Antibody Isolation in C. neoformans.

The importance of humoral immunity to fungal infections remains to be elucidated. In cryptococcosis, patients that fail to generate antibodies against antigens of the fungus Cryptococcus neoformans are more susceptible to the disease, demonstrating the importance of these molecules to the antifungal immune response. Historically, antibodies against C. neoformans have been applied in diagnosis, therapeutics, and as important research tools to elucidate fungal biology. Throughout the process of generating monoclonal antibodies (mAbs) from a single B-cell clone and targeting a single epitope, several immunization steps might be required for the detection of responsive antibodies to the antigen of interest in the serum. This complex mixture of antibodies comprises the polyclonal antibodies. To obtain mAbs, B-lymphocytes are harvested (from spleen or peripheral blood) and fused with tumor myeloma cells, to generate hybridomas that are individually cloned and specifically screened for mAb production. In this chapter, we describe all the necessary steps, from the immunization to polyclonal antibody harvesting, hybridoma generation, and mAb production and purification. Additionally, we discuss new cutting-edge approaches for generating interspecies mAbs, such as humanized mAbs, or for similar species in distinct host backgrounds.

Anti-Saccharomyces cerevisiae Antibody in Pediatric Crohn's Disease Patients without Mucosal Healing Is a Useful Marker of Mucosal Damage.

Background/Aims: We evaluated whether anti-Saccharomyces cerevisiae antibody (ASCA) titers are associated with diagnostic findings, disease activity, Paris classification phenotypes, and persistence after infliximab (IFX) treatment in children with Crohn's disease (CD). We also investigated the role of ASCA as a predictor of mucosal healing (MH) and clinical remission (CR). Methods: This study included 61 CD patients aged 19 years or younger who were diagnosed and treated between September 2010 and January 2019 and followed for at least 1 year. ASCA was regularly measured at the diagnosis of CD and at least 1 year after IFX therapy. Results: The average follow-up period was 3.8±3.4 years (range, 1.0 to 7.2 years). Regression analysis showed that the ASCA titer was the only factor associated with Simple Endoscopic Score for Crohn's Disease (SES-CD) or CR among all the parameters. In patients who had achieved MH (SES-CD=0), ASCA immunoglobulin G (IgG) was not associated with MH, but in patients without MH, ASCA IgG was associated with SES-CD (p=0.005) and CR (p<0.001). The cutoff value of ASCA IgG in patients with CR was 21.8 units. However, there was no difference in the relapse rate between the ASCA IgG-positive and -negative groups during the follow-up period. Conclusions: In patients who have not achieved MH, ASCA IgG is closely related to mucosal damage and CR. Unlike Western studies, ASCA IgG may be more helpful in predicting prognosis than immunoglobulin A in Korean patients, but it is not an appropriate indicator to predict the relapse of CD.

Conservation of Mannan Synthesis in Fungi of the Zygomycota and Ascomycota Reveals a Broad Diagnostic Target.

