The Experimental And Clinical Study Of GM Monoclonal Antibody Of Aspergillus Fumigatus In Diagnosis Of Invasive Aspergillosis

Aspergilli is an important pathogenic fungus which cause the deep part fungous infection for immunosuppression patients .The patients of immune hypofunction caused by malignant tumou, granulocytopenia, glucocorticosteroid and immunosuppressant are the risk factor of invasive aspergillosis(IA).When patients of immunosuppression breathe in Aspergillus spore , the spore invade lungs through bronchus and alveoli and caused invasion infection and disseminated to other organ. In recent ten years, aspergillus has become the second important pathogenic fungus after candid causing severe and usually fatal invasive infection in immunocompromised hosts. The fatality of Invasive Aspergillosis is 56%-88.1%. Clinic studies have showed 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 for aspergillosis. Although blood culture and tissue biopsy are regarded as golden standard for laboratory diagnosis of aspergillosis, they are not able to meet clinic need .Because positive rate of blood culture is very low and the tissue of body is difficult to be gained. Early diagnosis about 1A is difficulty. The drugs of anti-eumycete can't be used in time and the best opportunity of treatment is delayed. So, develop a rapid, high specific, sensitive diagnosis method for IA is very necessary.When Aspergilli infect body, its antigen compositions delivery to body fluid and excite organism generate specificity antibody. The aspergilli antigen and antibody can be detected by specific serological detection assay. Patient of IA usually have severe immunesuppression and can't generate enough antibody.On the other side,the generation of antibody need about two weeks,it make the early diagnosis of IA impossible. In addition, some organ in human body may have been contaminated by Aspergilli. The antibody of Aspergilli may be positive in body fluid in some degree. So, the antibody assay positive for Aspergilli can't prove Aspergilli infection. In recent years, people put focus on detection antigen of Aspergilli. Galactomannan(GM). It is the antigenic component of Aspergilli cell wall. The ratio of its galactose and carubinose is 1:1.17, its molecular weight is between 25000-75000. Epitope located in extremital galacto-furanose. Aspergillus fumigatus is the most common and main pathogenic agent among Aspergilli. In our research,we extract the GM antigenic component of Aspergillus fumigatus cell wall, prepare the monoclonal antibody of Aspergillus fumigatus, establish double antibody sandwich ELISA to determine the GM antigen of Aspergillus fumigatus in blood serum.We aim at to develop a early, rapid, sensitivity, specificity generally method to detect Aspergillus fumigatus antigen. The mainly studies as follows:1. Preparation and identification of GM monoclonal antibody against Aspergillus fumigatus. Eestablish double antibody sandwich ELISA to detect the GM antigen of Aspergillus fumigatus in blood serum.2. Establish animal model of IA infection. Using double antibody sandwich ELISA detecting GM antigen of Aspergillus fumigatus in serum specimen.Using immunoenzyme histochemical method detect GM antigen in tissue. Validate and evaluate application value of GM—McAb in animal model of IA infection.3. Using double antibody sandwich ELISA detect GM—McAb of Aspergillus fumigatus in serum specimen of IA patients. Evaluate GM—McAb application value in clinic.Section 1: Preparation and identification of monoclonal antibody against Aspergillus fumigatusCultivate Aspergillus fumigatus and extract GM-antigen, and then immune BALB/c mouse with GM-antigen. Confluens mouse spleen cell and myeloma cell, develop hybridoma cell. Gain four strain 8A2A17,7E11A1,9D47A1,2D27A6 stable excrete anti-aspergillus fumigatus McAb hybridoma cell strains. Immunofluorescence detect valence of antibody. 8A2A17,8A7A8,2D24A2 are 1: 32000 and 2D27A6 is 1: 2000.8A7A8,2D24A2,8A2A17and 2D27A6 can bind with hepar and counteract antigenic component of spore. There are cross reaction between Aspergillus fumigatus and Aspergillus flavus, Aspergillus terreus, black mold. It is showed that mycetes exist a common antigen. Use GM—McAb of Aspergillus fumigatus not only detect Aspergillus fumigatus specially but detect other kind of Aspergillus strains. This will have more generally practicability in clinical diagnosis. In generally, people don't emphasis specific identification of Aspergillus strains. Double antibody sandwich ELISA detect GM antigen of Aspergillus fumigatus: Chose two strains hypsi-affinity and different antigenic determinant McAb 7E11A1,9D47A1,as invest antibody and enzyme labelled antibody respectively. Using AFMP1 (GM recombination protein) as standard detection sample and BSA as negative control establish double antibody sandwich ELISA. Using OD₄₅₀ as Y-axis and density of protein as X-axis draw a standard curve.The results show that the highest sensitivity is 0.1ng/ml. AFMP1 standard detection sample density range of 0.1～60 ng/ml is align above standard curve. The result shows that the survey is range at 0.1～60ng/ml. Section 2:Establish animal model of IA infection and detection of antigen12 New Zealand white rabbits