Negative Immunodiffusion Test Results Obtained with Sera of Paracoccidioidomycosis Patients May Be Related to Low-Avidity Immunoglobulin G2 Antibodies Directed against Carbohydrate Epitopes

Sera.In a previous study, Blotta et al. described a specificity of 100% and a sensitivity of 87% for ID tests performed (3). For this study, we selected 28 serum samples from ID^neg PCM patients and 18 from ID^pos PCM patients (9 serum samples from patients with the JF and 9 serum samples from patients with the AF). The diagnosis was confirmed by histopathological examinations of skin or of ganglionic or pulmonary biopsy specimens or by the finding of the fungus in scrapings of skin lesions, in material obtained from lymph nodes, or in sputum (direct examination). All 28 ID^neg serum samples were from patients with the unifocal (pulmonary) AF of the disease.

ID test.The ID test was performed with a crude P. brasiliensis exoantigen as previously described (8).

Preparation of CFA. P. brasiliensis B-339 was grown on Sabouraud glucose agar at 35°C for 3 days. The fungal growth from three randomly selected tubes (about 300 mg [wet weight]) was collected by gently scraping the surface. The cell mass was suspended in 1 ml of 0.15 M phosphate-buffered saline (PBS) (pH 7.4), mixed for 30 s in a Vortex mixer, and immediately centrifuged at 10,000 × g in an Eppendorf tabletop centrifuge for 60 s. The resulting supernatant fluid contained cell-free antigen (CFA). The protein concentration in CFA was determined by the method of Bradford (5).

Preparation of P. brasiliensis gp43 antigen.Purified gp43 was obtained by affinity chromatography of the crude exoantigen of P. brasiliensis B-339 on Affigel-10 (Bio-Rad, Hercules, Calif.) coupled with anti-gp43 monoclonal antibody (7). gp43 was eluted from the column with 0.1 M glycine-HCl (pH 2.8), immediately neutralized with 2 M Tris (pH 9.0), concentrated in an Amicon 10K apparatus, and analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). The protein content was measured by the method of Bradford (5).

SDS-PAGE.Crude exoantigen or CFA obtained as described above was mixed with reducing sample buffer, containing 62.5 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 10% 2-mercaptoethanol, and 0.05% bromophenol blue. The polypeptides were separated by SDS-PAGE on 10% acrylamide gels in a Mini Protean II apparatus (Bio-Rad). Protein standards (Sigma, St. Louis, Mo.) with the following molecular masses were used: triosephosphate isomerase, 26.6 kDa; lactic dehydrogenase, 36.5 kDa; ovalbumin, 45 kDa; pyruvate kinase, 58 kDa; fructose-6-phosphate kinase, 84 kDa; and β-galactosidase, 116 kDa.

Immunoblotting for IgG and IgG subclasses.Immunoblotting was performed as previously described (2), with a few modifications. Briefly, after electrophoresis, proteins were transferred to nitrocellulose paper (NCP) by using a Mini Trans-Blot transfer cell (Bio-Rad). Prior to immunological staining, the free sites on the NCP were blocked with 5% nonfat milk in Tris-buffered saline containing 0.1% bovine serum albumin (BSA) and 0.05% Tween 20 (TBS-BSA-T) (pH 7.3) for 2 h at room temperature. The NCP was sliced vertically, and the strips were incubated individually for 1 h at room temperature with sera obtained from patients and diluted 1:200 in TBS-BSA-T. The strips were washed six times for 10 min each time with TBS-BSA-T and then incubated with peroxidase- anti-human IgG (1:1,000 dilution) (A-8667; Sigma) in TBS-BSA-T for 1 h. The strips were washed again and incubated with the substrate (3,3′-diaminobenzidine · 4HCl [Sigma] at 0.5 mg/ml and 10 μl of H₂O₂ in PBS). After color development, the strips were rinsed extensively with distilled water.

For the IgG subclass determination, after incubation with sera diluted 1:50 for 2 h, the strips were washed as described above. Then, antisubclass antibodies (anti-human IgG1 [I9388], IgG2 [I5635], IgG3 [I9763], or IgG4 [I9888]; Sigma) diluted 1:2,000 were added. After 1 h of incubation at 37°C, the strips were washed. Then, sheep anti-mouse IgG- peroxidase (1:1,000 dilution) (A6782; Sigma) was added. The subsequent steps were the same as those described for IgG.

