
RESULTS Inhibition of IgG signal with TP antigens. The new PREMIER Coccidioides EIA, which uses microwells adsorbed with CCA, can detect both IgG and IgM antibodies. Table 1 illustrates the results when 26 serum specimens and 2 cerebrospinal fluid (CSF) specimens which contained antibodies to coccidioidal antigens were assayed for both IgM and IgG. The fraction of TP-specific antibody was estimated in each serum specimen by preincubating the diluted serum with 2.4 p.g of partially purified PCAB or 120-kDa antigen per ml and performing a competitive inhibition assay. In a majority of cases (19 of 28), greater than 75% of the IgG response was inhibited by TP antigens. A similar percentage of IgM responses (12 of 18) was inhibited (>75%) by the TP antigen. In four serum specimens (specimens 6, 9, 11, and 16), the IgM signal (1.0) was high, yet the IgG responses were inhibited by >90% by the addition of the TP antigen.
Specimen
TP only
% Loss
Periodate-treated TP
IgG
1gM
IgG
IgM
IgG
IgM
Serum 12 9 2 26
0.516 2.330 0.879 2.554
1.129 0.076 0.354 1.205
0.012 0.012 0.012 0.023
0.007 0.023 0.012 0.011
98 99 99 99
99 NAa 97 99
CSF 1 2
0.012 0.011
0.013 ND
NDb ND
ND ND
ND ND
ND ND
a
NA, not calculated, because the control value was negative in the assay. determined because of an insufficient volume of specimen.
b ND, not
The results in Table 1 were obtained by using the PCAB antigen preparation as a TP antigen. The purified 120-kDa protein obtained from G. Cole was used to confirm that the inhibition results in Table 1 reflect a true TP response rather than a contaminant in the PCAB preparation. When the CAB preparation obtained from J. Galgiani was used as a third antigen, the results were also very similar (data not shown). Direct binding of sera to TP-coated microwells. Since IgG inhibition by TP antigens was greater than expected, a direct binding procedure was used to confirm that the IgG signals were directed to the PCAB and the 120-kDa antigens. Both the 120-kDa protein and PCAB antigens were used to coat microwells, and the basic EIA protocol described above was repeated. A large IgG signal was observed when microwells coated with these antigens were tested with the four serum specimens (Table 2). Pretreatment of the TP antigen with periodate prior to coating eliminated greater than 99% of the IgG signal when serum specimens 2, 9, 12, and 26 were assayed. This result is consistent with reports that carbohydrates are the antigenic sites on the TP antigen (2-4, 14) and is consistent with the binding of IgG from these sera to the TP antigen. Interestingly, two CSF specimens (specimens 1 and 2) yielded IgG signals that were uninhibited by the PCAB antigen (Table 1) and that did not bind to PCAB-coated microwells (Table 2). The IgG signals obtained with CCAcoated microwells (0.722 and 0.633) were .50-fold higher than those obtained from assays with microwells coated with the PCAB or the 120-kDa antigen (0.012 and 0.011). Separation of IgG and IgM components. Several serum specimens were chromatographed to separate the IgG and IgM fractions by using the Isolab Quik-Sep II system. Figure 1 illustrates the elution profiles when three different serum specimens were chromatographed and assayed by the EIA procedure (by using the CCA coating on the microwells). Figure la also shows the typical protein profile for serum chromatographed in this way. Assays of the fractions with both conjugates confirmed that the first peak contained primarily IgG (i.e., it was not contaminated with IgM) and identified the second peak as containing the IgM fraction. Figure 2 illustrates the binding profiles of peak IgG and IgM fractions from serum specimen 26 when microwells coated with the PCAB or the 120-kDa antigen were compared with the CCA-coated microwells. Notice that substantial IgG binding occurred even when no CF antigen or other
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GADE ET AL.
