Establishment Of Aspergillus Antigen Capture Method And Its Preliminary Application In Diagnosis Of Invasive Aspergillosis

Invasive aspergillosis(IA)is a systemic disseminated fungal infectious diseases,Aspergillus fumigatus(A.fumigatus)is the main pathogen,accounting for more than 90%.In recent years,due to the use of immunosuppressants,hormones and other drugs,the number of patients with low immunity has increased,leading to an increase in the incidence rate and mortality of invasive aspergillosis year by year.However,the onset of IA is hidden,and the clinical manifestation does not affect the specificity,making early diagnosis of IA is extremely difficult.At present,the gold standard for IA diagnosis is pathological diagnosis of sterile tissue,but the disadvantage is that sampling is invasive.Molecular diagnostic methods are sensitive,but due to difficulties in extracting fungal DNA and standardizing reagents from different manufacturers,they have not yet been approved for clinical use.Fungal cell walls are rich in polysaccharides and glycoproteins,which have been used for the diagnosis of various fungal infections.The clinical diagnosis IA commercialization kit mainly targets Afumigatus galactomannan(GM)or glycoprotein antigens and their antibodies.However,its detection performance varies greatly among different patient populations,and it is still difficult to solve the current situation of a large number of clinical infected patients and difficulties in early diagnosis.In the early stage of this experiment,various attempts were made to develop diagnostic reagents for Aspergillus.On the one hand,antibodies were prepared and antigen detection systems were established for the diagnostic value of A.fumigatus specific mannoprotein(Afmp1p)by prokaryotic and eukaryotic expression.However,the recombinant Afmplp was difficult to achieve the glycosylation level of complex fungal natural glycoproteins,and therefore,an effective system for detecting clinical samples was not established.On the other hand,an attempt was made to extract antigens from A.fumigatus after 7 days of cultivation.After immunizing mice,monoclonal antibodies(MAb)targeting Aspergillus antigens were obtained,and a detection system capable of detecting A.fumigatus culture supernatant was established.Although it has good specificity,it cannot detect antigens in patient serum.Compared with the development process of commercial reagents,it is speculated that the possible reason is that the immunogen used is a natural antigen of clinically isolated Afumigatus,which has been cultured for 7 days,and there are differences in the early swelling spores and hyphal structure of Afumigatus.Therefore,our project aims to use the standard strain of A.fumigatus purchased by ATCC,extract the early lysis antigen of A.fumigatus cultured for 24 hours as the immunogen,and obtain mouse MAb targeting the early antigen.Next,we will establish an enzyme linked immunosorbent assay(ELISA)system for Aspergillus antigen capture and preliminarily evaluate its clinical application value,in order to obtain a possible early diagnostic kit.Part Ⅰ:Preparation and identification of MAb against A fumigatus.We used A.fumigatus(ATCC strain)lysate antigen cultured for 24 hours as an immunogen and immunized BALB/c mice with various regimens including subcutaneous,intraperitoneal,and intramuscular injections.Select the mouse with the highest serum antibody titer to extract spleen cells and fuse them with mouse NS-1 cells.The fused hybridoma cells were screened by ELISA and Indirect immunofluorescence(IFA),and 18 strains(F1-F18)of hybridoma cells stably secreting anti A.fumigatus antibodies were obtained.We purified the ascites produced by 17 cell strains,and ultimately obtained 15 strains of anti A.fumigatus MAb,which were identified for their immunological characteristics.In ELISA,We found that 7 strains of anti A.fumigatus MAb were IgG1.They specifically target early lysis antigens of A.fumigatus cultured for 24 hours.These antibodies do not cross react with 18 clinically common funga1 lysis antigens(includes Candida albicans,Cryptococcus neoformans,Talaromyces marneffei(TM,yeast phase),Aspergillus niger,Aspergillus flavus,Aspergillus terreus,Aspergillus nidulans,Fusarium,Mucor,Rhizomucor minimus,Candida portugalis,Candida