ELISA for the determination of specific IgG.For the IgG enzyme-linked immunosorbent assay (ELISA), 96-well plates (Greiner, Kremsmünster, Austria) were coated with P. brasiliensis gp43 at 2 μg/ml. The assays were performed as described previously (20). The samples were run in duplicate. The cutoff was determined to be the mean plus 2 standard deviations of the absorbance obtained with sera from 23 healthy individuals. Samples were considered positive when the absorbances were higher than 0.320 at a 1:800 serum dilution. The IgG titer was established as the highest dilution of serum with an absorbance higher than the cutoff.

ELISAs for the determination of IgG subclasses.Ninety-six-well plates were coated with P. brasiliensis gp43 at 2 μg/ml in 0.1 M carbonate buffer (pH 9.6) overnight at 4°C. The plates were washed with PBS- 0.05% Tween 20 (PBS-T). The remaining binding sites were blocked with PBS-T- 5% nonfat dry milk for 2 h at 37°C. After three washes with PBS-T, serum samples (1:50 dilution) were added in duplicate, and the plates were incubated for 2 h at 37°C. The wells were washed as described above, and antisubclass antibodies (anti-human IgG1, IgG2, IgG3, or IgG4; Sigma) were added. After 1 h of incubation at 37°C, the plates were washed again, and sheep anti-mouse IgG- peroxidase (1:1,000 dilution) was added. After 1 h of incubation at 37°C and three washes with PBS-T, the substrate solution (o-phenylenediamine in 0.1 M citrate-phosphate buffer [pH 5.0] and 10 μl of 30% H₂O₂) was added, and the reaction was stopped by the addition of 2 N H₂SO₄. The optical densities (ODs) were read with an ELISA reader (SLT Spectra; SLT Instruments, Salzburg, Austria) at 492 nm. Results were expressed as the absorbance index (AI), a numerical value calculated by dividing the net absorbance of each test serum sample by the net absorbance of a positive reference serum pool on each plate and multiplying the resulting value by 100. The AI is an arbitrary value that is linearly related to the antibody concentration and allows the comparison of sera tested on different plates and in different experiments (20). The reference serum pool was composed of a mixture of four sera: two from JF and two from AF patients with PCM. The same pool was used throughout the study.

Treatment of gp43 with sodium metaperiodate.The treatment of gp43 with sodium metaperiodate was previously described by Puccia et al. (26), who showed the elimination of cross-reactions with samples from patients with other mycoses due to the removal of periodate-sensitive carbohydrate epitopes containing galactosyl residues. After overnight coating with gp43 (2 μg/ml) at 4°C, half of the ELISA plate was treated with 40 mM sodium metaperiodate in 1 M citrate buffer (pH 4.5) for 30 min in the dark. Individual serum samples were added to the corresponding wells on both the metaperiodate-treated and the untreated halves of the plate. The subsequent steps were the same as those described above. The titers of the samples were determined, and the decrease in the absorbance of metaperiodate-treated samples was calculated by dividing the result for the metaperiodate-treated well by the result for the untreated well and multiplying the resulting value by 100.

Determination of avidity of anti-gp43 antibodies.The avidity of anti-gp43 antibodies was estimated from their capacity to remain bound to the antigen in the presence of 6.0 M urea and was measured as described by Jenum et al. (17). Each serum sample was analyzed in a twofold dilution range (rows A and B, starting at 1:200). After incubation, row A was washed with PBS-T containing 6 M urea, whereas row B was washed with washing solution not containing urea. The subsequent steps of the reaction were the same as those of the standard ELISA. For each serum sample, two end-point titers (rows A and B) were calculated with the following formula: titer = dilution_x⁻¹ × 10ⁿ; in this equation, dilution_x is the highest dilution giving an OD of >0.32 (cutoff) and n is equal to log₂ × [(OD_x − 0.32)/(OD_x − OD_y)], where OD_x is the OD at dilution_x and OD_y is the OD at the next higher dilution from dilution_x. The IgG avidity was calculated with the following formula: avidity = (titer in row A/titer in row B) × 100.

Statistical methods.The nonparametric Mann-Whitney or Kruskal-Wallis test was used to compare the variables between groups, and the nonparametric Wilcoxon test was used to compare the response of antibodies against the metaperiodate-treated antigen to that of antibodies against the untreated antigen. Significance was defined as a P value of ≤0.05.