Ascomycetes and zygomycetes account for the majority of (i) fungi responsible for cutaneous, subcutaneous, and invasive human fungal infections, (ii) plant fungal pathogens, (iii) fungi that threaten global biodiversity, (iv) fungal agents of agricultural spoilage, and (v) fungi in water-damaged buildings. Rapid recognition of fungal infection (or contamination) enables early treatment (or remediation). A bioinformatics search found homologues of Saccharomyces cerevisiae Mnn9p present in members of the Zygomycota and Ascomycota phyla and absent in members of the Chytridiomycota and Basidiomycota. Mnn9p is a component of the yeast mannan polymerization complex and is necessary for α-1,6 mannan production. A monoclonal antibody (2DA6) was produced that was reactive with purified mannans of Mucor, Rhizopus, Aspergillus, Fusarium, and Candida species. Experimentation using a 2DA6 antigen capture enzyme-linked immunosorbent assay (ELISA) and extracts of fungi from the four phyla found agreement between the presence or absence of Mnn9p homologues and production or lack of production of mannan reactive with 2DA6. Studies of cell extracts from yeast mannan mutants identified α-1,6 mannan as the epitope recognized by 2DA6. To translate this finding into a point-of-use diagnostic, a 2DA6 lateral flow immunoassay was constructed that detected mannan in (i) extracts of dermatophytes and fungi that produce trauma-related infection and (ii) tissue from plants infected with Grosmannia clavigera or Sclerotium cepivorum These studies (i) revealed that the conservation of α-1,6-linked mannan in fungi of the Zygomycota and Ascomycota can be exploited as a broad diagnostic target and (ii) have provided a means to detect that target in an immunoassay platform that is well suited for clinic or field use.IMPORTANCE A key question asked when faced with an infection, an infestation, or environmental damage is whether it is a fungus. Identification of fungi as the cause of the problem can lead to remediation or treatment. Zygomycetes and ascomycetes account for the vast majority of fungal causes of human, animal, and plant disease, large-scale biodiversity loss, agricultural spoilage, and contamination of water-damaged buildings. These studies revealed the conservation of a common cell wall structural component of zygomycetes and ascomycetes to be a diagnostic target applicable to multiple pathogenic fungi and have leveraged that insight for practical use. Monoclonal antibodies reactive with this pan-fungal structure were produced and used to construct immunoassays (including ELISA and lateral flow assay) for detection of a broad range of pathogenic fungi.

Screening Coccidioides serology in kidney transplant recipients: A 10-year cross-sectional analysis.

BACKGROUND: Kidney transplant recipients (KTRs) are at risk for reactivation and complicated infection due to Coccidioides. Pre-transplant serological screening should provide benefit for patients from endemic areas. We evaluated Coccidioides seroprevalence by area of residence in KTRs at a major transplant program in Los Angeles. METHODS: We performed cross-sectional analyses of adult KTRs who underwent transplantation at UCLA between 2007-2016. Patients with Coccidioides serology by enzyme immunoassay (EIA) before or within 14 days from transplantation were included. Patients were classified as living in highly, established, suspected, or not endemic areas by their residential zip code. RESULTS: Overall prevalence of Coccidioides IgG and IgM were 1.4% and 2.8%, respectively. Of patients with positive serology, 31.4% had isolated IgG and 66.3% isolated IgM. Patients from established and highly endemic areas had IgG seropositivity of 3.7% versus 1.3% for patients living in suspected endemic areas(P < .01). Rates of IgM seropositivity were 3.7% compared to 2.8% respectively (P = .28). No patients from non-endemic areas had positive screening serology. CONCLUSIONS: Pre-transplant serological screening for Coccidioides is recommended in kidney transplant candidates from endemic areas. We observed high seroprevalence among patients from highly and established endemic areas, for whom universal prophylaxis is recommended. For residents from less well-established areas of endemicity, serological screening showed benefit in identifying patients at risk. In patients with isolated EIA IgM, performing repeat and confirmatory tests is recommended. Patients from non-endemic areas had low risk of infection, however, a thorough social history is necessary to evaluate risk.

Novel mouse monoclonal antibodies specifically recognize Aspergillus fumigatus galactomannan.

