| Background and objectivesThe continuous improvement of medical technology allows many patients to survive with their existing diseases. At the same time, invasive fungal infections (IFIs) have also increased significantly. Of the current IFIs, Aspergillus and non-albicans Candida species infections have tended to increase. However, infections by Candida albicans(C. albicans) have still been dominant. Globally, C. albicans is a major pathogen, causing 50–70% of all invasive fungal infection cases.Invasive infection by C. albicans mainly involves lung, blood, cerebral tissues, kidney, and intestines and has no special symptoms but it progresses rapidly and has a poor prognosis. Thus, early and quick diagnosis is critical for effective treatment of the infection. Unfortunately, the various current methods cannot allow us to diagnose the invasive infection by C.albicans earlier, more quickly, and more accurately. Current diagnosis relies on culture-based methods, which is lack of sensitivity and delays the diagnosis. Histopathological examination is also limited because it is invasive and may induce bleeding and other complications. Radiological examinations, such as CT, PET/CT, and chest radiography, exhibit variable manifestations and have no obvious specificity. Thus, they do not clearly distinguish bacterial infections, tuberculosis infections, fungal infections and inflammatory pseudo-tumors. Serological tests consist of the detection of fungal cell wall components, such as mannan, galactomannan andβ-(1,3)-D-glucan, which are helpful for diagnosing invasive infection by C. albicans, but the risks of cross-contamination and the need for reagent standardization are still present. By comparison, PCR-based methods are more flexible, but this process is insufficiently standardized, and its sensitivity is inconsistent across different reports. In addition, this method does not help judge infection or colonization, and the test can be easily prone to false-positive and false-negative results. Some have tried to find a diagnostic by testing C. albicans–specific metabolites, but their clinical applications are restricted because the metabolites produced by different strains vary considerably. The metabolites from the body and other microorganisms generate interference, and this method requires comparatively sophisticated equipment .In the field of radiopharmaceutics, some new types of tracers have been developed for diagnosing C. albicans infections, such as radiolabeled antimicrobial peptides, 99mTc-labeled poly liposomes, radiolabeled fluconazole , and 99mTc-labeled chitin-binding protein (CBP21) . The first two methods lack the ability to distinguish between C. albicans and bacterial infections. Although the latter two methods can distinguish C. albicans from bacterial infections, like the other methods of diagnosis, these two methods cannot tell whether the detected C. albicans is an opportunistic pathogen or it naturally exists in most normal people. Therefore, even if C. albicans were detected, one could not determine whether they are an infection or a colony. In summary, C.albicans infection can not be accurately detected up to now.C. albicans is a diphasic fungus with two forms, spores and hyphae. Under certain conditions, C. albicans can transform into hyphae from ellipsoidal single-cell blastospores, which are also termed yeast. In the early stages of the transformation, a germ tube is formed, which enhances the ability of C. albicans to colonize and invade the organism and evade host resistance. It is generally agreed that the formation of the germ tube is the marker of C. albicans transformation from a colonizing into a pathogenic fungus.Previously, our research team has successfully prepared a specific monoclonal antibody for the C. albicans germ tube (MAb03.2C1-C2), and preliminary results have confirmed that this antibody binds to specific target antigens in the tracheid wall of the outer membrane of the C. albicans germ tube. However, it does not recognize the spore-phase antigen and does not cross-react with other Candida and Aspergillus. Thus, it is assumed that the pecificity of MAb03.2C1-C2 might plays an important role in detecting and fighting C. albicans invasion. As we know that the whole monoclonal antibody has a high molecular weight, it