ABSTRACT
PURPOSE: Small size of recombinant scFv antibody has many advantages such as rapid blood clearances and improved targeting antibodies to tumor region. On the other hand owing to small size, number of amino group is insufficient in conjugation with chelator and fluorescence labeling. This study is to introduce poly lysine tag to the C-terminal end of scFv lym-1 sequence for fluorescence chelator conjugation. MATERIALS AND METHODS: Poly lysine scFv lym-1 gene, cloned into pET-22b (+) vector, was expressed in E. coli BL21 (DE3) strain. Antibody purification was performed with Ni-NTA column and then size exclusion column chromatography. Expression and purification levels of poly lysine tagged scFv lym-1 antibody were confirmed by western blot analysis. I-124, I-125, I-131 and Tc-99m were used for radiolabeling of purified poly lysine scFv lym-1. Flow cytometry analysis of FITC conjugated poly lysine scFv lym-1 was performed for confirmation of immunoreactivity of human Burkitt`s lymphoma cells. RESULTS: Poly lysine scFv lym-1 antibody was purified through two steps and identified as molecular weight of 48 KDa. Radiolabeling yields of I-124, I-125, I-131 and Tc-99m into poly lysine scFv lym-1 were >99%, >99%, >95% and >99%, respectively. Flow cytometry analysis of poly lysine scFv and scFv lym-1 was showed similar immunoreactivity to human Burkitt`s lymphoma cells. CONCLUSION: Poly lysine tag was useful for the sufficient number of amino groups to scFv lym-1 antibody for chelator conjugation with minimizing loss of immunoreactivity.
Subject(s)
Humans , Antibodies , Blotting, Western , Chromatography , Clone Cells , Flow Cytometry , Fluorescein-5-isothiocyanate , Fluorescence , Hand , Lymphoma , Lysine , Molecular Weight , Sprains and StrainsABSTRACT
PURPOSE: The purpose of this study is to image metastaic lung melanoma model with optimal pre-conditions for animal handling by using [18F]FDG small animal PET and clinical CT. MATERIALS AND METHODS: The pre-conditions for lung region tumor imaging were 16-22 h fasting and warming temperature at 30 degrees C. Small animal PET image was obtained at 60 min postinjection of 7.4 MBq [18F]FDG and compared pattern of [18F]FDG uptake and glucose standard uptake value (SUVG) of lung region between Ketamine/Xylazine (Ke/Xy) and Isoflurane (Iso) anesthetized group in normal mice. Metastasis tumor mouse model to lung was established by intravenous injection of B16-F10 cells in C57BL/6 mice. In lung metastasis tumor model, [18F]FDG image was obtained and fused with anatomical clinical CT image. RESULTS: Average blood glucose concentration in normal mice were 128.0+/-23.87 and 86.0+/-21.65 mg/dL in Ke/Xy group and Iso group, respectively. Ke/Xy group showed 1.5 fold higher blood glucose concentration than Iso group. Lung to Background ratio (L/B) in SUVG image was 8.6+/-0.48 and 12.1+/-0.63 in Ke/Xy group and Iso group, respectively. In tumor detection in lung region, [18F]FDG image of Iso group was better than that of Ke/Xy group, because of high L/B ratio. Metastatic tumor location in [18F]FDG small animal PET image was confirmed by fusion image using clinical CT. CONCLUSION: Tumor imaging in small animal lung region with [18F]FDG small animal PET should be considered pre-conditions which fasting, warming and an anesthesia during [18F]FDG uptake. Fused imaging with small animal PET and CT image could be useful for the detection of metastatic tumor in lung region.
Subject(s)
Animals , Animals , Mice , Anesthesia , Blood Glucose , Fasting , Glucose , Injections, Intravenous , Isoflurane , Lung , Melanoma , Neoplasm MetastasisABSTRACT
PURPOSE: Ethylenediamine-tetramethylenephosphonic acid (EDTMP) has widely used chelator for the labeling of bone seeking radiopharmaceuticals complexed with radiometals. 153Sm can be produced by the HANARO reactor at the Korea Atomic Energy Research Institute, Taejon, Korea. 153Sm has favourable radiation characteristics T1/2=46.7 h, beta max=0.81 MeV (20%), 0.71 MeV (49%), 0.64 MeV (30%) and gamma=103 keV (30%) emission which is suitable for imaging purposes during therapy. We investigated the labeling condition of 153Sm-EDTMP and imaging of 153Sm-EDTMP in normal rats. MATERIALS AND METHODS: EDTMP 20 mg was solved in 0.1 mL 2 M NaOH. 153SmCl3 was added to EDTMP solution and pH of the reaction mixtures was adjusted to 8 and 12, respectively. Radiochemical purity was determined with paper chromatography. After 30 min. reaction, reaction mixtures were neutralized to pH 7.4, and the stability was estimated upto 120 hrs. Imaging studies of each reaction were perfomed in normal rats (37 MBq/0.1 mL). RESULTS: The labeling yield of 153Sm-EDTMP was 99%. The stability of pH 8 reaction at 60, 96 and 120 hr was 99%, 95%, 89% and that of pH 12 at 36, 60, 96 and 120 hr was 99%, 95%, 88%, 66%, respectively. The 153Sm-EDTMP showed constantly higher bone uptake from 2 to 48 hr after injection. CONCLUSION: 153Sm-EDTMP, labeled at pH 8 reaction condition, has been stably maintained. Image of 153Sm-EDTMP at 2, 24, 48 hr after injection, demonstrate that 153Sm-EDTMP is a good bone seeking radiopharmaceuticals.