A panel of specific monoclonal antibodies (mAbs) against synthetic pentasaccharide ß-D-Galf-(1→5)-[ß-D-Galf-(1→5)]3-α-D-Manp, structurally related to Aspergillus fumigatus galactomannan, was generated using mice immunized with synthetic pentasaccharide-BSA conjugate and by hybridoma technology. Two selected mAbs, 7B8 and 8G4, could bind with the initial pentasaccharide with affinity constants of approximately 5.3 nM and 6.4 nM, respectively, based on surface plasmon resonance-based biosensor assay. The glycoarray, built from a series of synthetic oligosaccharide derivatives representing different galactomannan fragments, demonstrated that mAb 8G4 could effectively recognize the parental pentasaccharide while mAb 7B8 recognizes its constituting trisaccharide parts. Immunofluorescence studies showed that both 7B8 and 8G4 could stain A. fumigatus cells in culture efficiently, but not the mutant strain lacking galactomannan. In addition, confocal microscopy demonstrated that Candida albicans, Bifidobacterium longum, Lactobacillus plantarum, and numerous gram-positive and gram-negative bacteria were not labeled by mAbs 7B8 and 8G4. The generated mAbs can be considered promising for the development of a new specific enzyme-linked assay for detection of A. fumigatus, which is highly demanded for medical and environmental controls.

Identification of secreted and membrane-bound bat immunoglobulin using a Microchiropteran-specific mouse monoclonal antibody.

Bat immunity has received increasing attention because some bat species are being decimated by the fungal disease, White Nose Syndrome, while other species are potential reservoirs of zoonotic viruses. Identifying specific immune processes requires new specific tools and reagents. In this study, we describe a new mouse monoclonal antibody (mAb) reactive with Eptesicus fuscus immunoglobulins. The epitope recognized by mAb BT1-4F10 was localized to immunoglobulin light (lambda) chains; hence, the mAb recognized serum immunoglobulins and B lymphocytes. The BT1-4F10 epitope appeared to be restricted to Microchiropteran immunoglobulins and absent from Megachiropteran immunoglobulins. Analyses of sera and other E. fuscus fluids showed that most, if not all, secreted immunoglobulins utilized lambda light chains. Finally, mAb BT1-4F10 permitted the identification of B cell follicles in splenic white pulp. This Microchiropteran-specific mAb has potential utility in seroassays; hence, this reagent may have both basic and practical applications for studying immune process.

Production of an anti-dermatophyte monoclonal antibody and its application: immunochromatographic detection of dermatophytes.

Tinea refers to superficial infection with one of three fungal genera-Microsporum, Epidermophyton, or Trichophyton-that are collectively known as dermatophytes. These infections are among the most common diseases worldwide and cause chronic morbidity. They are usually diagnosed by direct microscopy and fungal culture, which are burdensome to perform in the clinical setting. To supplement conventional methods, we developed a new method that employs an immunochromatography test for detection of dermatophyte infections. First, anti-Trichophyton monoclonal antibodies (mAb) were produced in mice using a Trichophyton allergen solution as an immunogen. The mAb specificity was assessed by immunostaining alcohol fixed slide cultures and formalin fixed paraffin-embedded microbial samples. Both alcohol- and formalin-fixed samples of all seven species of Trichophyton tested displayed positive immunostaining. Immunochromatography test strips were created using the anti-Trichophyton mAb. The efficiency of the test strip was assessed in patients diagnosed with tinea unguium and in healthy volunteers. Of the 20 patient nails tested, 19 tested positive and one tested negative, whereas of the 17 volunteer nails, only one tested positive. However, KOH microscopic examination of the volunteer nail that tested positive revealed the existence of Trichophyton hyphae. Although the number of nails assayed was small, since the assay had a sensitivity of 95.0% (19/20) and a specificity of 94.1% (16/17), the obtained results were considered to be promising. Thus, while further investigation with a greater number of samples is necessary, this method could potentially be employed as a new diagnostic tool for Trichophyton in the future.

Nonculture Diagnostics in Fungal Disease.

Fungal diagnostics that utilize antibody, antigen or nucleic acid detection offer several advantages that supplement traditional culture-based methods. As a group, nonculture assays can help identify patients with invasive fungal infection (IFI) sooner than is possible with culture, are often more sensitive, and can be used to guide early interventions. Challenges associated with these techniques include the possibility for contamination or cross-reactivity as well as the potential for false negative tests. This review summarized the test characteristics and clinical utility of nonculture-based laboratory methods.