J. CLIN. MICROBIOL.
.5.UUU-
.uuu- o- oIgG Profile/PCAU Ag 0- lgG Profile/i120 kDa Ag 2.500 - -A IgM Profile/PCAB Ag Ag v IgM Profile/120 kDa
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FIG. 1. Elution profiles of three serum specimens fractionated on Quik-Sep columns and assayed by using CCA-coated microwells. (a) Data from serum specimen 26; (b) data from serum specimen 9; (c) data from serum specimen 20.
CCAs were present. The profile was essentially the same when either the PCAB or the more purified 120-kDa protein was adsorbed to the microwell. Cross-reaction of the IgM conjugate with the IgG conjugate. The possibility that IgG signals reflected a cross-reaction between the IgG conjugate and the IgM conjugate was also investigated. Purified human IgM (Jackson ImmunoResearch Laboratories) was added to the IgG conjugate prior to the assay of known positive serum specimens; the assay demonstrated that IgM inhibition results in modest decreases of less than 15% of the IgG signal. When the same ratio of IgM:conjugate was added to the IgM conjugate, the inhibition was greater than 99%.
A
5
10
15 20 25 Fraction Number
30
35
40
FIG. 2. Elution profiles of serum specimen 26 fractionated on Quik-Sep columns by using the direct TP-binding assay with either the 120-kDa protein or PCAB coated on the microwells. Ag, antigen.
IDTP and IDCF assays with the IgG fraction. ID assays used to confirm that IgG fractions are capable of precipitating both IDTP and IDCF antigens. A relatively sharp band of identity with IDTP-positive control standards was observed when concentrated IgG fractions from the sera were assayed with IDTP and IDCF reagents from the commerical kit. Concentrations of TP antigens required for inhibition. The antigen concentrations required to inhibit peak fractions of two different serum specimens (specimens 20 and 26) are illustrated in Fig. 3. The IgG peak from serum specimen 26 was totally (>99%) inhibited by both antigen preparations (PCAB and the 120-kDa antigen) at the highest concentration (19.2 ,ug/ml). Even at the intermediate antigen concentrations (1.9 ,ug/ml), the inhibition of serum specimen 26 was >95%. By contrast, the intermediate concentrations of antigen inhibited the IgG responses of serum specimen 20 by approximately 50%. The difference in inhibition between the two antigen preparations at lower concentrations could be accounted for by purity, molecular mass differences (the 120-kDa antigen is a purified glycoprotein [4], whereas the PCAB preparation primarily contains mannose-containing carbohydrates and no detectable protein), and quantitation differences (the 120-kDa antigen was quantified by a protein assay and the PCAB preparation was quantified by a carbohydrate assay [16]). The binding of the IgM peaks was also virtually eliminated by both antigen preparations at the high concentrations. Several other possible inhibitors were also added to the sample diluent and tested with sera known to contain antibodies to coccidioidal antigens. When 200-,.g/ml concentrations of mannose and casein were added to the sample diluent, inhibition was less than 5%. The same concentration of galactose caused inhibition of as high as 12%. When preparations of H. capsulatum and B. dermatitidis antigens were added to the sample diluent (2% [vol/voll), the inhibition was less than 20%. These concentrations were shown to inhibit sera which contained antibodies specific for those two pathogens by greater than 90%. When 10 ,l of a IDCF preparation (a 75% ammonium sulfate pellet from the IDCF reagent) per ml was added to the sample diluent, the inhibition was greater than 90%. Not all IgG responses were inhibited as much by TP antigens. The IgG responses from serum specimens 1, 10, 20, and 25 were inhibited by less than 60% by the addition of the PCAB antigen. The IgG peak of serum specimen 20 were
1.500
1.000-
0.5000-
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'0
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VOL. 30, 1992 a 2.500 0 U
l
NEW EIA FOR COCCIDIOIDES ANTIGENS T
l signal/PCAB Ag IgG
i
=JIgG signal/i120 kDa Ag CM IgG control signal M IgM control signal =IgM signal/i 20 kDa Ag M IggM signal/PCAB Ag
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a
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(ug/ml)
FIG. 3. Inhibition of IgG and IgM signals from serum specimens 26 (a) and 20 (b) with various concentrations of two TP antigen (Ag)
preparations.
