Sample records for anti-cd45 antibody labeled

  1. Biodistribution of Yttrium-90-Labeled Anti-CD45 Antibody in a Nonhuman Primate Model

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    Nemecek, Eneida; Hamlin, Donald K.; Fisher, Darrell R.; Krohn, Kenneth A.; Pagel, John M.; Applebaum, F. R.; Press, Oliver W.; Matthews, Dana C.


    Radioimmunotherapy may improve the outcome of hematopoietic cell transplantation for hematologic malignancies by delivering targeted radiation to hematopoietic organs while relatively sparing nontarget organs. We evaluated the organ localization of yttrium-90-labeled anti-CD45 (90Y-anti-CD45) antibody in macaques, a model that had previously predicted iodine-131-labeled anti-CD-45 (131I-anti-CD45) antibody biodistribution in humans. Experimental Design: Twelve Macaca nemestrina primates received anti-CD45 antibody labeled with 1 to 2 mCi of 90Y followed by serial blood sampling and marrow and lymph node biopsies, and necropsy. The content of 90Y per gram of tissue was determined by liquid scintillation spectrometry. Time-activity curves were constructed using average isotope concentrations in each tissue at measured time points to yield the fractional residence time and estimate radiation absorbed doses for each organ per unit of administered activity. The biodistribution of 90Y-anti-CD45 antibody was then compared with that previously obtained with 131I-anti-CD45 antibody in macaques. Results: The spleen received 2,120, marrow 1,060, and lymph nodes 315 cGy/mCi of 90Y injected. The liver and lungs were the nontarget organs receiving the highest radiation absorbed doses (440 and 285 cGy/mCi, respectively). Ytrrium-90-labeled anti-CD45 antibody delivered 2.5- and 3.7-fold more radiation to marrow than to liver and lungs, respectively. The ratios previously observed with 131I-antiCD45 antibody were 2.5-and 2.2-fold more radiation to marrow than to liver and lungs, respectively. Conclusions: This study shows that 90Y-anti-CD45 antibody can deliver relatively selective radiation to hematopoietic tissues, with similar ratios of radiation delivered to target versus nontarget organs, as compared with the 131I immunoconjugate in the same animal model.

  2. Bismuth 213-labeled anti-CD45 radioimmunoconjugate to condition dogs for nonmyeloablative allogeneic marrow grafts

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    Sandmaier, B M.(Fred Hutchinson Cancer Research Center, Seattle, WA); Bethge, W A.(Fred Hutchinson Cancer Research Center, Seattle, WA); Wilbur, D. Scott (Washington, Univ Of); Hamlin, Donald K.(Washington, Univ Of); Santos, E B.(Fred Hutchinson Cancer Research Center, Seattle, WA); Brechbiel, M W.(National Cancer Institute, National Institutes of Health, Bethesda, MD); Fisher, Darrell R.(BATTELLE (PACIFIC NW LAB)); Storb, R. (Fred Hutchinson Cancer Research Center)


    To lower treatment-related mortality and toxicity of conventional marrow transplantation, a nonmyeloablative regimen using 200 cGy total-body irradiation (TBI) and mycophenolate mofetil (MMF) combined with cyclosporine (CSP) for postgrafting immunosuppression was developed. To circumvent possible toxic effects of external- beam gamma irradiation, strategies for targeted radiation therapy were investigated. We tested whether the short-lived (46 minutes) alpha-emitter Bi-213 conjugated to an anti-CD45 monoclonal antibody (mAb) could replace 200 cGy TBI and selectively target hematopoietic tissues in a canine model of nonmyeloablative DLA-identical marrow transplantation. Biodistribution studies using iodine 123-labeled anti-CD45 mAb showed uptake in blood, marrow, lymph nodes, spleen, and liver. In a dose-escalation study, 7 dogs treated with the Bi-213-anti-CD45 conjugate (Bi-213 dose, 0.1-5.9 mCi/kg[3.7-218 MBq/kg]) without marrow grafts had no toxic effects other than a mild, reversible suppression of blood counts. On the basis of these studies, 3 dogs were treated with 0.5 mg/kg Bi-213-labeled anti-CD45 mAb (Bi-213 doses, 3.6, 4.6, and 8.8 mCi/kg[133, 170, and 326 MBq/kg]) given in 6 injections 3 and 2 days before grafting of marrow from DLA-identical littermates. The dogs also received MMF (10 mg/kg subcutaneously twice daily the day of transplantation until day 27 afterward) and CSP (15 mg/kg orally twice daily the day before transplantation until 35 days afterward). Therapy was well tolerated except for transient elevations in levels of transaminases in 3 dogs, followed by, in one dog, ascites. All dogs achieved prompt engraftment and stable mixed hematopoietic chimerism, with donor contributions ranging from 30% to 70% after more than 27 weeks of follow-up. These results form the basis for additional studies in animals and the design of clinical trials using Bi-213 as a nonmyeloablative conditioning regimen with minimal toxicity.

  3. 131I-Anti-CD45 Antibody Plus Busulfan and Cyclophosphamide before Allogeneic Hematophoietic Cell Transplantation for Treatment of Acute Myeloid Leukemia in First Remission

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    Pagel, John M.; Appelbaum, Frederick R.; Eary, Janet F.; Rajendran, Joseph G.; Fisher, Darrell R.; Gooley, Ted; Ruffner, Katherine; Nemecek, Eneida; Sickle, Eileen; Durack, Larry; Carreras, Jeanette; Horowitz, Mary; Press, Oliver W.; Gopal, Ajay K.; Martin, Paul J.; Bernstein, Irwin D.; Matthews, Dana C.


    In an attempt to improve outcomes for patients with acute myeloid leukemia (AML) after allogeneic hematopoietic cell transplantation (HCT), we conducted a Phase I/II study in which targeted irradiation delivered by 131I-anti-CD45 antibody was combined with targeted busulfan (BU; area-under-curve, 600-900 ng/ml) and cyclophosphamide (CY; 120 mg/kg). Fifty-two of 59 patients (88%) receiving a trace 131I-labeled dose of 0.5 mg/kg anti-CD45 murine antibody had higher estimated absorbed radiation in bone marrow and spleen than in any other organ. Forty-six patients were treated with 102-298 mCi 131I delivering an estimated 5.3-19 (mean 11.3) Gy to marrow, 17-72 (mean 29.7) Gy to spleen, and 3.5 Gy (n=4) to 5.25 Gy (n=42) to the liver. The estimated 3-year non-relapse mortality and disease-free survival (DFS) were 21% and 61%, respectively. These results were compared to those from 509 similar International Bone Marrow Transplant Registry patients transplanted using BU/CY alone. After adjusting for differences in age and cytogenetics-risk, the hazard of mortality among all antibody-treated patients was 0.65 times that of the Registry patients (95% CI 0.39-1.08; p=.09). The addition of targeted hematopoietic irradiation to conventional BU/CY is feasible and well tolerated, and Phase II results are sufficiently encouraging to warrant further study.

  4. Pretargeted Radioimmunotherapy Using Anti-CD45 Monoclonal Antibodies to Deliver Radiation to Murine Hematolymphoid Tissues and Human Myeloid Leukemia

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    Pagel, John M.; Matthews, Dana C.; Kenoyer, Aimee L.; Hamlin, Donald K.; Wilbur, D. Scott; Fisher, Darrell R.; Gopal, Ajay K.; Lin, Yukang; Saganic, Laura; Appelbaum, Frederick R.; Press, Oliver W.


    The efficacy of radioimmunotherapy (RIT) for treatment of patients with hematological malignancies frequently fails because of disease recurrence. We therefore conducted pretargeted RIT studies to augment the efficacy in mice of therapy using a pretargeted anti-human (h)CD45 antibody (Ab)-streptavidin (SA) conjugate followed by delivery of a biotinylated clearing agent and radiolabeled-DOTA-biotin. Tumor-to-blood ratios at 24 hours were 20:1 using pretargeted anti-hCD45 RIT and <1:1 with conventional RIT. In vivo imaging studies confirmed that the pretargeted RIT approach provided high-contrast tumor images with minimal blood-pool activity, whereas directly-labeled anti-hCD45 Ab produced distinct tumor images but the blood pool retained a large amount of labeled antibody for a prolonged time. Therapy experiments demonstrated that 90Y-DOTA-biotin significantly prolonged survival of mice treated pretargeted with anti-hCD45 Ab-SA compared to mice treated with conventional RIT using 90Y-labeled anti-hCD45 Ab at the maximally tolerated dose (400 µCi). Since human CD45 antigens are confined to xenograft tumor cells in this model, and all murine tissues are devoid of hCD45 and will not bind anti-hCD45 Ab, we also compared one-step and pretargeted RIT using an anti-murine (m)CD45 Ab (A20 ) in a model where the target antigen is present on normal hematopoietic tissues. After 24 hours, 27.3 ± 2.8% of the injected dose of radionuclide was delivered per gram (% ID/g) of lymph node using 131I-A20-Ab compared with 40.0 ± 5.4% ID/g for pretargeted 111In-DOTA-biotin (p value). These data suggest that multi-step pretargeted methods for delivering RIT are superior to conventional RIT when targeting CD45 for the treatment of leukemia and may allow for the intensification of therapy, while minimizing toxicities.

  5. Radiolabeled anti-CD45 antibody with reduced-intensity conditioning and allogeneic transplantation for younger patients with advanced acute myeloid leukemia or myelodysplastic syndrome. (United States)

    Mawad, Raya; Gooley, Ted A; Rajendran, Joseph G; Fisher, Darrell R; Gopal, Ajay K; Shields, Andrew T; Sandmaier, Brenda M; Sorror, Mohamed L; Deeg, Hans Joachim; Storb, Rainer; Green, Damian J; Maloney, David G; Appelbaum, Frederick R; Press, Oliver W; Pagel, John M


    We treated patients under age 50 years with iodine-131 ((131)I)-anti-CD45 antibody combined with fludarabine and 2 Gy total body irradiation to create an improved hematopoietic cell transplantation (HCT) strategy for advanced acute myeloid leukemia or high-risk myelodysplastic syndrome patients. Fifteen patients received 332 to 1561 mCi of (131)I, delivering an average of 27 Gy to bone marrow, 84 Gy to spleen, and 21 Gy to liver. Although a maximum dose of 28 Gy was delivered to the liver, no dose-limiting toxicity was observed. Marrow doses were arbitrarily capped at 43 Gy to avoid radiation-induced stromal damage; however, no graft failure or evidence of stromal damage was observed. Twelve patients (80%) developed grade II graft-versus-host disease (GVHD), 1 patient developed grade III GVHD, and no patients developed grade IV GVHD during the first 100 days after HCT. Of the 12 patients with chronic GVHD data, 10 developed chronic GVHD, generally involving the skin and mouth. Six patients (40%) are surviving after a median of 5.0 years (range, 4.2 to 8.3 years). The estimated survival at 1 year was 73% among the 15 treated patients. Eight patients relapsed, 7 of whom subsequently died. The median time to relapse among these 8 patients was 54 days (range, 26 to 1364 days). No cases of nonrelapse mortality were observed in the first year after transplantation. However, 2 patients died in remission from complications of chronic GVHD and cardiomyopathy, at 18 months and 14 months after transplantation, respectively. This study suggests that patients may tolerate myeloablative doses >28 Gy delivered to the liver using (131)I-anti-CD45 antibody in addition to standard reduced-intensity conditioning. Moreover, the arbitrary limit of 43 Gy to the marrow may be unnecessarily conservative, and continued escalation of targeted radioimmunotherapy doses may be feasible to further reduce relapse.

  6. Anti-CD45 radioimmunotherapy using 211At with bone marrow transplantation prolongs survival in a disseminated murine leukemia model

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    Orozco, Johnnie J.; Back, Tom; Kenoyer, Aimee L.; Balkin, Ethan R.; Hamlin, Donald K.; Wilbur, D. Scott; Fisher, Darrell R.; Frayo, Shani; Hylarides, Mark; Green, Damian J.; Gopal, Ajay K.; Press, Oliver W.; Pagel, John M.


    Anti-CD45 Radioimmunotherapy using an Alpha-Emitting Radionuclide 211At Combined with Bone Marrow Transplantation Prolongs Survival in a Disseminated Murine Leukemia Model ABSTRACT Despite aggressive chemotherapy combined with hematopoietic cell transplant (HCT), many patients with acute myeloid leukemia (AML) relapse. Radioimmunotherapy (RIT) using antibodies (Ab) labeled primarily with beta-emitting radionuclides has been explored to reduce relapse.

  7. Allogeneic hematopoietic cell transplantation after conditioning with I-131-anti-CD45 antibody plus fludarabine and low-dose total body irradiation for elderly patients with advanced acute myeloid leukemia or high-risk myelodysplastic syndrome.

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    Pagel, John M.; Gooley, T. A.; Rajendran, Joseph G.; Fisher, Darrell R.; Wilson, Wendy A.; Sandmaier, B. M.; Matthews, D. C.; Deeg, H. Joachim; Gopal, Ajay K.; Martin, P. J.; Storb, R.; Press, Oliver W.; Appelbaum, Frederick R.


    We conducted a study to estimate the maximum tolerated dose (MTD) of I-131-anti-CD45 antibody (Ab; BC8) that can be combined with a standard reduced-intensity conditioning regimen before allogeneic hematopoietic cell transplantation. Fifty-eight patients older than 50 years with advanced acute myeloid leukemia (AML) or high-risk myelodysplastic syndrome (MDS) were treated with (131)I-BC8 Ab and fludarabine plus 2 Gy total body irradiation. Eighty-six percent of patients had AML or MDS with greater than 5% marrow blasts at the time of transplantation. Treatment produced a complete remission in all patients, and all had 100% donor-derived CD3(+) and CD33(+) cells in the blood by day 28 after the transplantation. The MTD of I-131-BC8 Ab delivered to liver was estimated to be 24 Gy. Seven patients (12%) died of nonrelapse causes by day 100. The estimated probability of recurrent malignancy at 1 year is 40%, and the 1-year survival estimate is 41%. These results show that CD45-targeted radiotherapy can be safely combined with a reduced-intensity conditioning regimen to yield encouraging overall survival for older, high-risk patients with AML or MDS. This study was registered at as #NCT00008177.

  8. Allogeneic hematopoietic cell transplantation after conditioning with 131I-anti-CD45 antibody plus fludarabine and low-dose total body irradiation for elderly patients with advanced acute myeloid leukemia or high-risk myelodysplastic syndrome. (United States)

    Pagel, John M; Gooley, Theodore A; Rajendran, Joseph; Fisher, Darrell R; Wilson, Wendy A; Sandmaier, Brenda M; Matthews, Dana C; Deeg, H Joachim; Gopal, Ajay K; Martin, Paul J; Storb, Rainer F; Press, Oliver W; Appelbaum, Frederick R


    We conducted a study to estimate the maximum tolerated dose (MTD) of (131)I-anti-CD45 antibody (Ab; BC8) that can be combined with a standard reduced-intensity conditioning regimen before allogeneic hematopoietic cell transplantation. Fifty-eight patients older than 50 years with advanced acute myeloid leukemia (AML) or high-risk myelodysplastic syndrome (MDS) were treated with (131)I-BC8 Ab and fludarabine plus 2 Gy total body irradiation. Eighty-six percent of patients had AML or MDS with greater than 5% marrow blasts at the time of transplantation. Treatment produced a complete remission in all patients, and all had 100% donor-derived CD3(+) and CD33(+) cells in the blood by day 28 after the transplantation. The MTD of (131)I-BC8 Ab delivered to liver was estimated to be 24 Gy. Seven patients (12%) died of nonrelapse causes by day 100. The estimated probability of recurrent malignancy at 1 year is 40%, and the 1-year survival estimate is 41%. These results show that CD45-targeted radiotherapy can be safely combined with a reduced-intensity conditioning regimen to yield encouraging overall survival for older, high-risk patients with AML or MDS. This study was registered at as #NCT00008177.

  9. Anti-CD45 radioimmunotherapy with 90Y but not 177Lu is effective treatment in a syngeneic murine leukemia model.

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    Johnnie J Orozco

    Full Text Available Radioimmunotherapy (RIT for treatment of hematologic malignancies has primarily employed monoclonal antibodies (Ab labeled with 131I or 90Y which have limitations, and alternative radionuclides are needed to facilitate wider adoption of RIT. We therefore compared the relative therapeutic efficacy and toxicity of anti-CD45 RIT employing 90Y and 177Lu in a syngeneic, disseminated murine myeloid leukemia (B6SJLF1/J model. Biodistribution studies showed that both 90Y- and 177Lu-anti-murine CD45 Ab conjugates (DOTA-30F11 targeted hematologic tissues, as at 24 hours 48.8 ± 21.2 and 156 ± 14.6% injected dose per gram of tissue (% ID/g of 90Y-DOTA-30F11 and 54.2 ± 9.5 and 199 ± 11.7% ID/g of 177Lu-DOTA-30F11 accumulated in bone marrow (BM and spleen, respectively. However, 90Y-DOTA-30F11 RIT demonstrated a dose-dependent survival benefit: 60% of mice treated with 300 µCi 90Y-DOTA-30F11 lived over 180 days after therapy, and mice treated with 100 µCi 90Y-DOTA-30F11 had a median survival 66 days. 90Y-anti-CD45 RIT was associated with transient, mild myelotoxicity without hepatic or renal toxicity. Conversely, 177Lu- anti-CD45 RIT yielded no long-term survivors. Thus, 90Y was more effective than 177Lu for anti-CD45 RIT of AML in this murine leukemia model.

  10. Anti-CD45 Pretargeted Radioimmunotherapy using Bismuth-213: High Rates of Complete Remission and Long-Term Survival in a Mouse Myeloid Leukemia Xenograft Model

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    Pagel, John M; Kenoyer, Aimee L; Back, Tom; Hamlin, Donald K; Wilbur, D Scott; Fisher, Darrell R; Park, Steven I; Frayo, Shani; Axtman, Amanda; Orgun, Nural; Orozoco, Johnnie; Shenoi, Jaideep; Lin, Yukang; Gopal, Ajay K; Green, Damian J; Appelbaum, Frederick R; Press, Oliver W


    Pretargeted radioimmunotherapy (PRIT) using an anti-CD45 antibody (Ab)-streptavidin (SA) conjugate and DOTA-biotin labeled with β-emitting radionuclides has been explored as a strategy to decrease relapse and toxicity. α-emitting radionuclides exhibit high cytotoxicity coupled with a short path-length, potentially increasing the therapeutic index and making them an attractive alternative to β-emitting radionuclides for patients with Acute Myeloid Leukemia (AML). Accordingly, we have used 213Bi in mice with human leukemia xenografts. Results demonstrated excellent localization of 213Bi-DOTA-biotin to tumors with minimal uptake into normal organs. After 10 minutes, 4.5 ± 1.1% of the injected dose of 213Bi was delivered per gram of tumor. α imaging demonstrated uniform radionuclide distribution within tumor tissue 45 minutes after 213Bi-DOTA-biotin injection. Radiation absorbed doses were similar to those observed using a β-emitting radionuclide (90Y) in the same model. We conducted therapy experiments in a xenograft model using a single-dose of 213Bi-DOTA-biotin given 24 hours after anti-CD45 Ab-SA conjugate. Among mice treated with anti-CD45 Ab-SA conjugate followed by 800 μCi of 213Bi- or 90Y-DOTA-biotin, 80% and 20%, respectively, survived leukemia-free for >100 days with minimal toxicity. These data suggest that anti-CD45 PRIT using an α-emitting radionuclide may be highly effective and minimally toxic for treatment of AML.

  11. A comparison of the biodistribution and biokinetics of {sup 99m}Tc-anti-CD66 mAb BW 250/183 and {sup 99m}Tc-anti-CD45 mAb YTH 24.5 with regard to suitability for myeloablative radioimmunotherapy

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    Buchmann, Inga [Department of Nuclear Medicine, University Hospital, Ulm (Germany); Klinik und Poliklinik fuer Nuklearmedizin, Gebaeude 210, Johannes-Gutenberg-Universitaet Mainz, 55101, Mainz (Germany); Kull, Thomas; Glatting, Gerhard; Kotzerke, Jorg; Rattat, Dirk; Reske, Sven N. [Department of Nuclear Medicine, University Hospital, Ulm (Germany); Bunjes, Donald; Dohner, Hartmut [Department of Hematology, University Hospital, Ulm (Germany); Hale, Geoffrey [Sir William Dunn School of Pathology, Oxford (United Kingdom)


    Radioimmunotherapy (RIT) with radiolabelled monoclonal antibodies (mAbs) is an effective method of achieving myeloablation in leukaemia patients prior to stem cell transplantation (SCT). We wished to compare the approaches of specific binding to leukaemic blasts and non-specific binding to benign red marrow cells, which results in a myeloablative ''cross-fire'' effect. Therefore, we prospectively evaluated the biodistribution and biokinetics of the anti-CD45 mAb YTH 24.5 and the anti-CD66 mAb BW 250/183 with regard to their suitability for myeloablative RIT. The red marrow selective anti-CD66 mAb BW 250/183 (IgG1) binds to normal granulopoietic cells. In contrast, the anti-CD45 mAb YTH 24.5 (IgG2b) binds to 85-90% of acute leukaemic blasts and almost all haematopoietic white cells. Patients with leukaemic blast infiltration of the marrow <25% and assigned for RIT and SCT were included. Twelve patients (eight male, four female; median age 46{+-}7 years) with AML (5), CML (5) or ALL (2) were examined. Both mAbs were labelled with technetium-99m. Within 48 h, 906{+-}209 MBq {sup 99m}Tc-anti-CD66 mAb and 760{+-}331 MBq {sup 99m}Tc-anti-CD45 mAb were injected consecutively. Scintigraphic and urinary measurements were performed 1, 2, 4 and 24 h after injection. Serum activities were evaluated 2, 5, 10, 15, 30 and 60 min and 2, 4 and 24 h after injection. Compared with the anti-CD45 mAb, the anti-CD66 mAb showed an approximately fourfold higher accumulation in the red marrow, a 2.5-fold lower accumulation in the liver and similar accumulation in the kidneys. The serum activity (% of the injected dose) initially decreased faster for the anti-CD45 mAb but was similar for the two mAbs 24 h after injection: 3.3%{+-}1.2% (anti-CD66 mAb) and 2.4%{+-}1.1% (anti-CD45 mAb). The cumulated urinary excretion was 17%{+-}6.6% (anti-CD66 mAb) and 27.3%{+-}7.9% (anti-CD45 mAb) 24 h after application. In these patients with low tumour load, the anti-CD66 mAb BW 250

  12. PLGA-PEG Nanoparticles Coated with Anti-CD45RO and Loaded with HDAC Plus Protease Inhibitors Activate Latent HIV and Inhibit Viral Spread (United States)

    Tang, Xiaolong; Liang, Yong; Liu, Xinkuang; Zhou, Shuping; Liu, Liang; Zhang, Fujina; Xie, Chunmei; Cai, Shuyu; Wei, Jia; Zhu, Yongqiang; Hou, Wei


    Activating HIV-1 proviruses in latent reservoirs combined with inhibiting viral spread might be an effective anti-HIV therapeutic strategy. Active specific delivery of therapeutic drugs into cells harboring latent HIV, without the use of viral vectors, is a critical challenge to this objective. In this study, nanoparticles of poly(lactic-co-glycolic acid)-polyethylene glycol diblock copolymers conjugated with anti-CD45RO antibody and loaded with the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) and/or protease inhibitor nelfinavir (Nel) were tested for activity against latent virus in vitro. Nanoparticles loaded with SAHA, Nel, and SAHA + Nel were characterized in terms of size, surface morphology, zeta potential, entrapment efficiency, drug release, and toxicity to ACH-2 cells. We show that SAHA- and SAHA + Nel-loaded nanoparticles can target latently infected CD4+ T-cells and stimulate virus production. Moreover, nanoparticles loaded with SAHA + NEL were capable of both activating latent virus and inhibiting viral spread. Taken together, these data demonstrate the potential of this novel reagent for targeting and eliminating latent HIV reservoirs.

  13. The antibody approach of labeling blood cells

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    Srivastava, S.C.


    Although the science of blood cell labeling using monoclonal antibodies directed against specific cellular antigens is still in its early stages, considerable progress has recently been accomplished in this area. The monoclonal antibody approach offers the promise of greater selectivity and enhanced convenience since specific cell types can be labeled in vivo, thus eliminating the need for complex and damaging cell separation procedures. This article focuses on these developments with primary emphasis on antibody labeling of platelets and leukocytes. The advantages and the shortcomings of the recently reported techniques are critically assessed and evaluated.

  14. The antibody approach of labeling blood cells

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    Srivastava, S.C.


    Although the science of blood cell labeling using monoclonal antibodies directed against specific cellular antigens is still in its early stages, considerable progress has recently been accomplished in this area. The monoclonal antibody approach offers the promise of greater selectivity and enhanced convenience since specific cell types can be labeled in vivo, thus eliminating the need for complex and damaging cell separation procedures. This article focuses on these developments with primary emphasis on antibody labeling of platelets and leukocytes. The advantages and the shortcomings of the recently reported techniques are criticality assessed and evaluated.

  15. The antibody approach of labeling blood cells

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    Srivastava, S.C.


    Although the science of blood cell labeling using monoclonal antibodies directed against specific cellular antigens is still in its early stages, considerable progress has recently been accomplished in this area. The monoclonal antibody approach offers the promise of greater selectivity and enhanced convenience since specific cell types can be labeled in vivo, thus eliminating the need for complex and damaging cell separation procedures. This article focuses on these developments with primary emphasis on antibody labeling of platelets and leukocytes. The advantages and the shortcomings of the recently reported techniques are criticality assessed and evaluated.

  16. Extracorporeal adsorption therapy: A Method to improve targeted radiation delivered by radiometal-labeled monoclonal antibodies.

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    Nemecek, Eneida R.; Green, Damian J.; Fisher, Darrell R.; Pagal, John M.; Lin, Yukang; Gopal, A. K.; Durack, Lawrence D.; Rajendran, Joseph G.; Wilbur, D. S.; Nilsson, Rune; Sandberg, Bengt; Press, Oliver W.


    Many investigators have demonstrated the ability to treat hematologic malignancies with radiolabeled monoclonal antibodies targeting hematopoietic antigens such as anti-CD20 and anti-CD45. [1-5] Although the remission rates achieved with radioimmunotherapy (RIT) are relatively high, many patients subsequently relapse presumably due to suboptimal delivery of enough radiation to eradicate the malignancy. The dose-response of leukemia and lymphoma to radiation has been proven. Substantial amounts of radiation can be delivered by RIT if followed by hematopoietic cell transplantation to rescue the bone marrow from myeloablation.[ref] However, the maximum dose of RIT that can be used is still limited by toxicity to normal tissues affected by nonspecific delivery of radiation. Efforts to improve RIT focus on improving the therapeutic ratios of radiation in target versus non-target tissues by removing the fraction of radioisotope that fails to bind to target tissues and circulates freely in the bloodstream perfusing non-target tissues. Our group and others have explored several alternatives for removal of unbound circulating antibody. [refs] One such method, extracorporeal adsorption therapy (ECAT) consists of removing unbound antibody by a method similar to plasmapheresis after critical circulation time and distribution of antibody into target tissues have been achieved. Preclinical studies of ECAT in murine xenograft models demonstrated significant improvement in therapeutic ratios of radioactivity. Chen and colleagues demonstrated that a 2-hour ECAT procedure could remove 40 to 70% of the radioactivity from liver, lung and spleen. [ref] Although isotope concentration in the tumor was initially unaffected, a 50% decrease was noted approximately 36 hours after the procedure. This approach was also evaluated in a limited phase I pilot study of patients with refractory B-cell lymphoma. [ref] After radiographic confirmation of tumor localization of a test dose of anti-CD20

  17. Reduction-mediated technetium-99m labeling of monoclonal antibodies

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    Mather, S.J.; Ellison, D. (St. Bartholomews Hospital, London (England))


    A simple and generally applicable method for labeling antibodies with technetium-99m ({sup 99m}Tc) is described. Following reduction of intrinsic disulphide bonds, the antibody is labeled with {sup 99m}Tc in the presence of a weak competing ligand methylene diphosphonate. High labeling efficiencies (greater than 97%), in a final labeling step taking only a few minutes, can be routinely obtained with high in-vitro stability over 24 hr. No effect upon antibody reactivity is seen.

  18. Rapamycin combined with anti-CD45RB mAb and IL-10 or with G-CSF induces tolerance in a stringent mouse model of islet transplantation.

