A new monoclonal antibody‐based enzyme immunoassay (Innogenetics) for the detection and quantification of p24 core antigens of HIV‐1 (group M and group O) and of HIV‐2 was evaluated on 2745 serum samples and 18 culture supernatants and compared with a reference (Coulter) HIV‐1 p24 antigen assay. Positive results were confirmed by neutralization with the reagents of the respective tests. As demonstrated by dilution series of HIV cocultures, the new test recognizes p24 antigen of the most common HIV genetic subtypes, including group O and HIV‐2. Titres ranged from 729 to 531441. Therefore p24 antigen assay is but very weakly reactive with HIV‐2 (titres from 9 to 81).

A double-direct sandwich enzyme-linked immunosorbent assay that uses a rat anti-galactomannan monoclonal antibody as the acceptor and detector antibody was designed. This immunoassay, which detects less than 1 ng of galactomannan per ml, was assessed in a retrospective study with samples from patients with invasive aspergillosis. Serum is more appropriate than urine for use in the search for circulating galactomannan. Antigenemia does not have a transient character. Galactomannan can be detected at least 39 days before the death of the patients.

The performance of a direct sandwich enzyme-linked immunosorbent assay (ELISA) for detecting Aspergillus galactomannan was compared with that of the Pastorex Aspergillus antigen latex agglutination (LA) test by using 532 serum samples from 61 patients at risk for invasive aspergillosis. The ELISA gave positive results earlier in the course of infection than did the LA test. A sensitivity of 70% and a specificity of 86% were obtained for the LA test and corresponding values of 90 and 84% were obtained for the ELISA when a series of serum samples was employed.

Early diagnosis of invasive aspergillosis is of utmost importance but difficult to achieve. Serological methods were developed mainly because all the other diagnostic approaches had major drawbacks. The detection of antibodies against Aspergillus antigens provided little interesting information, since anti-Aspergillus antibodies were commonly found in healthy people, while, on the other hand, immunocompromised patients often failed to raise antibodies. Several groups of investigators showed that the detection in serum or urine by RIA or ELISA of Aspergillus antigens, was a highly specific indication of invasive disease. The sensitivities of the techniques, however, were moderate, partially due to the low antigen concentrations and the transient character of antigenemia. The only commercially available test is a latex agglutination test detecting 15 ng/ml galactomannan. It has a high specificity but reported sensitivities varied between 42% and 94.7%. Results with an experimental double-sandwich ELISA detecting about 1 ng/ml galactomannan suggest that the improvement of detection limit also greatly increases the clinical value of the test: patients become and remain positive 2 to 10 weeks earlier. If these results are confirmed, antigen detection could become a essential element in the management of an immunocompromised patient population.

Serum and urine samples from cattle with experimental and spontaneous systemic mycotic infections were tested for the presence of galactomannan and the 18 kDa antigen from Aspergillus fumigatus by an inhibition ELISA and immunoblotting, respectively. High levels of galactomannan (approximately 80 ng/ml.) were detected in serum from two of three calves experimentally infected with A. fumigatus. In two out of three cows with spontaneous acquired aspergillosis a similar amount of galactomannan was detected. Galactomannan was not found in serum samples of 20 cows which aborted due to either experimental or spontaneous placental aspergillosis. Twenty-four of forty urine samples from normal cattle reacted positively in the ELISA, consequently, the assay was not applicable on bovine urine. The 18 kDa antigen from A. fumigatus was detected in the urine from one calf out of three calves experimentally infected with aspergillosis and in the urine from one cow with spontaneous aspergillosis. It is concluded that detection of galactomannan in serum and the 18 kDa antigen in urine may be used as an aid for the diagnosis of disseminated bovine aspergillosis with the exception of placental and probably gastrointestinal localization.