following fractionation was inhibited only 47% by 2.4-,ug/ml concentrations of the PCAB or the 120-kDa antigen preparations (Fig. 3). It is interesting that neither CSF specimen included in this investigation was inhibitable by the PCAB or the 120-kDa antigen preparations and neither CSF specimen bound to microwells coated with either PCAB or the 120kDa antigen (Table 2). However, IgG signals from the four serum specimens listed in Table 2 were reduced by 98 to 99% when the PCAB or the 120-kDa antigen preparations were treated with periodate prior to coating the microwells. DISCUSSION The EIA data presented in this report indicate that the IgG fractions from several positive serum specimens contained large amounts of antibodies specific for the TP antigen. The IgM fractions also contained anti-TP activity, as expected. However, the percentage of IgG signal which could be inhibited in the EIA by the TP antigens or which directly bound to TP antigens was significantly larger than expected. This observation, which was determined by using the PREMIER Coccidioides EIA, is similar to the findings of other investigators (3, 4, 7) who have used EIA technology to measure the IgG and IgM responses to different antigen preparations, including the 120-kDa preparation. Indeed, the traditional precipitation reactions may primarily be IgM; however, a significant number of specimens appear to contain IgG antibodies which react with the TP-IDTP antigens in the EIA format.
1911
The PREMIER EIA kit provides microwells which are coated with a combination of antigens (CCAs). When these CCA-coated microwells were used to assay serum specimens 3 and 26, which were IDTP positive and IDCF negative, there was a significant IgG signal (Table 1). Inhibition experiments demonstrated that a surprisingly high percentage of the IgG signal in most serum specimens was inhibited by preincubation with the PCAB antigen. This occurred even when highly purified TP antigen (120-kDa protein) was used as the inhibitor. In this investigation, chromatographic methods were used to physically separate the IgG and IgM fractions prior to many experiments. The fractions were analyzed for protein content and by the EIA to detect antibody binding to various Coccidioides antigens. When the column fractions were assayed by using TP-coated microwells (either the PCAB or the 120-kDa antigen), the profile was similar to those generated with the microwells coated with CCA preparations (compare Fig. 1 and 2). Again, a substantial amount of IgG was bound to microwells which were coated with the PCAB or the 120-kDa antigen. The classical assays for TP antigen (or IDTP) are frequently interpreted as indicating an early coccidioides infection and the IgM response (14, 15, 17). The recent observation of significant IgG responses to purified TP antigen relates to the technology shift from precipitin reactions to the more sensitive EIA technology, which can detect both responses equally. In the present investigation, we demonstrated a substantial IgG response to PCAB and the 120-kDa antigen and verified that isolated IgG antibodies could yield a band of identity in an IDTP assay system. It is interesting that serum specimen 26 was IDCF negative and IDTP positive and that the IgG signal was essentially eliminated following preincubation with the TP antigen. This was the earliest specimen obtained from the patient. Assays of subsequent specimens described the standard transition to IDCF positive and IDTP positive and then IDCF positive and IDTP negative. Unfortunately, these subsequent specimens were not available for this EIA investigation. This case history is consistent with the observations that antibodies directed to the TP antigen are typically the early or the transient response and that the' antibodies specific for other antigens (including CF) are the later responses. The diagnosis of the early or acute phase of disease on the basis of TP or IDTP detection of IgM remains valid, although there is also an IgG response to the same antigen, as shown by EIA. REFERENCES 1. Bradford, M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254. 