The Construction And Evaluation Of Immunoassays By Targeting Candida Albicans Specific Csa2and Cryptococcus Neoformans Specific Cpl1

With the increasing of immunocompromised patients, pathogenic funga infection become more and more common. Candida albicans is the most frequent pathogen in all mycosis. Cryptococcus neoformans infection is also increasing because of the HIV infection patients occurrence and increasing. The early diagnosis of Candida albicans and Cryptococcus neoformans infection is very important for the early disease treatment and the survival rate of patients to raise.The relationship between Candida albicans and human is complicated. In usually, Candida albicans is mainly existed in the surface of human buff and mucous membrane and have no pathopoiesis that is called colonization. In some conditions, for example, host immune system defence decreasing, mucous membrane damage, alteration of intestinal flora, Candida albicans can cause surface and deep mucous membrane infection that is called candidiasis. Whe the host immune system is serious damaged, for instance, HIV infection, cancer chemotherapy, organ transplantation, Candida albicans can infect deep tissue and organs, even have chance to access blood stream caused severe, life-threatening candidemia and systemic infection that is called invasive candidiasis. Accurate discrimination of colonization and infection of Candida albicans can prevent antifungal agent abuse and help clinicians to take antifungal treatment in time.Traditional methods have some limitations in candidiasis diagnosis. Firstly, the clinical manifestation of candidiasis is nonspecific, clinicians can not diagnose candidiasis by the patient infection symptom. Secondly, open samples for example, sputum, urine and secretion direct microscopic examination has low detection rates, and the culture of these samples can lead to false positive because of contamination and colonization. Positive result has less importance to diagnose candidias. Pathological examination of deep tissue has high risk of causing patients severe complication. Thirdly, blood culture is considered the "gold standard" to diagnose invasive candidiasis, but50%-30%of invasive candidiasis patients confirmed by pathological examination has negative result by blood culture, and strain identification and susceptibility test require a few days and is hard to early diagnose candidiasis.The early diagnosis of candidiasis is mainly depend on the surface antigen and antibody of candida albicans. At present, the main commercial kits of diagnosis candidiasis includes β-D-glucan (BDG), mannan (Mn) and anti-mannan (A-Mn). BDG is fungal cell wall reconstituent except for Cryptococcus and Zygomycetes, and is a panfungal marker to diagnosis invasive mycosis. BDG has no species-specificity to diagnosis candidiasis and usually combine with galactomannan (GM), when BDG is positive and GM is negative, it hints candida infection, on the contrary, when BDG and GM are both positive, it hints Aspergillus infection. BDG is false positive when patient is on hemodialysis, severe mucositis, systemic bacterial infection and antibiotic treatment. Mn is one of the main components of candida cell wall and also is main discharging antigen after candida accessing human systemic circulatory system. Mn and A-Mn can not discriminate the colonization and infection of candida. In studies reported on Mn and A-Mn diagnostic value, the difference of sensitivity and specificity are significant because of the chosed patients difference. Most studies of Mn and A-Mn is on invasive candidiasis, but these studies are mostly case control trials and lack of randomized controlled trials. So, it is hard to evaluate accurately the diagnosis effection of invasive candidiasis.With the development of molecular biology techniques, gene detection is used to diagnosis candidiasis. But there are some problems needed to be solved, for examples, the discriminate colonization and infection, the false positive caused by sample exogenous contamination, the false negative caused by cell wall interfere with cell lysates and DNA releasing. In addition, the problems are also including the standardization of techniques and operation, experiment environment disposition, personnel requirement. Because of those problem, it is hard to use gene detection as routine method.Therefore, finding new markers to discriminate colonization and infection is the key and difficult problem to diagnosis candidiasis. Csa2is one member of CFEM (Common in several Fungal Extracellular Membrane proteins) family. There are8 cysteines of CFEM domain in its molecular structure. It is a small molecular weight and secreting protein without signal peptide structure, and abundante in hyphal culture supernant, Unapparent in yeast culture supernant. Those suggest that Csa2is abundantly expressed on the surface of hypha and released into blood circulation. The function of Csa2is unclear at present. The most important virulence factor is the