Alt a 15 is a new cross-reactive minor allergen of Alternaria alternata.

Alternaria alternata is one of the most common saprophytes worldwide that is clinically and epidemiologically associated with severe asthma. Therefore, the identification and characterization of all A. alternata allergens are of major clinical importance. This study describes a new cross-reactive A. alternata allergen that was officially named Alt a 15 by the official Allergen Nomenclature Subcommittee. The complete coding region for Alt a 15 was amplified using 5' and 3' rapid amplification of cDNA ends and PCR. The recombinant protein was produced in Escherichia coli as a 65-kDa fusion protein, and the protein sequence exhibits high homology with several important fungal allergens. Immunoblotting analyses revealed that IgE antibodies from A. alternata-sensitized patients (n=59) bound to rAlt a 15 with a prevalence of 10.2%. All patients who presented sIgE to rAlt a 15 were apparently poly-sensitized to A. alternata and C. lunata. The extensive cross-reactivity between A. alternata and C. lunata serine proteases was confirmed using immunoblotting inhibition assays. Overall, Alt a 15 is an important new cross-reactive allergen of A. alternata that explains some allergies to A. alternata without Alt a 1 sensitization and initial diagnostic errors for allergies to Alternaria. This molecule may improve the accuracy of the diagnosis, the understanding, and the management of IgE-mediated fungal diseases.

Humoral immunity links Candida albicans infection and celiac disease.

OBJECTIVE: The protein Hwp1, expressed on the pathogenic phase of Candida albicans, presents sequence analogy with the gluten protein gliadin and is also a substrate for transglutaminase. This had led to the suggestion that C. albicans infection (CI) may be a triggering factor for Celiac disease (CeD) onset. We investigated cross-immune reactivity between CeD and CI. METHODS: Serum IgG levels against recombinant Hwp1 and serological markers of CeD were measured in 87 CeD patients, 41 CI patients, and 98 healthy controls (HC). IgA and IgG were also measured in 20 individuals from each of these groups using microchips sensitized with 38 peptides designed from the N-terminal of Hwp1. RESULTS: CI and CeD patients had higher levels of anti-Hwp1 (p=0.0005 and p=0.004) and anti-gliadin (p=0.002 and p=0.0009) antibodies than HC but there was no significant difference between CeD and CI patients. CeD and CI patients had higher levels of anti-transglutaminase IgA than HC (p=0.0001 and p=0.0039). During CI, the increase in anti-Hwp1 paralleled the increase in anti-gliadin antibodies. Microchip analysis showed that CeD patients were more reactive against some Hwp1 peptides than CI patients, and that some deamidated peptides were more reactive than their native analogs. Binding of IgG from CeD patients to Hwp1 peptides was inhibited by γIII gliadin peptides. CONCLUSIONS: Humoral cross-reactivity between Hwp1 and gliadin was observed during CeD and CI. Increased reactivity to Hwp1 deamidated peptide suggests that transglutaminase is involved in this interplay. These results support the hypothesis that CI may trigger CeD onset in genetically-susceptible individuals.

A monoclonal antibody against glycoproteins of Aspergillus fumigatus shows anti-adhesive potential.

There has been an increase in the cases of fungal resistance against many antifungal drugs and an effective alternative mode in the form of immunotherapy is being considered as new hope. The adhesion of Aspergillus fumigatus conidia to the host components is one of the prime factors to cause aspergillosis. Carbohydrate components or glycoproteins present on the cell surface play an important role in interaction of the organism to the host and leads to adhesion. Any substance which is capable of disrupting this interaction may be a vital tool for the fungal clearance and hence may protect the host from infections caused by the fungus. In this study, a murine monoclonal antibody IgM generated against the secretory antigens of A. fumigatus, was found to be specific to a common epitope containing glyco-moieties of the various proteins and exhibited anti-adhesive potential in vitro.