To improve the immunohistopathological diagnosis of systemic bovine mycoses we have evaluated the utility of antifungal polyclonal and monoclonal antibodies, and peroxidase and alkaline phosphatase staining techniques. A rabbit polyclonal antibody to mannan from Candida albicans was specific for candidosis. The diagnosis of aspergillosis was accomplished using a rat monoclonal antibody to the galactofuran side chains of Aspergillus galactomannan. A murine monoclonal antibody reacting with weakly Con-A binding 41 and 46 kDa somatic antigens from Absidia corymbifera was used for immunostaining of zygomycetic hyphae. Peroxidase antiperoxidase (PAP) and alkaline phosphatase antialkaline phosphatase (APAAP) complexes were visualized using aminoethylcarbazole and fast red substrates. A green staining of PAP reactions with dioctyl sulfosuccinate sodium and 3,3',5,5'-tetramethylbenzidine (DONS/TMB) was effective for the demonstration of fungi in dual and triple infections. Tissue sections of experimentally infected mice were used to determine the sensitivity and specificity of the antibodies. Tisssues obtained from 161 bovine mycotic lesions previously studied by indirect immunofluorescence staining were further evaluated using the three antibodies. In all of 45 lesions solely affected by aspergillosis and in three solely affected by candidosis the diagnoses were confirmed by the new evaluation. In 85 of 96 cases of single infections with zygomycetes the diagnosis was confirmed, while none of the antibodies reacted with fungal elements in the remaining 11 lesions. Aspergillus hyphae were detected in all three lesions with dual aspergillosis and zygomycosis, whereas zygomycetic material was confirmed in only two of these cases. A mixed infection of candidosis and zygomycosis in a lymph node was confirmed too. In 13 cases in which a diagnosis had not hitherto been obtained, aspergillosis and zygomycosis were recorded each in three cases.

The guinea-pig model of invasive aspergillosis was used to study the effect of the intensity of tissue invasion and of antifungal treatment on galactomannan levels in plasma. In untreated animals, galactomannan titres, determined with Pastorex Aspergillus, steadily increased and reached a maximum shortly before death. There was a significant correlation (P < 0.05) between this increase and that of the mean colony forming units of Aspergillus fumigatus in muscle, kidney, brain, peritoneum, eye and spleen, but not in skin, liver and lung. Pastorex Aspergillus detected galactomannan in 19/20 (95%) of the infected untreated animals. Uninfected guinea-pigs (160 samples) remained negative. In animals treated with itraconazole or amphotericin B, striking differences in antigenemia were observed between surviving and non-surviving animals. Only 5/25 surviving animals had detectable amounts of galactomannan in plasma, all on day 2 and one also on day 5, suggesting that successful treatment rapidly eradicated A.fumigatus or reduced the fungus to a level too low to release sufficient amounts of galactomannan. Antigenemia in treated non-surviving guinea-pigs resembled more closely the results in untreated animals. However, the number of positive animals (21/29 or 72.5%) was lower, suggesting that unsuccessful antifungal treatment could also affect levels of circulating galactomannan.

A specific immunodominant 54-kDa antigen was purified from a culture filtrate of Nocardia asteroides by immunoaffinity chromatography. The chromatography column was prepared with immunoglobulin G obtained from sera from patients with lepromatous leprosy. Unbound solutes consisted of specific, partially purified N. asteroides antigens, primarily a 54-kDa band, accompanied by two others of 31 and 62 kDa. The Western blot (immunoblot) technique was applied to detecting the immunologic response to nocardiae in the serum of nocardiosis patients. Each of the serum samples from immunosuppressed or immunocompetent patients infected with N. asteroides reacted with the 54-kDa band, and two reacted with the 31- and 62-kDa bands. There was no reaction to either the 54- or the 31-kDa antigen with all serum samples obtained from patients with tuberculosis, except for one, with all serum samples obtained from patients with leprosy, or with all sera obtained from healthy controls. The partially purified 54-kDa antigen, specific for N. asteroides, was used as the immunogen to generate monoclonal antibodies (mAbs) and two mAbs were selected. As determined by Western blot, both mAbs reacted with the 54-kDa band. Using indirect immunofluorescence or enzyme immunoassay with whole N. asteroides micro-organisms, the mAbs did not react with N. asteroides cells. No cross-reactivity with mycobacterial antigens, either culture-filtrate antigens or tuberculin, was exhibited with any of the two mAbs. These mAbs are candidates to be used for the development of a sensitive and specific diagnostic test for nocardiosis.