has difficulty entering cells, and the resulting images would have poor quality. To this end, Fab'fragment, which has a low molecular weight and retains the specificity of the monovalent antigen, is preferred. In this study, we labeled Fab'fragment with 99mTc,which still recognizes C. albicans germ tube specifically. We studied the biodistribution and performed radionuclide imaging in a rat model of invasive C. albicans infection. Our results suggest that this would be a new specific experimental radiopharmaceutical for the diagnosis of invasive C. albicans infection.Methods:1.Preparation and Characterization of a Monoclonal Antibody Against the C. albicans Germ Tube (MAb03.2C1-C2) and the Fab'Fragment:The MAb was purified by caprylic acid–ammonium sulfate precipitation (CA-AS) and DEAE-52 chromatography as described before (Raweerith and Ratanabanangkoon 2003). The Fab'fragment was then obtained by digestion with pepsin and 2-mercaptoethanol (2-ME). The titers and immunological activities of the antibody fragment were then measured by indirect immunofluorescence. The purity of the antibody fragment was determined by SDS-PAGE.2. Preparation of 99Tcm- MAb03.2C1-C2 Fab':MAb03.2C1-C2 Fab'was modified with 2 - IT and labelled with 99mTc - GH. Labeling yield , radiochemical purity were determined by ascending paper chromatography and high-pressure liquid chromatography (HPLC).3.In vitro stability identification of 99Tcm- MAb03.2C1-C2 Fab': Evaluate the stability of 99Tcm- MAb03.2C1-C2 Fab'in vitro through a series of experiment such as stability at room temperature.4. Tracer kinetics analysis in vivo of 99Tcm- MAb03.2C1-C2 Fab'in healthy rabbits: Each of six rabbits was injected 37 MBq 99Tcm- MAb03.2C1-C2 Fab', then draw blood from ear vein at the following time: 1.5, 3, 5, 10, 30, 60, 120, 240, 480 min. These blood sample was weighed and measured byγ-immuno-counter, and then we calculated the radioactive concentration of every sample after correction by measurement efficiency ofγ-immuno-counter. At last, we evaluated the best compartmental model and parameter by virtue of pharmacokinetics analytical software.5. Body distribution and imaging study of 99Tcm- MAb03.2C1-C2 Fab'in vivo in healthy animals: 35 adult Kunming mice were involved in the study of tissue distribution, which were divided to 5 groups by random digits table. 7.4 MBq 99Tcm- MAb03.2C1-C2 Fab'was administrated via caudal vein of the mice. At 10, 30, 60, 120, 240 min after injection, mice were sacrificed and the blood, heart, lungs, liver, kidneys, intestines, muscles, bone and brain were excised, weighed and counted for radioactivity by a gamma counter. And then we calculated the percentage of the injected radioactive dose per gram of tissue wet weigh (%ID/g) after correction by reference source. In the study of imaging in healthy animals, we injected 37 MBq 99Tcm- MAb03.2C1-C2 Fab'into ear vein of rabbits. The dynamic imaging was observed by the scintigraphy with SPECT for 60 minutes and then static imaging at 90, 120, 180 and 240 min.6. The suitability of the 99mTc-labelled Fab'(99mTc-MAb03.2C1-C2 Fab') for imaging of C. albicans infection in vitro: suspensions of C. albicans, Aspergillus fumigatus (A. fumigatus), Staphylococcus aureus (S. aureus), Escherichia coli (E. coli)and Human lung carcinoma cell line A549 were incubated with 99mTc-MAb03.2C1-C2 Fab'and radioactivity retention was measured in a gamma counter.7. SPECT imaging and competitive imaging by MAb03.2C1-C2 Fab': In the study of imaging, 37MBq 99Tcm- MAb03.2C1-C2 Fab'was injected into the caudal vein of the rats with infected with fugus and bacteria in thigh muscles and 18.5MBq 99Tcm- MAb03.2C1-C2 Fab'was injected into the caudal vein of the tumor-bearing nude. The dynamic distribution was observed by the scintigraphy with SPECT at 30, 60, 90, 120, 180 and 240 min. In the study of competitive imaging, 0.5 mg MAb03.2C1-C2 Fab'was administrated into the caudal vein of the rats with intramuscular infections of C. albicans in the right thigh muscle and heat-killed C.albicans in the left thigh muscle, after 1h, 37 MBq 99Tcm- MAb03.2C1-C2 Fab'was administrated, then competitive imaging inhibited by MAb03.2C1-C2 Fab'was observed.Results:1. Preparation of MAb03.2C1-C2 Fab': Mab03.2C1-C2 Fab'was successfully prepared. Its purity is as