transformation from yeast to hypha. The losing of the transformation ability in Candida albicans cause virulence disappear. Candida albicans hypha can adhere and invade host tissue more easily, resulting in deep infection. Transformation from yeast to hypha can make Candida albicans escape phagocytosis. Gene related with hypha formation can participate in the formation of biofilm which is related on the antifungal drug tolerance. Candida albicans hypha is the mainly pathogenic morphous during infection. In this study, we constructed antigen captured and antibody captured ELISAs by Csa2as marker to hope increase the sensitivity and specificity of diagnosis candidiasis, to avoid colonization interference and overcome the shortage of BDG, Mn and A-Mn in diagnosis candidiasis.Cryptococcosis mostly happened in immunocompromised patients, especially in HIV infecting patients. Cryptococcus neoformans most often affects to the lung and brain, occasionally cause disseminated infection in other organs. The fatality rate of Cryptococcus neoformans on HIV infecting patients is about10%-40%. It can save the patients’lives for early diagnosis of Cryptococcosis.At present, the diagnosis of Cryptococcosis is mainly Indian ink capsule stain or mucicarmine stained encapsulated yeasts in multinucleated giant cells of granulomata of tissue sections searched by microscope; the gold standard for a definitive diagnosis is a positive isolation of Cryptococcus neoformans by fungal culture from the cerebrospinal fluid, blood, tissue biopsies, respiratory secretions or other clinical specimens of patients. But the two methods both have their limitations. The first method has a low detection rate, and the gold standard method often takes a few days.Therefore the diagnosis is often made by a positive antigen detection from the cerebrospinal fluid or sera of these patients. Currently both the reference latex agglutination immunoassay and an antigen capture sandwich enzyme linked immunosorbent assay are commercially available. Though the classical latex agglutination assay was produced with antibodies harvested from rabbits immunized by whole yeast cells, nowadays both types of assays use mono-specific polyclonal antibody directed against the glucuronoxylomannan (GXM) as the target antigen because this antigen was abundant and present in all major serotypes of Cryptococcus neoformans of A, B, C and D. However these serotypes differ in the structure of GXM by the levels of O-acetylation on the mannan backbone and the extent of xylose substitution. Given the immunodominance of the O-acetyl dependent epitopes on GXM, it is not surprising that there are reports of reduced sensitivity of these immunoassays for Cryptococcus neoformans serotype C because this serotype has least O-acetylation and greatest xylose substitution. Though such deficiency may be overcome by a batch of monoclonal antibodies targeting all4serotypes, a conserved protein antigen target which is abundantly secreted by the fungus would be a better choice for Cryptococcosis diagnosis.In2008, Liu OW et al firstly discovered CPL1gene by whole gene sequencing analysis of Cryptococcus neoformans and homologous gene replacement. They also confirmed that CPL1has relationship with capsule formation. The thick capsule is one of the most important phenotype characters and it also an important virulence factor of Cryptococcus neoformans. The virulence of mutant strain with thin or disappearing capsule is significantly weekened or disappeared. Knocking out CPL1gene, the capsule became thin and the virulence disappear. Because there is a signal peptide sequence in CPL1gene, Liu OW et al presumed Cpll is a secretory protein. In our study, we confirm Cpll is a specific secretory protein and construct Cpll antigen and antibody captured ELISA.There are two section in our study, their content mainly below:Part Ⅰ the construction of Csa2antigen and antibody captured ELISAs and their application in Candiduria patients.1. The construction and evaluation of candida albicans specific Csa2antigen captured ELISA.Csa2is a secretory protein in candida albicans hypha by mass chromatographic analysis. CSA2gene is orthologues with the CFEM domain of Candida tropicals, coccidioides immitis and Cryptococcus neoformans. Csa2is family member of CFEM. We constructed recombinate expression vector of pPIC9K-CSA2, CSA2gene were expressed by P. pastoris strain GS115to obtain recombinant Csa2(rCsa2). Recombinant Csa2were purified by Ni-NTA affinity chromatography and its molecular weight is about13.3kDa. Purified rCsa2were immunized in new Zealand white rabbits and guinea pig to prepare polyclonal antibodies. The rabbit antibody sera were purified, and the concentration of rabbit polyclonal IgG is20.5mg/mL. We choosed RPMI1640to culture Candida albicans hypha in4kinds of different medias. Candida albicans hypha culture supernant were obtained in18h,40h,64h,88h,112h,148h, and160h. The rCsa2concentration is the highest in112h supernant and became dynamic