Two latex agglutination tests for the detection of Candida antigens, Pastorex Candida (Sanofi Diagnostics Pasteur, Marnes-la-Coquette, France) and Cand-Tec (Ramco Laboratories, Inc., Houston, Tex.), were applied to 79 serum samples from 19 patients who were retrospectively selected on the basis of mycological and clinical evidence of C. albicans infection and the availability of serial serum samples taken near the date of a positive culture. The specificity in 60 control individuals was 100% for Pastorex and 98.3% for Cand-Tec. The tests scored positive for 10 (52.6%) and 9 (47.4%) patients, respectively. Pastorex detected antigen in only 3 of 12 patients (25%) with positive antibody detection tests, but was positive for all 7 patients (100%) who produced no or a low antibody response, suggesting that the test performs better in the absence of antibodies. However, the sensitivity of Pastorex also increased with the number of samples available per patient, which was lower for high-antibody-responder patients (2.8 versus 5.7). If the patients who provided only one or two serum samples were eliminated, the sensitivity of Pastorex rose to 76.9%. For the Cand-Tec, the sensitivity was not related to the presence of antibodies, nor was it related to the number of samples per patient. The observed antigenemia was transient with both Pastorex and Cand-Tec. Only 12.5% of the positive reactions occurred on the same serum sample, confirming that the two tests react with different antigens. A positive antigen test preceded other diagnostic indications for 6 of 10 Pastorex-positive patients and 5 of 9 Cand-Tec-positive patients.

We present an experimental study on the immunohistochemical identification of Penicillium marneffei in paraffin-embedded, formalin-fixed tissue. The monoclonal antibody EB-A1 detects a specific galactomannan that appears to have at least one epitope identical in P. marneffei and Aspergillus sp. This immunohistochemical approach could be useful in the diagnosis of a rare diseae, penicilliosis marneffei, which proves to be difficult to identify by conventional microscopy.

Monoclonal antibodies (MAbs) against Aspergillus fumigatus galactomannan were produced in rats. Seven of them, EB-A1 through EB-A7, were characterized in more detail. They were all immunoglobulin M antibodies, reacting in an indirect enzyme-linked immunosorbent assay with purified A. fumigatus galactomannan, with avidity constants of between 2 x 10(9) and 5 x 10(9)/M. Enzyme-linked immunosorbent assay inhibition experiments with modified galactomannan and synthetic oligomers of beta (1----5)galactofuranose demonstrated that the MAbs bound to an epitope located on the beta(1----5)galactofuranose-containing side chains of the galactomannan molecule. An identical or similar epitope also seemed to be present in other fungi. Immunofluorescence and immunoelectron microscopy experiments with EB-A2 revealed the presence of the antigen in the fungal wall and inside the cell. Immunoblotting experiments demonstrated that the epitope recognized by the MAbs was a common oligosaccharide moiety of a wide range of intracellular and extracellular glycoproteins in A. fumigatus. The characteristics of the MAbs justify their use in the diagnosis of invasive aspergillosis by antigen detection.

Two monoclonal antibodies (MAbs) of the immunoglobulin G2A isotype, reacting with a Nocardia-specific 54-kDa antigen, were generated. As determined by Western blot (immunoblot), both MAbs reacted only with the 54-kDa band. As determined by indirect immunofluorescence or enzyme immunoassay with whole microorganisms, the MAbs did not react with Nocardia cells. One of the MAbs showed weak cross-reactivity with mycobacterial antigens, while the other showed no cross-reactivity.

The monoclonal antibody EB-A1 to galactomannan is apparently specific for detecting Aspergillus species and Penicillium marneffei in formalin-fixed, paraffin-embedded tissues. It reveals hyphae, remnants of filaments, and organisms in the cytoplasm of some phagocytic cells.

Aspergillus species and dermatophytes are medically important fungi (Bodey and Vartivarian, 1989; Grappel et al., 1974). Both fungal groups produce exocellular antigens containing galactomannan molecules. Chemical characterization of these polysaccharidic antigens remain incomplete. However, the presence of non-reducing galactofuranose end units have been demonstrated unequivocally in both Aspergillus and dermatophyte species (Latgé et al., chapter 11; de Haan et al.,chapter 28). Polyclonal antibodies have been produced against Aspergillus galactomannans for the detection of antigens either in serum of patients with invasive aspergillosis or in foodstuffs, contaminated by fungi (Bennett et al., 1985; Notermans et al.,1988; Banks et al., 1990). These studies have shown that the galactofuran side chains of the galactomannan are immunodominant. Cross-reaction has been observed among these antisera and other fungi, including dermatophytes. Such cross-reaction may be due to structural relatedness of the polysaccharides of different fungal species but also to the polyclonal character of these antisera. Therefore, monoclonal antibodies (MAbs), directed against a single epitope, would be more usçful. A recent paper by Ste-Marie et al. (1990) describes two murine MAbs which react to Aspergillus galactomannan epitopes, which cross-reacted with fungi from other genera. In this paper we describe the production of rat MAbs against Aspergillus galactomannan and the initial characterization of rat MAbs against exocellular antigens of the dermatophyte Trichophyton rubrum.