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    Nicola Gagliani

    Full Text Available BACKGROUND: A large pool of preexisting alloreactive effector T cells can cause allogeneic graft rejection following transplantation. However, it is possible to induce transplant tolerance by altering the balance between effector and regulatory T (Treg cells. Among the various Treg-cell types, Foxp3(+Treg and IL-10-producing T regulatory type 1 (Tr1 cells have frequently been associated with tolerance following transplantation in both mice and humans. Previously, we demonstrated that rapamycin+IL-10 promotes Tr1-cell-associated tolerance in Balb/c mice transplanted with C57BL/6 pancreatic islets. However, this same treatment was unsuccessful in C57BL/6 mice transplanted with Balb/c islets (classified as a stringent transplant model. We accordingly designed a protocol that would be effective in the latter transplant model by simultaneously depleting effector T cells and fostering production of Treg cells. We additionally developed and tested a clinically translatable protocol that used no depleting agent. METHODOLOGY/PRINCIPAL FINDINGS: Diabetic C57BL/6 mice were transplanted with Balb/c pancreatic islets. Recipient mice transiently treated with anti-CD45RB mAb+rapamycin+IL-10 developed antigen-specific tolerance. During treatment, Foxp3(+Treg cells were momentarily enriched in the blood, followed by accumulation in the graft and draining lymph node, whereas CD4(+IL-10(+IL-4(- T (i.e., Tr1 cells localized in the spleen. In long-term tolerant mice, only CD4(+IL-10(+IL-4(- T cells remained enriched in the spleen and IL-10 was key in the maintenance of tolerance. Alternatively, recipient mice were treated with two compounds routinely used in the clinic (namely, rapamycin and G-CSF; this drug combination promoted tolerance associated with CD4(+IL-10(+IL-4(- T cells. CONCLUSIONS/SIGNIFICANCE: The anti-CD45RB mAb+rapamycin+IL-10 combined protocol promotes a state of tolerance that is IL-10 dependent. Moreover, the combination of rapamycin+G-CSF induces

  19. Monoclonal anti-elastin antibody labelled with technetium-99m

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    Oliveira, Marcia B.N. de; Silva, Claudia R. da; Araujo, Adriano C. de; Bernardo Filho, Mario [Universidade do Estado, Rio de Janeiro, RJ (Brazil). Instituto de Biologia Roberto Alcantara Gomes. Lab. de Radiofarmacia; Porto, Luis Cristovao M.S.; Gutfilen, Bianca [Universidade do Estado, Rio de Janeiro, RJ (Brazil). Instituto de Biologia Roberto Alcantara Gomes; Souza, J.E.Q. [Instituto Nacional do Cancer, Rio de Janeiro, RJ (Brazil). Centro de Pesquisa Basica; Frier, Malcolm [University Hospital, Nottingham (United Kingdom). Dept. of Medical Physics


    Technetium-99m ({sup 99m} Tc) is widely employed in nuclear medicine due to its desirable physical, chemical and biological properties. Moreover, it is easily available and normally is inexpensive. A reducing agent is necessary to label cells and molecules with {sup 99m} Tc and stannous chloride (Sn C L{sub 2}) is usually employed. Elastin is the functional protein component of the elastic fiber and it is related with some diseases such as arteriosclerosis, pulmonary emphysema and others. The present study refers to the preparation of the {sup 99m} Tc labeled monoclonal anti-elastin antibody. The monoclonal antibody was incubated with an excess of 2-iminothiolane. The free thiol groups created, were capable of binding with the reduced technetium. Labeling was an exchange reaction with {sup 99m} Tc-glucoheptonate. The labeled preparation was left at 4 deg C for one hour. Then, it was passed through a Sephadex G50 column. Various fractions were collected and counted. A peak corresponding to the radiolabeled antibody was obtained. Stability studies of the labelled anti-elastin were performed at 0,3 6, 24 hours, at both 4 deg C or room temperature. The biodistribution pattern of the {sup 99m} Tc-anti-elastin was studied in healthy male Swiss mice. The immunoreactivity was also determined. An useful labeled-anti-elastin was obtained to future immunoscintigraphic investigations. (author) 4 refs., 7 figs., 6 tabs.

  20. Fluorescent labeling of antibody fragments using split GFP.

    Directory of Open Access Journals (Sweden)

    Fortunato Ferrara

    Full Text Available Antibody fragments are easily isolated from in vitro selection systems, such as phage and yeast display. Lacking the Fc portion of the antibody, they are usually labeled using small peptide tags recognized by antibodies. In this paper we present an efficient method to fluorescently label single chain Fvs (scFvs using the split green fluorescent protein (GFP system. A 13 amino acid tag, derived from the last beta strand of GFP (termed GFP11, is fused to the C terminus of the scFv. This tag has been engineered to be non-perturbing, and we were able to show that it exerted no effect on scFv expression or functionality when compared to a scFv without the GFP11 tag. Effective functional fluorescent labeling is demonstrated in a number of different assays, including fluorescence linked immunosorbant assays, flow cytometry and yeast display. Furthermore, we were able to show that this split GFP system can be used to determine the concentration of scFv in crude samples, as well an estimate of antibody affinity, without the need for antibody purification. We anticipate this system will be of widespread interest in antibody engineering and in vitro display systems.

  1. Cell-free measurements of brightness of fluorescently labeled antibodies (United States)

    Zhou, Haiying; Tourkakis, George; Shi, Dennis; Kim, David M.; Zhang, Hairong; Du, Tommy; Eades, William C.; Berezin, Mikhail Y.


    Validation of imaging contrast agents, such as fluorescently labeled imaging antibodies, has been recognized as a critical challenge in clinical and preclinical studies. As the number of applications for imaging antibodies grows, these materials are increasingly being subjected to careful scrutiny. Antibody fluorescent brightness is one of the key parameters that is of critical importance. Direct measurements of the brightness with common spectroscopy methods are challenging, because the fluorescent properties of the imaging antibodies are highly sensitive to the methods of conjugation, degree of labeling, and contamination with free dyes. Traditional methods rely on cell-based assays that lack reproducibility and accuracy. In this manuscript, we present a novel and general approach for measuring the brightness using antibody-avid polystyrene beads and flow cytometry. As compared to a cell-based method, the described technique is rapid, quantitative, and highly reproducible. The proposed method requires less than ten microgram of sample and is applicable for optimizing synthetic conjugation procedures, testing commercial imaging antibodies, and performing high-throughput validation of conjugation procedures.

  2. SU-E-I-14: Comparison of Iodine-Labeled and Indium-Labeled Antibody Biodistributions

    Energy Technology Data Exchange (ETDEWEB)

    Williams, L [Retired from City of Hope Medical Center, Arcadia, CA (United States)


    Purpose: It is often assumed that animal biodistributions of novel proteins are not dependent upon the radiolabel used in their determination. In units of percent injected dose per gram of tissue (%ID/g), organ uptake results (u) may be obtained using either iodine or metal as radioactive labels. Iodination is preferred as it is a one-step process whereas metal labeling requires two chemical procedures and therefore more protein material. It is important to test whether the radioactive tag leads to variation in the uptake value. Methods: Uptakes of 3antibodies to Carcinoembryonic Antigen (CEA) were evaluated in a nude mouse model bearing 150 to 300 mg LS174T human colon cancer xenografts. Antibodies included diabody (56 kDa), minibody (80kDa) and intact M5A (150 kDa) anti-CEA cognates. Both radioiodine and indium-111 labels were used with uptakes evaluated at 7 time(t) points out to 96 h. Ratios (R) of u(iodine-label)/u(indium-label) were determined for liver, spleen, kidneys, lung and tumor. Results: Hepatic loss was rapid for diabody and minibody; by 24 h their R values were only 2%; i.e., uptake of iodine was 2% of that of indium for these 2 antibodies. By contrast, R for the intact cognate was 50% at that time point. Splenic results were similar. Tumor uptake ratios did not depend upon the antibody type and were 50% at 24 h. Conclusions: Relatively rapid loss of iodine relative to indium in liver and spleen was observed in lower mass antibodies. Tumor ratios were larger and independent of antibody type. Aside from tumor, the R ratio of uptakes depended on the antibody type. R values decreased monotonically with time in all tissues and for all cognates. Using this ratio, one can possibly correct iodine-based u (t) results so that they resemble radiometal-derived biodistributions.

  3. Yttrium Y 90 Anti-CD45 Monoclonal Antibody BC8 Followed by Donor Stem Cell Transplant in Treating Patients With High-Risk Acute Myeloid Leukemia, Acute Lymphoblastic Leukemia, or Myelodysplastic Syndrome (United States)


    Acute Myeloid Leukemia Arising From Previous Myelodysplastic Syndrome; Chronic Myelomonocytic Leukemia; Previously Treated Myelodysplastic Syndrome; Recurrent Adult Acute Lymphoblastic Leukemia; Recurrent Adult Acute Myeloid Leukemia; Refractory Anemia With Excess Blasts; Secondary Acute Myeloid Leukemia

  4. Preparation and Biological Evaluation of 188Re Labeled Monoclonal Antibody TGLA

    Institute of Scientific and Technical Information of China (English)

    WEN; Kai; ZHANG; Jun-li; CHEN; Bao-jun; CUI; Hai-ping


    <正>Monoclonal antibody TGLA is a specific targeting CD20 chimeric antibody. It can kill tumor cells and inhibit tumor cells’ growth effectively, which has been applied to clinical therapy of lymphoma cell B. 188 Re is easy to get, and emits both β and γ rays. 188Re labeled monoclonal antibody TGLA can be used for the study of lymphoma therapy and imaging. This work got the product 188Re-TGLA by direct labeling

  5. Solid phase radioimmunoassay using labelled antibodies: a conceptual framework for designing assays

    Energy Technology Data Exchange (ETDEWEB)

    Kalmakoff, J.; Parkinson, A.J.; Crawford, A.M.; Williams, B.R.


    A simple theoretical model for the antigen-antibody reaction is presented and used to evaluate the optimum conditions for designing solid phase radioimmunoassay (RIA) using labelled antibodies. Both theoretical and experimental data are presented, using a wide variety of antigens and their corresponding antibodies. The types of RIA described include the direct, the indirect, and sandwich assays for detecting either antigen or antibody. The experimental results confirm in a semiquantitative manner that the greatest sensitivity of the RIA is achieved when the smallest amount of labelled antibody is used, that whenever possible the antigen/antibody ratio should be greater than unity (greater than 1), and that the formation of the antigen-antibody complex is dependent on the mass action effect.

  6. Pharmacokinetics of indium-111-labeled antimyosin monoclonal antibody in murine experimental viral myocarditis

    Energy Technology Data Exchange (ETDEWEB)

    Yamada, T.; Matsumori, A.; Watanabe, Y.; Tamaki, N.; Yonekura, Y.; Endo, K.; Konishi, J.; Kawai, C. (Kyoto Univ. (Japan))


    The pharmacokinetics of indium-111-labeled antimyosin monoclonal antibody Fab were investigated with use of murine experimental viral myocarditis as a model. The biodistribution of indium-111-labeled antimyosin antibody Fab on days 3, 5, 7, 14, 21 and 28 after encephalomyocarditis virus inoculation demonstrated that myocardial uptake increased significantly on days 5, 7 and 14 (maximum on day 7) in infected versus uninfected mice (p less than 0.001). In vivo kinetics in infected mice on day 7 demonstrated that the heart to blood ratio reached a maximum 48 h after the intravenous administration of indium-111-labeled antimyosin Fab, which was considered to be the optimal time for scintigraphy. The scintigraphic images obtained with indium-111-labeled antimyosin Fab demonstrated positive uptake in the cardiac lesion in infected mice. The pathologic study demonstrated that myocardial uptake correlated well with pathologic grades of myocardial necrosis. High performance liquid chromatography revealed the presence of an antigen-antibody complex in the circulation of infected mice after the injection of indium-111-labeled antimyosin Fab. This antigen bound to indium-111-labeled antimyosin Fab in the circulation might be whole myosin and this complex may decrease myocardial uptake and increase liver uptake. It is concluded that indium-111-labeled antimyosin monoclonal antibody Fab accumulates selectively in damaged heart tissue in mice with acute myocarditis and that indium-111-labeled antimyosin Fab scintigraphy may be a useful method for the visualization of acute myocarditis.

  7. Trace of antibody to myeloperoxidase with nanocrystal quantum dot labeled antibody recognizing activating neutrophils (United States)

    Hoshino, Akiyoshi; Nagao, Tomokazu; Yamamoto, Kenji; Suzuki, Kazuo


    It is assumed that activated neutrophils contribute to the development of anti-neutrophil cytoplasmic auto-antibody (ANCA)-associated vasculitis due to the association of myelopeoxidase(MPO)-ANCA with MPO expressed on the surface of activated neutrophils. FITC-labeled antibody (Ab) used widely are not suitable for neutrophil examination because of the labile fluorescence emission of FITC. Therefore, it is necessary to develop specific fluorescent probes for MPO detection in neutrophils in vivo. Recently, fluorescent nanocrystal quantum dots (QDs) have been used for biotechnological and medical applications because of their greater and far longer fluorescence in. QDs have several advantages over organic fluorophores: high luminescence, far longer stability against photobleaching, and a range of fluorescence wavelengths from blue to infrared, depending on particle size. Thus, we examined the role of MPO and the Ab to MPO in the pathogenesis of glomerulonephritis associated with MPO-ANCA in experimental glomerulonephritis mice using QDs. We demonstrated the QD-conjugated anti-MPO Ab visualized the expression of MPO on the neutrophil surface after stimulation with proinflammatory cytokines. In addition, QD immuno-conjugates with anti-recombinant murine MPO (rmMPO) Ab revealed the trafficking of MPO-ANCA in vivo. Deceleration of blood flow in kidney vessels occurred in model mice, in which serum proteins including anti-rmMPO Ab were leaked out from collapsed glomeruli into the proximal tubule. Thus, sustained MPO expression on the neutrophil surface was significantly related to glomerulonephritis. These results indicate that the expressed MPO on the activated neutrophils with anti-MPO Ab may coordinately play essential roles in the initial steps for the development of glomerulonephritis.

  8. Development and evaluation of an enzyme-labeled antibody test for the rapid detection of hog cholera antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Saunders, G.C.


    A rapid enzyme-labeled antibody (ELA) microtechnique for the screening of swine for hog cholera antibodies was developed and evaluated with a blind study, using a 640-sample hog cholera serum bank. The total time to run a group of 22 samples was approximately 1 hour. The ELA test results correlated >99% with hog cholera serum-neutralization test results on the same serums. Test results also indicated that the ELA test shares with the hog cholera serum-neutralization test the problem of cross reactions between the antibodies of hog cholera and bovine viral diarrhea.

  9. Clinical comparison of three labeled-antibody immunoassays of free triiodothyronine. (United States)

    Piketty, M L; d'Herbomez, M; Le Guillouzic, D; Lebtahi, R; Cosson, E; Dumont, A; Dilouya, A; Helal, B O


    Three labeled-antibody immunoassays of free triiodothyronine (FT3) were studied in hyperthyroid patients, patients with nonthyroidal illness, and patients being treated with amiodarone; we also studied sera presenting known interferences (n for all groups = 465). The results were compared with those of a one-step labeled-analog assay. The precision of the two automated assays were similar to that of the manual assays. The three labeled-antibody FT3 assays demonstrated a satisfactory diagnostic performance for confirming hyperthyroidism and robustness to interference; nevertheless, two assays displayed unusual behavior in some patients with nonthyroidal illness, with chronic renal failure, or after amiodarone therapy.

  10. Biodistribution Analyses of a Near-Infrared, Fluorescently Labeled, Bispecific Monoclonal Antibody Using Optical Imaging. (United States)

    Peterson, Norman C; Wilson, George G; Huang, Qihui; Dimasi, Nazzareno; Sachsenmeier, Kris F


    In recent years, biodistribution analyses of pharmaceutical compounds in preclinical animal models have become an integral part of drug development. Here we report on the use of optical imaging biodistribution analyses in a mouse xenograft model to identify tissues that nonspecifically retained a bispecific antibody under development. Although our bispecific antibody bound both the epidermal growth factor receptor and insulin growth factor 1 receptor are expressed on H358, nonsmall-cell lung carcinoma cells, the fluorescence from labeled bispecific antibody was less intense than expected in xenografted tumors. Imaging analyses of live mice and major organs revealed that the majority of the Alexa Fluor 750 labeled bispecific antibody was sequestered in the liver within 2 h of injection. However, results varied depending on which near-infrared fluorophore was used, and fluorescence from the livers of mice injected with bispecific antibody labeled with Alexa Fluor 680 was less pronounced than those labeled with Alexa Fluor 750. The tissue distribution of control antibodies remained unaffected by label and suggests that the retention of fluorophores in the liver may differ. Given these precautions, these results support the incorporation of optical imaging biodistribution analyses in biotherapeutic development strategies.

  11. Direct 99mTc labeling of monoclonal antibodies: radiolabeling and in vitro stability. (United States)

    Garron, J Y; Moinereau, M; Pasqualini, R; Saccavini, J C


    Direct labeling involves 99mTc binding to different donor groups on the protein, giving multiple binding sites of various affinities resulting in an in vivo instability. The stability has been considerably improved by activating the antibody using a controlled reduction reaction (using 2-aminoethanethiol). This reaction generates sulfhydryl groups, which are known to strongly bind 99mTc. The direct 99mTc antibody labeling method was explored using whole antibodies and fragments. Analytical methods were developed for routine evaluation of radiolabeling yield and in vitro stability. Stable direct antibody labeling with 99mTc requires the generation of sulfhydryl groups, which show high affinity binding sites for 99mTc. Such groups are obtained with 2-aminoethanethiol (AET), which induces the reduction of the intrachain or interchain disulfide bond, with no structural deterioration or any loss of immunobiological activity of the antibody. The development of fast, reliable analytical methods has made possible the qualitative and quantitative assessment of technetium species generated by the radiolabeling process. Labeling stability is determined by competition of the 99mTc-antibody bond with three ligands, Chelex 100 (a metal chelate-type resin), free DTPA solution and 1% HSA solution. Very good 99mTc-antibody stability is obtained with activated IgG (IgGa) and Fab' fragment, which makes these substances possible candidates for immunoscintigraphy use.

  12. sup 99m Tc-labelled anti NCA-95 antibodies in prosthetic heart valve endocarditis

    Energy Technology Data Exchange (ETDEWEB)

    Bair, H.J.; Becker, W.; Wolf, F. (Dept. of Nuclear Medicine, Univ. Erlangen-Nuernberg, Erlangen (Germany)); Volkholz, H.J. (Dept. of Internal Medicine 1, Univ. of Erlangen-Nuernberg, Erlangen (Germany))


    A 54-y old women with earlier replacement of the mitral and aortic valves and clinical signs of localized endocarditis was studied with {sup 99m}Tc-labelled anti NCA-95 antibody. Whereas echocardiographic findings were negative, increased radionuclide uptake was observed left parasternal over the mitral valve as a sign of prosthetic valve endocarditis. This result could be confirmed by a similar study with leukocytes labelled in vitro with {sup 111}In-oxine. (orig.).

  13. Localization of colorectal carcinoma by rhenium-188-labeled B72.3 antibody in xenografted mice

    Energy Technology Data Exchange (ETDEWEB)

    Hosono, Masako N. [Osaka City Univ. (Japan). Medical School; Hosono, Makoto; Zamora, P.O.; Guhlke, S.; Haberberger, T.; Bender, H.; Knapp, F.F.R.; Biersack, H.J.


    In order to evaluate the feasibility of {sup 188}Re-labeled antibodies for radioimmunotargeting, monoclonal antibody B72.3, recognizing TAG-72, expressed on the surface membranes of colorectal cancer cells, was directly labeled with {sup 188}Re, obtained from a {sup 188}W/{sup 188}Re generator, using stannous tartrate and compared with {sup 125}I-labeled B72.3. As a control, a human IgG was also radiolabeled with {sup 188}Re and {sup 125}I. Prepared antibodies for {sup 188}Re labeling could be stored as kits. Biodistribution was determined in nude mice inoculated with human colorectal carcinoma LoVo. Labeling efficiency and immunoreactivity of {sup 188}Re-B72.3 were 80.3% and 64.7%, respectively. {sup 188}Re-B72.3 localized specifically in the LoVo tumors. Although the absolute tumor accumulation level of {sup 188}Re-B72.3 was lower than {sup 125}I-B72.3, {sup 188}Re-B72.3 demonstrated higher tumor-to-blood contrast than the {sup 125}I-labeled counterpart, 2.04{+-}0.44 vs. 1.05{+-}0.28 at 96 hours, because of fast clearance from the blood. {sup 188}Re-B72.3 seemed efficient for the imaging and therapy of colorectal carcinoma. (author)

  14. Flow cytometric measurement of RNA synthesis using bromouridine labelling and bromodeoxyuridine antibodies

    DEFF Research Database (Denmark)

    Jensen, P O; Larsen, J; Christiansen, J;


    Nuclear RNA synthesis can be analysed by flow cytometry of cells labelled with 5-bromouridine (BrUrd) and stained with anti-bromodeoxyuridine (BrdUrd) antibody and FITC-conjugated secondary antibody. A panel of 5 different commercially available anti-BrdUrd antibodies was tested on cells of a HL-...... the variation of RNA synthesis during the cell cycle. The BrUrd incorporation was high in the S and G2 phase, variable in G1, and negligible in mitosis. Similar results were obtained using other cell types....

  15. Labeling of antibodies with a /sup 67/Ga-phenolic aminocarboxylic acid chelate. Pt. 1. Chemistry and labeling technique

    Energy Technology Data Exchange (ETDEWEB)

    Schuhmacher, J.; Matys, R.; Hauser, H.; Maier-Borst, W.; Matzku, S.


    As a chelating agent for labeling antibodies (Abs) with metallic radionuclides, a propionic acid substituted ethylenediamine N, N'-di-((o-hydroxyphenyl) acetic acid) (P-EDDHA), which tighly complexes /sup 67/Ga, was synthetized. The /sup 67/Ga-P-EDDHA chelate was coupled in aqueous solution to IgG at a molar ratio of 1:1 via carbodiimide. The average coupling yield was 15%. A specific activity of 4 mCi/mg IgG could be obtained with commercially supplied /sup 67/Ga. In vitro stability was evaluated in human serum at 37/sup 0/C and showed a half-life of about 120 h for the release of /sup 67/Ga from the labeled Ab during the initial phase of incubation. This in vitro halflife is similar to that measured for /sup 111/In-DTPA labeled Abs. Because of the high stability of the /sup 67/Ga-P-EDDHA chelate, the in vivo formation of radioactive lebeled transferrin by transchelation, as described for /sup 111/In-DTPA labeled Abs, should, however, be reduced by this labeling technique.

  16. A new monoclonal antibody radiopharmaceutical for radioimmunoscintigraphy of breast cancer: direct labeling of antibody and its quality control

    Directory of Open Access Journals (Sweden)

    Mojtaba Salouti


    Full Text Available Radioimmunoscintigraphy (RIS has found widespread clinical application in tumor diagnosis. The antibody (Ab PR81 is a new murine anti-MUC1 monoclonal antibody (MAb against human breast carcinoma. In this study a very simple, rapid and efficient method for labeling of this MAb with 99mTc, particularly suitable for development of a ‘kit’is described. The reduction of Ab was performed with 2-mercaptoethanol (2-ME at a molar ratio of 2000:1 (2-ME:MAb and the reduced Ab was labeled with 99mTc via methylene diphosphonate (MDP as a transchelator. The labeling efficiency which was determined by instant thin layer chromatography (ITLC was 94.2%±2.3. Radiocolloides measured by cellulose nitrate electrophoresis were 2.5%±1.7. In vitro stability of the labeled product in human serum which was measured by gel filtration chromatography (FPLC was 70%±5.7 over 24 hr. The integrity of labeled MAb was checked by means of SDS-PAGE and no significant fragmentation was observed. The results of the cell-binding studies showed that both labeled and unlabeled PR81 were able to compete for binding to MCF 7 cells. Biodistribution studies were performed in normal BALB/c mice at 4 and 24 hrs post-injection and no important accumulation was observed in vital organs. These results show that the new radiopharmaceutical may be considered as a promising candidate for imaging of breast cancer.

  17. Differences in tumour and normal tissue concentrations of iodine- and indium-labelled monoclonal antibody. Pt. 2

    Energy Technology Data Exchange (ETDEWEB)

    Pimm, M.V.; Baldwin, R.W.; Perkins, A.C.


    The blood, tumour and whole-body levels and survivals of /sup 131/I- and /sup 111/In-labelled monoclonal antibody (791T/36) have been compared in mice with human tumour xenografts. The blood levels and survivals of /sup 131/I- and /sup 111/In-labelled antibody were similar when expressed as proportions of the injected doses. However, the whole-body survival of /sup 111/In following administration of /sup 111/In-labelled antibody was over twice as long as that of /sup 131/I after administration of /sup 131/I-labelled antibody, principally because free /sup 131/I was rapidly excreted but free /sup 111/In was retained, primarily in liver, spleen and kidneys. Consequently, when expressed in relation to the whole body, blood levels of /sup 111/In became progressively lower than those of /sup 131/I following administration of labelled antibodies. In mice with human tumour xenografts the proportion of the injected dose of /sup 111/In from /sup 111/In-labelled antibody deposited in tumour tissue was 4-5 times higher than that of /sup 131/I from /sup 131/I-labelled antibody. When compared with the whole-body levels of radiolabel the difference was less marked, although /sup 111/In accumulation in tumour was more rapid. The higher levels and longer retention of /sup 111/In in tumour produced tumour-to-blood ratios that were 7-8 times those achieved with /sup 131/I-labelled antibody.

  18. Demonstration of immunoglobulin G in normal human epidermis by peroxidase-labeled antibody.

    Directory of Open Access Journals (Sweden)



    Full Text Available Cytoplasmic immunoglobulin G (IgG in normal human epidermis was defined by a peroxidase-labeled antibody method. A correlation between cytoplasmic staining and the serum level of IgG was found. Epidermal cells containing IgG were not present when the serum level of IgG was less than 1000 microgram/ml.

  19. Tumor scintigraphy by the method for subtracting the initial image with technetium-99m labeled antibody

    Energy Technology Data Exchange (ETDEWEB)

    Karube, Yoshiharu; Katsuno, Kentaro; Ito, Sanae; Matsunaga, Kazuhisa; Takata, Jiro [Fukuoka Univ. (Japan). Faculty of Pharmaceutical Sciences; Kuroki, Masahide; Murakami, Masaaki; Matsuoka, Yuji


    The method for subtracting the initial image from the localization image was evaluated for radioimmunoscintigraphy of tumors with technetium-99m (Tc-99m) labeled antibodies. Monoclonal antibodies were parental mouse and mouse-human chimeric antibodies to carcinoembryonic antigen (CEA), designated F11-39 and ChF11-39, respectively, both of which have been found to discriminate CEA in tumor tissues from the CEA-related antigens. After reduction of the intrinsic disulfide bonds, these antibodies were labeled with Tc-99m. In vivo studies were performed on athymic nude mice bearing the human CEA-producing gastric carcinoma xenografts. Though biodistribution results showed selective and progressive accumulation of Tc-99m labeled antibodies at the tumor site, high radioactivity in blood was inappropriate for scintigraphic visualization of the tumors within a few hours. We examined the subtraction of the initial Tc-99m image from the Tc-99m localization image after a few hours. Subtracted images of the same count reflected the in vivo behavior of the Tc-99m radioactivity. The subtracted scintigrams revealed excellent tumor images with no significant extrarenal background. Visualization of the tumor site was dependent on antigen-specific binding and nonspecific exudation. These results demonstrate that a method of subtraction of the initial image may serve as a potentially useful diagnostic method for an abnormal site for agents with a low pharmacokinetic value. (author)

  20. Yttrium-90 labeled monoclonal antibody as a potential agent for radioimmunotherapy

    Energy Technology Data Exchange (ETDEWEB)

    Doherty, P.W.; Hnatowich, D.


