| Background and ObjectiveInvasive fungal infections have been represented an important cause of morbidity and mortality in an increasingly number of patients since the last three decades.Several reasons have been proposed for the increase in invasive fungal infections,including the use of antineoplastic and immunosuppressive agents, broad-spectrum antibiotics,and prosthetic devices and grafts,and more aggressive surgery.Patients with bums,leucopenia,HIV infections are also prone to fungal infections.Fungi are increasingly recognized as major pathogens in critically ill patients.The most frequent filamentous fungi(moulds) isolated are Aspergillus spp., but Penicillium spp.(especially Penicillium marneffei) are also increasing obviously. The morbidity is related with the status of human immunity and the risk of exposure (such as high concentration of fungi conidia).Aspergillus spp.,ubiquitous in nature,is one of the most common indoor fungal genera,and is the commonest contaminant of agricultural commodities.Meanwhile,they are the most commonly isolated invasive moulds.More than 10 of the 200 Aspergillus species are pathogenic to human, primarily A.fumigatus,A.flavus,less A.terreus,A.niger,and A.nidulans.The diseases caused by Aspergillus are very diverse,including from mycotoxicosis,allergic reactions in immunocompetent individuals,to systemic diseases with high mortality rates in immunocompromised patients,called invasive aspergillosis.P.marneffei is a thermal dimorphic pathogenic fungus that is endemic in tropical Asia,especially Thailand,northeastern India,southern parts of China(such as Guangdong,Guangxi, Hong Kong,Taiwan) and Vietnam.The organism is a relative recent addition to known Southeast Asian mycoses,being discovered in 1956 from bamboo rats.The rarity of human penicilliosis marneffei changed when the global AIDS pandemic arrived in Southeast Asia.Currently,the prevalence of human penicilliosis marneffei infection is increasing in areas where HIV infection is on the increase.The disease is the third most common opportunistic infection in patients with AIDS in Southeast Asia.Both Aspergillus spp.and P.marneffei can cause deadly infection in immunocomprised patients,in which the crude mortality from opportunistic fungal infections still exceeds 50%in most human studies.This is mainly due to the absence of early and accurate diagnosis and the toxic and side-effects of antifungal drugs.The increase of drug-resistance isolates is also one of the reasons.To reduce the mortality,early and accurate diagnosis is critical in improving the prognosis for patients though the delivery of more prompt antifungal therapy and lessening the unnecessary use of toxic antifungal drugs.However,early clinical diagnosis is often difficult since the signs and symptoms are non-specific,the definitive diagnosis of invasive fungal infections relies on laboratory tests.Currently, laboratory diagnosis of fungal infections is based on culture,histopathology, molecular and serological diagnostic technique.A culture yielding Aspergillus spp. and P.marneffei is a gold standard for the diagnosis of fungal infection,but it is time-consuming(the process takes at least 4 weeks),is relative insensitive,and requires specialized expertise for species determination.The molecular diagnosis provides more sensitive and rapid diagnosis than traditional culture method dose. However,this method requires experienced technicians and specialized laboratory equipments and ease to bring out false-positive results due to the ubiquitous existence of Aspergillus conidia.For histopathology,it is often difficult to obtain deep tissue specimens from critically ill patients.The specific antibody response that is usually induced in patients with invasive fungal infections can help in the diagnosis of the majority of mycoses.However,antibody detection may have important limitations. On one hand,the specificity of techiques used to detect antibodies may be low.For instance,due to the ease of dispersion of Aspergillus conidia,it is calculated that a person could inhale several hundred conidia of A.fumgitus per day,and each healthy individual has high specific antibody titers.On the other hand,antibody detection may lack sensitivity in severely immunocomprised patients who are not able to mount a detectable antibody response.While for P.marneffei infections,the high levels of specific antibodies were detected in some immunocomprised penicilliosis patients, but the sensitivity is low,and it was reported that serum antigen titers were higher in HIV-positive patients than serum antibody levels.Therefore,to develop a rapid,more sensitive and specific diagnostic test is necessary for the early diagnosis and prompt therapy of fungal infection,which may provide auxiliary diagnosis for AIDS.At present,most of the diagnostic methods of invasive fungal infection focus on detection of components in the fungal cells,especially polysaccharides.The availability of the polysaccharides(especial glycoproteins,such as mannan-protein complex),the major antigen in cell walls,which are also important and abundant structural components of fungal cell walls,has allowed to the development of diagnostic tests for Aspergillus spp.and P.marneffei infections.The occurrence of diagnostic tests kit Platelia Aspergillus is a commercial-available,double-sandwich ELISA allowing the detection of the circulating galactomannan antigen of Aspergillus in human serum,which indicates galactomannan may be the early diagnostic marker in fungal infections.But galactomannan is present in the cell wall of most Aspergillus and Penicillium species,and studies that have evaluated the Platelia Aspergillus assay have documented a high percentage of false-positive results in patients used antibiotics originate from Penicillium spp.