[Double-antigen sandwich ELISA for detecting Aspergillus fumigatus anti-Afmp1cr and Afmp2cr antibodies].

OBJECTIVE: To establish two double-antigen sandwich ELISA systems to detect anti-Afmp1cr and Afmp2cr antibodies of Aspergillus fumigatus. METHODS: Recombinant Afmp1cr and Afmp2cr proteins of A.fumigatus expressed in Pichia pastoris were obtained. Double-antigen sandwich ELISA systems for detecting specific anti-Afmp1cr and anti-Afmp2cr antibodies were developed after chessboard titrating to determine the appropriate concentrations of the recombinant proteins and HRP-labeled proteins. The sensitivity of the assay was evaluated using serum samples of rabbits immunized with Afmp1cr and Afmp2cr. The specificity of the assay was evaluated by detecting serum samples from healthy donors and patients with other pathogenic fungal and baterial infections. The performance of the two ELISA kits was furthered evaluated using serum samples from patients with suspected Aspergillus infection. RESULTS: The established ELISA kits were capable of detecting anti-Afmp1cr and anti-Afmp2cr antibodies in immunized rabbit serum at the maximum dilutions of 800 and 3200, respectively. No cross-reactivity was observed in detecting serum from patients with other pathogenic fungal or bactetial infections. Both of the two kits yielded positive results in sera from two established Aspergillus-infected cases and a suspected case. CONCLUSIONS: Two antibody-capture ELISA kits were developed for the laboratory diagnosis of A.fumigatus infection and can be potentially useful in the clinical diagnosis of Aspergillosis infections.

Fungal infections and treatment in cystic fibrosis.

PURPOSE OF REVIEW: This review summarizes some of the important recent findings concerning fungal airway infections in patients with cystic fibrosis (CF). For many years, both researchers and clinicians have focused on the problems in CF caused by chronic bacterial airway infection with organisms such as Haemophilus, Staphylococcus and Pseudomonas. However, until recently, the lack of sensitive culture techniques to isolate fungi in sputum, bronchoalveolar lavage fluid and other respiratory tract samples has limited the recognition of fungal species and their possible role in CF airway infections. RECENT FINDINGS: Recent studies using fungal-selective culture media and molecular techniques have shown a plethora of different fungal species in the sputum expectorated from CF patients. Cross-sectional studies have shown associations between Aspergillus and Candida in the sputum and worse lung function. The presence of allergic bronchopulmonary aspergillosis is likely to be a negative prognostic factor, but whether simple fungal colonization itself indicates future problems is not clear. Current research is now examining these epidemiological associations to try to determine the clinical implications. This will help determine whether fungal colonization/infection is associated with worse outcome in CF patients. SUMMARY: At present, there is no conclusive evidence that fungal organisms cause respiratory decline. Recent studies of antifungal therapy in CF patients have reported differing results, so further investigations in this area are needed.

Development and application of mold antigen-specific enzyme-linked immunosorbent assays (ELISA) to quantify airborne antigen exposure.

The aim of our study was to develop specific enzyme-linked immunosorbent assays (ELISAs) and apply these to assess mold antigen exposure in composting plants. Sandwich ELISAs based on polyclonal antibodies to Aspergillus fumigatus (Af), Penicillium chrysogenum (Pc), and Cladosporium herbarum (Ch) antigens were developed and validated. Reactivity to 18 different mold species was tested. To optimize extraction procedure, inhalable dust samples taken by a parallel sampler were extracted with or without homogenization. In 31 composting plants stationary pumps were installed at 4 sites to collect 124 inhalable dust samples. The newly developed ELISAs were used in addition to an anti ß-1,3-glucan ELISA to quantify mold antigens. The Cladosporium ELISA showed less than 0.04% reactivity to extracts from other fungal genera, while the Af ELISA demonstrated a reactivity of up to 3.6% and the Pc ELISA reacted up to 11% to other mold species. Extraction of parallel sampled filters gave higher antigen amounts with homogenization. The increase was highest for Pc-antigens, followed by Af-antigens, and lowest for Ch-antigens. Mean lower detection limits of homogenized inhalable dust samples were 5 ng/m(3) (Af), 0.6 ng/m(3) (Pc), 0.2 ng/m(3) (Ch), and 0.6 ng/m(3) (ß-1,3-glucan). The ELISAs were able to detect antigens in 43% (Af), 37% (Pc), 94% (Ch), or 100% (ß-1,3-glucan) of the 124 airborne dust samples. Inhalable dust, ß-1,3-glucan, and Af-, Pc-, and Ch-antigen concentrations were significantly correlated. The newly developed mold antigen ELISAs are thus able to measure airborne exposure levels in composting plants and differentiate between distinct fungi genera.