high as 91.6% by gel scanning imaging with Gel Imager, and its molecular weight is about 43 000 by electrophoresis determination and compared with the standard protein. Results of indirect immunofluorescence show that, Fab's still keeps the feature which can bine with surface antigen of C. albicans germ tube specifically,with a titre of 1:12000.2.Preparation of 99Tcm- MAb03.2C1-C2 Fab': 99Tcm- MAb03.2C1-C2 Fab'could be successfully prepared following the steps below: 30μl 2-IT-Fab'was added to the 74MBq(0.2ml)99mTc-GH solution. The reaction was performed under nitrogen atmosphere and 25°C for 30 min.The RCP of the labeled peptide was (84.92±2.46)%, After purification, the radiochemical purity was >95%. and its specific activity was (3.54±0.03)TBq/mmol. 3.Identification of stability of 99Tcm- MAb03.2C1-C2 Fab'in vitro: after 24 h, the RCP of 99Tcm- MAb03.2C1-C2 Fab'was (91.66士1.06)%; Cysteine replacement rate was less than 1% after adding cysteine of various concentration; trichloroacetic acid precipitation test revealed the labeled peptide couldn't combine with serum protein.4.Tracer kinetics analysis in vivo of 99Tcm- MAb03.2C1-C2 Fab'in healthy rabbits: tracer kinetics process in vivo of 99Tcm- MAb03.2C1-C2 Fab'in healthy rabbits followed two compartment model weighed by 1 and the main parameters t1/2α, , t1/2β, and CL were 1.21±0.77 min, 84.45±2.65min,and 1.00±0.00ml/min respectively.5.Tissue distribution and imaging study of 99Tcm- MAb03.2C1-C2 Fab'in vivo in healthy animals: 99Tcm- MAb03.2C1-C2 Fab'was cleared quickly from the mice, it mainly distributed in the kidneys,lung and liver.6. In vitro binding of 99mTc- MAb03.2C1-C2 Fab'to C. albicans, A. fumigatus, E. coil,S. aureus and A549 cells revealed that the 99mTc- MAb03.2C1-C2 Fab'preferentially binds to sporus with C. albicans Germ tubes (4.23±0.17 x 102 Bq per 1 x 107 for sporus with C. albicans Germ tubes). These values were significantly higher than those for A. fumigatus (0.38±0.01x 102 Bq per 1 x 107 cells for A. fumigatus,) and sporus with C. albicans (0.27±0.02 x 102 Bq per 1 x 107 cells for sporus with C. albicans) ( P < 0.05). The lowest values were found for 99mTc- MAb03.2C1-C2 Fab'binding to S. aureus(0.09±0.00x 102 Bq per 1 x 107 cells for S. aureus), heat-killed C. albicans(0.07±0.00 x 102 Bq per 1 x 107 cells for sporus with heat-killed C. albicans) , E. coli (0.06±0.00x 102 Bq per 1 x 107 cells for E. coli),and A549 cells(0.03±0.00 x 102 Bq per 1 x 107 cells for A549 cells).7.Tissue distribution and imaging study of 99Tcm- MAb03.2C1-C2 Fab'in vivo in rats: In the study of tissue distribution, There is large 99mTc-MAb03.2C1-C2-Fab'mass in the position where there is C.albicans infection, %ID/g is 11.71±1.64 at 120 min, which is obviously higher than the contralateral muscle tissues(0.62±0.18) or other organs(P<0.05). The %ID/g of99mTc-MAb03.2C1-C2-Fab'ratio in C.albicans infected muscle/contralateral muscle is about 6. Imaging study showed a significant radioactivity accumulation was viewed at the tissue with infections of C. albicans 0.5h after injection. The clear image could be observed one hour later, whereas the radioactivity accumulation could not be seen in the control thigh muscle injected A. fumigatus or E. coli or S. aureus or heat-killed C. albicans induced sterile in?ammation) and tumor. Furthermore, injecting of excess unlabelled Fab into the rats could notably reduce the radioactivity accumulation in the C. albicans infected position.Conclusions:1. MAb03.2C1-C2 Fab'was prepared successfully, it can specifically bound to the surface antigen of the C. albicans germ tube, not to the blastospores of C. albicans even at the titer of 1:12,000 .2.99Tcm- MAb03.2C1-C2 Fab'was successfully prepared with high RCP(>95%) and perfect stability in vitro and in vivo.3.99Tcm- MAb03.2C1-C2 Fab'had satisfactory characteristics in tracer kinetics and tissue distribution and was cleared quickly from blood and excreted quickly from kidneys and liver.4.99Tcm- MAb03.2C1-C2 Fab'could specifically accumulate in C. albicans infections tissues, but not in site of tumor or tissue infected with A. fumigatus or bacteria or in sterile inflammations. This study provides a new, specific radiopharmaceuticals for diagnosis of C. albicans infections. |
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