The Diagnostic Value Evaluation Of Fba1 And Hsp90 On Candida Albicans Infections Through Serum Detection

Candidiasis is one of the severe complications in the immunocompromised patients,such as people suffered with cancer,diabetes,AIDS and systemic lupus erythematosus(sle),deep and systemic candida infection has been the major reason to cause the death of patients.In candida infection,Candida albicans(C.albicans)is the most common fungal pathogen,which account for 45.6%,it is a commensal and opportunistic fungus,which normally exists in the oral cavity,upper respiratory tract,intestinal tract and vagina mucosa.It could interact with other fungi on the skin and mucosal surfaces and does not cause disease in normal condition.However,when immune system is broken,C.albicans could proliferate and invade the host in the form of mycelium and cause shallow or deep infections.Because no clinical symptoms could be found in early infection stage and less early diagnostic markers and detection methods have been reported,patients infected by these kinds of fungi could not be treated timely,which causes the increased mortality.Therefore,there is a pressing need to develop more effectively detection reagents for early diagnosis.The diagnostic methods commonly used in the laboratory include blood culture,pathological examination,and direct microscopy and so on.The blood culture method is currently considered to be the gold standard for clinical diagnosis,however,the sensitivity of this method is low and it is time-consuming,the optimal treatment time is usually delayed.The pathological examination of the tissue is a traumatic examination,and patients with severe diseases could hardly bear it.Serological test is an emerging strategy with several advantages,e.g.,safety,low cost,less samples,high efficiency.Thus,it is becoming the most popular method in laboratory conditions and has the potential to be used in clinic diagnosis after finding ideal antigen markers.In this study,two kinds of diagnostic markers,fructose diphosphatase(Fba1)and heat shock protein 90(Hsp90)were selected.Fba1 is present in the yeast cell wall and cytoplasm.When C.albican is in hypha growth phase,the expression of Fba1 increases obviously,indicating that Fba1 is associated with invasion and growth of C.albicans,and could be regarded as a specific hypha phase protein.Hsp90 mainly distributes in the cytoplasm,when systemic C.albican infection occurs,Hsp90 is capable to increase the drug resistance of C.albican,regulate protein synthesis and the transition of C.albican phases and induce cellular immune response.In addition to the previous studies in our laboratory,new diagnostic reagents was designed and prepared using phage display system,and the dual-display system was employed in this study.Using these reagents as antigens,a serological diagnostic method was established to detect the serum of cancer patients.The results are summarized as follows:1.Preparation of the detection reagents: four kinds of reagents were prepared in this study,(1)recombinant Fba1 protein,it was prepared and purified through prokaryotic expression system and Ni-NTA,respectively;(2)‘F-phage’,it was constructed by displaying Fba1 epitope on fd phage pVIII protein;3)‘phage-H’,it was constructed by displaying Hsp90 epitope on the fd phage pIII protein;(4)‘F-phage-H’,both epitopes of Fba1 and Hsp90 were simultaneously displayed on pIII and pVIII protein,respectively.2.Antigenicity examination of the detection reagents: In order to confirm the antigenicity of the detection reagents,rFba1 and the three phages mentioned above were separated by SDS-PAGE,and then recognized with polyclonal antibodies against Fba1 and Hsp90 proteins,western blot analysis showed that all the four detection reagents could react with the relative antibodies,indicating that they could be employed in the further serum detection as antigens.3.Serum detection of the cancer patients: Indirect ELISA method was carried out to detect the serum collect from healthy and patients suffered with cancer disease.After several cycles of repeated detections,the results demonstrated the highest sensitivity was found using the dual displayed phage,F-phage-H,as coated antigen,followed by rFba1 protein and the two single displayed phages,whereas no differences of specificity among the four reagents were observed Therefore,phage displayed epitopes from two different proteins on pIII and pVIII simultaneously could improve the sensitivity of detection.In conclusion,four reagents were prepared and subjected to serum detections,and the dual displayed phages was proved to have higher sensitivity,indicating that the combination of Fba1 and Hsp90 could improve the diagnostic sensitivity of C.albican infections,and the newly prepared F-phage-H has potential to be developed as a diagnostic reagent.