    The features that make /sup 90/Y an attractive candidate are: It is available as a generator (/sup 90/Sr-/sup 90/Y). It is a pure beta emitter (0.93 MeV); has a half-life of 2.7 days and is a metal ion which forms complexes with DTPA. Using an anti-CEA antibody (C-19) and DTPA (cyclic anhydride), the authors evaluated the labeling parameters, in vitro and in vivo stability, and functional integrity of the /sup 90/Y labeled C-19. The results were compared to those obtained with /sup 111/In labeled to the same antibody. Quality control showed that the /sup 90/Y complexed with free DTPA at a concentration necessary for protein labeling. Using a DTPA: antibody molar ratio of 2.5:1, a specific activity of of protein was achieved. There was no nonspecific protein binding. Affinity chromotography and HPLC analysis showed a dissociation of 15% of the /sup 90/Y from the C-19 over 72 hours in serum with preservation of functional integrity and without evidence for radiolysis. Binding to Lovo cells (known to have CEA on their surface) showed saturation kinetics. In vivo biodistribution in mice showed identical values for the /sup 90/Y and /sup 111/In labeled C-19 at 1 hour. At 24 hours, there was less kidney and liver uptake and more bone uptake in the case of /sup 90/Y. The authors conclude DTPA coupled antibodies can be stably labeled with /sup 90/Y at a specific activity appropriate for Radiotherapy.

  1. Immunoproteomics Using Polyclonal Antibodies and Stable Isotope–labeled Affinity-purified Recombinant Proteins* (United States)

    Edfors, Fredrik; Boström, Tove; Forsström, Björn; Zeiler, Marlis; Johansson, Henrik; Lundberg, Emma; Hober, Sophia; Lehtiö, Janne; Mann, Matthias; Uhlen, Mathias


    The combination of immuno-based methods and mass spectrometry detection has great potential in the field of quantitative proteomics. Here, we describe a new method (immuno-SILAC) for the absolute quantification of proteins in complex samples based on polyclonal antibodies and stable isotope–labeled recombinant protein fragments to allow affinity enrichment prior to mass spectrometry analysis and accurate quantification. We took advantage of the antibody resources publicly available from the Human Protein Atlas project covering more than 80% of all human protein-coding genes. Epitope mapping revealed that a majority of the polyclonal antibodies recognized multiple linear epitopes, and based on these results, a semi-automated method was developed for peptide enrichment using polyclonal antibodies immobilized on protein A–coated magnetic beads. A protocol based on the simultaneous multiplex capture of more than 40 protein targets showed that approximately half of the antibodies enriched at least one functional peptide detected in the subsequent mass spectrometry analysis. The approach was further developed to also generate quantitative data via the addition of heavy isotope–labeled recombinant protein fragment standards prior to trypsin digestion. Here, we show that we were able to use small amounts of antibodies (50 ng per target) in this manner for efficient multiplex analysis of quantitative levels of proteins in a human HeLa cell lysate. The results suggest that polyclonal antibodies generated via immunization of recombinant protein fragments could be used for the enrichment of target peptides to allow for rapid mass spectrometry analysis taking advantage of a substantial reduction in sample complexity. The possibility of building up a proteome-wide resource for immuno-SILAC assays based on publicly available antibody resources is discussed. PMID:24722731

  2. Immunoproteomics using polyclonal antibodies and stable isotope-labeled affinity-purified recombinant proteins. (United States)

    Edfors, Fredrik; Boström, Tove; Forsström, Björn; Zeiler, Marlis; Johansson, Henrik; Lundberg, Emma; Hober, Sophia; Lehtiö, Janne; Mann, Matthias; Uhlen, Mathias


    The combination of immuno-based methods and mass spectrometry detection has great potential in the field of quantitative proteomics. Here, we describe a new method (immuno-SILAC) for the absolute quantification of proteins in complex samples based on polyclonal antibodies and stable isotope-labeled recombinant protein fragments to allow affinity enrichment prior to mass spectrometry analysis and accurate quantification. We took advantage of the antibody resources publicly available from the Human Protein Atlas project covering more than 80% of all human protein-coding genes. Epitope mapping revealed that a majority of the polyclonal antibodies recognized multiple linear epitopes, and based on these results, a semi-automated method was developed for peptide enrichment using polyclonal antibodies immobilized on protein A-coated magnetic beads. A protocol based on the simultaneous multiplex capture of more than 40 protein targets showed that approximately half of the antibodies enriched at least one functional peptide detected in the subsequent mass spectrometry analysis. The approach was further developed to also generate quantitative data via the addition of heavy isotope-labeled recombinant protein fragment standards prior to trypsin digestion. Here, we show that we were able to use small amounts of antibodies (50 ng per target) in this manner for efficient multiplex analysis of quantitative levels of proteins in a human HeLa cell lysate. The results suggest that polyclonal antibodies generated via immunization of recombinant protein fragments could be used for the enrichment of target peptides to allow for rapid mass spectrometry analysis taking advantage of a substantial reduction in sample complexity. The possibility of building up a proteome-wide resource for immuno-SILAC assays based on publicly available antibody resources is discussed.

  3. {sup 68}Ga-labelled recombinant antibody variants for immuno-PET imaging of solid tumours

    Energy Technology Data Exchange (ETDEWEB)

    Eder, Matthias; Eisenhut, Michael [German Cancer Research Center, Radiopharmaceutical Chemistry, Heidelberg (Germany); Knackmuss, Stefan; Gall, Fabrice Le; Reusch, Uwe; Little, Melvyn [Affimed Therapeutics AG, Heidelberg (Germany); Rybin, Vladimir [European Molecular Biology Laboratory, Heidelberg (Germany); Haberkorn, Uwe; Mier, Walter [University of Heidelberg, Department of Nuclear Medicine, Heidelberg (Germany)


    Recombinant antibodies isolated from human antibody libraries have excellent affinities and high target specificity. As full-length IgGs are cleared inadequately slowly from the circulation, the aim of this work was to figure out which kind of recombinant antibody fragment proves to be appropriate for imaging epithelial cell adhesion molecule (EpCAM)-expressing tumours with the short-living radioisotope {sup 68}Ga. In order to combine the promising tumour targeting properties of antibodies with {sup 68}Ga, four antibody variants with the same specificity and origin only differing in molecular weight were constructed for comparison. Therefore, the binding domains of a single-chain fragment variable (scFv) isolated from a human naive antibody library were modified genetically to construct the respective full-length IgG, the tria- and diabody variants. These molecules were conjugated with the bifunctional chelating agent N,N{sup '}-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N{sup '}-diacetic acid (HBED-CC) to enable {sup 68}Ga labelling at ambient temperature and compared in biodistribution and immuno-PET imaging experiments. The antibody variants with identical specificity proved to have the correct molecular weight, high binding affinity and specificity to their antigen, EpCAM. Radiometal complexation was efficiently performed at room temperature leading to {sup 68}Ga-labelled antibodies with unchanged binding properties compared to the original antibody variants. The best targeting properties were obtained with the scFv and especially with the diabody. The triabody showed higher absolute tumour uptake but only moderate clearance from circulation. The antibody variants differed considerably in normal organ uptake, clearance from circulation and tumour accumulation. The data demonstrate the feasibility of imaging solid tumours with the {sup 68}Ga-labelled diabody format. This type of recombinant protein might be a promising carrier even for the

  4. Luminol/antibody labeled gold nanoparticles for chemiluminescence immunoassay of carcinoembryonic antigen

    Energy Technology Data Exchange (ETDEWEB)

    Yang Xiaoyan, E-mail: [Key Laboratory of Eco-chemical Engineering, Ministry of Education, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042 (China); Guo Yingshu; Wang Aiguo [Key Laboratory of Eco-chemical Engineering, Ministry of Education, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042 (China)


    A facile strategy by loading luminol and secondary antibody on gold nanoparticles (Au NPs) was described in the present work. The as-prepared luminol/antibody labeled Au NPs conjugates (LAAu NPs) were used as the chemiluminescent probe for the detection of carcinoembryonic antigen (CEA) in serum. The LAAu NPs were characterized by transmission electron microscopy (TEM), UV-vis spectrophotometry (UV-vis), and chemiluminescent method. Stable and efficient chemiluminescence (CL) was obtained when luminol molecules and secondary antibodies were coimmobilized on the Au NPs by using hydrogen peroxide (H{sub 2}O{sub 2}) as an oxidant, horseradish peroxidase (HRP) as a catalyst, and 4-(4'-iodo)phenylphenol (IPP) as an enhancer. The LAAu NPs were further evaluated via a sandwich-type CL immunoassay of CEA in serum. In this protocol, the CEA analyte was captured by the primary antibody immobilized on the surface of magnetic beads, and then was sandwiched by the secondary antibody loaded on luminol-labeled Au NPs. The chemiluminescent intensity was proportional to the concentration of CEA over the range of 5.0 x 10{sup -10} to 5.0 x 10{sup -8} g mL{sup -1} and 5.0 x 10{sup -9} to 2.0 x 10{sup -8} g mL{sup -1} by using HRP and Co{sup 2+} as catalysts, respectively. The present chemiluminescent immunoassay based on the luminol/antibody labeled Au NPs conjugates has offered great promise for simple, highly biocompatible, and cost-effective analysis of biological samples.

  5. Click-chemistry strategy for labeling antibodies with copper-64 via a cross-bridged tetraazamacrocyclic chelator scaffold. (United States)

    Kumar, Amit; Hao, Guiyang; Liu, Li; Ramezani, Saleh; Hsieh, Jer-Tsong; Öz, Orhan K; Sun, Xiankai


    We report a click-chemistry based modular strategy for antibody labeling with (64)Cu (t1/2 = 12.7 h; β(+) 0.656 MeV, 17.4%; β(-) 0.573 MeV, 39%; EC 43%) under ambient condition utilizing a cross-bridged tetraazamacrocyclic (CB-TE2A) analogue, which otherwise requires harsh conditions that make the CB-TE2A analogues under-utilized for protein labeling despite the fact that they form kinetically inert copper complexes with high in vivo stability. Our strategy involves prelabeling a CB-TE2A based scaffold (CB-TE2A-1C) with (64)Cu and its subsequent reaction with an antibody via the tetrazine-norbornene mediated click chemistry. The effectiveness of this strategy was demonstrated by labeling two monoclonal antibodies, an anti-PSMA antibody (YPSMA-1) and a chimeric anti-phosphatidylserine antibody (Bavituximab). The immunoreactivity of the antibodies remained unchanged after the tetrazine modification and click-chemistry (64)Cu labeling. To further demonstrate the practicality of the modular (64)Cu labeling strategy, we tested positron emission tomography (PET) imaging of tumor with the (64)Cu-labeled bavituximab in a mouse xenograft model. The tumor visualization and uptake of the labeled antibody exhibited the versatility of the click-chemistry strategy.

  6. Uptake of indium-111 in the liver of mice following administration of indium-111-DTPA-labeled monoclonal antibodies: Influence of labeling parameters, physiologic parameters, and antibody dose

    Energy Technology Data Exchange (ETDEWEB)

    Schuhmacher, J.; Klivenyi, G.; Matys, R.; Kirchgebner, H.; Hauser, H.; Maier-Borst, W.; Matzku, S. (Institute of Radiology and Pathophysiology, Heidelberg (Germany, F.R.))


    Liver uptake of indium-111 ({sup 111}In) in mice was investigated following administration of {sup 111}In-DTPA murine monoclonal antibodies ({sup 111}In-DTPA-MAbs) labeled by the cyclic anhydride method. Biodistribution of HPLC-purified {sup 111}In-DTPA-MAb preparations was checked with a low (0.2 micrograms) and a high (8.0 micrograms) MAb dose. Using Bio Gel P-30 for desalting the MAb-conjugates, {sup 111}In uptake in the liver amounted to 8%-9% of the injected dose (ID) and was independent from the MAb dose, the DTPA-to-MAb molar ratio, tumor growth and biologic variability (different MAbs and different strains of mice). Using Sephadex G-25 for desalting, 0.2 micrograms doses from 7 out of 26 preparations showed increased liver accumulation of {sup 111}In in non-tumor mice ranging from 15%-25% of ID. Corresponding high doses led to a normal value of 8%-9%. Increased liver uptake of the low dose could not be reduced by coadministration of the unconjugated MAb, but was normal after reinjection of in vivo filtered material. An inverse intracellular distribution of {sup 111}In activity between sediment and supernatant of liver homogenates, following the administration of the low and the high MAb dose, indicated an artifact of the labeling procedure rather than an inherent biological property of labeled MAbs.

  7. Dye Labelled Monoclonal Antibody Assay for Detection of Toxic Shock Syndrome Toxin -1 from Staphylococcus Aureus

    Directory of Open Access Journals (Sweden)

    V Javid Khojasteh


    Full Text Available Objective: The aim of study was to develop a rapid assay, dye labelled monoclonal antibody assay (DLMAA, using non-radioactive organic synthetic dyes for identification of Toxic Shock Syndrome Toxin-1 (TSST-1 producing strains of Staphylococcus aureus.Materials and Methods: The assay protocol required only two simple steps; addition of TSST-1 antigen to a nitrocellulose membrane and then adding a colloidal dye labelled antibody (D/A suspension detection reagent.Results: The sensitivity and specificity of the assay was determined relative to positive and negative strains compared to an ELISA assay. Overall 100% agreement was found between both assays. The sensitivity for detection of TSST-1 was 30 ng.Conclusion: The DLMAA did not require handling and disposal of radioactive materials. It is a rapid qualitative technique for detection of TSST-1 toxin at room temperature within a short time.

  8. Preparation and Characterization of {sup 177}Lu Labeled Antibody against Tyrosine Kinase Receptor Her2

    Energy Technology Data Exchange (ETDEWEB)

    Lee, So-Young; Hong, Young-Don; Choi, Sun-Ju [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)


    The tyrosine kinase receptor Her2, also known in humans as erbB2, is a member of the epidermal growth factor receptor (EGFR or erbB1) family. The Her2 is highly expressed in many cancer types and over expressed in approximately 30% of all primary breast cancer. Overexpression of Her2 is associated with a poor prognosis. Her2 is a suitable target because it involves an extracellular domain that can be targeted by antibodies produced by B cells. Based on these advantages, we tried to prepare the {sup 177}Lu labeled Her2 antibody. This radioimmunoconjugate could act by not only blocking the Her2 signalling pathway using antibody but also killing the tumour cell using {beta} energy of {sup 177}Lu.

  9. Comparison of the immunoreactivity of rituximab antibody labeled with either I-125 or Re-188 for radioimmunotherapy

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Tae Hyun; Chung, Hye Kyung; Lee, Tae Sup; Chung, Wee Sup; Woo, Kwang Sun; Lee, Myung Jin; Kim, So Yeon; Chung, Jae Ho; CHoi, Chang Woon; Lim, Sang Moo [KIRAMS, Seoul (Korea, Republic of); Darwati, Siti [National Nuclear Energy Agency, Tangerang (Indonesia)


    Monoclonal antibodies against tumor-associated antigens can be applied as delivery vehicles for radionuclides to treat tumors. The specificity of MAbs for tumor-associated antigens can be exploited to direct radionuclides selectively to tumor cells after systemic administration. In radioimmunotherapy, therapeutic efficacy depends on the choice of the radionuclide. The chemical characteristics of radioiodine and radiometals (Re-188) differ significantly with respect to labeling procedure and consequently the specificity of monoclonal antibody can be affected due to discrepancy of labeling condition. Rituximab is a genetically engineered, chimeric anti-CD20 monoclonal antibody with mouse variable and human constant region. The CD-20 itself plays an important role in human B-cell proliferation and is an effective target for immunotherapy. In the present study, we compared the immunoreactivity of I-125-labeled Rituximab with Re-188-labeled Rituximab according to radionuclide-optimized labeling condition in cell binding assay of Lindmo method.

  10. Targeting BCL1 lymphoma with anti-idiotype antibodies: biodistribution kinetics of directly labeled antibodies and bispecific antibody-targeted bivalent haptens. (United States)

    Manetti, C; Rouvier, E; Gautherot, E; Loucif, E; Barbet, J; Le Doussal, J M


    The mouse BCL1 lymphoma model has been used for evaluating immunotherapy with anti-idiotype (anti-Id) antibodies, including Id immunisation, IgG therapy and bispecific (Bs) antibody-targeted cytotoxicity. Here, we provide quantitative data on the targeting of small (25 +/- 12 mg) intrasplenic BCL1 tumours, using anti-Id IgG, F(ab')2 and anti-Id x anti-hapten BsF(ab')2 covalently labelled with 125iodine, as well as noncovalent complexes of BsF(ab')2 and 125I-labelled bivalent hapten. The results are the following: 1) up to 115% of the injected dose per gram (% ID/g) of spleen can be localised in the first hour, corresponding to approximately 600% ID/g of tumour; 2) localisation is specific for cell-surface Id; 3) optimal doses can overcome circulating Id; 4) circulating Id markedly increases the catabolism of IgG, thus impairing tumour localisation; 5) bivalent reagents are internalised by the target cells; 6) iodine covalently bound to bivalent antibodies [IgG, F(ab')2] is rapidly (T(1/2): 6-9 hr) released from the tumour; in contrast, the bivalent hapten is retained for a longer time (T(1/2): 25 hr); and 7) in the absence of bivalent hapten, the monovalent BsF(ab')2 is not rapidly internalised and dissociates from tumour cell-surface Id. Our results suggest that monovalent anti-Id, lacking Fc, can efficiently be targeted to the BCL1 tumour surface. For radioimmunotherapy, the intracellular targeting of catabolism-resistant 125I-labelled bivalent hapten provides optimal tissue selectivity.

  11. Antibody labelling of resilin in energy stores for jumping in plant sucking insects.

    Directory of Open Access Journals (Sweden)

    Malcolm Burrows

    Full Text Available The rubbery protein resilin appears to form an integral part of the energy storage structures that enable many insects to jump by using a catapult mechanism. In plant sucking bugs that jump (Hemiptera, Auchenorrhyncha, the energy generated by the slow contractions of huge thoracic jumping muscles is stored by bending composite bow-shaped parts of the internal thoracic skeleton. Sudden recoil of these bows powers the rapid and simultaneous movements of both hind legs that in turn propel a jump. Until now, identification of resilin at these storage sites has depended exclusively upon characteristics that may not be specific: its fluorescence when illuminated with specific wavelengths of ultraviolet (UV light and extinction of that fluorescence at low pH. To consolidate identification we have labelled the cuticular structures involved with an antibody raised against a product of the Drosophila CG15920 gene. This encodes pro-resilin, the first exon of which was expressed in E. coli and used to raise the antibody. We show that in frozen sections from two species, the antibody labels precisely those parts of the metathoracic energy stores that fluoresce under UV illumination. The presence of resilin in these insects is thus now further supported by a molecular criterion that is immunohistochemically specific.

  12. Detection of adriamycin cardiotoxicity with indium-111 labeled antimyosin monoclonal antibody imaging

    Energy Technology Data Exchange (ETDEWEB)

    Yamada, Takehiko; Matsumori, Akira; Tamaki, Nagara; Morishima, Shigeru; Watanabe, Yuji; Yonekura, Yoshiharu; Endo, Keigo; Konishi, Junji; Kawai, Chuichi (Kyoto Univ. (Japan). Faculty of Medicine)


    Myocardial imaging with indium-111 labeled antimyosin monoclonal antibody (antimyosin imaging) has been reported to be useful in the noninvasive detection of myocardial cell necrosis in dilated cardiomyopathy as well as in myocardial infarction and myocarditis. We used antimyosin imaging to detect myocardial damage in 2 patients with malignant lymphoma in whom adriamycin cardiotoxicity was suspected. Patients were injected with 74 MBq of indium-111 labeled antimyosin (Fab. fraction). Forty-eight hours later, planar imaging and single-photon emission computed tomography were performed using a gamma camera with a medium energy general purpose collimator. Antimyosin imaging demonstrated diffuse myocardial uptake not only in one patient with congestive heart failure but also in another patient at the early stage without congestive heart failure. Antimyosin imaging may be a sensitive method for noninvasive visualization of myocardial cell damage and useful in the early diagnosis of specific heart muscle disease. (author).

  13. {sup 90}Nb - a potential PET nuclide. Production and labeling of monoclonal antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Radchenko, V.; Roesch, F. [Mainz Univ. (Germany). Inst. of Nuclear Chemistry; Hauser, H.; Eisenhut, M. [German Cancer Research Center, Heidelberg (Germany). Radiopharmaceutical Chemistry; Vugts, D.J.; Dongen, G.A.M.S. van [VU University Medical Center, Amsterdam (Netherlands). Dept. of Nuclear Medicine and PET Research; VU University Medical Center, Amsterdam (Netherlands). Dept. of Otolaryngology/Head and Neck Surgery


    Fast progressing immuno-PET gives reasons to develop new potential medium-long and long-lived radioisotopes. One of the promising candidates is {sup 90}Nb. It has a half-life of 14.6 h, which allows visualizing and quantifying processes with medium and slow kinetics, such as tumor accumulation of antibodies and antibodies fragments or polymers and other nanoparticles. {sup 90}Nb exhibits a high positron branching of 53% and an optimal energy of {beta}{sup +} emission of E{sub mean} = 0.35 MeV only. Consequently, efficient radionuclide production routes and Nb{sup V} labeling techniques are required. {sup 90}Nb was produced by the {sup 90}Zr(p,n){sup 90}Nb nuclear reaction on natural zirconium targets. No-carrier-added (n.c.a.) {sup 90}Nb was separated from the zirconium target via a multi-step separation procedure including extraction steps and ion-exchange chromatography. Protein labeling was exemplified using the bifunctional chelator desferrioxamine attached to the monoclonal antibody rituximab. Desferrioxamine was coupled to rituximab via two different routes, by the use of N-succinyl-desferrioxamine (N-suc-Df) and by means of the bifunctional derivative p-isothiocyanatobenzyl-desferrioxamine B (Df-Bz-NCS), respectively. Following antibody modification, labeling with {sup 90}Nb was performed in HEPES buffer at pH 7 at room temperature. In vitro stability of the radiolabeled conjugates was tested in saline buffer at room temperature and in fetal calf serum (FCS) at 37 C. The selected production route led to a high yield of 145 {+-} 10 MBq/{mu}A h of {sup 90}Nb with high radioisotopic purity of > 97%. This yield may allow for large scale production of about 10 GBq {sup 90}Nb. The separation procedure resulted in 76-81% yield. The Zr/{sup 90}Nb decontamination factor reaches 10{sup 7}. Subsequent radiolabeling of the two different conjugates with {sup 90}Nb gave high yields; after one hour incubation at room temperature, more than 90% of {sup 90}Nb-Df-mAb was

  14. Realistic multi-cellular dosimetry for 177Lu-labelled antibodies: model and application (United States)

    Marcatili, S.; Pichard, A.; Courteau, A.; Ladjohounlou, R.; Navarro-Teulon, I.; Repetto-Llamazares, A.; Heyerdahl, H.; Dahle, J.; Pouget, J. P.; Bardiès, M.


    Current preclinical dosimetric models often fail to take account of the complex nature of absorbed dose distribution typical of in vitro clonogenic experiments in targeted radionuclide therapy. For this reason, clonogenic survival is often expressed as a function of added activity rather than the absorbed dose delivered to cells/cell nuclei. We designed a multi-cellular dosimetry model that takes into account the realistic distributions of cells in the Petri dish, for the establishment of survival curves as a function of the absorbed dose. General-purpose software tools were used for the generation of realistic, randomised 3D cell culture geometries based on experimentally determined parameters (cell size, cell density, cluster density, average cluster size, cell cumulated activity). A mixture of Monte Carlo and analytical approaches was implemented in order to achieve as accurate as possible results while reducing calculation time. The model was here applied to clonogenic survival experiments carried out to compare the efficacy of Betalutin®, a novel 177Lu-labelled antibody radionuclide conjugate for the treatment of non-Hodgkin lymphoma, to that of 177Lu-labelled CD20-specific (rituximab) and non-specific antibodies (Erbitux) on lymphocyte B cells. The 3D cellular model developed allowed a better understanding of the radiative and non-radiative processes associated with cellular death. Our approach is generic and can also be applied to other radiopharmaceuticals and cell distributions.

  15. Specific Fluorine Labeling of the HyHEL10 Antibody Affects Antigen Binding and Dynamics

    Energy Technology Data Exchange (ETDEWEB)

    Acchione, Mauro; Lee, Yi-Chien; DeSantis, Morgan E.; Lipschultz, Claudia A.; Wlodawer, Alexander; Li, Mi; Shanmuganathan, Aranganathan; Walter, Richard L.; Smith-Gill, Sandra; Barchi, Jr., Joseph J. (SAIC); (NCI)


    To more fully understand the molecular mechanisms responsible for variations in binding affinity with antibody maturation, we explored the use of site specific fluorine labeling and {sup 19}F nuclear magnetic resonance (NMR). Several single-chain (scFv) antibodies, derived from an affinity-matured series of anti-hen egg white lysozyme (HEL) mouse IgG1, were constructed with either complete or individual replacement of tryptophan residues with 5-fluorotryptophan ({sup 5F}W). An array of biophysical techniques was used to gain insight into the impact of fluorine substitution on the overall protein structure and antigen binding. SPR measurements indicated that {sup 5F}W incorporation lowered binding affinity for the HEL antigen. The degree of analogue impact was residue-dependent, and the greatest decrease in affinity was observed when {sup 5F}W was substituted for residues near the binding interface. In contrast, corresponding crystal structures in complex with HEL were essentially indistinguishable from the unsubstituted antibody. {sup 19}F NMR analysis showed severe overlap of signals in the free fluorinated protein that was resolved upon binding to antigen, suggesting very distinct chemical environments for each {sup 5F}W in the complex. Preliminary relaxation analysis suggested the presence of chemical exchange in the antibody-antigen complex that could not be observed by X-ray crystallography. These data demonstrate that fluorine NMR can be an extremely useful tool for discerning structural changes in scFv antibody-antigen complexes with altered function that may not be discernible by other biophysical techniques.

  16. Enzyme immunoassay of mumps virus in cell culture with peroxidase-labelled virus specific monoclonal antibodies and its application for determination of antibodies

    NARCIS (Netherlands)

    Tiel, F.H. van; Kraaijeveld, C.A.; Baller, J.; Harmsen, T.; Oosterlaken, T.A.M.; Snippe, H.


    Mumps neutralizing monoclonal antibodies (MAs) were purified and labelled with horseradish peroxidase and used to detect virus-infected Vero cells, which were seeded as monolayers in wells of 96-well plates. This direct enzyme immunoassay (EIA) in cell culture proved to be a sensitive method for det

  17. Safety and Efficacy of 188-Rhenium-Labeled Antibody to Melanin in Patients with Metastatic Melanoma

    Directory of Open Access Journals (Sweden)

    M. Klein


    Full Text Available There is a need for effective “broad spectrum” therapies for metastatic melanoma which would be suitable for all patients. The objectives of Phase Ia/Ib studies were to evaluate the safety, pharmacokinetics, dosimetry, and antitumor activity of 188Re-6D2, a 188-Rhenium-labeled antibody to melanin. Stage IIIC/IV metastatic melanoma (MM patients who failed standard therapies were enrolled in both studies. In Phase Ia, 10 mCi 188Re-6D2 were given while unlabeled antibody preload was escalated. In Phase Ib, the dose of 188Re-6D2 was escalated to 54 mCi. SPECT/CT revealed 188Re-6D2 uptake in melanoma metastases. The mean effective half-life of 188Re-6D2 was 12.4 h. Transient HAMA was observed in 9 patients. Six patients met the RECIST criteria for stable disease at 6 weeks. Two patients had durable disease stabilization for 14 weeks and one for 22 weeks. Median overall survival was 13 months with no dose-limiting toxicities. The data demonstrate that 188Re-6D2 was well tolerated, localized in melanoma metastases, and had antitumor activity, thus warranting its further investigation in patients with metastatic melanoma.