were separated to two groups.Group one: experiment group 6 examples; group two (negative control): common control is 4 examples and immune suppression control is 4 examples. Experiment group and immune suppression group intervenous drop infusion hydrocortisone 15mg/kg, cyclophosphamide 25mg/kg continuity 2 days. At the third day, intramuscular injection hydrocortisone 15mg/kg, experiment group intravenous injection 5×10⁶CFU/ml Aspergillus fumigatus spore suspl, 1ml. Immune suppression control group intravenous injection liquor natrii chloridi isotonicus 1ml. Collection blood samples of every group rabbits before infection and after infection at 24h, 48h, 72h, 96h respectively. Kill rabbits of these two groups at 96 hours after infection. Organs as lung, hepar, lien, kidney were get out and make these tissue as homogenate and others were put in liquor of 10%formaldehyde fixed 8 hours. Organ homogenate culture: cut lung, hepar,lien, kidney and take apart of them become tissue homogenate, inoculate in agar culture, 25℃, cultivate more than 96h,observe the growth condition of Aspergillus fumigatus. Detect Aspergillus fumigatus through Smear. Dyeing Organ tissue with HE: lung, hepar,lien, kidney were dyed with HE to detected the shape of Aspergillus fumigatus. Immunoenzyme histochemical stain: Immunoenzyme histochemical stain lung, hepar,lien, kidney use HRP-labeled McAb. Double antibody sandwich ELISA detection: Use anti-GM—McAb of Aspergillus fumigatus 7E11A1 and 9D47A1 as invest antibody and enzyme labeled antibody respectively to detect antigen of Aspergillus fumigatus in blood serum.Results: Organ tissue culture showed that experiment group can be seen the green colony, film preparation dyeing with medan can be seen spore and hypha, the hypha are institia like and have partitioning. The control group haven't colony. Organ tissue HE dyeing: the spore and hypha in experiment group were showed that the hypha are institia with partitioning. The control group can't see the spore and hypha. Immunoenzyme histochemical showed: Spore and hypha can be seen scattered or stacked leonine and lampros. The hypha are institia like and have partitioning. The control group can't be seen the spore and hypha. Double antibody sandwich ELISA detection: Aspergillus fumigatus antigen can be dectected in blood samples when the spore suspl.of Aspergillus fumigatus were injected after 24 hours. The number of OD were raised gradually after injection 24, 48, 72, 96h.Section 3:clinical application of GM—McAb in detection of Aspergillus fumigatus antigen15 cases with high risk of invasive aspergillus were collected, male 8 examples and female 7 examples. Age was range from three months to eighty years old.Among them, pathology diagnosis Aspergillus infection was 2 examples, highly suspect Aspergillus infection was 3 examples, questionable Aspergillus infection was 10 examples. Using GM—McAb of Aspergillus fumigatus 7E11A1,9D47A1 as the invest antigen and enzyme label antibody respectivly detected the Aspergillus fumigatus antigen in blood serum. The clinic data were collected including the basic condition of patient,diagnosis,pathogenetic condition evol, various kinds examination especially microbiological examination, pathology and screenage examination,treatment turnover and so on.Results: The method of double antibody sandwich ELISA was used to detect GM-antigen of patiens. 15 serum specimen of high risk Aspergillosis were detected. Among them 5 specimen were positive and the results is coincidence with the pathology diagnosis and highly suspect Aspergillus infection. One case of highly suspect Aspergillosis was collected two blood specimen preparation of acute stage and convalescence stage, the results show that serum detection strongly positive in crest-time by ELISA and convalescence stage is negative. aThree doubtful cases which have basic disease and sputum culture positive GM antigen is negative by ELISA.Above-mentioned study shows: We extracted GM antigen from the culture components of Aspergillus fumigatus, then immuned BALB/c mice. Using the hybridoma technique prepared GM—McAb of Aspergillus fumigatus. Get high specificity and affinity, aim directly at different antigen determinant hybridoma cell by screening; composed matched-pairs McAb, developed a rapid, high sensitivity and specificity double antibody sandwich ELISA method to detect GM-antigen. Established a rabbit animal model of IA infection and detected GM-antigen.The results showed that the serum detection was positive by double antibody sandwich ELISA. The results of clinical cases detection showed that double antibody sandwich ELISA detect GM-antigen is coincidence with the pathology final diagnosis and highly suspect Aspergillus infection case. So it is one of method to diagnose Aspergillus fumigatus GM antigen by detecting serum used GM—McAb of Aspergillus fumigatus.The kinesis monitoring may be one of diagnosis method of evaluating severity degree of IA and curative effect of antifungal agent. GM McAb-HRP can be used as immunoenzyme histochemical stain in Aspergillus infection cases. It is a rapid, direct-viewing, results facility judgement, and develop a new and specific diagnosis method of Aspergillus fumigatus histology examination.