Use of Monoclonal Antibodies in Diagnosis of Paracoccidioidomycosis: New Strategies for Detection of Circulating Antigens B
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JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 1997, p. 3278–3283 Vol. 35, No. 12 0095-1137/97/$04.0010 Copyright © 1997, American Society for Microbiology Use of Monoclonal Antibodies in Diagnosis of Paracoccidioidomycosis: New Strategies for Detection of Circulating Antigens B. L. GO´ MEZ,1 J. I. FIGUEROA,2 A. J. HAMILTON,2 B. ORTIZ,1 1 2 1 M. A. ROBLEDO, R. J. HAY, AND A. RESTREPO * Corporacio´n para Investigaciones Biolo´gicas, Medellı´n, Colombia,1 and St. John’s Institute of Dermatology, Guy’s Hospital, London SE19RT England2 Received 30 May 1997/Returned for modification 13 August 1997/Accepted 21 September 1997 The precise diagnosis of paracoccidioidomycosis, in most cases, is established by direct methods and indirect immunological tests. The latter method is reliant on the identification of the host’s humoral responses, which are usually impaired or absent in patients with severe juvenile forms of the disease and in immunocompro- mised patients. Determining disease activity or assessing treatment responses by measuring antibody levels is difficult, since antibody titer may remain elevated or persist at stationary levels, even in the presence of clinical improvement. Consequently, there is a need for alternative tests aimed at the identification of circulating antigens. A modification of the standard hybridoma production method was used to raise a panel of murine monoclonal antibodies (MAbs) against the yeast form of Paracoccidioides brasiliensis. Of these, MAb P1B, directed against an 87-kDa determinant, was used to develop an inhibition ELISA (inh-ELISA) capable of detecting as little as 5.8 ng of circulating antigen per ml of serum. Sera from 46 patients with paracoccidioid- omycosis or other mycoses and sera from healthy individuals were evaluated by the inh-ELISA; overall sensitivity was 80.4% (37 of 46 paracoccidioidomycosis patients tested positive), and specificity compared with that of normal controls from areas of endemicity was 81.4%. The inh-ELISA detected circulating antigen in 100% of patients with the acute form of paracoccidioidomycosis and in 83.3 and 60% of patients with the chronic multifocal and unifocal forms of paracoccidioidomycosis according to the patients’ clinical presenta- tion. These results indicate that the inh-ELISA with MAb P1B is effective in the detection of circulating antigen and that this test may be useful for monitoring responses to treatment and establishing disease prognoses. Paracoccidioidomycosis (PCM), a disorder caused by the source (7, 10) create more difficulties. Data from many differ- dimorphic fungus Paracoccidioides brasiliensis, is one of the ent studies make it evident that different tests with the same most important systemic mycoses in Central and South Amer- serum do not always give similar results. Consequently, it is ica (1). The disease primarily involves the lungs and then advisable to employ more than one test for the diagnosis of disseminates to other organs and systems. Secondary lesions PCM (39). frequently appear in the mucous membranes, skin, lymph A different diagnostic approach is desirable for immuno- nodes, and adrenals. Two main clinical forms are recognized: compromised individuals. Antibody titer may be lower or ab- the acute or subacute form (juvenile type) and the chronic sent in up to 50% of immunocompromised patients (18). The form (adult type) (16). Both the clinical presentation and the detection of P. brasiliensis circulating antigens in body fluids course of the disease vary from patient to patient (1, 40), could facilitate the early diagnosis of the mycosis and confirm impeding prompt clinical diagnosis. a preliminary diagnosis when antibody detection is nonconclu- The definitive diagnosis of PCM can be accomplished only sive. In addition, the monitoring of treatment response by by laboratory procedures, mainly by the direct visualization of the fungus and its isolation in culture; however, the time re- measuring antigen levels would be an asset, since antibody titer quired to isolate P. brasiliensis from clinical specimens repre- may remain elevated even after apparent clinical remission sents a hindrance to rapid diagnosis (1). Since its introduction (12, 34, 43). The detection of antigenemia has proved difficult by Moses in 1916, the detection of serum antibodies against in PCM patients, however, and it has been attempted by coun- components of P. brasiliensis has been one of the main tools for terimmunoelectrophoresis (41), immunoelectrophoresis-im- diagnosis and for monitoring patients’ responses to treatment munodiffusion (20), passive hemagglutination inhibition, in- (33). The most common serological tests used are complement verted linear immunoelectrophoresis (33), immunoradiometric fixation (11), immunodiffusion (4, 37), immunoenzymatic as- assay (13), a competitive enzyme-linked immunosorbent assay says (2, 3, 6, 30, 42), counterimmunoelectrophoresis (9), and (ELISA) (17), and immunoblotting (31). The majority of these immunoblotting (5, 32). Unfortunately, there is extensive an- reports indicate variations in sensitivity and specificity, proba- tigenic cross-reactivity between P. brasiliensis and other fungi bly due to the small number of patients evaluated. In this study (1, 8, 27, 30, 35), limiting the value of the tests that are cur- we describe the development of an inhibition ELISA (inh- rently employed. Cross-reactivity and the lack of antigen stan- ELISA) for the detection of circulating antigen with a mono- dardization due to variations in the isolates used as an antigen clonal antibody (MAb) produced by a modification of the standard hybridoma production technique. A representative number of serum samples from well-documented PCM pa- * Corresponding author. Mailing address: Laboratorio de Micolo- gia, Corporacio´n para Investigaciones Biolo´gicas (CIB); Carrera 72A tients was assessed together with sera from individuals with No. 78B141, AA 73-78 Medellı´n, Colombia. Phone: 57-4-4410855. Fax: other mycoses and sera from healthy individuals living in areas 57-4-4415514. E-mail: [email protected]. of endemicity or areas of nonendemicity. 3278 VOL. 35, 1997 USE OF MAbs IN THE DIAGNOSIS OF PCM 3279 TABLE 1. Sources of serum and urine specimens tested Two control mice did not receive cyclophosphamide. Twelve days later, the mice received an i.p. inoculation with P. brasiliensis CIB B-339 CYA (50 mg/mouse, No. of specimensa made up in a 1:1 ratio in Freund’s incomplete adjuvant). This inoculation was Group repeated on day 20, and in order to determine which mouse had the highest Sera Urine differential polyclonal response on day 22, mice were tail bled and antibody responses to P. brasiliensis and H. capsulatum CYAs were quantified by ELISA, PCM, all clinical forms 46 13 as previously described (14, 21). The chosen mouse received a further intrave- Acute 5 1 nous dose of 50 mgofP. brasiliensis CYA in 100 ml of sterile PBS, and its spleen Chronic multifocal 31 8 was used for the fusion protocol 3 days later. Cells of the murine myeloma line Chronic unifocal 10 4 Sp 2/0 were fused with spleen cells from the donor mouse as previously described (22). Hybridomas were screened by ELISA 7 days later, and colonies showing Other mycoses specificity against P. brasiliensis CYA were expanded onto 24-well plates and Histoplasmosis 10 12 subcloned twice by limiting dilutions. Pristane (Sigma)-primed BALB/c mice were injected i.p. with 104 cells from each hybridoma line, and ascitic fluid was Aspergillosis 10 collected 7 to 10 days later. Culture supernatants of the different hybridoma lines Cryptococcosis 10 were also collected and concentrated 100-fold by ammonium sulfate precipita- Sporotrichosis 9 tion (24). Characterization of MAbs. The specificity of MAbs was assessed by ELISA Tuberculosis 9 and Western blot assay, as described elsewhere (14, 22). MAbs were subclassed with a subclassing kit (Serotec, Kidlington, United Kingdom) as previously de- Healthy persons from areas of endemicity 49 20 scribed (22). Healthy persons from the United Kingdom 20 inh-ELISA. A modification of the methods described by Freitas da Silva and Roque-Barreira (17) and Le Pape and Deunff (28) was used. An inh-ELISA was developed for both serum and urine samples. The diluting buffer used in the Total 163 45 serum experiments consisted of a pool of NHS 1:10 in PBS 0.05%-Tween 20 (PBS-Tween), 20 mM MgCl , and 1% bovine serum albumin (Sigma Chemical a Each serum and urine sample is from a different patient. 2 Co., St. Louis, Mo.). The diluting buffer for experiments with urine was the same but contained a pool of normal human urine (NHU) instead of NHS. MAb P1B concentrated culture supernatant was used at a constant concentration of 1: 70,000, and all samples were tested 1:2 in diluting buffer. MATERIALS AND METHODS Inhibition plate. An inhibition standard curve was constructed by adding different concentrations of P. brasiliensis CIB 339 CYA (from 4 ng to 60 mg per Clinical samples. A total of 46 patients with mycologically confirmed (direct m m KOH examination, isolation by culture, and positive serological tests) and active ml) to 100 l of pooled NHS (or NHU) and then adding 100 l of the stan- PCM were used in this study. One serum sample was taken from each patient at dardized dilution of MAb P1B. NHS or NHU made up 1:2 in diluting buffer was the time of diagnosis. In addition, 13 of these patients each provided a single used as a negative control. All the standards, samples, and controls were tested in duplicate. Samples were plated on 96-well round microtiter plates (Nunc A/S, urine sample. Samples were collected between January 1988 and December 1996 m at the mycology laboratory, Corporacio´n para Investigaciones Biolo´gicas (CIB), Kamstrup, Denmark) previously blocked by incubation with 200 l of 5% bovine Medellı´n, Colombia. Patients were classified according to their respective clinical serum albumin per well made up in PBS-Tween, for2hat37°C. Plates were presentations (16, 39) (Table 1).