  18. Label-free interaction analysis as a tool to demonstrate biosimilarity of therapeutic monoclonal antibodies

    Directory of Open Access Journals (Sweden)

    Sinha-Datta U


    Full Text Available Uma Sinha-Datta, Srijit Khan, Dhananjay WadgaonkarFast Trak, GE Healthcare Life Science, John F Welch Technology Centre, Bangalore, IndiaAbstract: In the biosimilar eon, where various analytical platforms are needed to show biosimilarity, we demonstrate the use of surface plasmon resonance biosensor as a label-free interaction analysis tool to compare two therapeutic monoclonal antibodies (mAb1-i and mAb2-i with their biosimilars (mAb1-B and mAb2-B1, B2, B3 based on kinetics, affinity, and thermal stability studies. We calculate active analyte concentration using Biacore systems' calibration-free concentration analysis method and demonstrate its importance for kinetic evaluation. The kinetic constants (ka and kd and affinity constant (KD of the mAbs for binding to specific antigens were evaluated. It was found that the biosimilars were very similar to their innovator with respect to binding to its antigen demonstrating functional similarity. To further confirm biosimilarity to the originator molecules, we conducted a thermal stability analysis of both mAbs using differential scanning calorimetry. This analysis showed good structural similarity in between innovator antibodies and biosimilars, with major Tm as 84.1°C (mAb1 and 72.8°C (mAb2, demonstrating structural similarity.Keywords: surface plasmon resonance, Biacore, kinetics, low level of ligand immobilization, calibration-free concentration analysis, bivalent analytes, active concentration

  19. Fast and single-step immunoassay based on fluorescence quenching within a square glass capillary immobilizing graphene oxide-antibody conjugate and fluorescently labelled antibody. (United States)

    Shirai, Akihiro; Henares, Terence G; Sueyoshi, Kenji; Endo, Tatsuro; Hisamoto, Hideaki


    A single-step, easy-to-use, and fast capillary-type immunoassay device composed of a polyethylene glycol (PEG) coating containing two kinds of antibody-reagents, including an antibody-graphene oxide conjugate and fluorescently labelled antibody, was developed in this study. The working principle involved the spontaneous dissolution of the PEG coating, diffusion of reagents, and subsequent immunoreaction, triggered by the capillary action-mediated introduction of a sample solution. In a sample solution containing the target antigen, two types of antibody reagents form a sandwich-type antigen-antibody complex and fluorescence quenching takes place via fluorescence resonance energy transfer between the labelled fluorescent molecules and graphene oxide. Antigen concentration can be measured based on the decrease in fluorescence intensity. An antigen concentration-dependent response was obtained for the model target protein sample (human IgG, 0.2-10 μg mL(-1)). The present method can shorten the reaction time to within 1 min (approximately 40 s), while conventional methods using the same reagents require reaction times of approximately 20 min because of the large reaction scale. The proposed method is one of the fastest immunoassays ever reported. Finally, the present device was used to measure human IgG in diluted serum samples to demonstrate that this method can be used for fast medical diagnosis.

  20. Evaluation of near infrared fluorescent labeling of monoclonal antibodies as a tool for tissue distribution. (United States)

    Conner, Kip P; Rock, Brooke M; Kwon, Gayle K; Balthasar, Joseph P; Abuqayyas, Lubna; Wienkers, Larry C; Rock, Dan A


    The pharmacokinetic (PK) behavior of monoclonal antibodies (mAbs) is influenced by target-mediated drug disposition, off-target effects, antidrug antibody-mediated clearance, and interaction with fragment-crystallizable domain (Fc) receptors such as neonatal Fc receptor. All of these interactions hold the potential to impact mAb biodistribution. Near infrared (NIR) fluorescent probes offer an approach complementary to radionuclides to characterize drug disposition. Notably, the use of FDA-approved IRDye800 (IR800; LI-COR, Lincoln, NE) as a protein-labeling agent in preclinical work holds the potential for quantitative tissue analysis. Here, we tested the utility of the IR800 dye as a quantitative mAb tracer during pharmacokinetic analysis in both plasma and tissues using a model mouse monoclonal IgG1 (8C2) labeled with ≤1.5 molecules of IR800. The plasma PK parameters derived from a mixture of IR800-8C2 and 8C2 dosed intravenously to C57BL/6 mice at 8 mg/kg exhibited a large discrepancy in exposure depending on the method of quantitation [CLplasma = 8.4 ml/d per kilogram (NIR fluorescence detection) versus 2.5 ml/d per kilogram (enzyme-linked immunosorbent assay)]. The disagreement between measurements suggests that the PK of 8C2 is altered by addition of the IR800 dye. Additionally, direct fluorescence analysis of homogenized tissues revealed several large differences in IR800-8C2 tissue uptake when compared with a previously published study using [(125)I]8C2, most notably an over 4-fold increase in liver concentration. Finally, the utility of IR800 in combination with whole body imaging was examined by comparison of IR800-8C2 levels observed in animal sagittal cross-sections to those measured in homogenized tissues. Our results represent the first PK analysis in both mouse plasma and tissues of an IR800-mAb conjugate and suggest that mAb disposition is significantly altered by IR800 conjugation to 8C2.

  1. Label Free QCM Immunobiosensor for AFB1 Detection Using Monoclonal IgA Antibody as Recognition Element

    Directory of Open Access Journals (Sweden)

    Özlem Ertekin


    Full Text Available This study introduces the use of an IgA isotype aflatoxin (AF specific monoclonal antibody for the development of a highly sensitive Quartz Crystal Microbalance (QCM immunobiosensor for the detection of AF in inhibitory immunoassay format. The higher molecular weight of IgA antibodies proved an advantage over commonly used IgG antibodies in label free immunobiosensor measurements. IgA and IgG antibodies with similar affinity for AF were used in the comparative studies. Sensor surface was prepared by covalent immobilization of AFB1, using self assembled monolayer (SAM formed on gold coated Quartz Crystal, with 1-Ethyl-3-(3-dimethylaminopropyl carbodiimide/N-hydroxy succinimide (EDC/NHS method using a diamine linker. Nonspecific binding to the surface was decreased by minimizing the duration of EDC/NHS activation. Sensor surface was chemically blocked after AF immobilization without any need for protein blocking. This protein free sensor chip endured harsh solutions with strong ionic detergent at high pH, which is required for the regeneration of the high affinity antibody-antigen interaction. According to the obtained results, the detection range with IgA antibodies was higher than IgG antibodies in QCM immunosensor developed for AFB1.

  2. Screening for epitope specificity directly on culture supernatants in the early phase of monoclonal antibody production by an ELISA with biotin-labeled antigen

    DEFF Research Database (Denmark)

    Andersen, Ditte C; Jensen, Charlotte H; Gregersen, Annemette;


    This report describes an assay for comparison of epitope specificity in groups of monoclonal antibodies against a given antigen. The only prerequisite is the biotin-labeled antigen. One of the monoclonal antibodies is captured onto a plastic surface via a rabbit anti-mouse Ig, and the other...... preincubated with biotinylated antigen. When the two antibodies react with the same epitope subsequent binding of the biotin-labeled antigen is abolished (inhibition). In the cases where no inhibition was observed, the two antibodies were considered to react with distinct, independent epitopes. The obvious...

  3. Application of {sup 99m}Tc-labeled chimeric Fab fragments of monoclonal antibody A7 for radioimmunoscintigraphy of pancreatic cancer

    Energy Technology Data Exchange (ETDEWEB)

    Matsumura, Hiroomi [Kyoto Prefectural Univ. of Medicine (Japan)


    Pancreatic cancer is one of the most lethal diseases and its prognosis is still poor. To improve the survival rate, it is essential to develop new technologies for early and definitive diagnosis. In this study, chimeric Fab fragments of monoclonal antibody A7 were successfully radio-labeled with {sup 99m}Tc, preventing depression of the antigen-binding activity. {sup 99m}Tc-labeled monoclonal antibody A7, {sup 99m}Tc-labeled chimeric Fab fragments of monoclonal antibody A7, {sup 99m}Tc-labeled normal mouse IgG and {sup 99m}Tc-labeled Fab fragments of normal mouse IgG were injected intravenously into nude mice bearing human pancreatic cancer xenografts and the radioactivity was subsequently measured. The tumor accumulation was significantly higher with labeled monoclonal antibody A7 than with normal mouse IgG, and higher with chimeric Fab fragments of monoclonal antibody A7 than with Fab fragments of normal mouse IgG. The tumor/blood ratio of radioactivity increased rapidly over time with chimeric Fab fragments of monoclonal antibody A7. These results suggest that chimeric Fab fragments of monoclonal antibody A7 may be useful for diagnosing pancreatic cancer by means of radioimmunoscintigraphy. (author)

  4. {sup 99m}Tc-labeled chimeric anti-NCA 95 antigranulocyte monoclonal antibody for bone marrow imaging

    Energy Technology Data Exchange (ETDEWEB)

    Sarwar, M.; Higuchi, Tetsuya; Tomiyoshi, Katsumi [Gunma Univ., Maebashi (Japan). School of Medicine] [and others


    Chimeric mouse-human antigranulocyte monoclonal antibody (ch MAb) against non-specific cross-reacting antigen (NCA-95) was labeled with {sup 99m}Tc (using a direct method) and {sup 125}I (using the chloramine T method), and its binding to human granulocytes and LS-180 colorectal carcinoma cells expressing carcinoembryonic antigen on their surfaces, cross-reactive with anti-NCA-95 chimeric monoclonal antibody, increased in proportion to the number of cells added and reached more than 80% and 90%, respectively. In biodistribution studies, {sup 99m}Tc and {sup 125}I-labeled ch anti-NCA-95 MAb revealed high tumor uptake, and the tumor-to-blood ratio was 2.9 after 24 hours. The tumor-to-normal-organ ratio was also more than 3.0 in all organs except for the tumor-to-kidney ratio. Scintigrams of athymic nude mice confirmed the results of biodistribution studies that showed higher radioactivity in tumor and kidney of the mice administered with {sup 99m}Tc-labeled ch MAb. A normal volunteer injected with {sup 99m}Tc-labeled ch anti-NCA-95 antigranulocyte MAb showed clear bone marrow images, and a patient with aplastic anemia revealed irregular uptake in his lumbar spine, suggesting its utility for bone marrow scintigraphy and for the detection of hematological disorders, infections, and bone metastasis. (author)

  5. A method for triple fluorescence labeling with Vicia villosa agglutinin, an anti-parvalbumin antibody and an anti-G-protein-coupled receptor antibody. (United States)

    Bausch, S B


    The aim of the original study [S.B. Bausch, C. Chavkin, Vicia villosa agglutinin labels a subset of neurons coexpressing both the mu opioid receptor and parvalbumin in the developing rat subiculum, Dev. Brain Res., 97, 1996, 169-177] [3] was to develop a method for identifying a subset of mu opioid receptor-expressing interneurons in the rat subiculum for electrophysiological studies. Previous studies had shown that a subset of parvalbumin-positive neurons in the rat subiculum could be labeled with the lectin, Vicia villosa agglutinin (VVA) [C.T. Drake, K.A. Mulligan, T.L. Wimpey, A. Hendrickson, C. Chavkin, Characterization of Vicia villosa agglutinin-labeled GABAergic neurons in the hippocampal formation and in acutely dissociated hippocampus, Brain Res., 554, 1991, 176-185] [11], and that mu opioid receptor immunoreactivity (-IR) and parvalbumin-IR were colocalized in a subset of neurons in the hippocampus and dentate gyrus [S.B. Bausch, C. Chavkin, Colocalization of mu and delta opioid receptors with GABA, parvalbumin and a G-protein-coupled inwardly rectifying potassium channel in the rodent brain, Analgesia, 1, 1995, 282-285] [2]. We hypothesized that a subset of mu opioid receptor-expressing neurons in the subiculum also would express the calcium binding protein, parvalbumin, and could be labeled with VVA. Labeling of live neurons with VVA [11] then could be used to identify these neurons. This protocol was designed to triple-label neurons expressing the mu opioid receptor, parvalbumin and the carbohydrate group, N-acetylgalactosamine (which binds VVA [S.E. Tollefsen, R. Kornfeld, The B4 lectin from Vicia villosa seeds interacts with N-acetylgalactosamine residues alpha-linked to serine or threonine residues in cell surface glycoproteins, J. Biol. Chem., 258, 1983, 5172-5176][M.P. Woodward, W.W. Young, R.A. Bloodgood, Detection of monoclonal antibodies specific for carbohydrate epitopes using periodate oxidation, J. Immunol. Methods, 78, 1985, 143-153] [25

  6. Site-specific labeling of cysteine-tagged camelid single-domain antibody-fragments for use in molecular imaging. (United States)

    Massa, Sam; Xavier, Catarina; De Vos, Jens; Caveliers, Vicky; Lahoutte, Tony; Muyldermans, Serge; Devoogdt, Nick


    Site-specific labeling of molecular imaging probes allows the development of a homogeneous tracer population. The resulting batch-to-batch reproducible pharmacokinetic and pharmacodynamic properties are of great importance for clinical translation. Camelid single-domain antibody-fragments (sdAbs)-the recombinantly produced antigen-binding domains of heavy-chain antibodies, also called Nanobodies-are proficient probes for molecular imaging. To safeguard their intrinsically high binding specificity and affinity and to ensure the tracer's homogeneity, we developed a generic strategy for the site-specific labeling of sdAbs via a thio-ether bond. The unpaired cysteine was introduced at the carboxyl-terminal end of the sdAb to eliminate the risk of antigen binding interference. The spontaneous dimerization and capping of the unpaired cysteine required a reduction step prior to conjugation. This was optimized with the mild reducing agent 2-mercaptoethylamine in order to preserve the domain's stability. As a proof-of-concept the reduced probe was subsequently conjugated to maleimide-DTPA, for labeling with indium-111. A single conjugated tracer was obtained and confirmed via mass spectrometry. The specificity and affinity of the new sdAb-based imaging probe was validated in a mouse xenograft tumor model using a modified clinical lead compound targeting the human epidermal growth factor receptor 2 (HER2) cancer biomarker. These data provide a versatile and standardized strategy for the site-specific labeling of sdAbs. The conjugation to the unpaired cysteine results in the production of a homogeneous group of tracers and is a multimodal alternative to the technetium-99m labeling of sdAbs.

  7. Immunoscintigraphy and radioimmunotherapy in Cuba: experiences with labeled monoclonal antibodies for cancer diagnosis and treatment (1993-2013). (United States)

    Peña, Yamilé; Perera, Alejandro; Batista, Juan F


    INTRODUCTION The availability of monoclonal antibodies in Cuba has facilitated development and application of innovative techniques (immunoscintigraphy and radioimmunotherapy) for cancer diagnosis and treatment. Objective Review immunoscintigraphy and radioimmunotherapy techniques and analyze their use in Cuba, based on the published literature. In this context, we describe the experience of Havana's Clinical Research Center with labeled monoclonal antibodies for cancer diagnosis and treatment during the period 1993-2013. EVIDENCE ACQUISITION Basic concepts concerning cancer and monoclonal antibodies were reviewed, as well as relevant international and Cuban data. Forty-nine documents were reviewed, among them 2 textbooks, 34 articles by Cuban authors and 13 by international authors. All works published by the Clinical Research Center from 1993 through 2013 were included. Bibliography was obtained from the library of the Clinical Research Center and Infomed, Cuba's national health telematics network, using the following keywords: monoclonal antibodies, immunoscintigraphy and radioimmunotherapy. RESULTS Labeling the antibodies (ior t3, ior t1, ior cea 1, ior egf/r3, ior c5, h-R3, 14F7 and rituximab) with radioactive isotopes was a basic line of research in Cuba and has fostered their use as diagnostic and therapeutic tools. The studies conducted demonstrated the good sensitivity and diagnostic precision of immunoscintigraphy for detecting various types of tumors (head and neck, ovarian, colon, breast, lymphoma, brain). Obtaining different radioimmune conjugates with radioactive isotopes such as 99mTc and 188Re made it possible to administer radioimmunotherapy to patients with several types of cancer (brain, lymphoma, breast). The objective of 60% of the clinical trials was to determine pharmacokinetics, internal dosimetry and adverse effects of monoclonal antibodies, as well as tumor response; there were few adverse effects, no damage to vital organs, and a positive

  8. A Quick and Parallel Analytical Method Based on Quantum Dots Labeling for ToRCH-Related Antibodies

    Directory of Open Access Journals (Sweden)

    Li Ding


    Full Text Available Abstract Quantum dot is a special kind of nanomaterial composed of periodic groups of II–VI, III–V or IV–VI materials. Their high quantum yield, broad absorption with narrow photoluminescence spectra and high resistance to photobleaching, make them become a promising labeling substance in biological analysis. Here, we report a quick and parallel analytical method based on quantum dots for ToRCH-related antibodies including Toxoplasma gondii, Rubella virus, Cytomegalovirus and Herpes simplex virus type 1 (HSV1 and 2 (HSV2. Firstly, we fabricated the microarrays with the five kinds of ToRCH-related antigens and used CdTe quantum dots to label secondary antibody and then analyzed 100 specimens of randomly selected clinical sera from obstetric outpatients. The currently prevalent enzyme-linked immunosorbent assay (ELISA kits were considered as “golden standard” for comparison. The results show that the quantum dots labeling-based ToRCH microarrays have comparable sensitivity and specificity with ELISA. Besides, the microarrays hold distinct advantages over ELISA test format in detection time, cost, operation and signal stability. Validated by the clinical assay, our quantum dots-based ToRCH microarrays have great potential in the detection of ToRCH-related pathogens.

  9. A new application of scanning electrochemical microscopy for the label-free interrogation of antibody-antigen interactions

    Energy Technology Data Exchange (ETDEWEB)

    Holmes, Joanne L.; Davis, Frank; Collyer, Stuart D. [Cranfield Health, Cranfield University, Cranfield, MK43 0AL (United Kingdom); Higson, Seamus P.J., E-mail: [Cranfield Health, Cranfield University, Cranfield, MK43 0AL (United Kingdom)


    Within this work we present a 'proof of principle' study for the use of scanning electrochemical microscopy (SECM) to detect and image biomolecular interactions in a label-free assay as a potential alternative to current fluorescence techniques. Screen-printed carbon electrodes were used as the substrate for the deposition of a dotted array, where the dots consist of biotinylated polyethyleneimine. These were then further derivatised, first with neutravidin and then with a biotinylated antibody to the protein neuron specific enolase (NSE). SECM using a ferrocene carboxylic acid mediator showed clear differences between the array and the surrounding unmodified carbon. Imaging of the arrays before and following exposure to various concentrations of the antigen showed clear evidence for specific binding of the NSE antigen to the antibody derivatised dots. Non-specific binding was quantified. Control experiments with other proteins showed only non-specific binding across the whole of the substrate, thereby confirming that specific binding does occur between the antibody and antigen at the surface of the dots. Binding of the antigen was accompanied by a measured increase in current response, which may be explained in terms of protein electrostatic interaction and hydrophobic interactions to the mediator, thereby increasing the localised mediator flux. A calibration curve was obtained between 500 fg mL{sup -1} to 200 pg mL{sup -1} NSE which demonstrated a logarithmic relationship between the current change upon binding and antigen concentration without the need for any labelling of the substrate.

  10. A Novel PET Imaging Using 64Cu-Labeled Monoclonal Antibody against Mesothelin Commonly Expressed on Cancer Cells

    Directory of Open Access Journals (Sweden)

    Kazuko Kobayashi


    Full Text Available Mesothelin (MSLN is a 40-kDa cell differentiation-associated glycoprotein appearing with carcinogenesis and is highly expressed in many human cancers, including the majority of pancreatic adenocarcinomas, ovarian cancers, and mesotheliomas, while its expression in normal tissue is limited to mesothelial cells lining the pleura, pericardium, and peritoneum. Clone 11-25 is a murine hybridoma secreting monoclonal antibody (mAb against human MSLN. In this study, we applied the 11-25 mAb to in vivo imaging to detect MSLN-expressing tumors. In in vitro and ex vivo immunochemical studies, we demonstrated specificity of 11-25 mAb to membranous MSLN expressed on several pancreatic cancer cells. We showed the accumulation of Alexa Fluor 750-labeled 11-25 mAb in MSLN-expressing tumor xenografts in athymic nude mice. Then, 11-25 mAb was labeled with 64Cu via a chelating agent DOTA and was used in both in vitro cell binding assay and in vivo positron emission tomography (PET imaging in the tumor-bearing mice. We confirmed that 64Cu-labeled 11-25 mAb highly accumulated in MSLN-expressing tumors as compared to MSLN-negative ones. The 64Cu-labeled 11-25 mAb is potentially useful as a PET probe capable of being used for wide range of tumors, rather than 18F-FDG that occasionally provides nonspecific accumulation into the inflammatory lesions.

  11. Quinone-Based Polymers for Label-Free and Reagentless Electrochemical Immunosensors: Application to Proteins, Antibodies and Pesticides Detection

    Directory of Open Access Journals (Sweden)

    Minh-Chau Pham


    Full Text Available Polyquinone derivatives are widely recognized in the literature for their remarkable properties, their biocompatibility, simple synthesis, and easy bio-functionalization. We have shown that polyquinones present very stable electroactivity in neutral aqueous medium within the cathodic potential domain avoiding side oxidation of interfering species. Besides, they can act as immobilized redox transducers for probing biomolecular interactions in sensors. Our group has been working on devices based on such modified electrodes with a view to applications for proteins, antibodies and organic pollutants using a reagentless label-free electrochemical immunosensor format. Herein, these developments are briefly reviewed and put into perspective.

  12. A rapid radioimmunoassay using /sup 125/I-labeled staphylococcal protein A for antibody to varicella-zoster virus

    Energy Technology Data Exchange (ETDEWEB)

    Richman, D.D.; Cleveland, P.H.; Oxman, M.N.; Zaia, J.A.


    A sensitive radioimmunoassay for serum antibody to varicella-zoster virus is described; it uses 125I-labeled staphylococcal protein A and a specially designed immunofiltration apparatus. The assay accurately distinguishes between individuals who are susceptible and those who are immune to infection with varicella-zoster virus. In addition, it can detect passive antibody in recipients of varicella-zoster immune globulin. This radioimmunoassay also detects the heterologous antibody responses that occasionally occur in patients infected with herpes simplex virus, which also have been detected by other antibody assays. The particular advantages of this assay are the use of noninfectious reagents, the speed of execution (less than 3 hr), the requirement for only small quantities of serum (30 microliters), the objectivity of end-point determination, and the capability of screening large numbers of sera. Consequently, this radioimmunoassay is especially useful for the rapid identification of susceptible individuals, which is essential for the appropriate management of patients and hospital personnel after exposure to varicella.

  13. A CCD-based reader combined quantum dots-labeled lateral flow strips for ultrasensitive quantitative detection of anti-HBs antibody. (United States)

    Zhang, Xueqing; Li, Ding; Wang, Can; Zhi, Xiao; Zhang, Chunlei; Wang, Kan; Cui, Daxiang


    Herein we reported a CCD-based reader combined quantum dots-labeled lateral flow strips for ultrasensitive quantitative detection of anti-HBs antibody. The CdTe quantum dots were prepared, then were used to label Hepatitis B Virus surface antigen, and then were fabricated into lateral flow strips. The as-prepared lateral flow strips were used to test different concentration of anti-HBV surface antibodies. The CCD-based reader was designed and fabricated, the quantitative analysis software was compiled, and resultant CCD-based reader system was used for quantitative analysis of examined anti-HBs antibodies on the strips. Results showed that the quantum dots-labeled lateral flow strips could detect the anti-HBs antibody with the limitation concentration of 200 pg/mL, the CCD-based reader system could detect anti-HBs antibody with the sensitivity of 2 pg/mL. In conclusion, the prepared CCD-based reader combined quantum dots-labeled lateral flow strips can be used for quantitative detection of anti-HBs antibody in sera with the sensitivity of 2 pg/mL, and has great potential in applications such as ultrasensitive detection of HBV antigens or antibodies, and other tumor biomarkers in near future.

  14. Flow cytometric immunophenotyping of feline bone marrow cells and haematopoietic progenitor cells using anti-human antibodies. (United States)

    Araghi, Atefeh; Nassiri, Seyed Mahdi; Atyabi, Nahid; Rahbarghazi, Reza; Mohammadi, Elham


    There is a paucity of species-specific antibodies available for feline haematopoietic conditions. The purpose of this study was to broaden the panel of antibodies available for use in the immunophenotypic characterisation of feline haematopoietic cells by testing clones of anti-human monoclonal antibodies (mAbs) on normal, neoplastic and cultured feline haematopoietic progenitors to determine cross-reactivity to feline counterparts. In this study, 24 clones of anti-human mAbs were tested on normal or neoplastic feline bone marrow and peripheral blood cells. Six of these mAbs, including anti-cluster of differentiation (CD)61, anti-CD18, anti-CD14, anti-CD235a, anti-CD41 and anti-CD29, cross-reacted with normal feline bone marrow cells, whereas anti-CD33 and anti-CD117 cross-reacted with the blast cells in the bone marrow of two cats with myelodysplastic syndrome, and anti-CD71, anti-235a, anti-41 and anti-42 cross-reacted with immature erythroid cells in a cat with erythroleukaemia. In a feline immunodeficiency virus-positive cat, bone marrow cells were labelled with anti-CD33, anti-14 and anti-45. Anti-CD18, anti-CD14, anti-CD41 and anti-CD61 also reacted with the peripheral blood cells of the healthy cats. The feline haematopoietic progenitors formed colonies in the methylcellulose-based semisolid medium with significant enrichment of colony-forming unit-granulocyte, monocyte and burst-forming unit-erythroid. A panel of six anti-feline mAbs (anti-CD21-like, anti-T lymphocytes, anti-CD172a, anti-granulocyte, anti-CD45-like and anti-CD18) and eight anti-human antibodies (anti-CD71, anti-CD33, anti-CD235a, anti-CD41, anti-CD61, anti-CD117, anti-CD38 and anti-CD34) were used for the immunophenotypic characterisation of the feline bone marrow progenitors. CD45, CD33, CD235a and CD18 were expressed by the feline haematopoietic progenitor cells, with the highest expression level for CD45.

  15. Antibody (United States)

    An antibody is a protein produced by the body's immune system when it detects harmful substances, called antigens. Examples ... microorganisms (bacteria, fungi, parasites, and viruses) and chemicals. Antibodies may be produced when the immune system mistakenly ...

  16. In Vivo Molecular Imaging to Diagnose and Subtype Tumors through Receptor-Targeted Optically Labeled Monoclonal Antibodies

    Directory of Open Access Journals (Sweden)

    Yoshinori Koyama


    Full Text Available Molecular imaging of cell surface receptors can potentially diagnose tumors based on their distinct expression profiles. Using multifilter spectrally resolved optical imaging with three fluorescently labeled antibodies, we simultaneously imaged three different cell surface receptors to distinguish tumor types noninvasively. We selected tumors overexpressing different subtypes of EGFR receptor: HER-1 (A431 and HER-2 (NIH3T3/HER2+, or interleukin-2 receptor α-subunit receptor (IL-2Rα; SP2/Tac. After tumor establishment, a cocktail of three fluorescently labeled monoclonal antibodies was injected: cetuximab-Cy5 (targeting HER-1, trastuzumab-Cy7 (HER-2, daclizumab-AIexaFluor700 (IL-2Ra. Optical fluorescence imaging was performed after 24 hours with both a red filter set and three successive filter sets (yellow, red, deep red. Spectrally resolved imaging of 10 mice clearly distinguished A431, NIH3T3/HER2+, SP2-Tac tumors based on their distinct optical spectra. Three-filter sets significantly increased the signal-to-background ratio compared to a single-filter set by reducing the background signal, thus significantly improving the differentiation of each of the receptors targeted (P < .022. In conclusion, following multifilter spectrally resolved imaging, different tumor types can be simultaneously distinguished and diagnosed in vivo. Multiple filter sets increase the signal-to-noise ratio by substantially reducing the background signal, may allow more optical dyes to be resolved within the narrow limits of the near-infrared spectrum.

  17. An Ultrasensitive Electrochemical Immunosensor for Alpha-Fetoprotein Using an Envision Complex-Antibody Copolymer as a Sensitive Label

    Directory of Open Access Journals (Sweden)

    Lei Zheng


    Full Text Available A novel strategy is presented for sensitive detection of alfa-fetoprotein (AFP, using a horseradish peroxidase (HRP-functionalized Envision antibody complex (EVC as the label. The Envision-AFP signal antibody copolymer (EVC-AFP Ab2 was composed of a dextran amine skeleton anchoring more than 100 molecules of HRP and 15 molecules of secondary antibody, and acted as a signal tag in the immunosensor. The sensor was constructed using the following steps: First, gold electrode (GE was modified with nano-gold (AuNPs by electro-deposition in HAuCl4 solution. The high affinity of the AuNPs surface facilitates direct formation of a self-assembled thiolated protein G layer. Next, the coated GE was incubated in a solution of AFP capture antibody (AFP Ab1; these antibodies attach to the thiolated protein G layer through their non-antigenic regions, leaving the antigen binding sites for binding of target analyte. Following a sandwich immunoreaction, an EVC-AFP Ab2-AFP-AFP Ab1 immunocomplex was formed on the electrode surface, allowing large amounts of HRP on the complex to produce an amplified electrocatalytic current of hydroquinone (HQ in the presence of hydrogen peroxide (H2O2. Highly amplified detection was achieved, with a detection limit of 2 pg/mL and a linear range of 0.005–0.2 ng/mL for AFP in 10 μL undiluted serum; this is near or below the normal levels of most cancer biomarker proteins in human serum. Measurements of AFP in the serum of cancer patients correlated strongly with standard enzyme-linked immunosorbent assays. These easily fabricated EVC-modified immunosensors show excellent promise for future fabrication of bioelectronic arrays. By varying the target biomolecules, this technique may be easily extended for use with other immunoassays, and thus represents a versatile design route.

