Preparation And Characterization Of Monoclonal Antibody Of Aspergillus Fumigatus And Establishment Of Aspergillus Fumigatus GM Antigen-capture Assay

In recent years, due to various iatrogenic factors for example extensive use of broad-spectrum antibiotics, corticosteroids, immunosuppressants and anti-tumor drugs, and development of organic transplantation and many varieties of interventional catheterization, incidence by opportunistically invasive mycotic infection apparently increases. Aspergillus has become the second important pathogenic fungus after Candid, causing severe and usually fatal invasive infections in immunocompromised hosts. Invasive Aspergillosis (LA) caused by Aspergillus fumigatus is responsible for over 90% of the total LA patients, in whom the mortality rate is 80 to 90%, even when treated. Clinic studies have showned that early diagnosis of IA is closely associated with a better prognosis. But owing to unconspicuous behavior of Aspergillus infection and lackage of clinic features, it is very hard to achieve early diagnosis. Although blood culture and tissue biopsy are regarded as golden standard for laboratory diagnosis of Aspergillus infection, they are not able to meet clinic need because of long time for culture, low positive rate, false positive phenomena caused by operation contamination and limitation and trauma by biopsy. At present, serological and molecular diagnosis have already become concerns in the field. PCR technology is used to detect trace amount of fungal DNA in early infection and identify the fungus species type, which provides certain help for directing clinic administration. But due to high requirement tolaboratory and operator at primer design, anti-contamination treatment of clinic samples and judgement of results etc, its application still remains at small samples research. Specific serum antibody detection assay has been used in some investigations, but it also has shortcomings due to the presence of host antibodies does not always correlate with presence of invasive disease, especially for the poor immunological status of the host. Moreover, environmental surveys indicate that all humans will inhale at least several hundred A. fumigatus conidia per day, antibody to A. fumigatus extensively exists in the serum of health adults. So antibody detection is not suitable for early and specific detection. Now attention has been focused on fungal antigen detection. More than 100 antigen molecules have been found from extract of A. fumigatus, among which about 12 were purified into single antigen forms. It was confirmed that rich galactomannan (GM) in cell wall of A. fumigatus was its primary antigen component. GM was the first antigen detected in experimentally infected animals and patients with IA. Its content was high in the primary serum and body fluid, therefore detection of GM antigen in patients may be as an early diagnosis approach for A. fumigatus infection. Commercial kit for detecting GM protein has been developed by French Pasteur Laboratory, but its unsatisfactory performance in sensitivity and specificity greatly hampered its clinic application. To date, a sensitive and reliable early diagnosis method is still not available in the world.The present study is intended to establish a sensitive, specific and early detection method for GM protein of A. fumigatus based on monoclonal antibody technology. Three sections are performed in this research.Section 1: Preparation of rabbit polyclonal antibody against recombinant GM protein (AFMP1) and establishment of sandwich EL1SA to screen monoclonal antibody against natural epitope of Aspergillus fumigatusWhen employing the complex protein as immunogen to achieve anti-GM antibody, the primary problem is how to screen the monoclonal antibody. This part study aimed to establish an assay to screen the anti-GM antibody in that provides a basis for the following researches. The recombinant GM protein (designated as AFMP1) was expressed and purified, then was used to immunize the New Zealand rabbit to obtain high-titer polyclonal serum against AFMP1. Purified rabbit polyantibody was coated on the plate to capture GM protein exists in the culture of A. fumigatus for screening monoclonal antibody specifically binding natural sites of GM of A. fumigatus.Section 2: Preparation and identification of monoclonal antibody against Aspergillus fumigatusThe monoclonal antibodies against AFMP1 had been obtained in our lab by immunized the BALB/c mice with recombinant AFMP1 protein, while only part of these monoclonal antibodies reacted with the natural antigen of A. fumigatus validated by the immunofluorescence, indicating that some epitopes of the recombinant protein has changed. The trouble may be solved by immunized with natural GM protein, but considering the difficulties in purification of the natural form of GM protein, we selected the complex proteins which include the GM protein as immunogen. Different crude extract components from A. fumigatus were prepared and used to immunized the BALB/c mice, then the cell fusion technique was employed to obtain hybridoma cell strains, using the established method in section 1 to screen the monoclonal antibody against the natural epitope in GM protein of A. fumigatus. The section itself contains three parts.Part