| [Background]Aspergillosis is defined as a calss of infectious diseases, caused by Aspergillus spp with widespread presence in the environment. In recent decades, the incidence of Aspergillus infection has been significantly rising. According to the literature, Aspergillus fumigatus is one of the most common species associated with human disease. Aspergillus fumigatus cause mainly pulmonary infection diseases, such as allergic pulmonary aspergillosis (ABPA), pulmonary aspergilloma and invasive aspergillosis (IA). The mortality of IA in immunosuppressed patients with hematologic malignancies and bone marrow transplant can be as high as60%to100%. Aspergillus fumigatus is ubiquitous filamentous fungus in the environment, and can generate thousands of conidia. Humans inhale hundreds of Aspergillus fumigatus conidia daily. In immunocompetent individuals, inhaled conidia can be eliminated by innate immune system. In recent years, as the result of the gradually increasing of the organ transplantation, hematopoietic stem cell transplantation, hematologic malignancies, HIV infection, more and more patients immunocompromised are steadily expanding. In these patients, because of weakening the innate defense mechanisms, such conidia of Aspergillus fumigatus can escape from the defense line and reach the pulmonary alveoli through the terminal respiratory airways and swelling in the lungs, germinate hyphae, thereby causing pulmonary aspergilloma disease. In severe immunocompromised patients, hyphae are able to penetrate the lung tissue into the bloodstream, migrate to multiple body organs including liver, kidneys, spleen, eyes, brain,etc, causing IA. For immunocompromised individuals, once infected with Aspergillus fumigatus, even given current antifungal therapy, the mortality is still high. Therefore, early diagnosis and rational targeted anti-fungal treatment, avoiding blind empirical treatment for Aspergillus fumigatus infection disease outcome are particularly important.Since the symptoms of Aspergillus infection are nonspecific, eary clinical diagnosis of the infection is often difficult. Currently, the main methods of diagnosis of Aspergillus fumigatus infection are traditional diagnosis, serological diagnosis (specific antigen and antibody detection), gene diagnosis. Traditional methods include direct microscopy, sterile cavity fluid specimen culture, histopathology identification and imaging studies. The gold standard for diagnosis of Aspergillus fumigatus infection is to obtain positive direct microscopic examination or positive microbiological culture and to obtain histological evidence of mycelial growth in biopsy specimens. But Aspergillus fumigatus the positive rate of culture from blood is low, because the culture is vulnerable to pollution by the air Aspergillus, the specificity is questioned. Histological biopsy is often precluded from patients in critical conditions, sincethe patients are often too weak to receive this implement. Additionally patients are unwilling to accept the biopsy. Crescent sign and halo sign on imaging in patients with candidiasis or cytomegalovirus infection can also be observed, it is not as a basis for the diagnosis of Aspergillus infection. It is generally accepted that the commercial assay kits for detection of galactomannan antigen (GM) and (1,3)-β-D glucan antigen (BG, used in G test) presented in fungal cell wall have been used for diagnosis of fungal infection. According to the third edition of the China Index blood disease/cancer patients with invasive aspergillosis diagnostic criteria and treatment principles, GM is an indicator of clinical therapeutic efficacy, G test is an indicator of clinical diagnosis of invasive fungal infections. GM mainly from an Aspergillus cell wall galactomannan antigen can be used as an early diagnostic marker of Aspergillus infection. However, GM detection can not distinguish between Penicillium and Aspergillus infection because this antigen is also present on the cell wall of Penicillium marneffi. Moreover, there are the cross-reaction among GM antigen, semi-synthetic penicillin (piperacillin/tazobactam, etc.), dairy products containing GM ingredients high and intestinal parasitic bacteria wall phospholipid, which can make false-positive results in several populations includingpatients receiving semi-synthetic penicillin therapy,cancer patients receiving chemotherapy (because of gastrointestinal mucosal injury, leading to GM through the damaged intestinal mucosa into the blood) as well as neonatal patients. These people just are prone to be infected by invasive fungal, therefore, it is difficult to explain the GM-positive results for the clinical diagnosis. AsGM antigen released into the blood circulation can be removed rapidly and be uncapable of persisting, it may lead to false negative results. Theglucan (BG) of G trials detectionwidely present in fungal cell walls except Cryptococcus and Zygomycetes,, is a broad spectrum of fungi