balance during the following culturing time. By indirect fluorescence staining, we found that Csa2is mainly existed in the surface of Candida albicans hypha, and there are no Csa2expression on the surface of Candida albicans yeast and candida glabrata. Replacing rabbit antibody serum by PBS, we also found there was no non-specific fluorescence combination on the surface of Candida albicans hypha.The Csa2antigen captured ELISA is constructed by20ug/mL purified rabbit IgG antibody as captured antibody,1:500dilution guinea pig antibody serum as detecting antibody,1:1500dilution HRP labeled goat anti-guinea pig IgG as second detecting antibody. A series of known rCsa2concentrations and serially multiple proportion diluted culture supernant of the fungal pathogens including Candida albicans hypha, Candida tropicalis, Candida glabrata, Candida krusei, Canadida parapsilosis; Aspergillus fumigates, Aspergillus flavus, Aspergillus terreus, Aspergillus niger, Aspergillus nidulans, Penicillium marneffi and Cryptococcus neoformans. The detectable least concentration of rCsa2is240pg/mL and the highest dilution times of Candida albicans hypha is64. There were no cross reaction between Candida albicans hypha culture supernant and other fungal culture supernant.2. The construction and evaluation of candida albicans specific Csa2antibody captured ELISA.We prepared HRP labeled rCsa2(rCsa2-HRP), and constructed Csa2antibody captured ELISA by O.lug/mL rCsa2as captured antigen,1:1000dilution of rCsa2-HRP as detecting antigen. A serious of multiple proportion diluted rabbit antibody sera of rCsa2of candida albicans, rCpll of Cryptococcus neoformans, rAfmplp and rAfmp4p of Aspergillus fumigates, and rMplp of Penicillium marneffi and normal rabbit serum were detected by the constructed ELISA.16000diluted rabbit Csa2antibody serum can be detectable by the constructed ELISA. There were no cross reaction between rabbit Csa2antibody serum and other recombinant fungal protein immunizied rabbit sera.3. The application and evaluation of the two constructed ELISAs in Candiduria patients sera.The cutoff value(cutoff=mean±3×SD) of Csa2antigen captured ELISA was0.416by detecting324sera of healthy people.5sera were detected Csa2antigen positive in23sera of caniduria patients.2sera were detected Csa2antigen positive in324healthy people and1serum was detected Csa2antigen positive in patient with Candida tropicalis. The sera of7other candiduria are all Csa2antigen negative. Using SPSS13.0analysis, the sensitivity, specificity, positive predictive value, negative predictive value of Csa2antigen captured ELISA is21.74%(5/23),99.09%(328/331),71.43%(5/7), and94.80%(328/346).respectively.The cutoff value(cutoff=mean±3×SD) of Csa2antibody captured ELISA was0.072by detecting324sera of healthy people.15sera were detected Csa2antibody positive in23sera of caniduria patients.1serum was detected Csa2antibody positive in4sera of candiduria caused by Candida tropicalis. The sera of6other candiduria and324healthy people are all Csa2antibody negative. Using SPSS13.0analysis, the sensitivity, specificity, positive predictive value, negative predictive value of Csa2antibody captured ELISA is65.22%(15/23),99.70%(330/331),97.75%(15/16), and97.63%(330/338) respectively.16sera were detected Csa2antigen or antibody positive in23sera of caniduria patients.2sera were detected Csa2antibody positive in4sera of candiduria caused by Candida tropicalis and2sera were detected Csa2antigen positive in324healthy people. The sera of6other candiduria and322healthy people are all Csa2antigen and antibody negative. Using SPSS13.0analysis, the sensitivity, specificity, positive predictive value, negative predictive value of Csa2antibody captured ELISA is69.56%(16/23),98.79%(327/331),80.00%(16/20),97.90%(327/334) respectively.We constructed Csa2antibody inderect ELISA by1.0ug/mL rCsa2as captured antigen,1:5000dilution of HRP labeled anti-human IgG antibody as detecting antibody. The cutoff value(cutoff=mean±3×SD) of Csa2antibody inderect ELISA was0.574by detecting120sera of healthy people.15sera were detected Csa2antibody positive in22sera of caniduria patients.1sera were detected Csa2antibody positive in4sera of candiduria caused by Candida tropicalis and Candida parapsilosis,6sera were detected Csa2antibody positive in120healthy people. The sera of4other candiduria and114healthy people are all Csa2antibody negative. Using SPSS13.0analysis, the sensitivity, specificity, positive predictive value, negative predictive value of Csa2antibody captured ELISA is68.18%(15/22),93.65%(118/126),65.21%(15/22) and94.40%(118/125).respectively.Part Ⅱ the construction and evaluation of Cryptococcus neoformans Cpll and its antibody immunoassay1. The expression and immunized sera preparation of Cpll We search orthologues protein of Cpll among Candida, Aspergillus, Penicillium and Trichosporon, only Cryptococcus neoformans var grubi and Cryptococcus gattii have orthologues protein of Cpll. We constructed recombinate expression vector of