Subject(s)
Animals , Rats , Academies and Institutes , Chromatography, Paper , Hydrogen-Ion Concentration , Korea , Nuclear Energy , RadiopharmaceuticalsABSTRACT
PURPOSE: The herpes simplex virus type 1 thymidine kinase gene (HSV1-tk) is an attractive candidate as a reporter gene in noninvasive reporter gene monitoring system. The HSV1-tk gene was chosen as a reporter gene, because it has been extensively studied, and there are appropriate reporter probes, substrates of HSV1-tk gene product, to apply for HSV1-tk gene imaging. We used radiolabeled 5-iodovinyl-2'-deoxyuridine (IVDU) and 5-Iodovinyl-2'-fluoro-2'-deoxyuridine (IVFRU) as reporter probes for HSV1-tk gene monitoring system. MATERIALS AND METHODS: We prepared HSV1-tk gene transduced Morris hepatoma cell line using retroviral vector, MOLTEN containing HSV1-tk gene. And we confirmed the HSV1-tk gene expression by Northern blotting and Western blotting. We compared in vitro uptakes of radioiodinated IVDU and IVFRU to monitor HSV1-tk gene expression in Morris hepatoma cell line (MCA) and HSV1-tk gene tranduced MCA (MCA-tk) cells until 480 minutes. We also performed correlation analysis between percentage of HSV1-tk gene tranduced MCA cell % (MCA-tk%) and uptakes of radiolabeled IVDU or IVFRU. RESULTS: MCA-tk cell expressed HSV1-tk mRNA and HSV1-TK protein. Two compounds showed minimal uptake in MCA, but increased uptake was observed in MCA-tk. IVDU showed 4-fold higher accumulation than IVFRU at 480 min in MCA-tk (p 0.96) with increasing MCA-tk%. CONCLUSION: The radiolabeld IVDU and IVFRU showed higher specific accumulation in retrovirally HSV1-tk gene transfected Morris hepatoma cell line. Both IVDU and IVFRU could be used as good substrates for evaluation of HSV1-tk gene expression.
Subject(s)
Animals , Blotting, Northern , Blotting, Western , Cell Line , Gene Expression , Genes, Reporter , Genetic Therapy , Herpes Simplex , Herpesvirus 1, Human , Liver Neoplasms, Experimental , RNA, Messenger , Simplexvirus , Thymidine Kinase , Thymidine , ZidovudineABSTRACT
PURPOSE: There has been a renewal of interest in Macrophage migration inhibitory factor (MIF), especially correlation in pathogenesis of sepsis by many infectious diseases and in regulation of host inflammatory and immune response. We developed immunoradiometric assay (IRMA) to determine serum human MIF concentration. MATERIALS AND METHODS: The IRMA system utilizes solid phase bound monoclonal anti-recombinant human MIF (rhMIF) antibody as a capture antibody, biotinylated polyclonal anti-rhMIF antibody as a detector antibody. We applied with rhMIF that concentration of standard solutions increased from 0 ng/ml to 100 ng/ml. We used 125I-streptavidin (SA) as radiotracer to determination of rhMIF concentration. Streptavidin was labeled with 125I by Chloramine-T method and 125I-SA was purified by ultracentrifugation. 125I-SA stability was evaluated by ITLC analysis at 4 degrees C and room temperatures until 60days. To validate IRMA system for MIF, we experimented intra-assay and inter-assay coefficients of variation, recovery test and dilution test. RESULTS: Radiolabeling yield of 125I-SA was 87% and purified 125I-SA retained above 99% radiochemical purity. 125I-SA showed above 93% stability in 4 degrees C until 60days that it is good for immunoradiometric assay as radiotracer. Plotted standard dose response curve showed that increased concentration of rhMIF linearly correlated (R2=0.99) with bound radioactivity of 125I-SA. The highest intra- and inter-assay coefficients of variation were 5.5% and 7.6%, respectively. The average of recovery of MIF in samples was 102%. In dilution test, linear response curves were obtained (R2=0.97). CONCLUSION: Radioimmunoassay using 125I-SA as radiotracer thought to be useful for the determination of serum MIF concentration, and further, its data will be used to evaluate the correlation between clinical significance and serum MIF concentration in patients with various inflammatory diseases.