cycle markers. So, BG testing can not distinguish between Aspergillus with other fungal infections.Like as GM trials, G trials in patients with hemodialysis, hemolysis, jaundice, intravenous infusion of albumin or gamma globulin, and even some bacterial infections can lead to false-positive results. There are advantages and disadvantages about G trials and GM detection, but false-positive and false-negative are inevitable, which lead to some limitations in clinical applications. Therefore, it is necessary to find new antigne marker to develop specific and senseitive immunoassy. In recent years, biomolecular diagnosis DNA-based detection has gradually become a hot spot. With a whole-genome shotgun approach was finished and complete genomic sequence of Aspergillus fumigatus strain Af293was obtained,Many researchers pay their attention to develop biomolecular diagnosis of invasive aspergillosis by designing specific primers based on sequence alignment and genemis data. Biomolecular diagnosis has many advantages including the classification of different types in the same strain and the acquaintance of drug resistance. Besides of difficulties in standardization of methodology and experimental operation, there are still many problems of using biomolecular diagnosis in clinic. It is difficult for making genetic testing become a routine detection method of diagnosis about invasive aspergillosis. Since the clinical signs and symptoms of Aspergillus fumigatus infection are nonspecific, imaging features are absence, early diagnosis is still more difficult.Professor Yuen, et al cloned the AFMP1gene thatencodes the first antigenic cell wall galactomannoprotein in Aspergillus fumigatus. AFMP1codes for a protein (Afmplp) thatis a homolog of Mplp. This protein was observed in patients with aspergilloma and invasive aspergillosis due to A.fumigatus. Previously, we developed the antigen and antibody tests using E.coil expressed GST-Afmp1p and rabbits/guinea pig anti-GST-Afmplp polyclonal antibodies (Pabs). A sensitive enzyme-linked immunosorbent assay (ELISA) developed with antibodies against Afmplp is capable of detecting this protein from the cell culture supernatant of A. fumigatus. This Afmp1p antigen-based ELISA is also specific for A. fumigatus whereas the cell culture supernatants of the other eight fungi gave negative results. Finally, a clinical evaluation of sera from invasive aspergillosis patients indicates that8of15(53%) patients are positive for Afmp1p antigen test. Furthermore, an Afmp1p antibody test was performed with these serum specimens. We establish a recombinant antigenic protein-based indirect ELISA for the detection of specific antibodies in patients with aspergillosis, by which the high level of specific antibody against Afmp1p was detected in patients with invasive A. fumigatus infections. Therefore, the detection of specific antibodies may be used for diagnosis of invasive aspergillosis. It would provide a new choice for the diagnosis of aspergillosis.As series work,, Yuen, et al successfully cloned the Aspergillus fumigatus mannoprotein2(AFMP2) gene, which encodes a novel immunogenic protein (Afmp2p) of the antigenic mannoprotein superfamily, in A. fumigatus. Afmp2p is homologous to Afmp1p. Western blot analysis of Afmp2p in A. fumigatus fungal cell lysate and culture supernatant and immunogold staining and electron microscopy showed that Afmp2p is predominantly secreted into the culture supernatant, whereas only minimal amounts can be detected in the cell lysate and cell wall. Finally, it was observed that patients with aspergilloma and invasive aspergillosis due to A. fumigatus develop a specific antibody response against recombinant Afmp2p.Afmp2p is a good candidate for complementing Afmp1p in serodiagnosis of A. fumigatus infections.Professor Yuen, who in the course of expressing recombinant proteins Afmp1p and Afmp2p found the fragments of Afmp protein which were encoded by conserved sequence of genes(A.fumigatus galactomannoprotein1conserved regions, Afmplcr and A.fumigatus galactomannoprotein2conserved regions,Afmp2cr). So Afmp1cr and Afmp2cr have similar immunogenicity, which can induce production of specific antibodies with potential application in clinical.The objects of this study is to establish immunoassasys to detect specific antibodies of Aspergillus fumigatus with recombinant Afmp1cr and Afinp2cr.1. Expression of recombinant Afmp1cr and Afmp2cr protein and preparation of antisera against Afmp1cr and Afmp2cr by immunization of rabbits.Afmplcr and Afmp2cr are expressed in Pichia pastoris. The recombinant expressed Afmp1cr (14Kda) and Afmp2cr (13-25Kda) identified by SDS-PAGE, which match their molecular weights, indicating their expressions were successful. Indirect ELISA and Western blot were performed with the serum of rabbit immunizedwith A.fumigatus natural protein to further identify expressed Afmp1cr and Afmp2cr. The results showed that recombinant Afmp1cr and Afmp2cr