pPIC9K-CPL1, CPL1gene were expressed by P. pastor is strain GS115to obtain recombinant Cpll(rCpll). Recombinant Cpll were purified by Ni-NTA affinity chromatography and its molecular weight is about18.6kDa. Purified rCpll were immunized in new Zealand white rabbits and guinea pig to prepare polyclonal antibodies. The rabbit antibody sera were purified, and the concentration of rabbit polyclonal IgG is20.25mg/mL.2. The construction and evaluation of Cryptococcus neoformans specific Cpll antigen captured ELISAWe choosed YPM-R to culture capsule Cryptococcus neoformans in4kinds of different medias. the culture supernant were obtained in18h,40h,64h,88h,112h,148h, and160h. The rCpll concentration is the highest in148h supernant and became dynamic balance during the following culturing time. By indirect fluorescence staining, we found that Cpll protein is specifically located on the partial surfaces of Cryptococcus neoformans, especially the part of emerging bud, and there are no Cpll expression on the surface of Cryptococcus gattii and Trichosporon asahii.The Cpll antigen captured ELISA is constructed by20ug/mL purified rabbit IgG antibody as captured antibody,1:500dilution guinea pig antibody serum as detecting antibody,1:1000dilution HRP labeled goat anti-guinea pig IgG as second detecting antibody. A series of known concentrations rCpll and serially multiple proportion diluted culture supernant of the fungal pathogens including Cryptococcus neoformans, Cryptococcus neoformans var grubii, Cryptococcus gattii, Trichosporon asahii, Candida albicans, Candida glabrata, Candida krusei, Canadida parapsilosis; Aspergillus fumigates, Aspergillus flavus, Aspergillus terreus, Aspergillus niger, Aspergillus nidulans and Penicillium marneffi. The detectable least concentration of rCsa2is240pg/mL and the highest dilution times is64in Cryptococcus neoformans culture supernant,32in Cryptococcus neoformans var grubi culture supernant. There were no cross reaction between Cryptococcus neoformans and other fungal culture supernant.3. The construction and evaluation of Cryptococcus neoformans specific Cpll antibody captured ELISA.We prepared HRP labeled rCpll(rCpll-HRP), and constructed Cpll antibody captured ELISA by0.1ug/mL rCpl1as captured antigen,1:1000dilution of rCpl1-HRP as detecting antigen. A serious of multiple proportion diluted rabbit antibody sera of rCpll of Cryptococcus neoformans, rCsa2of candida albicans, rAfmplp and rAfmp4p of Aspergillus fumigates, and rMplp of Penicillium marneffi and normal rabbit serum were detected by the constructed ELISA.8000diluted rabbit Cpl1antibody serum can be detectable by the constructed ELISA. There were no cross reaction between rabbit Cpll antibody serum and other recombinant fungal protein immunizied rabbit sera.ConclusionsBy our study, the conclusions include as follow:1. We successfully expressed rCsa2in P. pastor is strain GS115. The purified rCsa2were immunized rabbit and guinea pig, and high titer antibody sera were obtained. Csa2antigen captured ELISA were constructed with purified rabbit antibody as capture antibody and guinea pig antibody as detecting antibody. This method had good detecting sensitivity and specificity.2. We successfully prepared rCsa2-HRP. Csa2double antigens sandwich ELISA were constructed with rCsa2as capture antigen and rCsa2-HRP as detecting antigen. This method had good detecting sensitivity and specificity. The Csa2inderect ELISA was constructed successfully based on the Csa2double antigens sandwich ELISA.3. the cutoff value were calculated by detecting324sera or120sera of healthy people. The three methods had high specificity and a little low sensitivity of detecting candiduria. Because of the interference of contaminate and colonization, the results should be systemicly analysed. It maybe a good hint of candida albicans infection development. When Csa2antigen positive and antibody negative, it means host is in the early infection; when Csa2antigen negative and antibody positive, it means host had been infected by candida albicans; when they are both positive, it means host are keeping in the infection. When they are both negative, it means host maybe had candida albicans colonization.4. We confirmed that Cpl1is a kind of specific secretory protein and successfully expressed rCpl1in P. pastoris strain GS115. The purified rCpl1were immunized rabbit and guinea pig, and high titer antibody sera were obtained rCpl1antigen captured ELISA were constructed with purified rabbit antibody as capture antibody and guinea pig antibody as detecting antibody. This method had good detecting sensitivity and specificity.5. We successfully prepared rCpll-HRP. Cpll antibody captured ELISA were constructed with rCpll as capture antigen and rCp11-HRP as detecting antigen. This method had good detecting sensitivity and specificity.Because we. can not obtain enough sera of invasive candidiasis and cryptococcosis, the effection of the constructed methods to diagnose the two diseases need to further confirmed.