2. Bronnimann, D. A., and J. N. Galgiani. 1989. Coccidioidomycosis. Eur. J. Clin. Microbiol. Infect. Dis. 8:466-473. 3. Cole, G. T., D. Kruse, and K. R. Seshan. 1991. Antigen complex of Coccidioides immitis which elicits a precipitin antibody response in patients. Infect. Immun. 59:2434-2446. 4. Cole, G. T., D. Kruse, S. Zhu, K. R. Seshan, and R. W. Wheat. 1990. Composition, serologic reactivity, and immunolocalization of a 120-kilodalton tube precipitin antigen of Coccidioides immitis. Infect. Immun. 58:179-188. 5. Cox, R. A., and L. A. Britt. 1986. Isolation of a coccidioidin component that reacts with immunoglobulin M precipitin antibody. Infect. Immun. 53:449-453. 6. Galgiani, J. N., K. 0. Dugger, J. I. Ito, and M. A. Wieden. 1984. Antigenemia in primary coccidioidomycosis. J. Trop. Med. Hyg. 33:645-649. 7. Galgiani, J. N., J. I. Ito, an,d K. 0. Dugger. 1985. Enzyme-linked
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immunosorbent assay detection of coccidioidal antigens in human serum. Coccidioidomycosis, p. 239-249. In H. Einstein and A. Catanzaro (ed.), Coccidioidomycosis, Proceedings of the 4th Internatational Conference. National Foundation for Infectious Diseases, Washington, D.C. 8. Green, J. H., W. K. Harrell, J. E. Johnson, and R. Benson. 1980.
9. 10.
11. 12.
13.
Isolation of an antigen from Blastomyces dermatitidis that is specific for the diagnosis of blastomycosis. Curr. Microbiol. 4:293-296. Huppert, M., and J. W. Bailey. 1965. The use of immunodiffusion tests in coccidioidomycosis. Am. J. Clin. Pathol. 44:364368. Huppert, M., and J. W. Bailey. 1965. The use of immunodiffusion tests in coccidioidomycosis. Am. J. Clin. Pathol. 44:369373. Kaufman, L. 1973. Value of immunodiffusion tests in the diagnosis of systemic mycotic diseases. Ann. Clin. Lab. Sci. 3:141-146. Kaufman, L., P. G. Standard, M. Huppert, and D. Pappagianis. 1985. Comparison and diagnostic value of the coccidioidin heat-stable (HS and tube precipitin) antigens in immunodiffusion. J. Clin. Microbiol. 22:515-518. Leonard, J., and R. Talbot. 1987. Indirect enzyme immunosorbent assay of coccidioidal 1gM antibody in serum. Abstr. 31st
J. CLIN. MICROBIOL. Ann. Coccidioides Study Group Meeting, p. 11. 14. Pappagianis, D., and B. L. Zimmer. 1990. Serology of coccidioidomycosis. Clin. Microbiol. Rev. 3:247-268. 15. Sawaki, Y., M. Iluppert, J. W. Bailey, and Y. Yagi. 1966. Patterns of human antibody reactions in coccidioidomycosis. J. Bacteriol. 91:422-427. 16. Scott, T. A., and E. H. Melvin. 1953. Determination of dextran with anthrone. Anal. Chem. 25:1656-1661. 17. Smith, C. E., M. T. Saito, and S. A. Simons. 1956. Pattern of 39,500 serologic tests in coccidioidomycosis. JAMA 160:546552. 18. Talbot, R. C., R. C. Johnson, and J. Leonard. 1986. Enzyme linked immunosorbent assay of coccidioidal antibody in CSF specimens. Abstr. 13th Ann. Coccidioides Study Group Meeting, p. 1. 19. Wieden, M. A., J. N. Galgiani, and D. Pappagianis. 1983. Comparison of immunodiffusion techniques with standard complement fixation assay for quantitation of coccidioidal antibodies. J. Clin. Microbiol. 18:529-534. 20. Zimmer, B. L., and D. Pappagianis. 1988. Characterization of a soluble protein of Coccidioides immitis with activity as an immunodiffusion-complement fixation antigen. J. Clin. Microbiol. 26:2250-2256.