protein efficiently combined with the rabbit antiserum against natural A.fumigatus protein. The purified recombinant Afmp1cr and Afmp2cr were injected into one rabbit to obtain anti-Afmp1cr and anti-Afmp2cr rabbit polyclonal antibodies, respectively. The antibodytiter of anti-Afmp1cr and anti-Afmp2cr rabbit polyclonal antibodies, were both1024000determined by indirect ELISA. All the three experiments of indirect ELISA, Western blot and immunofluorescence assay (IFA) showed that the polclonal antibodies against Afmp1cr anti-Afmp2cr can bind with the natural protein of A.fumigatus.2. Development of indirect ELISA immunoassasys to detect antibodies against Afmp1cr and Afmp2cr for diagnosis of Aspergillus fumigatus infectionAn indirect ELISA for detecting specific anti-Afmplcr antibodies was developed by optimizing concentrations of the recombinant Afmp1cr protein and HRP labelled goat anti-human IgG, and another EILSA system for detecting anti-Afmp2cr antibody was developed in a similar way. The specificity of the assay was evaluated by detecting serum samples from healthy donors and the patients of with other pathogenic fungi and bateria infection. The performance of the two systems were furthered evaluated with the serum samples from suspected patients of aspergillus infection(two cases of which were proved aspergillus infection by biopsy). The results were as follows:the cutoffs of indirect ELISA established by detecting94healthy people serum for detecting specific anti-Afmplcr antibodies0.189, the specificity of the assay was96.1%(99/103) in detecting other microbial infections, and cross-reactivity was observed in sera ofpatients with other pathogenic fungi(Candida, Mucor) infection; the assays showed positive results in detecting two proved aspergillus infected patients and a suspected one.; no differential OD values were observed between16patients and94health peoples, suggesting that the indirect ELISA used for detecting specific anti-Afmp2cr antibodies can not distinguish between healthy people and patients.3. Development of double-antigen sandwich ELISA to detect anti-Afmp1cr/Afmp2cr antibodies for diagnosis of Aspergillus fumigatus infection.A double-antigen sandwich ELISA was developed by optimizing concentrations of the recombinant Afmp1cr and HRP labelled Afmp1cr, and a similar system t for detecting anti-Afmp2cr antibody was also developed..The sensitivity of the assay was evaluated by detecting serum of rabbit that was immunized with Afmp1cr, Afinp2cr, respectively. The specificity of the assay was evaluated by detecting serum samples from healthy donors and the patients of with other pathogenic fungi and bateria infection. The performance of the two systemswere furthered evaluated with the serum samples from suspected patients of aspergillus infection(two cases of which were proved aspergillus infection by biopsy).. The results were as follows: The maximum dilutions of rabbit serum detected by our established anti-Afinplcr and anti-Afmp2cr antibodies capture double antigen sandwich ELISA is800and 3200, respectively. the cutoffs of indirect ELISA established by detecting94healthy people serum for detecting specific anti-Afmplcr antibody and anti-Afmp2cr were0.129and0.101, respectively; The specificity of each assay detection of other microbial infections was100%.Both of the two ELISA systems showed positive results in detecting two established aspergillus infected patients and a suspected one.Summary1. Two potential bio-makers of A.fumigatus infection-galactomannoprotein, Afmp1cr and Afmp2cr were expressed in Pichia pastor is. We preparad the rabbit antisera by immunization with recombinant purified proteins Afmp1cr and Afmp2cr respectively.2. Development of two indirect ELISAs immunoassasys by detecting anti-Afmp1cr/Afmp2cr antibodies for Aspergillus fumigatus infection. For detection of The anti-Afinp1cr antibody,the assays showed positive results in detecting two proved aspergillus infected patients and a suspected one, and no false positive in healthy donors and patients with other microbial infection was observed. The indirect ELISA for detecting specific anti-Afmp2cr antibody can not distinguish between healthy people and patients.3. Development of two double-antigen sandwich ELISAs used for detecting anti-Afmplcr/Afmp2cr antibodies.. Two double-antigen sandwich ELISAs for detecting specific anti-Afmp1cr/Afmp2cr antibodies:The specificity of two assays detection of other microbial infections were100%(0/103). Both of the two sandwich ELISA system showed positive results in detecting two established aspergillus infected patients and a suspected one, no false positive in103patiens with other microbial infection was observed. Moreover, no cross-reactivity was observed in seraof patients with other pathogenic fungi(Candida, Cryptococcus neoformans, Mucor) or bactetia(Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus) infections. |
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