  18. Pretargetted imaging of colorectal cancer recurrences using an 111In-labelled bivalent hapten and a bispecific antibody conjugate. (United States)

    Chetanneau, A; Barbet, J; Peltier, P; Le Doussal, J M; Gruaz-Guyon, A; Bernard, A M; Resche, I; Rouvier, E; Bourguet, P; Delaage, M


    In 11 patients recurrence of colorectal cancer was suspected by a rise in serum carcinoembryonic antigen (CEA) (nine cases), by a subocclusive clinical situation (one case) or by endoscopy (on an anastomosis, one case). Two-step tumour targetting was performed by a first injection of 0.1 mg kg-1 of unlabelled bispecific antibody conjugate (an anti-CEA Fab' fragment chemically coupled to an anti-diethylene triamine pentaacetate (DTPA)-indium fragment) followed 4 to 5 days later by injection of the bivalent DTPA hapten labelled with 5 to 8 mCi 111In. Planar scintigraphy, single photon emission computed tomographic (SPECT) 360 degrees acquisitions and whole-body scans were obtained 4.5 and 24 h after injection of the radiolabelled hapten. Biodistribution was determined for eight patients at 48 h. The final diagnosis was confirmed histologically in nine patients (eight by second-look surgery, one by laparotomy). Overall, results were one true negative (1-year follow-up) and 10 true positive; however, for the three large liver metastases (3 to 6 cm), only the periphery of the metastasis had high uptake compared to normal liver. For pelvic recurrences, immunoscintigraphic (IS) contrast was better for small tumours. The highest tumour uptake was found for a 1 cm diameter pelvic recurrence (7.2% i.d. kg-1). Mean tumour-to-blood ratios were 6.4. Thus, this two-step tumour targetting technique, which uses a bispecific antibody conjugate and an 111In-labelled bivalent hapten injected sequentially without chasing the excess bispecific antibody, provided satisfactory results in this preliminary clinical trial for detection of recurrent colorectal cancers.

  19. Immunoscintigraphy using /sup 111/In-DTPA labeled monoclonal antibodies: Comparison between ETC and planar imaging

    Energy Technology Data Exchange (ETDEWEB)

    Happ, J.; Baum, R.P.; Frohn, J.; Weimer, B.; Hoer, G.; Halbsguth, A.; Lochner, B.; Brandhorst, I.


    The present study was done in order to examine if the use of /sup 111/In-DTPA-labeled MAb fragments in place of /sup 131/I-labeled MAb fragments increases the sensitivity of tomographic immunoscintigraphy to reach the level of that of planar imaging techniques. In 11 patients with various primary tumors, local recurrences or metastases (colorectal carcinoma (n=7), ovarian carcinoma (n=2), papillary thyroid carcinoma (n=1), undifferentiated carcinoma of the lung (n=1)), immuniscintigraphy (IS) was carried out using /sup 111/In-DTPA-labeled F(ab')/sub 2/ fragments of various MAbs (anti-CEA, OC 125, anti-hTG) and planar and tomographic imaging were compared intraindividually. By conventional diagnostic procedures, the presence of a tumor mass was confirmed (transmission computer tomography, ultrasound) or verified (/sup 131/I whole-body scintigraphy, histology) in all cases. Immunoscintigraphy was positive in 9 out of 11 cases by ECT and in 10 out of 11 cases by planar imaging. When using /sup 111/In-labeled MAb fragments, intraindividual comparison of ECT and planar imaging resulted in a similar sensitivity. The increased sensitivity of ECT using this tracer in contrast to /sup 131/I-labeled MAb fragments may be attributed to the fact that the physical properties of /sup 111/In are much more suitable for the gamma cameras most commonly used (single detector, 38'' crystal); using /sup 111/In-labelled MAb fragments, count rates sufficient for ECT can be obtained within a reasonable acquisition time. This allows to combine IS with the advantages of ECT regarding tumour localization and prevention of artefacts due to superposition of background.

  20. Au-labeled antibodies to enhance the sensitivity of a refractometric immunoassay: detection of cocaine. (United States)

    Wenger, Bernard; Kugelbrey, Karl; Gao, Hui; Sigrist, Hans; Voirin, Guy


    An integrated platform for a very sensitive detection of cocaine based on a refractometric biosensor is demonstrated. The system uses a waveguide grating biosensor functionalized with a cocaine multivalent antigen-carrier protein conjugate. The immunoassay scheme consists of the competitive binding of cocaine-specific antibodies to the immobilized conjugates. A 1000-fold enhancement of the sensor's sensitivity is achieved when using gold conjugated monoclonal antibodies instead of free antibodies. Together with the optimization of the assay conditions, the setup is designed for a quick identification of narcotics using automated sampling. The results show that the presence of cocaine in a liquid sample could be identified down to a concentration of 0.7 nM within one minute. This value can be reduced even further when longer binding time is allowed (0.2 nM after 15 min). Application of the system to detection of narcotics at airport security control points is discussed.

  1. Sequential radioimmunotherapy with 177Lu- and 211At-labeled monoclonal antibody BR96 in a syngeneic rat colon carcinoma model

    DEFF Research Database (Denmark)

    Eriksson, Sophie E; Elgström, Erika; Bäck, Tom


    for small, established tumors. A combination of such radionuclides may be successful in regimens of radioimmunotherapy. In this study, rats were treated by sequential administration of first a 177Lu-labeled antibody, followed by a 211At-labeled antibody 25 days later. METHODS: Rats bearing solid colon...... carcinoma tumors were treated with 400 MBq/kg body weight 177Lu-BR96. After 25 days, three groups of animals were given either 5 or 10 MBq/kg body weight of 211At-BR96 simultaneously with or without a blocking agent reducing halogen uptake in normal tissues. Control animals were not given any 211At-BR96....... Myelotoxicity, body weight, tumor size, and development of metastases were monitored for 120 days. RESULTS: Tumors were undetectable in 90% of the animals on day 25, independent of treatment. Additional treatment with 211At-labeled antibodies did not reduce the proportion of animals developing metastases...

  2. Demonstration of dnp groups on the draining lymph node cells of guinea pigs following skin painting with DNCB by peroxidase labelled antibody method.

    Directory of Open Access Journals (Sweden)



    Full Text Available The distribution of 2,4-dinitrophenyl (DNP groups in the draining lymph nodes of guinea pigs 12 h after painting the skin with 2,4-dinitrochlorobenzene (DNCB was examined by a peroxidase labelled antibody method using antibody against DNP groups. DNP groups were detected on cells that were found mainly in the subcapsular sinus of the lymph nodes. Electron microscopic examination showed DNP groups distributed on the surface of lymphocytes. The significance of these findings is discussed.

  3. Comparative distribution study of /sup 111/In-labelled DTPA and TTHA monoclonal antibody conjugates in a choriocarcinoma xenograft model

    Energy Technology Data Exchange (ETDEWEB)

    Buckley, R.G.; Barnett, P.; Searle, F.; Pedley, R.; Boden, J.A.


    Conjugates of the chelating agents DTPA and TTHA with a monoclonal anti-HCG were prepared. The tissue distribution of the /sup 111/In-Labelled conjugates and also /sup 111/In-citrate was studied in mice bearing human choriocarcinoma xenografts. The antibody conjugates both gave high liver and spleen radionuclide accumulation. Elevated femur levels were observed for the TTHA conjugate and /sup 111/In-citrate. Generally the DTPA conjugate showed the highest tumor-tissue ratios, although its tumor/blood ratio was lower than the other two materials. The results infer that the DTPA conjugate has the greatest utility as an imaging agent but that it would require a background subtraction technique.

  4. A Label-Free Electrochemical Immunosensor for Carbofuran Detection Based on a Sol-Gel Entrapped Antibody

    Directory of Open Access Journals (Sweden)

    Qingqing Li


    Full Text Available In this study, an anti-carbofuran monoclonal antibody (Ab was immobilized on the surface of a glassy carbon electrode (GCE using silica sol-gel (SiSG technology. Thus, a sensitive, label-free electrochemical immunosensor for the direct determination of carbofuran was developed. The electrochemical performance of immunoreaction of antigen with the anti-carbofuran monoclonal antibody was investigated by cyclic voltammetry (CV and electrochemical impedance spectroscopy (EIS, in which phosphate buffer solution containing [Fe(CN6]3−/4− was used as the base solution for test. Because the complex formed by the immunoreaction hindered the diffusion of [Fe(CN6]3−/4− on the electrode surface, the redox peak current of the immunosensor in the CV obviously decreased with the increase of the carbofuran concentration. The pH of working solution, the concentration of Ab and the incubation time of carbofuran were studied to ensure the sensitivity and conductivity of the immunosensor. Under the optimal conditions, the linear range of the proposed immunosensor for the determination of carbofuran was from 1 ng/mL to 100 μg/mL and from 50 μg/mL to 200 μg/mL with a detection limit of 0.33 ng/mL (S/N = 3. The proposed immunosensor exhibited good high sensitivity and stability, and it was thus suitable for trace detection of carbofuran pesticide residues.

  5. A label-free electrochemical immunosensor for carbofuran detection based on a sol-gel entrapped antibody. (United States)

    Sun, Xia; Du, Shuyuan; Wang, Xiangyou; Zhao, Wenping; Li, Qingqing


    In this study, an anti-carbofuran monoclonal antibody (Ab) was immobilized on the surface of a glassy carbon electrode (GCE) using silica sol-gel (SiSG) technology. Thus, a sensitive, label-free electrochemical immunosensor for the direct determination of carbofuran was developed. The electrochemical performance of immunoreaction of antigen with the anti-carbofuran monoclonal antibody was investigated by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), in which phosphate buffer solution containing [Fe(CN)(6)](3-/4-) was used as the base solution for test. Because the complex formed by the immunoreaction hindered the diffusion of [Fe(CN)(6)](3-/4-) on the electrode surface, the redox peak current of the immunosensor in the CV obviously decreased with the increase of the carbofuran concentration. The pH of working solution, the concentration of Ab and the incubation time of carbofuran were studied to ensure the sensitivity and conductivity of the immunosensor. Under the optimal conditions, the linear range of the proposed immunosensor for the determination of carbofuran was from 1 ng/mL to 100 μg/mL and from 50 μg/mL to 200 μg/mL with a detection limit of 0.33 ng/mL (S/N = 3). The proposed immunosensor exhibited good high sensitivity and stability, and it was thus suitable for trace detection of carbofuran pesticide residues.

  6. Treatment of (131)I-labeled anti-CD147 monoclonal antibody in VX2 carcinoma-induced liver tumors. (United States)

    Niu, Huanzhang; Wang, Ruihua; Cheng, Jingliang; Gao, Shegan; Liu, Baoping


    Hepatocellular carcinoma (HCC) is a major health problem worldwide. CD147 has been reported to be overexpressed in HCC and blocking CD147 expression can decrease tumor growth. (131)I is often used in combination with other drugs to treat HCC and yields positive results. In this study, we combined the (131)I and CD147 monoclonal antibody to treat HCC in a rabbit VX2 animal model. In the (131)I-labeled CD147 antibody ((131)I-CD147-Ab) treatment group, the animals lived considerably longer than the animals in the other treatment groups. Metastasis and tumor growth in the (131)I-CD147-Ab treatment group were also inhibited. MMP2 and CD31 expression were significantly lower in the treatment group, whereas Tunel staining was overexpressed. These findings suggest that (131)I-CD147-Ab is a promising drug in the treatment of HCC, by inhibiting metastasis and growth and by decreasing the expression of MMP2 and CD31 or by inducing tumor necrosis. After testing the biochemical parameters, (131)I-CD147-Ab caused fewer side-effects in the animals.

  7. In vitro evaluation of avidin antibody pretargeting using 211At-labeled and biotinylated poly-L-lysine as effector molecule

    DEFF Research Database (Denmark)

    Frost, Sofia H L; Jensen, Holger Lau; Lindegren, Sture


    Pretargeting is an approach for enhancing the therapeutic index of radioimmunotherapy by separating the administrations of tumor-targeting substance and radiolabel. In this study, a pretargeting model system of avidin-conjugated monoclonal antibody trastuzumab and biotinylated, (211)At-labeled po...

  8. Biodistribution of [sup 125]I labeled monoclonal antibody against gamma seminoprotein in the nude mice bearing human benign prostatic hyperplasia xenografts

    Energy Technology Data Exchange (ETDEWEB)

    Fujino, Awato (Kitasato Univ., Sagamihara, Kanagawa (Japan). School of Medicine)


    The biodistribution of [sup 125]I labeled monoclonal antibody against gamma seminoprotein ([gamma]-Sm) in the nude mice bearing human benign prostatic hyperplasia xenograft was evaluated by whole body autoradiography and by counting of radioactivity in organs. The monoclonal antibody (murine Ig G[sub 1], K) to [gamma]-Sm which was established in this institute and its F(ab')[sub 2] fragment were radioiodinated using Iodogen method. The autoradiograms demonstrated specific uptake of [sup 125]I-intact Ig G as well as [sup 125]I-F(ab')[sub 2] within the prostatic adenoma xenografts 4 days after intravenous administrations. Radioactivity in the xenograft was relatively higher than those in the liver, kidney or lung. These results suggest that radio-labeled monoclonal antibody against [gamma]-Sm might be applicable for radioimmunodetection of prostatic tumors which produce [gamma]-Sm. (author).

  9. Quantitative PET imaging of Met-expressing human cancer xenografts with {sup 89}Zr-labelled monoclonal antibody DN30

    Energy Technology Data Exchange (ETDEWEB)

    Perk, Lars R.; Stigter-van Walsum, Marijke; Vosjan, Maria J.W.D.; Leemans, C.R. [VU University Medical Centre, Department of Otolaryngology-Head and Neck Surgery, De Boelelaan 1117, P.O. Box 7057, Amsterdam (Netherlands); Visser, Gerard W.M.; Kloet, Reina W. [VU University Medical Centre, Nuclear Medicine and PET Research, Amsterdam (Netherlands); Giaccone, Giuseppe [National Cancer Institute, Medical Oncology Branch CCR, Bethesda, MD (United States); Albano, Raffaella; Comoglio, Paolo M. [University of Turin Medical School, Division of Molecular Oncology, Institute for Cancer Research and Treatment (IRCC), Turin (Italy); Dongen, Guus A.M.S. van [VU University Medical Centre, Department of Otolaryngology-Head and Neck Surgery, De Boelelaan 1117, P.O. Box 7057, Amsterdam (Netherlands); VU University Medical Centre, Nuclear Medicine and PET Research, Amsterdam (Netherlands)


    Targeting the c-Met receptor with monoclonal antibodies (MAbs) is an appealing approach for cancer diagnosis and treatment because this receptor plays a prominent role in tumour invasion and metastasis. Positron emission tomography (PET) might be a powerful tool for guidance of therapy with anti-Met MAbs like the recently described MAb DN30 because it allows accurate quantitative imaging of tumour targeting (immuno-PET). We considered the potential of PET with either {sup 89}Zr-labelled (residualising radionuclide) or {sup 124}I-labelled (non-residualising radionuclide) DN30 for imaging of Met-expressing tumours. The biodistribution of co-injected {sup 89}Zr-DN30 and iodine-labelled DN30 was compared in nude mice bearing either the human gastric cancer line GLT-16 (high Met expression) or the head-and-neck cancer line FaDu (low Met expression). PET images were acquired in both xenograft models up to 4 days post-injection (p.i.) and used for quantification of tumour uptake. Biodistribution studies in GTL-16-tumour-bearing mice revealed that {sup 89}Zr-DN30 achieved much higher tumour uptake levels than iodine-labelled DN30 (e.g. 19.6%ID/g vs 5.3%ID/g, 5 days p.i.), while blood levels were similar, indicating internalisation of DN30. Therefore, {sup 89}Zr-DN30 was selected for PET imaging of GLT-16-bearing mice. Tumours as small as 11 mg were readily visualised with immuno-PET. A distinctive lower {sup 89}Zr uptake was observed in FaDu compared to GTL-16 xenografts (e.g. 7.8%ID/g vs 18.1%ID/g, 3 days p.i.). Nevertheless, FaDu xenografts were also clearly visualised with {sup 89}Zr-DN30 immuno-PET. An excellent correlation was found between PET-image-derived {sup 89}Zr tumour uptake and ex-vivo-assessed {sup 89}Zr tumour uptake (R {sup 2} = 0.98). The long-lived positron emitter {sup 89}Zr seems attractive for PET-guided development of therapeutic anti-c-Met MAbs. (orig.)

  10. A comparison of 67Cu- and 131I-labelled forms of monoclonal antibodies SEN7 and SWA20 directed against small-cell lung cancer. (United States)

    Smith, A; Zangemeister-Wittke, U; Waibel, R; Schenker, T; Schubiger, P A; Stahel, R A


    The intact anti-SCLC monoclonal antibody (MAb) SEN7 and its F(ab')2 were labelled with the beta-emitting isotope 67Cu. Both materials retained their biological activity in vitro as determined by the Lindmo assay. In a direct comparison of in vivo distribution in a xenograph model, 131I- and 67Cu-labelled intact SEN7 showed similar absolute tumour accumulation. Blood levels were markedly lower in the case of the 67Cu-labelled antibody, resulting in improved tumour:blood ratios which reached a maximum of 13:1 compared with only 4.5:1 for 131I-SEN7. In the case of the 67Cu-labelled F(ab')2, very high accumulation of the nuclide was observed in the kidney. Levels of radio copper in liver and spleen were also found to be significantly raised when compared with radio iodine. SWA20, a MAb which had previously failed to show any selective in vivo accumulation in tumour xenografts when labelled with radio iodine showed higher and more stable tumour accumulation when labelled with 67Cu.

  11. Label-free capture of breast cancer cells spiked in buffy coats using carbon nanotube antibody micro-arrays (United States)

    Khosravi, Farhad; Trainor, Patrick; Rai, Shesh N.; Kloecker, Goetz; Wickstrom, Eric; Panchapakesan, Balaji


    We demonstrate the rapid and label-free capture of breast cancer cells spiked in buffy coats using nanotube-antibody micro-arrays. Single wall carbon nanotube arrays were manufactured using photo-lithography, metal deposition, and etching techniques. Anti-epithelial cell adhesion molecule (EpCAM) antibodies were functionalized to the surface of the nanotube devices using 1-pyrene-butanoic acid succinimidyl ester functionalization method. Following functionalization, plain buffy coat and MCF7 cell spiked buffy coats were adsorbed on to the nanotube device and electrical signatures were recorded for differences in interaction between samples. A statistical classifier for the ‘liquid biopsy’ was developed to create a predictive model based on dynamic time warping to classify device electrical signals that corresponded to plain (control) or spiked buffy coats (case). In training test, the device electrical signals originating from buffy versus spiked buffy samples were classified with ˜100% sensitivity, ˜91% specificity and ˜96% accuracy. In the blinded test, the signals were classified with ˜91% sensitivity, ˜82% specificity and ˜86% accuracy. A heatmap was generated to visually capture the relationship between electrical signatures and the sample condition. Confocal microscopic analysis of devices that were classified as spiked buffy coats based on their electrical signatures confirmed the presence of cancer cells, their attachment to the device and overexpression of EpCAM receptors. The cell numbers were counted to be ˜1-17 cells per 5 μl per device suggesting single cell sensitivity in spiked buffy coats that is scalable to higher volumes using the micro-arrays.

  12. Label-free capture of breast cancer cells spiked in buffy coats using carbon nanotube antibody micro-arrays. (United States)

    Khosravi, Farhad; Trainor, Patrick; Rai, Shesh N; Kloecker, Goetz; Wickstrom, Eric; Panchapakesan, Balaji


    We demonstrate the rapid and label-free capture of breast cancer cells spiked in buffy coats using nanotube-antibody micro-arrays. Single wall carbon nanotube arrays were manufactured using photo-lithography, metal deposition, and etching techniques. Anti-epithelial cell adhesion molecule (EpCAM) antibodies were functionalized to the surface of the nanotube devices using 1-pyrene-butanoic acid succinimidyl ester functionalization method. Following functionalization, plain buffy coat and MCF7 cell spiked buffy coats were adsorbed on to the nanotube device and electrical signatures were recorded for differences in interaction between samples. A statistical classifier for the 'liquid biopsy' was developed to create a predictive model based on dynamic time warping to classify device electrical signals that corresponded to plain (control) or spiked buffy coats (case). In training test, the device electrical signals originating from buffy versus spiked buffy samples were classified with ∼100% sensitivity, ∼91% specificity and ∼96% accuracy. In the blinded test, the signals were classified with ∼91% sensitivity, ∼82% specificity and ∼86% accuracy. A heatmap was generated to visually capture the relationship between electrical signatures and the sample condition. Confocal microscopic analysis of devices that were classified as spiked buffy coats based on their electrical signatures confirmed the presence of cancer cells, their attachment to the device and overexpression of EpCAM receptors. The cell numbers were counted to be ∼1-17 cells per 5 μl per device suggesting single cell sensitivity in spiked buffy coats that is scalable to higher volumes using the micro-arrays.

  13. Renal uptake of bismuth-213 and its contribution to kidney radiation dose following administration of actinium-225-labeled antibody

    Energy Technology Data Exchange (ETDEWEB)

    Schwartz, J; O' Donoghue, J A; Humm, J L [Department of Medical Physics, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10065 (United States); Jaggi, J S [Bristol-Myers Squibb, Plainsboro, NJ (United States); Ruan, S; Larson, S M [Nuclear Medicine Service Department of Radiology, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10065 (United States); McDevitt, M; Scheinberg, D A, E-mail: [Molecular Pharmacology and Chemistry, Sloan-Kettering Institute, 1275 York Avenue, New York, NY 10065 (United States)


    Clinical therapeutic studies using {sup 225}Ac-labeled antibodies have begun. Of major concern is renal toxicity that may result from the three alpha-emitting progeny generated following the decay of {sup 225}Ac. The purpose of this study was to determine the amount of {sup 225}Ac and non-equilibrium progeny in the mouse kidney after the injection of {sup 225}Ac-huM195 antibody and examine the dosimetric consequences. Groups of mice were sacrificed at 24, 96 and 144 h after injection with {sup 225}Ac-huM195 antibody and kidneys excised. One kidney was used for gamma ray spectroscopic measurements by a high-purity germanium (HPGe) detector. The second kidney was used to generate frozen tissue sections which were examined by digital autoradiography (DAR). Two measurements were performed on each kidney specimen: (1) immediately post-resection and (2) after sufficient time for any non-equilibrium excess {sup 213}Bi to decay completely. Comparison of these measurements enabled estimation of the amount of excess {sup 213}Bi reaching the kidney ({gamma}-ray spectroscopy) and its sub-regional distribution (DAR). The average absorbed dose to whole kidney, determined by spectroscopy, was 0.77 (SD 0.21) Gy kBq{sup -1}, of which 0.46 (SD 0.16) Gy kBq{sup -1} (i.e. 60%) was due to non-equilibrium excess {sup 213}Bi. The relative contributions to renal cortex and medulla were determined by DAR. The estimated dose to the cortex from non-equilibrium excess {sup 213}Bi (0.31 (SD 0.11) Gy kBq{sup -1}) represented {approx}46% of the total. For the medulla the dose contribution from excess {sup 213}Bi (0.81 (SD 0.28) Gy kBq{sup -1}) was {approx}80% of the total. Based on these estimates, for human patients we project a kidney-absorbed dose of 0.28 Gy MBq{sup -1} following administration of {sup 225}Ac-huM195 with non-equilibrium excess {sup 213}Bi responsible for approximately 60% of the total. Methods to reduce renal accumulation of radioactive progeny appear to be necessary for the

  14. Application of an enzyme-labeled antigen method for visualizing plasma cells producing antibodies against Strep A, a carbohydrate antigen of Streptococcus pyogenes, in recurrent tonsillitis. (United States)

    Onouchi, Takanori; Mizutani, Yasuyoshi; Shiogama, Kazuya; Inada, Ken-ichi; Okada, Tatsuyoshi; Naito, Kensei; Tsutsumi, Yutaka


    Streptococcus pyogenes is the main causative pathogen of recurrent tonsillitis. Histologically, lesions of recurrent tonsillitis contain numerous plasma cells. Strep A is an antigenic carbohydrate molecule on the cell wall of S. pyogenes. As expected, plasma cells in subjects with recurrent tonsillitis secrete antibodies against Strep A. The enzyme-labeled antigen method is a novel histochemical technique that visualizes specific antibody-producing cells in tissue sections by employing a biotin-labeled antigen as a probe. The purpose of the present study was to visualize plasma cells producing antibodies reactive with Strep A in recurrent tonsillitis. Firstly, the lymph nodes of rats immunized with boiled S. pyogenes were paraformaldehyde-fixed and specific plasma cells localized in frozen sections with biotinylated Strep A. Secondly, an enzyme-labeled antigen method was used on human tonsil surgically removed from 12 patients with recurrent tonsillitis. S. pyogenes genomes were PCR-detected in all 12 specimens. The emm genotypes belonged to emm12 in nine specimens and emm1 in three. Plasma cells producing anti-Strep A antibodies were demonstrated in prefixed frozen sections of rat lymph nodes, 8/12 human specimens from patients with recurrent tonsillitis but not in two control tonsils. In human tonsils, Strep A-reactive plasma cells were observed within the reticular squamous mucosa and just below the mucosa, and the specific antibodies belonged to either IgA or IgG classes. Our technique is effective in visualizing immunocytes producing specific antibodies against the bacterial carbohydrate antigen, and is thus a novel histochemical tool for analyzing immune reactions in infectious disorders.

  15. RE-186 labeled 16.88 IgM and 88BV59 IgG human antibody studies to assess potential for radioimmunotherapy

    Energy Technology Data Exchange (ETDEWEB)

    Breitz, H.; Seiler, C.; Weiden, P. [Virginia Mason Medical Center, Seattle, WA (United States)]|[NeoRx Corp, Seattle, WA (United States)]|[Organon Teknike/Biotechnology Research Institute, Rockville, MD (United States)] [and others


    Two studies with Re-186-MAG{sub 2}GABA labeled human antibodies were carried out to assess feasibility for radioimmunotherapy. Antibodies 16.88 and 88BV59 react with different epitopes of CTA 16.88, a tumor associated antigen of colorectal carcinoma. In a phase I dose escalation study, 14 patients received 60 mg/m{sup 2} 16.88 IgM MoAb. The dose of Re-186 ranged from 25 mCi/m{sup 2} to 210 mCi/m{sup 2} divided into 3 weekly infusions. In a pilot study with 88BV59, a human IgG3k MoAb, 20 mg antibody was labeled with 25 mCi/m{sup 2} Re-186 and administered to 4 patients with colon carcinoma. Tumor targeting was seen in 12 of 14 patients with 16.88 and all 4 patients with 88BV59. Retention of antibody at the tumor was longer with 88BV59. One patient developed a rash. No other acute or delayed toxicities were observed. Human anti-human antibody did not develop in any patient. The slower metabolism of the 88BV59 IgG suggests that this form of immunoconjugate merits further investigation for use in radioimmunotherapy.

  16. Establishment of intein-mediated protein ligation under denaturing conditions: C-terminal labeling of a single-chain antibody for biochip screening. (United States)

    Sydor, Jens R; Mariano, Maria; Sideris, Steve; Nock, Steffen


    Intein-mediated protein ligation is a recently developed method that enables the C-terminal labeling of proteins. This technique requires a correctly folded intein mutant that is fused to the C-terminus of a target protein to create a thioester, which allows the ligation of a peptide with an N-terminal cysteine (1, 2). Here we describe the establishment of this method for the labeling, under denaturing conditions, of target proteins that are expressed insolubly as intein fusion proteins. A GFPuv fusion protein with the Mycobacterium xenopi gyrA intein was expressed in inclusion bodies in Escherichia coli and initially used as a model protein to verify intein cleavage activity under different refolding conditions. The intein showed activity after refolding in nondenaturing and slightly denaturing conditions. A construct of the same intein with an anti-neutravidin single-chain antibody was also expressed in an insoluble form. The intein-mediated ligation was established for this single chain antibody-intein fusion protein under denaturing conditions in 4 M urea to prevent significant precipitation of the fusion protein during the first refolding step. Under optimized conditions, the single-chain antibody was labeled with a fluorescent peptide and used for antigen screening on a biochip after final refolding. This screening procedure allowed the determination of binding characteristics of the scFv for avidin proteins in a miniaturized format.