(i.e.ampicillin-sulbactam, piperacillin-tazobactam,and amoxicillin- clavulanic acid).By contrast,there is no diagnostic kit for P.marneffei infections.Therefore,an accurate and specific diagnosis of Aspergillus and P.marneffei infection is necessary.Methods and ResultsThis study consists of three parts,as follows:Ⅰ.The detection of glycoproteins specific in Aspergillus genus and A.fumigatus Our preliminary research has prepared dozens of monoclonal antibodies(mAbs) which are specific for glycoprotein extracted from A.fumigatus and recombinant glycoprotein(the first antigenic cell wall galactomannanoprotein specific in A.fumigatus,named Afmp1p) of A.fumigatus respectively,and has established two immuno-enzymatic assays based on two groups of mAbs above.This study focuses on the specificity and broad-spectrum of detection of the two methods.The two different double mAb ELISA assays were performed to detect antigens in the cell culture supernatants of several species of Aspergillus,which are isolated from clinical patients and environment.The results indicated that the mAbs prepared with crude antigen of A.fumigatus were specific for antigens in both clinical isolates and environmental isolates of Aspergillus.spp,whereas the other group was proved to be specific for the antigen of clinical and environmental isolates of A.fumigatus.And no cross reaction with the cell culture of P.marneffei and Candidas were observed with the two methods.The ELISA assays,which can detect both of the clinical and environmental isolates of Aspergillus,or differentiate A.fumigatus from other species of Aspergillus spp.,provide us with new tools for monitoring Aspergillus in indoor environment and contamination both in agricultural commodities and food,and are also useful for early diagnosis of patients with aspergillosis.Ⅱ.Preparation and characterization of monoclonal antibodies and rabbit hyperimmune serum against the glycoproteins of P.marneffeiIt was reported that some penicilliosis patients were mannoprotein positive, which shows that mannoprotein is a serological diagnosis marker.In this study,we expressed a recombinant mannoprotein(named Mp1p) in P.pastoris which was cloned from the cDNA display phage,and the protein was used to immunize mice for the production of hybridomas.Then the splenocytes of the immunized mice were fused with myeloma cells to produce hybridoma cell line,which could secret anti-Mp1p protein antibodies.18 hybridomas that produced mAbs against the Mp1p protein of P.marneffei were successfully selected by indirect immunofluorescence assay,ELISA,and Western blot assay.Among of these,10 mAbs were of IgG1 isotype,3 were IgG2a,3 were IgG2b,and 2 were IgG.Competitive inhibition assay showed that the 18 mAbs recognized at least 4 distinct epitopes of Mp1p antigen.Rabbits were initially immunized with subcutaneous injections of purified Mp1p protein emulsified in an equal volume of complete Freund's adjuvant at multi-sites. And the rabbit immune serum against Mp1p protein of P.marneffei was obtained with higher antibodies titer of 1×10-7.The specificity of mAbs and rabbit hyperimmune serum against Mp1p protein of P.marneffei was further identified.Both the western blot assay and indirect immunofluorescence assay showed that the 18 mAbs and rabbit immune serum strongly reacted with the yeast cells which are the only form existing in human rather than mycelium cells of P.marneffei,with no recognition of the other Penicillium and some common isolates of Aspergillus.These results showed hybridomas producing high specificity mAbs to Mp1p protein of P.marneffei which bind to distinct epitopes and thus select mAb pairs that can be used to develop a highly sensitivity of antigen capture assay.Ⅲ.Development and optimization of antigen-capture assay based on monoclonal antibodies against the glycoproteins of P.marneffeiIn this study,we developed antigen capture ELISA for detection of Mp1p protein of P.marneffei.To establish optimal antigen capture assay,capturing and detecting antibodies were selected by sandwich ELISA paired the mAbs and polyclonal antibodies one by one.The best coating antibody and biotin-conjugate detection antibody selected ultimately to construct the antigen capture ELISA was 1F53A7 and 3C10A9-Biotin.Recombinant Mp1p protein was used as a standard to establish a detection sensitivity of approximated 31pg/ml for Mp1p antigen-capture ELISA. Furthermore,this mAb-based antigen capture ELISA is specific for Mp1p of P.marneffei,and has no cross reactivity with culture supernatant of other species of Penicillium.All these results indicate that the established test is sensitive and specific which may pave a road for providing a novel laboratory diagnosis tool for P.marneffei infection.Conclusions 1.Two mAb-based antigen-capture ELISA for detection Aspergillus genus specific antigen and A.fumigatus specific antigen were successfully established, which will provide new approach for early diagnosis of aspergillosis and monitoring contamination of Aspergillus spp.in environment,agricultural commodities and food.2.Preparation of mAbs specific to yeast cells of P.marneffei,which are useful for development of diagnostic kit,study on the function of the Mp1p protein and pathogenesis in P.marneffei infection.3.A P.marneffei yeast cells antigen specific mAb-based antigen-capture ELISA has been developed.It has high detection specificity to P.marneffei without cross-reactivity with other Penicllium species.This characteristic may be useful for the early diagnosis of P.marneffei infection.4.On the basis of mannoprotein,we developed antigen-capture ELISA which can differentiate Aspergillus and P.marneffei successfully. |
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