Subject(s)
Humans , Communicable Diseases , Immunoradiometric Assay , Macrophages , Radioactivity , Radioimmunoassay , Sepsis , Streptavidin , UltracentrifugationABSTRACT
No abstract available.
ABSTRACT
PURPOSE: We investigated the direct labeling method of antibody with 99mTc and 188Re and examined the stability and function of these labeled compounds in in vitro and in vivo. MATERIALS AND METHODS: Disulfide bond of nonspecific human IgG was reduced to -SH group by 2-mercaptoethanol. Stannous ion was used to reduce 99mTc and 188Re. The stability of 99mTc-IgG and 188Re-IgG was estimated upto 24 hrs. Biodistribution was evaluated in abscess bearing rats at 4 and 24 hr post-injection of 99mTc or 188Re labeled IgG. RESULTS: The number of -SH group per reduced IgG molecule was 2.34. The labeling yield of 99mTc-IgG and 188Re-IgG were 90% and 95%, respectively. The stability of 99mTc-IgG at 1, 4, 6 and 24 hr was 91%, 83%, 78%, 7% and that of 188Re-IgG, high uptake was found on kidney, blood, stomach and abscess (9.42+/-0.68, 1.43+/-0.24, 0.86+/-0.18, 0.72+/-0.10 %ID/g, respectively). The uptakes at 24 hr were kidney, abscess, stomach, and blood in descending order. In case of 188Re-IgG, high uptake at 4 hr post injection appeared on kidney, blood, abscess and stomach (3.92+/-0.62, 1.32+/-0.08, 0.88+/-0.01, 0.26+/-0.06, respectively). The upatkes at 24 hr were kidney, abscess, blood abd stomach in descending order. The abscess to blood uptake ratio of 99mTc-IgG was 0.5 at 4 hr and 2.02 at 24 hr and that of 188Re-IgG was 0.67 and 1.29. CONCLUSION: 99mTc-IgG and 188Re-IgG and 188Re-IgG canbe labeled efficiently with direct labeling method. However, 99mTc-IgG and 188Re-IgG, labeled with direct method, was unstable. Further study in needed to enhance the stability of the antibody labeling.
Subject(s)
Animals , Humans , Rats , Abscess , Immunoglobulin G , Kidney , Mercaptoethanol , StomachABSTRACT
PURPOSE: The aim of this sutdy was to evaluate the feasibility of 3-[131I]Iodo-O-methyl-L-a-methyltyrosine ([131I]OMINT) as an agent for tumor image. MATERIALS AND METHODS: After synthesis of 4-O-methyl-L-a-methyltyrosine (OMAMT), OMAMT was labeled with 131I using Iodogen method. In viro cellular uptake study was performed using 9 L gliosarcoma cells at various time points upto 4 hr. The biodistribution (five rats implanted with the 9 L gliosarcoma cells per group) was evaluated at 30 min, 2 hr, 24 hr after iv injection of 3.7 MBq [131I]OMIMT or L-3-[131I]iodo-a-methyltyrosine ([131I]IMT). Gamma camera images were obtained at 30min, 2 hr, and 24 hr. RESULTS: [131I]OMINT uptake was 3.3 times and 2.5 times higher than [131I]IMT uptake at 30 min and 60 min, respectively and same after 2 hr in in vitro sutdy using 9L gliosarcoma cells. Maximum accumulation in tumor occurred at 30 min for both [131IOMINT and [131I]IMT in tumor bearing rats. The tumor uptake of [131I]OMINT was significantly higher than that of [131I]IMT in tumor bearing rats. The tumor uptake of [131I]OMIMT was significantly higher than that of [131I]IMT at early time point studied (3.74 +/- 0.48 vs 0.38 +/- 0.17% ID/g at 30 min and 2.40 +/- 0.17 vs 0.24 +/- 0.03% ID/g at 2 hr, respectively, p<0.01). However, the tumor uptake of both radiolabels were not significantly different at 24 hr (0.04 +/- 0.01 vs 0.05 +/- 0.01% ID/g). Tumor was visualized as early as at 30 min in gamma camera images. CONCLUSION: These data suggested that [131I]OMIMT might be a useful tumor imaging agent and has more advantage for the tumor imaging compared to [131I]IMT.