  17. Label free checkerboard assay to determine overlapping epitopes of Ebola virus VP-40 antibodies using surface plasmon resonance. (United States)

    Anderson, George P; Liu, Jinny L; Zabetakis, Dan; Legler, Patricia M; Goldman, Ellen R


    Immunoassay formats, in which antibodies provide sensitivity and specificity, are often utilized to provide rapid and simple diagnostic tests. Surface plasmon resonance is frequently used to evaluate the suitability of antibodies by determining binding kinetics to agents or surrogate antigens. We used SPR to evaluate a number of commercial monoclonal antibodies as well as single domain antibodies produced in-house. All the antibodies targeted the Ebola virus viral protein 40 (VP40). We determined the ability of each antibody to bind to immobilized VP40, and ensured they did not bind Ebola glycoprotein or the nucleoprotein. A subset of the monoclonal antibodies was immobilized to characterize antigen capture in solution. It can be advantageous to utilize antibodies that recognize distinct epitopes when choosing reagents for detection and diagnostic assays. We determined the uniqueness of the epitope recognized by the anti-VP40 antibodies using a checkerboard format that exploits the 6×6 array of interactions monitored by the Bio-Rad ProteOn XPR36 SPR instrument. The results demonstrate the utility of surface plasmon resonance to characterize monoclonal and recombinant antibodies. Additionally, the analysis presented here enabled the identification of pairs of anti-VP40 antibodies which could potentially be utilized in sandwich type immunoassays for the detection of Ebola virus.

  18. Simultaneous detection and semiquantification of DNA damage in normal and apoptotic cells: triple-immunofluorescent labeling using DAPI, antibodies, and TUNEL. (United States)

    Agrawal, Anant; Godar, Dianne E


    We developed a triple-labeling immunofluorescence technique that simultaneously identifies total DNA (DAPI), DNA damage (antibodies), and dead cells [terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive cells] and a method that semiquantifies DNA damage in paraffin-embedded tissues. Using this technique in combination with our analysis method, scientists can now simultaneously detect and compare the relative amounts of DNA damage of almost any kind (except single-strand and double-strand breaks), using indirect fluorescent antibody labeling, in both normal and dying cells of different tissues. Simultaneous labeling of DNA damage and dead or TUNEL-positive cells can reduce processing costs and analysis time, and can lead to discoveries concerning how cells die from different DNA damages. We used increasing doses of UV (290 to 400 nm) radiation to create DNA damage in the form of cyclobutane pyrimidine dimers and 6-4 photoproducts that kill some of the cells in 3-dimensional tissue-engineered skin and vaginal samples. We describe a protocol that reliably detects and semiquantifies DNA damage in both normal and apoptotic cells. We show this triple-labeling immunofluorescence technique and analysis method yields linear UV dose response curves for damage to DNA bases that allows semiquantification of cyclobutane pyrimidine dimers and calculation of its repair rate (T=1 and 24 h), whereas TUNEL allows quantification of the number of apoptotic cells. Scientists can now create beautiful fluorescent pictures that simultaneously detect DNA damage in both normal and apoptotic cells to assess and semiquantify the damage to understand better how different insults lead to the cell's demise.

  19. Highly sensitive enzyme-free immunosorbent assay for porcine circovirus type 2 antibody using Au-Pt/SiO2 nanocomposites as labels. (United States)

    Wu, Long; Yin, Wenmin; Tang, Kun; Shao, Kang; Li, Qin; Wang, Pan; Zuo, Yunpeng; Lei, Xiaomin; Lu, Zhicheng; Han, Heyou


    Improving the performance of conventional enzyme-linked immunosorbent assay (ELISA) is of great importance to meet the demand of early clinical diagnosis of various diseases. Herein, we report a feasible enzyme-free immunosorbent assay (EFISA) system using antibody conjugated Au-Pt/SiO2 nanocomposites (APS NCs) as labels. In this system, Au-Pt/SiO2 nanospheres (APS NPs) were first synthesized by wet chemical method and exhibited intrinsic peroxidase and catalase-like activity with excellent water-solubility. Then APS NCs were utilized as labels to replace HRP conjugated antibody, and Fe3O4 magnetic beads (MBs) to entrap the analyte. To discuss the performance of EFISA system, Human IgG was served as a model analyte, and porcine circovirus type 2 (PCV2) serums as real samples. The system boosted the detection limit of HIgG to 75pgmL(-1) with a RSD below 5%, a 264-fold improvement as compared with conventional ELISA. This is the first time that APS NCs have been used and successfully optimized for the sensitive dilution detection of PCV2 antibody (5:10(7)) in ELISA. Besides, APS NCs have advantages related to low cost, easy preparation, good stability and tunable catalytic activity, which make them a potent enzyme mimetic candidate and may find potential applications in bioassays and clinical diagnostics.

  20. Visualization of high-throughput and label-free antibody-polypeptide binding for drug screening based on microarrays and surface plasmon resonance imaging (United States)

    Chen, Shengyi; Deng, Tao; Wang, Tongzhou; Wang, Jia; Li, Xin; Li, Qiang; Huang, Guoliang


    This work presents a visualization method for the high-throughput monitoring of antibody-polypeptide binding by integrating a microarray chip with surface plasmon resonance imaging (SPRi). A prism-coupled SPRi system with smart images processing software and a 5×5 polypeptide microarray was developed. The modeling analysis was performed to optimize the system and the materials of prism and chip, looking for the optimal incident wavelength and angle of incidence for dynamic SPRi detection in solution. The system can dynamically monitor 25 tunnels of biomolecule interactions in solution without secondary tag reactants. In addition, this system can determine the specific profile of antibody-polypeptide binding in each tunnel and yield a visual three-dimensional histogram of dynamic combinations in all microarray tunnels. Furthermore, the detection limit of the label-free antibody-polypeptide binding reached 1 pg/μL in a one-step binding test, and an ultrasensitive detection of 10 fg/μL was obtained using three-step cascade binding. Using the peptide microarray, the amount of sample and reagents used was reduced to 80 nL per tunnel, and 20×20 tunnels of biomolecule interactions could be analyzed in parallel in a 7 mm×7 mm microreaction cells. This device and method offer a potential platform for high-throughput and label-free dynamic monitoring multiple biomolecule interactions for drug discovery and basic biomedical research.

  1. Label-free antibody detection using band edge fringes in SOI planar photonic crystal waveguides in the slow-light regime. (United States)

    García-Rupérez, Jaime; Toccafondo, Veronica; Bañuls, María José; Castelló, Javier García; Griol, Amadeu; Peransi-Llopis, Sergio; Maquieira, Ángel


    We report experimental results of label-free anti-bovine serum albumin (anti-BSA) antibody detection using a SOI planar photonic crystal waveguide previously bio-functionalized with complementary BSA antigen probes. Sharp fringes appearing in the slow-light regime near the edge of the guided band are used to perform the sensing. We have modeled the presence of these band edge fringes and demonstrated the possibility of using them for sensing purposes by performing refractive index variations detection, achieving a sensitivity of 174.8 nm/RIU. Then, label-free anti-BSA biosensing experiments have been carried out, estimating a surface mass density detection limit below 2.1 pg/mm2 and a total mass detection limit below 0.2 fg.

  2. Application of the indirect enzyme-labeled antibody microtest to the detection and surveillance of animal diseases. [Brucellosis, cholera, and trichinosis in cattle and swine

    Energy Technology Data Exchange (ETDEWEB)

    Saunders, G.C. Clinard, E.H.; Bartlett, M.L.; Sanders, W.M.


    The rapid, indirect enzyme-labeled antibody (ELA) microplate test has been developed as a diagnostic and surveillance tool to aid in the control of animal disease. Data are presented, which illustrate the application of the test to viral (hog cholera), parasitic (trichinosis), and bacterial (brucellosis) diseases of animals. A greater than 95 percent correlation was observed between the hog cholera ELA test and the hog cholera serum neutralization test performed on over 2000 mixed hog cholera positive and negative field samples obtained during the 1976 New Jersey epizootic. Of 56 swine naturally infected with Trichinella spiralis at a level considered dangerous to man, all were ELA positive, while only one of 360 T. spiralis negative packing house sera was ELA positive. Preliminary experiments with bovine brucellosis (Brucella abortus) indicate that the ELA test is more sensitive than other test methods currently in use. ELA procedures should soon become tests of choice for the detection of antibodies to animal disease agents.

  3. Preparation and Evaluation of 99mTc-labeled anti-CD11b Antibody Targeting Inflammatory Microenvironment for Colon Cancer Imaging. (United States)

    Cheng, Dengfeng; Zou, Weihong; Li, Xiao; Xiu, Yan; Tan, Hui; Shi, Hongcheng; Yang, Xiangdong


    CD11b, an active constituent of innate immune response highly expressed in myeloid-derived suppressor cells (MDSCs), can be used as a marker of inflammatory microenvironment, particularly in tumor tissues. In this research, we aimed to fabricate a (99m)Tc-labeled anti-CD11b antibody as a probe for CD11b(+) myeloid cells in colon cancer imaging with single-photon emission computed tomography (SPECT). In situ murine colon tumor model was established in histidine decarboxylase knockout (Hdc(-/-)) mice by chemicals induction. (99m)Tc-labeled anti-CD11b was obtained with labeling yields of over 30% and radiochemical purity of over 95%. Micro-SPECT/CT scans were performed at 6 h post injection to investigate biodistributions and targeting of the probe. In situ colonic neoplasma as small as 3 mm diameters was clearly identified by imaging; after dissection of the animal, anti-CD11b immunofluorescence staining was performed to identify infiltration of CD11b+ MDSCs in microenvironment of colonic neoplasms. In addition, the images displayed intense signal from bone marrow and spleen, which indicated the origin and migration of CD11b(+) MDSCs in vivo, and these results were further proved by flow cytometry analysis. Therefore, (99m)Tc-labeled anti-CD11b SPECT displayed the potential to facilitate the diagnosis of colon tumor in very early stage via detection of inflammatory microenvironment.

  4. In vivo amyloid-β imaging in the APPPS1-21 transgenic mouse model with a 89Zr- labeled monoclonal antibody.

    Directory of Open Access Journals (Sweden)

    Ann-Marie eWaldron


    Full Text Available Introduction: The accumulation of amyloid-β is a pathological hallmark of Alzheimer’s disease and is a target for molecular imaging probes to aid in diagnosis and disease monitoring. This study evaluated the feasibility of using a radiolabeled monoclonal anti-amyloid-β antibody (JRF/AβN/25 to non-invasively assess amyloid-β burden in aged transgenic mice (APPPS1-21 with μPET imaging.Methods: We investigated the antibody JRF/AβN/25 that binds to full-length Aβ. JRF/AβN/25 was radiolabeled with a [89Zr]-desferal chelate and intravenously injected into 12-13 month aged APPPS1-21 mice and their wild-type (WT controls. Mice underwent in vivo μPET imaging at 2, 4 and 7 days post injection and were sacrificed at the end of each time point to assess brain penetrance, plaque labeling, biodistribution and tracer stability. To confirm imaging specificity we also evaluated brain uptake of a non-amyloid targeting [89Zr]-labeled antibody (Trastuzumab as a negative control, additionally we performed a competitive blocking study with non-radiolabeled Df-Bz-JRF/AβN/25 and finally we assessed the possible confounding effects of blood retention. Results: Voxel-wise analysis of μPET data demonstrated significant [89Zr]-Df-Bz-JRF/AβN/25 retention in APPPS1-21 mice at all time points investigated. With ex vivo measures of radioactivity, significantly higher retention of [89Zr]-Df-Bz-JRF/AβN/25 was found at 4 and 7 day pi in APPPS1-21 mice. Despite the observed genotypic differences, comparisons with immunohistochemistry revealed that in vivo plaque labeling was low. Furthermore, pre-treatment with Df-Bz-JRF/AβN/25 only partially blocked [89Zr]-Df-Bz-JRF/AβN/25 uptake indicative of a high contribution of non-specific binding. Conclusion: Amyloid plaques were detected in vivo with a radiolabeled monoclonal anti-amyloid antibody. The low brain penetrance of the antibodies in addition to non-specific binding prevented an accurate estimation of plaque

  5. Intraindividual comparison of {sup 99m}Tc-labelled anti-SSEA-1 antigranulocyte antibody and {sup 99m}Tc-HMPAO labelled white blood cells for the imaging of infection

    Energy Technology Data Exchange (ETDEWEB)

    Gratz, S.; Behr, T.; Herrmann, A.; Becker, W. [Goettingen Univ. (Germany). Abt. fuer Nuklearmedizin; Dresing, K.; Stuermer, K.M. [Department of Trauma, Plastic and Reconstructive Surgery, University of Goettingen (Germany); Tarditi, L.; Franceschini, R. [SORIN Biomedica, Saluggia (Italy); Rhodes, B. [RHOMED, Albuquerque, New Mexiko (United States)


    The aim of this study was to compare the diagnostic accuracy of the {sup 99m}Tc-anti-SSEA-1 Mab with that of {sup 99m}Tc-HMPAO labelled white blood cells (WBCs). To this end, 17 patients with 23 proven infectious foci were examined with 555 MBq {sup 99m}Tc-anti-SSEA-1 MAb and with 370 MBq {sup 99m}Tc-HMPAO labelled autologous leucocytes within a period of 7 days. All the infections were confirmed by culturd of 7 days. Whole-body images and planar spot views with the antibody were performed at 1-h, 4-h and 24-h post injection; the biodistribution of the antibody was quantified, absorbed radiation doses were calculated and the diagnostic results were compared with the {sup 99m}Tc-HMPAO WBC images. Human anti-mouse antibody (HAMA) evaluation was performed in all patients before and 3 months after antibody injection. Blood was drawn at different times after {sup 99m}Tc-anti-SSEA-1 MAb injection to determine the amount of granulocyte-associated radioactivity and to calculate recovery. {sup 99m}Tc-anti-SSEA-1 MAb scintigraphy detected all 23 lesions, while 21 were detected with {sup 99m}Tc-HMPAO WBC scan. In this small group of patients, the sensitivity and specificity of {sup 99m}Tc-anti-SSEA-1 MAb scintigraphy were 95% and 96% respectively, as compared with 91% and 82% respectively for {sup 99m}Tc-HMPAO WBC scan. An increasing uptake of the injected activity in the lesion at different time points was indicative of high affinity and of specific PMN binding.There was no HAMA formation. In four of five patients investigated, a transient mild leukopenia was found at 15 min p.i. There was increased uptake of the antibody in liver and spleen and normal uptake in kidneys and bone marrow.The estimated radiation doses for the whole body and the red bone marrow were 1.1 x 10{sup -2} cGy/37 MBq and 5.3 x 10{sup -2} cGy/37 MBq, respectively. The activity associated to the PMNs in vivo was 33.5%, 30.6%, 21.3% and 9% at 5, 15, 30 and 45 min. post-injection, respectively

  6. Cell Wall Growth and Modulation Dynamics in a Model Unicellular Green Alga—Penium margaritaceum: Live Cell Labeling with Monoclonal Antibodies

    Directory of Open Access Journals (Sweden)

    David S. Domozych


    Full Text Available Penium margaritaceum is a unicellular charophycean green alga that possesses cell wall polymers similar to those of land plants. Several wall macromolecules of this alga are recognized by monoclonal antibodies specific for wall polymer epitopes of land plants. Immunofluorescence protocols using these antibodies may be employed to label specific cell wall constituents of live cells. Fluorescent labeling persists for several days, and this attribute allows for tracing of wall epitopes in both long- and short-term studies of cell development. Quantitative analysis of surface area covered by cell wall polymers is also easily performed. We show that significant cell expansion caused by incubation of cells in low levels of osmotically active agents like mannitol, glucose, or sucrose results from the inability of cells to undergo cytokinesis but does not result in significant changes to the amount of new cell wall. We also demonstrate that cells can be maintained for long periods of time in culture medium supplemented with specific cell wall-degrading enzymes where notable changes to wall infrastructure occur. These results demonstrate the great potential value of Penium in elucidating fundamental events during cell wall synthesis and modulation in plant cells.

  7. Granulocyte-specific monoclonal antibody technetium-99m-BW 250/183 and indium-111 oxine-labelled leukocyte scintigraphy in inflammatory bowel disease

    Energy Technology Data Exchange (ETDEWEB)

    Segarra, I.; Roca, M.; Ricart, Y.; Mora, J.; Puchal, R.; Martin-Comin, J. (Hospital de Bella Vista, Barcelona (Spain). Servicio de Medicina Nuclear); Baliellas, C.; Vilar, L. (Hospital de Bella Vista, Barcelona (Spain). Servicio de Gastroenterologia)


    Thirty-three patients suspected of suffering from inflammatory bowel disease were studied. Autologous leukocytes were labelled with indium 111 oxine and re-injected simultaneously with 0.3-0.5 mg of {sup 99m}Tc-labelled granulocyte-specific monoclonal antibody BW 250/183. Two scans were obtained, the early scan 3-4 h post-injection (p.i.) and the late scan 18-24 h p.i. Using the endoscopy study as standard, the diagnostic accuracy of both agents was determined. Sensitivity, specificity and accuracy of {sup 111}In-scans was 88.8%, 100.0% and 93.7% at 4 h and 94.7%, 100.0% and 96.9% and 24 h, respectively. Concerning the results using antibodies, the values were 61.1%, 100.0% and 78.1% at 4 h and 78.9%, 92.8% and 84.8% at 24 h, respectively. Segmental analysis showed concordance in 89.3% and 93.3% of the cases at 4 and 24 h, respectively. Though less sensitive and less accurate than scanning employing indium 111 leukocytes, the BW 250/183 granulocyte-specific scintigraphy can be used for inflammatory bowel disease diagnosis and localization. (orig.).

  8. Radioimmunotherapy of carcinoma of colon with [131I]-labeled recombinant chimeric monoclonal antibodies to carcinoembryonic antigen

    Institute of Scientific and Technical Information of China (English)

    Qiu-jun LU; Guang-xing BIAN; Yuan-yuan CHEN; Min ZHANG; Shao-ming GUO; Li-qing WEN


    Aim: To study the distribution of [131I]-labeled anti-CEA MoAbs and its therapeutic effect on the human colonic cancer model in nude mice. Methods: A nude mice model of human colonic cancer was established. [131I]-labeled anti-CEA MoAbs were injected intravenously into mice. The distribution of the MoAbs was then determined and the effect of RIT on human colonic cancer was observed. Results:The [131I]-labeled anti-CEA MoAbs had a specific distribution after injection.Tumor/non-tumor ratios for [131I]-labeled anti-CEA MoAbs were 10-20 times higher than [131I]-labeled IgG 96 h after injection. Thirty days after injection, significant inhibition of the volume and weight of tumor was observed in the treated mice compared with the control. The tumor growth inhibition rate of 3.1 mCi/kg CEA MoAbs group (LS 180, LS 174T, SW1116) was 47.8%-64.0%. This was 69.6%-78.6%in the 6.25 mCi/kg CEA MoAbs group, and 81.8%-86.2% in the 12.5 mCi/kg [131I]-labeled anti-CEA MoAbs group. The plasma CEA level was also lower in treated mice. Conclusion: The results indicate that [131I]-labeled anti-CEA MoAbs can be effective in RIT on colonic cancers.

  9. Comparative studies of antibody anti-CD20 labeled with {sup 188}Re; Estudo comparativo da marcacao do anticorpo anti-CD20 com {sup 188}Re

    Energy Technology Data Exchange (ETDEWEB)

    Dias, Carla Roberta de Barros Rodrigues


    Nuclear Medicine is an unique and important modality in oncology and the development of new tumor-targeted radiopharmaceuticals for both diagnosis and therapy is an area of interest for researchers. Rituximab (RTX) is a quimeric monoclonal antibody (mAb) (IgG 1) that specifically binds to CD20 antigen with high affinity and has been successfully used for the treatment of Non-Hodgkin Lymphoma (NHL) of cell B. The CD20 antigen is expressed over more than 90% of cell B NHL. Technetium-99m ({sup 99m}Tc) and rhenium-188 ({sup 188}Re) are an attractive radionuclide pair for clinical use due to their favorable decay properties for diagnosis ({sup 99m}Tc: T{sub 1/2} = 6 h, {gamma} radiation = 140 keV) and therapy ({sup 188}Re: T{sub 1/2} = 17 h, maximum {beta} energy = 2.12 MeV) and to their availability in the form of {sup 99}Mo/{sup 99}mTc and {sup 188}W/{sup 188}Re generators. The radionuclides can be conjugated to mAb using similar chemical procedures. The aim of this work was to study the labeling of anti-CD20 mAb (RTX) with {sup 188}Re using two techniques: the direct labeling method [{sup 188}Re(V)] and the labeling method via the carbonyl nucleus [{sup 188}Re(I)]. Besides the quality control, the radiolabeled mAb was submitted to in vivo, in vitro and ex vivo biological studies. For the direct labeling, RTX was reducing by incubation with 2-mercaptoethanol for generating sulphydryl groups (-SH) and further labeled with {sup 188}Re(V), in a study of several parameters in order to reach an optimized formulation. The labeling via the carbonyl nucleus both {sup 99}mTc and {sup 188}Re were employed through 2 different procedures: (1) labeling of intact RTX with {sup 99}mTc(I) and (2) reduced RTX (RTX{sub red}) labeled with {sup 99}mTc(I)/{sup 188}Re(I). Also a parameter study was performed to obtain an optimized formulation. The quality control method for evaluating the radiochemical purity showed a good labeling yield (93%) for the direct method. The labeling method

  10. Imaging cancer using PET - the effect of the bifunctional chelator on the biodistribution of a {sup 64}Cu-labeled antibody

    Energy Technology Data Exchange (ETDEWEB)

    Dearling, Jason L.J., E-mail: jason.dearling@childrens.harvard.ed [Division of Nuclear Medicine and Department of Radiology, Children' s Hospital Boston, 300 Longwood Avenue, Boston, MA 02115 (United States); Harvard Medical School, Boston, MA 02115 (United States); Voss, Stephan D. [Division of Nuclear Medicine and Department of Radiology, Children' s Hospital Boston, 300 Longwood Avenue, Boston, MA 02115 (United States); Harvard Medical School, Boston, MA 02115 (United States); Dunning, Patricia; Snay, Erin [Division of Nuclear Medicine and Department of Radiology, Children' s Hospital Boston, 300 Longwood Avenue, Boston, MA 02115 (United States); Fahey, Frederic [Division of Nuclear Medicine and Department of Radiology, Children' s Hospital Boston, 300 Longwood Avenue, Boston, MA 02115 (United States); Harvard Medical School, Boston, MA 02115 (United States); Smith, Suzanne V. [Australian National Science and Technology Organisation (ANSTO), New Illawarra Road, PMB1, Menai, New South Wales 2234 (Australia); Huston, James S. [EMD Serono Research Center, 45A Middlesex Turnpike, Billerica, MA 01821-3936 (United States); Boston Biomedical Research Institute, Watertown, MA 02472-2899 (United States); Meares, Claude F. [Department of Chemistry, University of California, One Shields Avenue, Davis, CA 95616-5295 (United States); Treves, S. Ted; Packard, Alan B. [Division of Nuclear Medicine and Department of Radiology, Children' s Hospital Boston, 300 Longwood Avenue, Boston, MA 02115 (United States); Harvard Medical School, Boston, MA 02115 (United States)


    Introduction: Use of copper radioisotopes in antibody radiolabeling is challenged by reported loss of the radionuclide from the bifunctional chelator used to label the protein. The objective of this study was to investigate the relationship between the thermodynamic stability of the {sup 64}Cu-complexes of five commonly used bifunctional chelators (BFCs) and the biodistribution of an antibody labeled with {sup 64}Cu using these chelators in tumor-bearing mice. Methods: The chelators [S-2-(aminobenzyl)1,4,7-triazacyclononane-1,4,7-triacetic acid (p-NH{sub 2}-Bn-NOTA): 6-[p-(bromoacetamido)benzyl]-1, 4, 8, 11-tetraazacyclotetradecane-N, N', N'', N'''-tetraacetic acid (BAT-6): S-2-(4-aminobenzyl)-1,4,7,10-tetraazacyclododocane tetraacetic acid (p-NH{sub 2}-Bn-DOTA): 1,4,7,10-tetraazacyclododocane-N, N', N', N''-tetraacetic acid (DOTA): and 1-N-(4-aminobenzyl)-3,6,10,13,16,19-hexaazabicyclo[6.6.6]eicosane-1, 8-diamine (SarAr)] were conjugated to the anti-GD2 antibody ch14.18, and the modified antibody was labeled with {sup 64}Cu and injected into mice bearing subcutaneous human melanoma tumors (M21) (n = 3-5 for each study). Biodistribution data were obtained from positron emission tomography images acquired at 1, 24 and 48 hours post-injection, and at 48 hours post-injection a full ex vivo biodistribution study was carried out. Results: The biodistribution, including tumor targeting, was similar for all the radioimmunoconjugates. At 48 h post-injection, the only statistically significant differences in radionuclide uptake (p < 0.05) were between blood, liver, spleen and kidney. For example, liver uptake of [{sup 64}Cu]ch14.18-p-NH{sub 2}-Bn-NOTA was 4.74 {+-} 0.77 per cent of the injected dose per gram of tissue (%ID/g), and for [{sup 64}Cu]ch14.18-SarAr was 8.06 {+-} 0.77 %ID/g. Differences in tumor targeting correlated with variations in tumor size rather than which BFC was used. Conclusions: The results of this

  11. [3H]Azidodantrolene photoaffinity labeling, synthetic domain peptides and monoclonal antibody reactivity identify the dantrolene binding sequence on RyR1

    Energy Technology Data Exchange (ETDEWEB)

    Paul-Pletzer, Kalanethee; Yamamoto, Takeshi; Bhat, Manju B.; Ma, Jianjie; Ikemoto, Noriaki; Jimenez, Leslie S.; Morimoto, Hiromi; Williams, Philip G.; Parness, Jerome


    Dantrolene is a drug that suppresses intracellular Ca2+ release from sarcoplasmic reticulum in normal skeletal muscle and is used as a therapeutic agent in individuals susceptible to malignant hyperthermia. Though its precise mechanism of action has not been elucidated, we have identified the N-terminal region (amino acids 1-1400) of the skeletal muscle isoform of the ryanodine receptor (RyR1), the primary Ca2+ release channel in sarcoplasmic reticulum, as a molecular target for dantrolene using the photoaffinity analog [3H]azidodantrolene(1). Here, we demonstrate that heterologously expressed RyR1 retains its capacity to be specifically labeled with [3H]azidodantrolene,indicating that muscle specific factors are not required for this ligand-receptor interaction. Synthetic domain peptides of RyR1, previously shown to affect RyR1 function in vitro and in vivo, were exploited as potential drug binding site mimics and used in photoaffinity labeling experiments. Only DP1 and DP1-2, peptide s containing the amino acid sequence corresponding to RyR1 residues 590-609, were specifically labeled by [3H]azidodantrolene. A monoclonal anti-RyR1 antibody which recognizes RyR1 and its 1400 amino acid N-terminal fragment, recognizes DP1 and DP1-2 in both Western blots and immunoprecipitation assays, and specifically inhibits [3H]azidodantrolene photolabeling of RyR1 and its N-terminal fragment in sarcoplasmic reticulum. Our results indicate that synthetic domain peptides can mimic a native, ligand binding conformation in vitro, and that the dantrolene binding site and the epitope for the monoclonal antibody on RyR1 are equivalent and composed of amino-acids 590-609.

  12. Improved labelling of DTPA- and DOTA-conjugated peptides and antibodies with 111In in HEPES and MES buffer.


    Brom, Maarten; Joosten, Lieke; Oyen, Wim JG; Gotthardt, Martin; Boerman, Otto C.


    ABSTRACT: BACKGROUND: In single photon emission computed tomography [SPECT], high specific activity of 111In-labelled tracers will allow administration of low amounts of tracer to prevent receptor saturation and/or side effects. To increase the specific activity, we studied the effect of the buffer used during the labelling procedure: NaAc, NH4Ac, HEPES and MES buffer. The effect of the ageing of the 111InCl3 stock and cadmium contamination, the decay product of 111In, was also examined in th...