jiemonica,Candida glabrata,Candida kerrula,Candida parapsilosis,Candida tropicalis,Sedospora apicalis,Arthrospora polypodata).The other 8 strains are IgM,which react with early(cultured for 24 hours)and late(cultured for 4 days)antigens of A.fumigatus,and have certain cross reactions with various different fungi(mainly combined with A.flavus,A.terreus,A.niger,and A.nesting).We consider them to be Aspergillus specific antibodies.Western blotting(WB)further confirmed that 7 IgGl type antibodies bind to early lysis antigens of A.fumigatus.The main binding bands are all 25kDa.And 8 IgM type antibodies can bind to both early(37100kDa)and late(50-250kDa)antigens of A.fumigatus,with a binding spectrum similar to R6 from PlateliaTM Aspergillus Ag/Bio-Rad.In IFA,thirteen of the antibodies were bound to the swollen spores of A.fumigatus and the somatic cell cell wall of the early hyphae to varying degrees.Next,we labeled the MAb with biotin and measured its potency.This suggests that we have successfully obtained MAb that can recognize different antigenic properties and different antigenic epitopes,and provide the possibility for the subsequent establishment of antibody pairing for Aspergillus antigen capture ELISAPart Ⅱ:Establishment and optimization of Aspergillus antigen capture ELISAUsing the 15 antibodies obtained from the first part of the experiment,combined with their immunological characteristics and labeled antibody titers,the MAb were paired and optimized.By detecting the supernatants of ATCC and clinical isolates of Afumigatus(referred to as HK)cultured in early and late stages,IgM MAb F1,which recognizes glycoprotein epitopes,was obtained as the capture antibody.Biotin labeled F1(biotin-F1)was used as the optimal pairing for antibody detection.Finally,the capture antibody F1 coating concentration was determined to be 10μg/ml,biotinF1 and Streptavidin-HRP working ratio is 1:1000,the sensitivity of detecting ATCC strain supernatant and HK strain supernatant in early and late culture is 1:10000～1:100000,which is higher than that of Aspergillus antigen detection system established in the early laboratory.There was no cross reaction with the culture supernatant of common clinical infections such as A.terreus,TM,Cryptococcus neoformans,and Candida albicans.However,there was a weak cross reaction with A.flavus and relatively rare clinical infections such as Specific Penicillium and A.nesting.This suggests that our newly established ELISA system specifically targets Aspergillus infection,especially A.fumigatus(accounting for 90%of pathogenic Aspergillus).Part Ⅲ:Preliminary clinical evaluation of Aspergillus secreted antigen capture ELISADue to the difficulty in obtaining clinical specimens for IA,our study aims to preliminarily evaluate the sensitivity of the detection system by simulating Aspergillus infection serum,and determine the impact of sample pretreatment on the detection effect to determine whether pretreatment of patient serum is necessary.We mixed the culture supernatant of A.fumigatus with different dilution ratios and mixed serum from normal individuals to create a simulated Aspergillus infection serum.The serum was treated without heating,incubated at 37℃ for 30 minutes after mixing,and treated with reference to R7(EDTA acidic solution)from PlateliaTM Aspergillus Ag.The test results showed that the A450 value of the serum treated with R7 was higher than that of the other two methods.The sensitivity of the simulated serum detection was 1:100～1:1000.It was found that the sensitivity of our newly established system was inferior to PlateliaTM Aspergillus Ag(1:1000).We detected 120 normal human serum(R7 treated)samples using F1/biotin-F1 to determine a cutoff of 0.462.Furthermore,we tested the serum of 74 patients with invasive aspergillosis using F1/biotin-F1(including 2 proven,51 probable,and 21 possible),and found that the test results were all negative.It is speculated that the differences may be related to serum antigen levels,sample collection and preservation,and serum pretreatment.In the future,we can improve the antibody labeling method and amplify the detection effect to improve detection sensitivity.In addition,we can also make improvements in the selection of immunogens to obtain antibodies with higher sensitivity.