Toward developing a universal treatment for fungal disease using radioimmunotherapy targeting common fungal antigens.

BACKGROUND: Previously, we demonstrated the ability of radiolabeled antibodies recognizing the cryptococcal polysaccharide capsule to kill Cryptococcus neoformans both in vitro and in infected mice. This approach, known as radioimmunotherapy (RIT), uses the exquisite ability of antibodies to bind antigens to deliver microbicidal radiation. To create RIT reagents which would be efficacious against all major medically important fungi, we have selected monoclonal antibodies (mAbs) to common surface fungal antigens such as heat shock protein 60 (HSP60), which is found on the surface of diverse fungi; beta (1,3)-glucan, which is a major constituent of fungal cell walls; ceramide which is found at the cell surface, and melanin, a polymer present in the fungal cell wall. METHODS: MAbs 4E12, an IgG2a to fungal HSP60; 2G8, an IgG2b to beta-(1,3)-glucan; and 6D2, an IgM to melanin, were labeled with the alpha particle emitting radionuclide 213-Bismuth ((213)Bi) using the chelator CHXA". B11, an IgM antibody to glucosylceramide, was labeled with the beta emitter 188-Rhenium ((188)Re). Model organisms Cryptococcus neoformans and Candida albicans were used to assess the cytotoxicity of these compounds after exposure to either radiolabeled mAbs or controls. RESULTS: (213)Bi-mAbs to HSP60 and to the beta-(1,3)-glucan each reduced the viability of both fungi by 80-100%. The (213)Bi-6D2 mAb to melanin killed 22% of C. neoformans, but did not kill C. albicans. B11 mAb against fungal ceramide was effective against wild-type C. neoformans, but was unable to kill a mutant lacking the ceramide target. Unlabeled mAbs and radiolabeled irrelevant control mAbs caused no killing. CONCLUSION: Our results suggest that it is feasible to develop RIT against fungal pathogens by targeting common antigens and such an approach could be developed against fungal diseases for which existing therapy is unsatisfactory.

Antibodies Against Sporothrix schenckii Enhance TNF-α Production and Killing by Macrophages.

Sporotrichosis is a chronic granulomatous mycosis caused by the dimorphic fungus Sporothrix schenckii. The immunological mechanisms involved in the prevention and control of sporotrichosis suggest that cell-mediated immunity plays an important role in protecting the host against S. schenckii. Nonetheless, recent data strongly support the existence of protective Abs against this pathogenic fungus. In a previous study, we showed that passive Ab therapy led to a significant reduction in the number of colony forming unit in the organs of mice when the MAb was injected before and during S. schenckii infection. The ability of opsonization to enhance macrophage damage to S. schenckii and subsequent cytokine production was investigated in this work. Here we show that the fungicidal characteristics of macrophages are increased when the fungus is phagocytosed in the presence of inactivated serum from mice infected with S. schenckii or mAb anti-gp70. Additionally, we show an increase in the levels of pro-inflammatory cytokines such as TNF-α and IL-1ß. This study provides additional support for the importance of antibodies in protecting against S. schenckii and concludes that opsonization is an important process to increase TNF-α production and fungus killing by macrophages in experimental sporotrichosis.