  13. Improved labelling of DTPA- and DOTA-conjugated peptides and antibodies with 111In in HEPES and MES buffer.

    NARCIS (Netherlands)

    Brom, M.; Joosten, L.; Oyen, W.J.G.; Gotthardt, M.; Boerman, O.C.


    ABSTRACT: BACKGROUND: In single photon emission computed tomography [SPECT], high specific activity of 111In-labelled tracers will allow administration of low amounts of tracer to prevent receptor saturation and/or side effects. To increase the specific activity, we studied the effect of the buffer

  14. Improved flow cytometric identification of myelopoiesis by the simultaneous labelling with CD13, CD14 and CD66 monoclonal antibodies

    DEFF Research Database (Denmark)

    Bonde, J; Meyer, K; Broe, M K;


    The aim of the present study was to increase our knowledge of myelopoiesis evaluated by flow cytometry. We therefore designed a triple-marker assay employing monoclonal antibodies against the CD13 (immature), the CD14 (monocytic), and the CD66 (mature myeloid) antigens using three-colour immunofl...

  15. Antibody-functionalized nanoparticles for imaging cancer: influence of conjugation to gold nanoparticles on the biodistribution of 89Zr-labeled cetuximab in mice. (United States)

    Karmani, Linda; Labar, Daniel; Valembois, Vanessa; Bouchat, Virginie; Nagaswaran, Praveen Ganesh; Bol, Anne; Gillart, Jacques; Levêque, Philippe; Bouzin, Caroline; Bonifazi, Davide; Michiels, Carine; Feron, Olivier; Grégoire, Vincent; Lucas, Stéphane; Vander Borght, Thierry; Gallez, Bernard


    Antibody-labeled gold nanoparticles represent a promising novel tool regarding cancer imaging and therapy. Nevertheless, the characterization of biodistribution of such immunonanocarriers has been poorly documented. In this study, the biodistribution of (89)Zr-labeled cetuximab before and after the coupling reaction to gold nanoparticles (AuNPs) was compared and the quantitative imaging performance of (89)Zr immuno-PET was evaluated. Cetuximab was functionalized with the desferal moiety and labeled with (89)Zr ((89)Zr-Df-Bz-NCS-cetuximab). AuNPs with a mean diameter of 5 nm were synthesized according a new method developed in the laboratory, and conjugated to (89)Zr-Df-Bz-NCS-cetuximab using carbodiimide chemistry (AuNPs-PPAA-cetuximab-(89)Zr). The two tracers were injected in A431 xenograft-bearing mice. Tumor and liver uptakes were assessed at different times after injection using quantitative PET imaging. The in vivo specificity of the binding was investigated using a saturating dose of unlabeled cetuximab. Radiolabeled cetuximab was conjugated to AuNPs with a coupling reaction yield >75%. All conjugates were stable in vitro and to a lesser extent in plasma. In vivo distribution studies revealed no significant difference in tumor uptake for cetuximab conjugated to nanoparticles up to 72 h after injection, compared with unconjugated cetuximab. Immuno-PET studies showed that AuNPs-PPAA-cetuximab-(89)Zr provided high tumor-to-background ratio. The liver uptake of AuNPs-PPAA-cetuximab-(89)Zr was higher, compared with (89)Zr-Df-Bz-NCS-cetuximab. In vivo blocking experiments demonstrated selective tumor targeting after coupling reaction. This study showed that the conjugation of AuNPs to cetuximab did not affect its tumor accumulation and that the efficacy of EGFR-targeted nanoparticles was unaltered. The (89)Zr-labeled cetuximab-targeted gold nanoparticles could be a valuable tool for theranostic purposes.

  16. Distribution of cells labelled by a novel somatic stem cell-recognizing antibody (A3) in pulmonary genesis and bleomycin induced pulmonary fibrosis in rats. (United States)

    Hori, M; Juniantito, V; Izawa, T; Ichikawa, C; Tanaka, M; Tanaka, K; Takenaka, S; Kuwamura, M; Yamate, J


    Stem cells play important roles in organogenesis and remodelling after tissue injury. A monoclonal antibody (A3) has been produced against rat somatic stem cells. The present study investigated the distribution of cells labelled by A3 in the lung of fetal, neonatal and adult rats, as well as in the lung of rats with bleomycin (BLM) induced pulmonary fibrosis. In developing fetal lungs, A3(+) interstitial cells were present around the bronchi/bronchioles and arterioles, while in neonatal and adult lungs, the A3 reactivity of the interstitial cells gradually disappeared and instead, vascular endothelial cells in alveolar capillaries and arterioles expressed A3. By double immunofluorescence labelling, the A3(+) interstitial cells also expressed vimentin (a mesenchymal marker) and CD34 (a marker of immature mesenchymal cells), indicating that the interstitial cells were immature mesenchymal cells concentrated in organs as precursors to cells of connective tissues. A3(+)endothelial cells were co-expressed RECA-1 (a marker of rat endothelial cells) and A3 was localized to the cell membrane and cytoplasm of these cells by immunoelectron microscopy. In BLM induced fibrotic lesions, there were many A3(+) cells, which also expressed vimentin or RECA-1 by dual immunofluorescence labelling. There were few CD34(+)/A3(+) double positive cells. No cells co-expressed A3 and α-smooth muscle actin (a marker of well-differentiated myofibroblastic cells). Although the detailed properties of cells labelled by A3 remain to be discovered, A3 would appear to be a useful marker of immature mesenchymal cells and vascular endothelial cells in developing lungs and in pulmonary fibrosis.

  17. Detection of myocarditis with indium-111-labelled monoclonal antimyosin antibodies. A review; Znaczenie antymiozyn znakowanych indem-111 w diagnostyce zapalenia miesnia sercowego

    Energy Technology Data Exchange (ETDEWEB)

    Wojnicz, R. [Slaskie Centrum Chorob Serca, Zabrze (Poland)


    Indium-111 labelled monoclonal antimyosin antibody (AMA-In111) is a sensitive immunoscintigraphic agent for detecting of myocyte necrosis. Due to some limitations including low specificity for myocarditis (MCI) and a very high cost of the study, this agent should be used in only part of patients with clinically suspected MCI. Non-invasive and invasive (coronary angiography) diagnostic procedures should precede AMA-In111 scintigraphy in these patients. The utility of AMA-In111 scintigraphy seems to be greatest in following clinical conditions characterized by myocyte necrosis: 1. diagnosis of MCI in patients with myocardial infarction demonstrated to have normal coronary arteries by selective angiography, 2. detection and quantification of anthracycline-induced myocardial damage, 3. assessment of myocardial damage in cardiac rejection after one post-transplant year. (author)

  18. Electrogenerated chemiluminescence biosensing for the detection of prostate PC-3 cancer cells incorporating antibody as capture probe and ruthenium complex-labelled wheat germ agglutinin as signal probe

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Haiying [Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an 710062 (China); Department of Chemistry, Yuncheng University, Yuncheng 044300 (China); Li, Zhejian; Shan, Meng; Li, Congcong; Qi, Honglan; Gao, Qiang [Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an 710062 (China); Wang, Jinyi [College of Science and College of Veterinary Medicine, Northwest A& F University, Yangling 712100 (China); Zhang, Chengxiao, E-mail: [Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an 710062 (China)


    Highlights: • A novel biosensor was developed for the detection of prostate cancer cells. • The selectivity of the biosensor was improved using antibody as capture probe. • The biosensor showed the low extremely detection limit of 2.6 × 10{sup 2} cells mL{sup −1}. • The ruthenium complex-labelled WGA can be transported in the cell vesicles. - Abstract: A highly selective and sensitive electrogenerated chemiluminescence (ECL) biosensor for the detection of prostate PC-3 cancer cells was designed using a prostate specific antibody as a capture probe and ruthenium complex-labelled wheat germ agglutinin as a signal probe. The ECL biosensor was fabricated by covalently immobilising the capture probe on a graphene oxide-coated glassy carbon electrode. Target PC-3 cells were selectively captured on the surface of the biosensor, and then, the signal probe was bound with the captured PC-3 cells to form a sandwich. In the presence of tripropylamine, the ECL intensity of the sandwich biosensor was logarithmically directly proportion to the concentration of PC-3 cells over a range from 7.0 × 10{sup 2} to 3.0 × 10{sup 4} cells mL{sup −1}, with a detection limit of 2.6 × 10{sup 2} cells mL{sup −1}. The ECL biosensor was also applied to detect prostate specific antigen with a detection limit of 0.1 ng mL{sup −1}. The high selectivity of the biosensor was demonstrated in comparison with that of a lectin-based biosensor. The strategy developed in this study may be a promising approach and could be extended to the design of ECL biosensors for highly sensitive and selective detection of other cancer-related cells or cancer biomarkers using different probes.

  19. Pharmacokinetics, biodistribution and dosimetry of 99mTc-labeled anti-human epidermal growth factor receptor humanized monoclonal antibody R3 in rats. (United States)

    Iznaga Escobar, N; Morales, A M; Ducongé, J; Torres, I C; Fernández, E; Gómez, J A


    The pharmacokinetics, biodistribution and dosimetry of 99mTc-labeled anti-human epidermal growth factor receptor (anti-hEGF-r) humanized monoclonal antibody (MAb) R3 was investigated following intravenous injection in normal Wistar rats. Serum disappearance curves were best fit by a two-compartment model having a mean distribution half-life (t 1/2alpha) of 0.250 h and a mean elimination (t 1/2beta) of 13.89 h. Among the various organs, a little accumulation of the radiolabeled antibody was found only in kidneys. Biodistribution and dosimetry studies in humans were performed by extrapolation of the animal data to humans. Absorbed dose to normal organs and the remainder of the whole body were estimated using the medical internal radiation dose formula, and dose contributions from radioactivity in transit through the gastrointestinal tract were estimated using a compartment model. Extrapolated values of radiation absorbed dose to normal organs in rads per millicurie administered were whole body, 0.0085; lower large intestine wall, 0.0898; small intestine, 0.0530; upper large intestine wall, 0.0731; and kidneys, 0.0455. The effective dose equivalent predicted was 0.0162 rem/mCi and the effective dose was found to be 0.015 rem/mCi. On the basis of the pharmacokinetics, biodistribution and internal radiation dosimetry information obtained in this study, a diagnostic phase I clinical trial with 99mTc-labeled humanized MAb R3 conjugate in patients should be supported.

  20. A Highly Selective and Sensitive Fluorescence Detection Method of Glyphosate Based on an Immune Reaction Strategy of Carbon Dot Labeled Antibody and Antigen Magnetic Beads. (United States)

    Wang, Duo; Lin, Bixia; Cao, Yujuan; Guo, Manli; Yu, Ying


    A sensitive fluorescence detection method for glyphosate (GLY) was established based on immune reaction. First, carbon dot labeled antibodies (lgG-CDs) which were able to specifically identify glyphosate were prepared with the environmentally friendly carbon dots (CDs) and glyphosate antibody (lgG). lgG-CDs could be used to in situ visualize the distribution of glyphosate in plant tissues. In order to eliminate the effects of excess lgG-CDs on the determination of GLY, antigen magnetic beads Fe3O4-GLY based on magnetic nanoparticles Fe3O4 and glyphosate were constructed and utilized to couple with the excess lgG-CDs. After magnetic separation to remove antigen magnetic beads, there was a linear relationship between the fluorescence intensity of lgG-CDs and the logarithmic concentration of glyphosate in the range of 0.01-80 μg/mL with a detection limit of 8 ng/mL. The method was used for the detection of glyphosate in Pearl River water, tea, and soil samples with satisfactory recovery ratio between 87.4% and 103.7%.

  1. Detection of anthrax protective antigen (PA) using europium labeled anti-PA monoclonal antibody and time-resolved fluorescence. (United States)

    Stoddard, Robyn A; Quinn, Conrad P; Schiffer, Jarad M; Boyer, Anne E; Goldstein, Jason; Bagarozzi, Dennis A; Soroka, Stephen D; Dauphin, Leslie A; Hoffmaster, Alex R


    Inhalation anthrax is a rare but acute infectious disease following adsorption of Bacillus anthracis spores through the lungs. The disease has a high fatality rate if untreated, but early and correct diagnosis has a significant impact on case patient recovery. The early symptoms of inhalation anthrax are, however, non-specific and current anthrax diagnostics are primarily dependent upon culture and confirmatory real-time PCR. Consequently, there may be a significant delay in diagnosis and targeted treatment. Rapid, culture-independent diagnostic tests are therefore needed, particularly in the context of a large scale emergency response. The aim of this study was to evaluate the ability of monoclonal antibodies to detect anthrax toxin proteins that are secreted early in the course of B. anthracis infection using a time-resolved fluorescence (TRF) immunoassay. We selected monoclonal antibodies that could detect protective antigen (PA), as PA83 and also PA63 and LF in the lethal toxin complex. The assay reliable detection limit (RDL) was 6.63×10(-6)μM (0.551ng/ml) for PA83 and 2.51×10(-5)μM (1.58ng/ml) for PA63. Despite variable precision and accuracy of the assay, PA was detected in 9 out of 10 sera samples from anthrax confirmed case patients with cutaneous (n=7), inhalation (n=2), and gastrointestinal (n=1) disease. Anthrax Immune Globulin (AIG), which has been used in treatment of clinical anthrax, interfered with detection of PA. This study demonstrates a culture-independent method of diagnosing anthrax through the use of monoclonal antibodies to detect PA and LF in the lethal toxin complex.

  2. Antibody functionalized graphene biosensor for label-free electrochemical immunosensing of fibrinogen, an indicator of trauma induced coagulopathy. (United States)

    Saleem, Waqas; Salinas, Carlos; Watkins, Brian; Garvey, Gavin; Sharma, Anjal C; Ghosh, Ritwik


    An antibody, specific to fibrinogen, has been covalently attached to graphene and deposited onto screen printed electrodes using a chitosan hydrogel binder to prepare an inexpensive electrochemical fibrinogen biosensor. Fourier Transform Infrared (FT-IR) spectroscopy has been utilized to confirm the presence of the antibody on the graphene scaffold. Electrochemical Impedance Spectroscopy (EIS) has been utilized to demonstrate that the biosensor responds in a selective manner to fibrinogen in aqueous media even in the presence of plasminogen, a potentially interfering molecule in the coagulopathy cascade. Furthermore, the biosensor was shown to reliably sense fibrinogen in the presence of high background serum albumin levels. Finally, we demonstrated detection of clinically relevant fibrinogen concentrations (938-44,542μg/dL) from human serum and human whole blood samples using this biosensor. This biosensor can potentially be used in a point-of-care device to detect the onset of coagulopathy and monitor response following therapeutic intervention in trauma patients. Thus this biosensor may improve the clinical management of patients with trauma-induced coagulopathy.

  3. Semiautomatic Landmark-Based Two-Dimensional—Three-Dimensional Image Fusion in Living Mice: Correlation of Near-Infrared Fluorescence Imaging of Cy5.5-Labeled Antibodies with Flat-Panel Volume Computed Tomography

    Directory of Open Access Journals (Sweden)

    Christian Dullin


    Full Text Available Connecting fluorescence signals with anatomic structures enhances our ability to monitor biologic processes in mice. Here, we present a semiautomated approach to correlate two-dimensional (2D noninvasive near-infrared fluorescence (NIRF imaging with three-dimensional (3D, high-resolution, flat-panel volume computed tomography (fpVCT. We developed an algorithm to colocalize fluorescence signals of NIRF-labeled antibodies directed against matriptase and urokinase plasminogen activator receptor (uPAR to orthotopic carcinomas in mice visualized by fpVCT. For this purpose, mice were anesthetized and fixed on a multimodality animal bed containing fiducial markers filled with iodine-containing contrast agent and fluorescent dye. After intravenous administration of contrast agent and Cy5.5-labeled antibodies, NIRF and fpVCT images were obtained, without repositioning the mice. Binding of Cy5.5-labeled matriptase-specific antibody to pancreatic tumors and Cy5.5-labeled uPAR-specific antibody to mammary carcinomas was assessed by time-domain NIRF imaging measuring the location of fluorescence intensity and its lifetime. In summary, we developed a novel 2D-3D registration technique for image fusion with NIRF imaging and fpVCT to provide complementary information in tumor models on the in vivo association of functional information with anatomic structures. The combination of fpVCT with NIRF imaging will now allow targeted and effective monitoring of preclinical tumor therapies.

  4. Antithyroglobulin antibody (United States)

    Thyroglobulin antibody; Thyroiditis - thyroglobulin antibody; Hypothyroidism - thyroglobulin antibody; Thyroiditis - thyroglobulin antibody; Graves disease - thyroglobulin antibody; Underactive thyroid - thyroglobulin antibody

  5. High contrast tumor imaging with radio-labeled antibody Fab fragments tailored for optimized pharmacokinetics via PASylation. (United States)

    Mendler, Claudia T; Friedrich, Lars; Laitinen, Iina; Schlapschy, Martin; Schwaiger, Markus; Wester, Hans-Jürgen; Skerra, Arne


    Although antigen-binding fragments (Fabs) of antibodies constitute established tracers for in vivo radiodiagnostics, their functionality is hampered by a very short circulation half-life. PASylation, the genetic fusion with a long, conformationally disordered amino acid chain comprising Pro, Ala and Ser, provides a convenient way to expand protein size and, consequently, retard renal filtration. Humanized αHER2 and αCD20 Fabs were systematically fused with 100 to 600 PAS residues and produced in E. coli. Cytofluorimetric titration analysis on tumor cell lines confirmed that antigen-binding activities of the parental antibodies were retained. The radio-iodinated PASylated Fabs were studied by positron emission tomography (PET) imaging and biodistribution analysis in mouse tumor xenograft models. While the unmodified αHER2 and αCD20 Fabs showed weak tumor uptake (0.8% and 0.2% ID/g, respectively; 24 h p.i.) tumor-associated radioactivity was boosted with increasing PAS length (up to 9 and 26-fold, respectively), approaching an optimum for Fab-PAS400. Remarkably, 6- and 5-fold higher tumor-to-blood ratios compared with the unmodified Fabs were measured in the biodistribution analysis (48 h p.i.) for αHER2 Fab-PAS100 and Fab-PAS200, respectively. These findings were confirmed by PET studies, showing high imaging contrast in line with tumor-to-blood ratios of 12.2 and 5.7 (24 h p.i.) for αHER2 Fab-PAS100 and Fab-PAS200. Even stronger tumor signals were obtained with the corresponding αCD20 Fabs, both in PET imaging and biodistribution analysis, with an uptake of 2.8% ID/g for Fab-PAS100 vs. 0.24% ID/g for the unmodified Fab. Hence, by engineering Fabs via PASylation, plasma half-life can be tailored to significantly improve tracer uptake and tumor contrast, thus optimally matching reagent/target interactions.

  6. Experimental studies for immunoscintigraphy of adenocarcinoma of the prostate with [sup 125]I-labeled monoclonal antibody to [gamma]-seminoprotein

    Energy Technology Data Exchange (ETDEWEB)

    Fujino, Awato; Suyama, Kazuho; Shidara, Toshiya; Ao, Teruaki; Ishibashi, Akira; Koshiba, Ken (Kitasato Univ., Sagamihara, Kanagawa (Japan). School of Medicine)


    We established LNCaP tumor in male nude mice after inoculation of LNCaP cells. The tumor produces the organ-specific glycoproteins in the human prostate, [gamma]-seminoprotein ([gamma]-Sm). The LNCaP tumor is composed of fluid and solid portions. The fluid portion contains much more [gamma]-Sm than the solid part. Monoclonal antibody against [gamma]-Sm (murine IgG[sub 1], K) was radiolabeled with iodine-125 by lactoperoxidase method followed by passage over column chromatography. Finally the conjugates with specific activity of 49.8-56.6 KBq/[mu]g were obtained. Biodistribution of [sup 125]I- labeled monoclonal antibody to [gamma]-Sm ([sup 125]I-[gamma]MAb) was evaluated by whole-body autoradiography (ARG) and by determination of label uptake (DLU) by the LNCaP tumor using the well type gamma counter. The ARG were obtained 1-9 days after intravenous administration of 32-38 [mu]g of [sup 125]I-[gamma]MAb to 12 nude mice bearing LNCaP tumor. The DLU were evaluated 3, 4 and 7 days after intravenous administration of 18-25 [mu]g of [sup 125]I- [gamma]MAb to 9 nude mice bearing LNCaP tumor. The DLU by organs (fluid portion, solid portion, liver, kidney, spleen, lung, etc.) were expressed as F-values (%) which were calculated by the following formula: F=radioactivity per gram of tissue (dpm/g) / injected dose per gram of animal (dpm/g)x100 and tissue-to-blood ratios (T/B). The specific uptake by the LNCaP tumors, especially in the fluid portion, was observed on ARG, with optimal images of the tumor evaluated 4-7 days after administration. The F-values and T/B of the LNCaP tumors were always higher than those of other organs. Especially T/B of fluid portion were of 6.7 and 6.3, evaluated 4 and 7 days after administration of [sup 125]I-[gamma]MAb respectively. These results suggest that the radiolabeled [gamma] MAb is applicable for immunoscintigraphy of adenocarcinoma of the prostate. (author).

  7. Improved renal clearance and tumor targeting of {sup 99m}Tc-labeled anti-Tac monoclonal antibody Fab by chemical modifications

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Meyoung-kon; Jeong, Hyeh-Jean; Kao, Chih-Hao K.; Yao, Zhengsheng; Paik, David S.; Pie, Jae Eun; Kobayashi, Hisataka; Waldmann, Thomas A.; Carrasquillo, Jorge A.; Paik, Chang H. E-mail:


    This study was undertaken to improve the renal clearance and tumor targeting properties of {sup 99m}Tc-labeled humanized anti-Tac (HuTac) monoclonal antibody Fab fragments using two chemical approaches: 1) labeling with a renal secretion agent {sup 99m}Tc-mercaptoacetyltriglycine (MAG3) and 2) lowering its isoelectric point (pI) by acylation. HuTac Fab (3.3 mg/mL) was reacted with a trifluorophenyl ester (TFP) of {sup 99m}Tc-MAG3 alone or was additionally reacted with TFP-glycolate to reduce the pI. In Balb/c mice, {sup 99m}Tc-MAG3-Fab (pI>9.3) rapidly accumulated in the kidneys (177% injected dose [ID]/g at 15 min) and then gradually cleared out of the kidneys. In contrast, the glycolation (pI 4.6{approx}6.6) drastically reduced the renal uptake (31% ID/g) and also the whole-body retention (82% ID vs 101% for the nonglycolated) at 15 min, indicating that the glycolated {sup 99m}Tc-MAG3-Fab (pI 4.6{approx}6.6) was rapidly excreted. The glycolated remained in the blood longer than the nonglycolated (1.2% vs 0.3% ID/g at 360 min), but this effect was less drastic than the effect shown on the renal uptake. In nude mice bearing receptor-positive (ATAC4) tumors, the glycolated {sup 99m}Tc-MAG3-Fab increased the peak tumor uptake to 14.8% ID/g from 8.3% ID/g for {sup 99m}Tc-MAG3-Fab, whereas the glycolation resulted in a drastic reduction of the renal uptake at 15 min. We demonstrated that the renal clearance and the tumor targeting of Fab could be optimized by chemical modifications.

  8. Quantitative PET of EGFR expression in xenograft-bearing mice using {sup 64}Cu-labeled cetuximab, a chimeric anti-EGFR monoclonal antibody

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    Cai, Weibo; Chen, Kai; He, Lina; Cao, Qizhen; Chen, Xiaoyuan [Stanford University School of Medicine, The Molecular Imaging Program at Stanford (MIPS), Department of Radiology and Bio-X Program, Stanford, CA (United States); Koong, Albert [Stanford University School of Medicine, Department of Radiation Oncology, Stanford, CA (United States)


    Cetuximab, a chimeric monoclonal antibody targeting epidermal growth factor receptor (EGFR) on the surface of cancer cells, was approved by the FDA to treat patients with metastatic colorectal cancer. It is currently also in advanced-stage development for the treatment of several other solid tumors. Here we report for the first time the quantitative positron emission tomography (PET) imaging of EGFR expression in xenograft-bearing mice using {sup 64}Cu-labeled cetuximab. We conjugated cetuximab with macrocyclic chelating agent 1,4,7,10-tetraazadodecane-N,N',N'',N'''-tetraacetic acid (DOTA), labeled with {sup 64}Cu, and tested the resulting {sup 64}Cu-DOTA-cetuximab in seven xenograft tumor models. The tracer uptake measured by PET was correlated with the EGFR expression quantified by western blotting. The estimated human dosimetry based on the PET data in Sprague-Dawley rats was also calculated. MicroPET imaging showed that {sup 64}Cu-DOTA-cetuximab had increasing tumor activity accumulation over time in EGFR-positive tumors but relatively low uptake in EGFR-negative tumors at all times examined (<5%ID/g). There was a good correlation (R {sup 2} = 0.80) between the tracer uptake (measured by PET) and the EGFR expression level (measured by western blotting). Human dosimetry estimation indicated that the tracer may be safely administered to human patients for tumor diagnosis, with the dose-limiting organ being the liver. The success of EGFR-positive tumor imaging using {sup 64}Cu-DOTA-cetuximab can be translated into the clinic to characterize the pharmacokinetics, to select the right population of patients for EGFR-targeted therapy, to monitor the therapeutic efficacy of anti-EGFR treatment, and to optimize the dosage of either cetuximab alone or cetuximab in combination with other therapeutic agents. (orig.)

  9. Radioimmunotherapy in medullary thyroid cancer using bispecific antibody and iodine 131-labeled bivalent hapten: preliminary results of a phase I/II clinical trial. (United States)

    Kraeber-Bodéré, F; Bardet, S; Hoefnagel, C A; Vieira, M R; Vuillez, J P; Murat, A; Ferreira, T C; Bardiès, M; Ferrer, L; Resche, I; Gautherot, E; Rouvier, E; Barbet, J; Chatal, J F


    The toxicity and therapeutic efficacy of escalating doses of anti-carcinoembryonic antigen x anti-N alpha-(diethylenetriamine-N,N,N',N''-tetraacetic acid)-In bispecific monoclonal antibody (F6-734) and iodine 131-labeled bivalent hapten were determined in a Phase I/II trial. A total of 26 patients with recurrences of medullary thyroid cancer documented by imaging and a rise in serum thyrocalcitonin were enrolled. Twenty to 50 mg of F6-734 and 40-100 mCi of 131I-hapten were injected 4 days apart. Quantitative scintigraphy was performed after the second injection for dosimetry estimations in eight cases. Clinical, biological, and morphological follow-up was carried out for 1 year after treatment. The mean percentage of injected activity per gram of tumor at the time of maximum uptake was 0.08% (range, 0.003-0.26%). The tumor biological half-life ranged from 3 to 95 days, and tumor doses ranged from 2.91 to 184 cGy/mCi. The estimated tumor-to-nontumor dose ratios were 43.8 x 53.4, 29.6 x 35.3, 10.9 x 13.6, and 8.4 x 10.0 for total body, red marrow, liver, and kidney, respectively. Grade III/IV hematological toxicity was observed in seven patients, most of them with bone metastases. Among the 17 evaluable patients, 4 pain reliefs, 5 minor tumor responses, and 4 biological responses with decrease of thyrocalcitonin were observed. Nine patients developed human anti-mouse antibody. Dose-limiting toxicity was hematological, and maximum tolerated activity was 48 mCi/m2 in this group of patients, most of whom had suspected bone marrow involvement. The therapeutic responses observed in patients mainly with a small tumor burden are encouraging for the performance of a Phase II trial with minimal residual disease.