I . Four crude extract culture components were prepared according to the different morphological characteristic of A. fumigatus at different growth stages: 1. Cultured conidia of A. fumigatus using Sabouraud plate, inactivated the conidiaand homogenized, obtained culture cell extract of A. fumigatus.2. Cultured conidia of A. fumigatus using Sabouraud plate, and inactivated conidia under aseptic condition. Then obtained conidia of A. fumigatus for immunization.3. Inoculated conidia of A. fumigatus into 1640 culture medium for amplifying A. fumigatus. After inactivated and homogenized, the homogenized supernatant of A. fumigatus hyphae was obtained.4. Filtered and concentrated 1640 culture of A. fumigatus to obtain filtrate of A. fumigatus cultural supernatant.Part II. Immunized BALB/c mice with the above four culture components respectively, and identified mice serum titer employing the 3 methods below:1. Coated plate using rabbit polyclonal antibody against AFMPl, which captured GM protein in culture substance of A. fumigatus, thus used sandwich ELISA to identify antibody titer of mice serum.2. Coated plate using recombinant GM protein (AFMPl) and employed indirect ELISA to detect antibody titer of mice serum.3. Spreaded conidia and hyphae of A. fumigatus onto slides and used immunofluorescent assay to identify antibody titer of mice serum.Part III. Chose the BALB/c mouse with the highest serum titer to GM for preparation of the monoclonal antibodies.The immunized splenocytes of the chosen BALB/c mouse were fused with the NS-1 myeloma cells, the hybridoma cells were seleted by 3 methods mentioned aboved. Thereafter, the positive hybridoma cell strains were subcloned with limited dilution and screened, finally obtained four hybridomas producing mAbs steadily. Three of the mAbs were IgG3 isotype, and the other was IgGl. Immunofluorescentassay showed the four mAbs all cross reacted to other Aspergillus species including Aspergillus flavus, Aspergillus terreus and Aspergillus niger.Section 3: Establishment and optimization of antigen-capture assay for dectection of Aspergillus fumigatus GMIt was found that from study of section 2, monoclonal antibodies, prepared from BALB/c mice immunized with crude extract from culture components of A. fumigatus, had cross-reactions with other Aspergillus species indicating theirs specificity was not high. While the previously obtained antibodies immunized with the recombinant GM protein had strong specificity against GM protein of A. fumigatus and had no cross-reaction with other Aspergillus. Based on the mAbs specially against GM, this experiment was aimed to establish and optimize a detection method for A. fumigatus.Take four mAbs against GM of A. fumigatus to further identify their reaction properties to conidia and hyphae of A. fumigatus using immunofluorescent assay, the result showed three mAbs, 7A18A4, 7C1B3 and 7D3B2, recognized the hyphae of A. fumigatus. The competitive inhibition test was employed to confirm the binding epitopes between the three mAbs. Results indicated 7A18A4 entirely not share epitope with the other two mAbs, while 7C1B3 and 7D3B2 shared some common epitopes. Coated the three mAbs separately or by the combined form, then paired with the rabbit anti-GM serum to development the sandwich ELISA for detecting GM, it was found the sensitivity and specificity was the best coated together by 7A18A4 and 7C1B3. The optimal working concentration of rabbit antiserum and enzyme labeled goat anti rabbit IgG as well as the reaction time were determined by chessboard titration. In this study, two different mAbs recognizing different epitope were employed for capture, and rabbit polyantibody for indirect detection, forming the reaction complex as capture antibody- antigen- second antibody- enzymedconjugated anti second antibody, due to the increase layer of the polyantibody compared to the former established direct double mAb sandwich ELISA, the reaction efficiency and the detection sensitivity were both improved remarkably. The newly developed method also had a more wide linear detection range.Summarization: we used recombinant GM protein to immunize New Zealand white rabbit to obtain polyclonal antibody against GM, then established indirect ELISA to detect antibody against GM of A. fumigatus. Using the hybridoma technique, four mAbs against A. fumigatus were successfully prepared from the BALB/c mice immunized with crude extract antigen of A. fumigatus. Although these four mAbs had cross reaction with other Aspergillus strains, they may be help in the broad-spectrum Aspergillus detection. A sandwich ELISA for detection GM of A. fumigatus were established by pairing the former obtained mAbs with the rabbit anti-GM polyantibody, which was able to specially detect the GM protein existed in the culture supernatant of A. fumigatus, without cross reaction to other Aspergillus strains. The lower limit of detection of recombinant GM for the sandwich ELISA was 1.5 pg per ml, while its detection sensitivity and specificity should need further confirmation by clinic studies. Successful establishment of the detection assay for GM antigen of A. fumigatus will provide a new tool for early diagnosis of A. fumigatus infection and have important clinical meanings to the prevention and cure of the disease.