Identification of fungal DNA in BALF from patients with home-related hypersensitivity pneumonitis.

BACKGROUND: In Japan, a major type of home-related hypersensitivity pneumonitis (HP) is summer-type HP, which is caused by Trichosporon asahii (T. asahii) or Trichosporon mucoides. Some patients with home-related HP test negative for antibodies against Trichosporon; yet, a causative mold antigen cannot be identified. METHODS: We analyzed 19 patients with home-related HP, 8 healthy volunteers, and 35 patients with other diseases. We extracted DNA from cell pellets of bronchoalveolar lavage fluid (BALF), amplified the DNA by PCR using Trichosporon-specific primers or other fungus-specific primers, and cloned as well as sequenced the PCR amplicon. Other primers used were specific for Acremonium chrysogenum, Aspergillus fumigatus, Aspergillus niger, Fusarium napiforme, Humicola fuscoatra, Penicillium corylophilum, and Pezizia domiciliana. RESULTS: We detected Trichosporon DNA (n = 17) and F. napiforme DNA (n = 2) by PCR in 19 patients with home-related HP; however, these species were not identified in healthy volunteers. After sequencing of the PCR amplicon for Trichosporon species, we identified T. asahii (n = 11), Trichosporon japonicum (n = 1), and Cryptococcus uzbekistanesis (n = 4). CONCLUSION: We could detect fungal DNA in BALF cell pellets from patients with home-related HP. These data suggest that this method might be useful to detect antigens responsible for home-related HP.

Detection of antibodies against Paracoccidioides brasiliensis melanin in in vitro and in vivo studies during infection.

Several cell wall constituents, including melanins or melanin-like compounds, have been implicated in the pathogenesis of a wide variety of microbial diseases caused by diverse species of pathogenic bacteria, fungi, and helminthes. Among these microorganisms, the dimorphic fungal pathogen Paracoccidioides brasiliensis produces melanin in its conidial and yeast forms. In the present study, melanin particles from P. brasiliensis were injected into BALB/c mice in order to produce monoclonal antibodies (MAbs). We identified five immunoglobulin G1 (IgG1) κ-chain and four IgM melanin-binding MAbs. The five IgG1 κ-chain isotypes are the first melanin-binding IgG MAbs ever reported. The nine MAbs labeled P. brasiliensis conidia and yeast cells both in vitro and in pulmonary tissues. The MAbs cross-reacted with melanin-like purified particles from other fungi and also with commercial melanins, such as synthetic and Sepia officinalis melanin. Melanization during paracoccidioidomycosis (PCM) was also further supported by the detection of IgG antibodies reactive to melanin from P. brasiliensis conidia and yeast in sera and bronchoalveolar lavage fluids from P. brasiliensis-infected mice, as well as in sera from human patients with PCM. Serum specimens from patients with other mycoses were also tested for melanin-binding antibodies by enzyme-linked immunosorbent assay, and cross-reactivities were detected for melanin particles from different fungal sources. These results suggest that melanin from P. brasiliensis is an immunologically active fungal structure that activates a strong IgG humoral response in humans and mice.

[Characterization of specific monoclonal antibodies to Aspergillus conidia by flow cytometry].

OBJECTIVE: To characterize the specific monoclonal antibodies to Aspergillus conidia. METHODS: Flow cytometry was used to examine the reactivity of the specific monoclonal antibodies to Aspergillus conidia. RESULTS: Both the monoclonal antibodies MA3 and Con2 showed specific reactivity to Aspergillus conidia suspensions. MA3 was capable of binding to the conidia of A.fumigatus, A.flavus, A.niger and A.terreus, while Con2 was reactive only to the conidia of A.fumigatus. CONCLUSION: Two specific monoclonal antibodies (MA3 and Con2) to Aspergillus conidia have been obtained.