  10. Administration guidelines for radioimmunotherapy of non-Hodgkin's lymphoma with (90)Y-labeled anti-CD20 monoclonal antibody. (United States)

    Wagner, Henry N; Wiseman, Gregory A; Marcus, Carol S; Nabi, Hani A; Nagle, Conrad E; Fink-Bennett, Darlene M; Lamonica, Dominick M; Conti, Peter S


    90Y-ibritumomab tiuxetan is a novel radioimmunotherapeutic agent recently approved for the treatment of relapsed or refractory low-grade, follicular, or CD20+ transformed non-Hodgkin's lymphoma (NHL). (90)Y-ibritumomab tiuxetan consists of a murine monoclonal antibody covalently attached to a metal chelator, which stably chelates (111)In for imaging and (90)Y for therapy. Both health care workers and patients receiving this therapy need to become familiar with how it differs from conventional chemotherapy and what, if any, safety precautions are necessary. Because (90)Y is a pure beta-emitter, the requisite safety precautions are not overly burdensome for health care workers or for patients and their families. (90)Y-ibritumomab tiuxetan is dosed on the basis of the patient's body weight and baseline platelet count; dosimetry is not required for determining the therapeutic dose in patients meeting eligibility criteria similar to those used in clinical trials, such as shielding during dose preparation and administration; primary lead shielding should be avoided because of the potential exposure risk from bremsstrahlung. Because there are no penetrating gamma-emissions associated with the therapy, (90)Y-ibritumomab tiuxetan is routinely administered on an outpatient basis. Furthermore, the risk of radiation exposure to patients' family members has been shown to be in the range of background radiation, even without restrictions on contact. There is therefore no need to determine activity limits or dose rate limits before patients who have been treated with (90)Y radioimmunotherapy are released, as is necessary with patients who have been treated with radiopharmaceuticals that contain (131)I. Standard universal precautions for handling body fluids are recommended for health care workers and patients and their family members after (90)Y-ibritumomab tiuxetan administration. In summary, (90)Y-ibritumomab tiuxetan introduces (90)Y into clinical practice and expands the role

  11. An antibody against a conserved C-terminal consensus motif from plant alternative oxidase (AOX) isoforms 1 and 2 label plastids in the explosive dwarf mistletoe (Arceuthobium americanum, Santalaceae) fruit exocarp. (United States)

    Ross Friedman, Cynthia; Ross, Bradford N; Martens, Garnet D


    Dwarf mistletoes, genus Arceuthobium (Santalaceae), are parasitic angiosperms that spread their seeds by an explosive process. As gentle heating triggers discharge in the lab, we wondered if thermogenesis (endogenous heat production) is associated with dispersal. Thermogenesis occurs in many plants and is enabled by mitochondrial alternative oxidase (AOX) activity. The purpose of this study was to probe Arceuthobium americanum fruit (including seed tissues) collected over a 10-week period with an anti-AOX antibody/gold-labeled secondary antibody to determine if AOX could be localized in situ, and if so, quantitatively assess whether label distribution changed during development; immunochemical results were evaluated with Western blotting. No label could be detected in the mitochondria of any fruit or seed tissue, but was observed in fruit exocarp plastids of samples collected in the last 2 weeks of study; plastids collected in week 10 had significantly more label than week 9 (p = 0.002). Western blotting of whole fruit and mitochondrial proteins revealed a signal at 30-36 kD, suggestive of AOX, while blots of whole fruit (but not mitochondrial fraction) proteins showed a second band at 40-45 kD, in agreement with plastid terminal oxidases (PTOXs). AOX enzymes are likely present in the A. americanum fruit, even though they were not labeled in mitochondria. The results strongly indicate that the anti-AOX antibody was labeling PTOX in plastids, probably at a C-terminal region conserved in both enzymes. PTOX in plastids may be involved in fruit ripening, although a role for PTOX in thermogenesis cannot be eliminated.

  12. High-throughput analysis of therapeutic and diagnostic monoclonal antibodies by multicapillary SDS gel electrophoresis in conjunction with covalent fluorescent labeling. (United States)

    Szekrényes, Ákos; Roth, Udo; Kerékgyártó, Márta; Székely, Andrea; Kurucz, István; Kowalewski, Karen; Guttman, András


    Capillary gel electrophoresis (CGE) in the presence of sodium dodecyl sulfate (SDS) is a well-established and widely used protein analysis technique in the biotechnology industry, and increasingly becoming the method of choice that meets the requirements of the standards of International Conference of Harmonization (ICH). Automated single channel capillary electrophoresis systems are usually equipped with UV absorbance and/or laser-induced fluorescent (LIF) detection options offering general applicability and high detection sensitivity, respectively; however, with limited throughput. This shortcoming is addressed by the use of multicapillary gel electrophoresis (mCGE) systems with LED-induced fluorescent detection (LED-IF), also featuring automation and excellent detection sensitivity, thus widely applicable to rapid and large-scale analysis of biotherapeutics, especially monoclonal antibodies (mAb). The methodology we report in this paper is readily applicable for rapid purity assessment and subunit characterization of IgG molecules including detection of non-glycosylated heavy chains (NGHC) and separation of possible subunit variations such as truncated light chains (Pre-LC) or alternative splice variants. Covalent fluorophore derivatization and the mCGE analysis of the labeled IgG samples with multi-capillary gel electrophoresis are thoroughly described. Reducing and non-reducing conditions were both applied with and without peptide N-glycosidase F mediated deglycosylation.

  13. In vitro and in vivo evaluation of direct rhenium-188-labeled anti-CD52 monoclonal antibody alemtuzumab for radioimmunotherapy of B-cell chronic lymphocytic leukemia

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    Decker, Mario de [Department of Radiopharmacy, Faculty of Pharmaceutical Sciences, Ghent University, Ghent (Belgium); Department of Nuclear Medicine and Molecular Imaging, University Medical Centre Groningen, Groningen (Netherlands)], E-mail:; Bacher, Klaus; Thierens, Hubert [Department of Medical Physics and Radiation Protection, Ghent University, Ghent (Belgium); Slegers, Guido [Department of Medical Imaging of Domestic Animals, Ghent University, Ghent (Belgium); Dierckx, Rudi A. [Department of Nuclear Medicine and Molecular Imaging, University Medical Centre Groningen, Groningen (Netherlands); Vos, Filip de [Department of Radiopharmacy, Faculty of Pharmaceutical Sciences, Ghent University, Ghent (Belgium)


    Alemtuzumab (Campath, Berlex) is a humanized IgG1 rat monoclonal antibody directed against the cell surface CD52 antigen, found on lymphocytes and monocytes. It is being developed for the treatment of chronic lymphocytic leukemia (CLL), autoimmune disease and for the prevention of transplant rejection. This study focused on synthesis, quality control, in vitro evaluation and biodistrubution of {sup 188}Re-labeled alemtuzumab for radioimmunotherapy of B-cell CLL. {sup 188}Re-alemtuzumab was synthesized using a direct radiolabeling method. Reduction of the intramolecular disulfide bonds of the antibody was performed with tris-(carboxyethyl)-phosphine (Pierce), using a 1:60 molar excess. Reaction took place at room temperature for 20 min. A PD-10 desalting column was used to purify the reduced antibody from excess phospine. Complexation and transchelation of {sup 188}ReO{sub 4}{sup -} was achieved using sodium gluconate as weak chelator and SnCl{sub 2} as reducing agent. Quality control was done using instant thin-layer chromatography. Binding assays were performed on a CD52-positive cell line (HuT-78). Female NMRI mice were injected intravenously with 20 {mu}g radiolabeled alemtuzumab and killed at preset time intervals for biodistribution studies. Tissues were dissected, weighed and counted for determination of radioactivity. Data were expressed as percentage injected activity per gram of tissue (% IA/g tissue) or as percentage injected activity (% IA). {sup 188}Re-alemtuzumab was prepared achieving high radiochemical yields. Labeling efficiency of more than 95% can be obtained using optimal reaction conditions. {sup 188}Re-alemtuzumab showed good in vitro stability, remaining intact at 24 h after radiolabeling. In mice, {sup 188}Re-alemtuzumab showed high uptake in the blood (25.10{+-}1.36% IA at 1 h p.i.), followed by a biexponential clearance (t{sub 1/2{alpha}}=4.790 h and t{sub 1/2{beta}}=55.45 h). Increased uptake was observed in kidneys and heart (9

  14. An anti-CD45RO immunotoxin eliminates T cells latently infected with HIV-1 in vitro


    McCoig, Cynthia; Dyke, Gregory; Chou, Chin-Sheng; Picker, Louis J.; Ramilo,Octavio; Vitetta, Ellen S.


    Despite the success of highly active antiretroviral therapy (HAART) in lowering circulating HIV-1 to undetectable levels in most infected individuals, several studies have documented the presence of a small reservoir of latently infected cells in HAART patients, the majority of which are CD45RO+ memory T cells. We previously have demonstrated that latently infected, replication-competent cells can be generated in vitro after eliminating CD25+ cells with an immunotoxin (IT). The present study ...

  15. The study of conjugation of anti-CD20 monoclonal antibody for labeling with metallic or lanthanides radionuclides; Estudo de conjugacao do anticorpo anti-CD20 para marcacao com radionuclideos metalicos ou lantanideos

    Energy Technology Data Exchange (ETDEWEB)

    Akanji, Akinkunmi Ganiyu


    Lymphomas are malignancies or cancers that start from the malign transformation of a lymphocyte in the lymphatic system. Generally, lymphomas start from the lymph nodes or from the agglomeration of the lymphatic tissues, organs like stomach, intestines, in some cases it can involve the bone marrow and the blood, it can also disseminate to other organs. Lymphomas are divided in two major categories: Hodgkin lymphoma and non-Hodgkin lymphoma (NHL). Patient with NHL are generally treated with radiotherapy alone or combined with immunotherapy using monoclonal antibody rituximab (MabThera Registered-Sign ). Currently, monoclonal antibodies (Acm) conjugated with bifunctional chelate agents and radiolabeled with metallic or lanthanides radionuclides are a treatment reality for patients with NHL by the principle of radioimmunotherapy (RIT). This study focused on the conditions of conjugation of Acm rituximab (MabThera Registered-Sign ) with bifunctional chelating agents DOTA and DTPA. Various parameters were studied: method of Acm purification, conditions of Acm conjugation, the method for determination of number of chelate agent coupled to the Acm, method for purification of the conjugated antibody Acm, conditions of labeling of the conjugated antibody with lutetium-177, method of purification of the radiolabeled immuno conjugate, method of radiochemical purity (RP), specific binding in vitro Raji cells (Human Burkitt) and biological distribution performed in normal Balb-c mouse. The three methodologies employed in pre-purification of Acm (dialysis, size exclusion chromatograph and dial filtration) demonstrated to be efficient; they provided sample recovery exceeding 90%. However, the methodology of dial filtration presents minimal sample loss, and gave the final recovery of the sample in micro liters; thereby facilitating sample use in subsequent experiments. Numbers of chelators attached to the Acm molecule was proportional to the molar ratio studied. When we evaluated

  16. Evaluation of (89Zr-labeled human anti-CD147 monoclonal antibody as a positron emission tomography probe in a mouse model of pancreatic cancer.

    Directory of Open Access Journals (Sweden)

    Aya Sugyo

    Full Text Available INTRODUCTION: Pancreatic cancer is an aggressive cancer and its prognosis remains poor. Therefore, additional effective therapy is required to augment and/or complement current therapy. CD147, high expression in pancreatic cancer, is involved in the metastatic process and is considered a good candidate for targeted therapy. CD147-specfic imaging could be useful for selection of appropriate patients. Therefore, we evaluated the potential of a fully human anti-CD147 monoclonal antibody 059-053 as a new positron emission tomography (PET probe for pancreatic cancer. METHODS: CD147 expression was evaluated in four pancreatic cancer cell lines (MIA Paca-2, PANC-1, BxPC-3, and AsPC-1 and a mouse cell line A4 as a negative control. Cell binding, competitive inhibition and internalization assays were conducted with (125I-, (67Ga-, or (89Zr-labeled 059-053. In vivo biodistribution of (125I- or (89Zr-labeled 059-053 was conducted in mice bearing MIA Paca-2 and A4 tumors. PET imaging with [(89Zr]059-053 was conducted in subcutaneous and orthotopic tumor mouse models. RESULTS: Among four pancreatic cancer cell lines, MIA Paca-2 cells showed the highest expression of CD147, while A4 cells had no expression. Immunohistochemical staining showed that MIA Paca-2 xenografts also highly expressed CD147 in vivo. Radiolabeled 059-053 specifically bound to MIA Paca-2 cells with high affinity, but not to A4. [(89Zr]059-053 uptake in MIA Paca-2 tumors increased with time from 11.0±1.3% injected dose per gram (ID/g at day 1 to 16.9±3.2% ID/g at day 6, while [(125I]059-053 uptake was relatively low and decreased with time, suggesting that 059-053 was internalized into tumor cells in vivo and (125I was released from the cells. PET with [(89Zr]059-053 clearly visualized subcutaneous and orthotopic tumors. CONCLUSION: [(89Zr]059-053 is a promising PET probe for imaging CD147 expression in pancreatic cancer and has the potential to select appropriate patients with CD147

  17. R-phycoerythrin标记抗猪瘟病毒荧光抗体的制备及其与FITC标记荧光抗体检测的比较研究%Preparation of R-phycoerythrin-labeled Anti-CSFV Fluorescence Antibody and Comparison with FITC-labeled Anti-CSFV Fluorescence Antibody

    Institute of Scientific and Technical Information of China (English)

    赵守山; 颜世敢; 朱丽萍; 崔道实


    用摩尔比100∶1的化学交联剂SPDP分别将RPE、抗猪瘟病毒抗体衍生,两种衍生物再以摩尔比1∶1液相交联,HPLC纯化制备出RPE标记的抗猪瘟病毒荧光抗体.对制备的荧光抗体从荧光特性、抗体活性、稳定性及纯度等方面进行鉴定.以澳化氰活化的琼脂糖微球为固相载体,采用固相免疫荧光检测法,以制备的RPE、FITC分别标记的荧光抗体检测猪瘟病毒细胞毒.结果表明,RPE标记的荧光抗体检测阳性微球在蓝绿光激发下发射桔黄色荧光,荧光明亮,无本底光干扰,检测灵敏度高达2.67×10-6mg·mL-1,是FITC标记的荧光抗体检测灵敏度的10倍.RPE可替代FTTC或与FITC等荧光染料一起用于多色荧光免疫检测.%R-phycoerythrin from Polysiphonia uneolata was an excellent fluorescent dye with orange-yellowed bright fluorescence. Both of them have the same absorption peak, but the maximum fluorescence emission of R-phycoerythrin was SO nm larger than that of FITC. Purified R-phycoerythrin and multiple-clone antibody of Classical Swine Fever virus was modified by SPDP at the molar ratio of 100 to 1, and then conjugated with each other at the molar ratio of 1 to 1, subsequently purified with HPLC. R-phycoerythrin-labeled antibody was applied to detect CSFV by use of solid-phase immunofluorescence detection method on a CNBr-activated sepharose spheres. Hie system showed orange-yellowed emission fluorescence with high sensitivity and high specificity, the detection sensitivity attained 2.67 x 10"6 mg ? mL~', which was ten times more sensitive than that of FITC-labeled antibody. R-phycoerythrin can substitute for FITC or be used in multiple immunofluorescence detection combined with other dyes.

  18. Specific tumor labeling enhanced by polyethylene glycol linkage of near infrared dyes conjugated to a chimeric anti-carcinoembryonic antigen antibody in a nude mouse model of human pancreatic cancer (United States)

    Maawy, Ali A.; Hiroshima, Yukihiko; Zhang, Yong; Luiken, George A.; Hoffman, Robert M.; Bouvet, Michael


    Labeling of metastatic tumors can aid in their staging and resection of cancer. Near infrared (NIR) dyes have been used in the clinic for tumor labeling. However, there can be a nonspecific uptake of dye by the liver, lungs, and lymph nodes, which hinders detection of metastasis. In order to overcome these problems, we have used two NIR dyes (DyLight 650 and 750) conjugated to a chimeric anti-carcinoembryonic antigen antibody to evaluate how polyethylene glycol linkage (PEGylation) can improve specific tumor labeling in a nude mouse model of human pancreatic cancer. The conjugated PEGylated and non-PEGylated DyLight 650 and 750 dyes were injected intravenously into non-tumor-bearing nude mice. Serum samples were collected at various time points in order to determine serum concentrations and elimination kinetics. Conjugated PEGylated dyes had significantly higher serum dye concentrations than non-PEGylated dyes (p=0.005 for the 650 dyes and pdecreased their accumulation in lymph nodes, liver, and lung. These results demonstrate enhanced selective tumor labeling by PEGylation of dyes conjugated to a tumor-specific antibody, suggesting their future clinical use in fluorescence-guided surgery.

  19. Initial experience with locoregional radioimmunotherapy using {sup 131}I-labelled monoclonal antibodies against tenascin (BC-4) for treatment of glioma (WHO III and IV)

    Energy Technology Data Exchange (ETDEWEB)

    Poepperl, G.; Gildehaus, F.J.; Hahn, K.; Tatsch, K. [Klinik und Poliklinik fuer Nuklearmedizin, Klinikum Grosshadern, Muenchen (Germany); Goetz, C.; Reulen, H.J. [Klinik und Poliklinik fuer Neurochirurgie, Klinikum Grosshadern, Muenchen (Germany); Yousry, T.A. [Inst. fuer Neuroradiologie der LMU Muenchen, Klinikum Grosshadern, Muenchen (Germany)


    Aim: None of the established treatments (surgery, radiotherapy, chemotherapy) for malignant glioma has improved its very poor prognosis. Adjuvant locoregional radioimmunotherapy (RIT) represents a new therapeutic approach. We present our initial experience with this therapeutic tool with respect to adverse effects, biokinetics and clinical follow-up. Methods: Following surgery and radiotherapy, 12 patients with glioma (4, WHO stage III; 8, WHO stage IV) underwent 1-5 RIT-cycles (average dose 1100 MBq {sup 131}labelled monoclonal BC-4 antibodies) at six week intervals. Follow-up included serial FDG-PET and MRI investigations. Evaluation of biokinetics included whole body scans, together with analysis of blood, urine and fluid from the tumor cavity. Results: Following RIT, four patients experienced temporary seizures, which, in one case, were associated with temporary aphasia. Eight patients developed HAMA (human anti-mouse anti-bodies) during follow-up. Mean biologic half-life of the radiopharmaceutical in the resection cavity was 3.9 d (range: 1.0-10.2 d) and remained stable intraindividually during further RIT-cycles. The antibody/radionuclide conjugate remain stable in the tumor cavity for at least 5 d. Median survival presently stands at 18.5 months compared to 9.7 months in a historical patient group (n=89) undergoing conventional therapeutic strategies. Five patients show no signs of recurrence. In three patients with post-surgical evidence of residual tumor, one patient showed partial remission, one stable disease, and one progressive disease during RIT. Four patients without evidence of residual tumor mass at the beginning of RIT developed recurrence during therapy. Conclusions: Initial experience demonstrates that locoregional RIT is a well tolerated treatment modality that may represent a promising new approach in the management of patients with malignant glioma. Advantages of local application include passage of the blood-brain barrier, high concentration

  20. A bifunctional HBED-derivative for labeling of antibodies with [sup 67]Ga, [sup 111]In and [sup 59]Fe. Comparative biodistribution with [sup 111]In-DPTA and [sup 131]I-labeled antibodies in mice bearing antibody internalizing and non-internalizing tumors. [Isothiocyanato-substituted phenolic polyaminocarboxylic acid (HBED-CI)

    Energy Technology Data Exchange (ETDEWEB)

    Schuhmacher, J.; Hull, W.E.; Matys, R.; Hauser, H.; Maier-Borst, W.; Matzku, S. (German Cancer Research Center, Heidelberg (Germany)); Klivenyi, G. (National ' ' FJC' ' Research Inst. for Radiobiology and Radiohygiene, Budapest (Hungary)); Kalthoff, H.; Schmiegel, W.H. (University Hospital, Eppendorf, Hamburg (Germany))


    To investigate whether bifunctional ligands containing chelating structures other than EDTA and DTPA and metallic radiotracers other than [sup 111]In will reduce the non-specific radioactivity uptake in the liver during immunoscintigraphy, we synthetized an isothiocyanato-substituted phenolic polyaminocarboxylic acid (HBED-CI) for labeling of MAbs with [sup 67]Ga, [sup 111]In and [sup 59]Fe. Biodistribution of HBED-CI-labeled MAbs was compared to that of [sup 131]I and [sup 111]In-DTPA labeled MAbs in nude mice bearing tumors, which differ with regard to intracellular internalization and catabolism of the corresponding MAb-antigen complex. In the liver a continuous radioactivity excretion for [sup 67]Ga-HBED-CI-labeled MAbs was observed with kinetics that parallel [sup 131]I clearance after administration of [sup 131]I-MAbs, while [sup 111]In-HBED-CI-labeling led to a constant [sup 111]In liver level quite similar to that of [sup 111]In-DTPA-MAbs. In tumors, [sup 67]Ga-HBED-CI-MAb uptake again paralleled that of [sup 131]I-MAbs. A much lower uptake was found in the case of MAb-antigen internalization. [sup III]In of [sup III]In-HBED-CI- and [sup III]In-DTPA-labeled MAbs continuously accumulated in both types of tumors. Compared with [sup III]In-DTPA-MAbs, an improvement in tumor-to-liver ratios, due to the reduced liver radioactivity associated with [sup 67]Ga-HBED-CI-labeled MAbs, could only be obtained with non-internalizing tumors. The time course of radioactivity distribution in the liver and in MAb-internalizing tumors after administration of [sup 67]Ga-HBED-CI-, [sup III]In-HBED-CI- and [sup III]In-DTPA-labeled MAbs further indicates a dominating influence of the metallic radiotracer rather than the ligand on retention or excretion of radioactivity in MAb-catabolizing tissues. (author).

  1. Surface plasmon resonance-enhanced fluorescence implementation of a single-step competition assay: demonstration of fatty acid measurement using an anti-fatty acid monoclonal antibody and a Cy5-labeled fatty acid. (United States)

    Vareiro, Margarida M L M; Tranchant, Isabelle; Maplin, Sandra; Zak, Kris; Gani, M M; Slevin, Christopher J; Hailes, Helen C; Tabor, Alethea B; Cameron, Petra J; Jenkins, A Toby A; Williams, David E


    The development of a single-step, separation-free method for measurement of low concentrations of fatty acid using a surface plasmon resonance-enhanced fluorescence competition assay with a surface-bound antibody is described. The assay behavior was unexpectedly complex. A nonlinear coverage-dependent self-quenching of emission from surface-bound fluorescent label was deduced from the response kinetics and attributed to a surface plasmon-mediated energy transfer between adsorbed fluorophores, modified by the effects of plasmon interference. Principles of assay design to avoid complications from such effects are discussed. An anti-fatty acid mouse monoclonal antibody reacting to the alkyl chain was prepared and supported on a gold chip at a spacing appropriate for surface-plasmon field-enhanced fluorescence spectroscopy (SPEFS), by applying successively a self-assembled biotinylated monolayer, then streptavidin, then biotinylated protein A, and then the antibody, which was crosslinked to the protein A. Synthesis of a fluorescently (Cy5) tagged C-11 fatty acid is reported. SPEFS was used to follow the kinetics of the binding of the labeled fatty acid to the antibody, and to implement a competition assay with free fatty acid (undecanoic acid), sensitive at the 1 microM scale, a sensitivity limit caused by the low affinity of antibodies for free fatty acids, rather than the SPEFS technique itself. Free fatty acid concentration in human serum is in the range 0.1-1mM, suggesting that this measurement approach could be applied in a clinical diagnostic context. Finally, a predictive, theoretical model of fatty acid binding was developed that accounted for the observed "overshoot" kinetics.

  2. The study of labeling with Iodine-131 of monoclonal antibody anti-CD20 used for the treatment of non-Hodgkin lymphoma; Estudo de marcacao com Iodo-131 de anticorpo monoclonal anti-CD20 na terapia de linfoma nao-Hodgkin

    Energy Technology Data Exchange (ETDEWEB)

    Akanji, Akinkunmi Ganiyu


    Lymphomas are malignancies of the lymphatic system, described by Thomas Hodgkin in 1932. Traditionally, lymphomas are classified in two basic groups: Hodgkin disease and non-Hodgkin lymphoma (NHL). Patients with NHL were earlier treated with radiotherapy alone or in combination with immunotherapy using monoclonal antibody anti-CD20 (ex., Rituximab-Mabthera, Roche). However, Radioimmunotherapy is a new modality of treatment for patients with NHL, in which cytotoxic radiation from therapeutic radioisotopes is delivered to tumors through monoclonal antibodies. This study focused on labeling conditions of monoclonal antibody anti-CD20 (Rituximab-Mabthera, Roche) with iodine-131, by direct radioiodination method using Chloramine-T as oxidizing agent. Labeling parameters investigated were: Radiochemical purity (RP), method of purification, incubation time, antibody mass, oxidative agent mass, stability in vitro, stability in vivo, immunoreactivity and biological distribution performed in normal Swiss mouse. Product of high radiochemical purity was obtained with no notable difference between the methods applied. No clear evidence of direct influence of incubation time on radiochemical purity of the labeled antibody was observed. Whereas, a clear evidence of direct influence of activity on radiochemical purity of the labeled antibody was observed when antibody mass was varied. After purification, the labeled product presented radiochemical purity of approximately 100 %. Product of superior radiochemical yield was observed when standard condition of labeling was used. The labeled product presented variation in radiochemical purity using five different stabilizer conditions. The condition in which gentisic acid was combined with freeze appears more suitable and capable of minimizing autoradiolysis of the antibody labeled with high therapeutic activity of iodine-131. The labeled product presented low immunoreactivity when compared to the literature. Biological distribution in

  3. {sup 90}Nb: potential radionuclide for application in immuno-PET. Development of appropriate production strategy and first in vivo evaluation of {sup 90}Nb-labeled monoclonal antibody

    Energy Technology Data Exchange (ETDEWEB)

    Radchenko, Valery


    Nuclear medicine is a modern and highly effective tool for the detection and treatment of oncological disease. Molecular imaging based on radiotracers includes single photon emission tomography (SPECT) and positron emission tomography (PET), which provide non-invasive tumor visualization on nano- and picomolar level, respectively. Currently, many novel tracers for more precise discovery of small tumors and metastases have been introduced and are under investigation. Many of them are protein-based biomolecules which nature herself produces as antigens for the eradication of tumor cells. Antibodies and antibody fragments play an important role in tumor diagnostics and treatment. PET imaging with antibodies and antibody fragments is called immuno-PET. The main issue that needs to be addressed is that appropriate radiotracers with half-lives related to the half-lives of biomolecules are needed. The development of novel radiotracers is a multistep, complicated task. This task includes the evaluation of production, separation and labeling strategy for chosen radionuclide. Finally, the biomolecule-radionuclide complex should be stable in time. An equally important factor is the economic suitability of the production strategy, which will lead to a key decision for future application of the developed radionuclide. In recent work, {sup 90}Nb has been proposed as a potential candidate for application in immuno-PET. Its half-life of 14.6 hours is suitable for application with antibody fragments and some intact antibodies. {sup 90}Nb has a relatively high positron branching of 53% and an optimal energy of β{sup +} emission of 0.35 MeV that can provide high quality of imaging with low dose of used radionuclide. First proof-of-principle studies have shown that {sup 90}Nb: (i) can be produced in sufficient amount and purity by proton bombardment of natural zirconium target (ii) can be isolated from target material with appropriate radiochemical purity (iii) may be used for

  4. 抗犬瘟热病毒荧光标记单抗的制备和初步鉴定%Preparation and Preliminary Identification of Fluorescein Labeled Monoclonal Antibody against Canine Distemper Virus

    Institute of Scientific and Technical Information of China (English)

    苏建青; 褚秀玲; 杨松涛; 夏咸柱; 岳妙姝


    [Objective] The aim of the present study was to develop a direct immunofluorescence method for the diagnosis of canine distemper (CD) with FITC-conjugated monoclonal antibodies (FITC-McAb). [Method] The McAb against CDV, designated as CE3, was purified with protein G and labeled with FITC through agitation method. After purification and identification, the optimal working concentration of FITC-labeled CE3 was determined. Then 61 clinical samples of suspected canine distemper were detected by direct immunofluorescence assay. [Result] The absorption test, blocking test and specificity test showed that the labeled antibody had high specificity and sensitivity, but didnt have cross reaction with canine parvovirus (CPV), canine parainfluenza virus (CPIV), canine adenovirus (CAV) and rabies virus (RV). The optimal working concentration was 1∶80. The positive rate of clinical suspected samples was 48%. [Conclusion] The direct immunofluorescence assay developed in this study was rapid, specific and convenient, and had great significance for the early diagnosis of canine distemper.

  5. Uptake of /sup 99m/Tc labelled (Fab')/sub 2/ fragments of monoclonal antibody 225. 28S by a benign ocular naevus

    Energy Technology Data Exchange (ETDEWEB)

    Bomanji, J.; Granowska, M.; Britton, K.E.; Hungerford, J.L.


    Malignant melanoma is one of the most common primary intraocular neoplasms. Recently, /sup 99m/Tc radiolabelled (Fab')/sub 2/ fragments of monoclonal antibody 225.28S raised against cutaneous melanomas have been used for imaging uveal melanomas. We report here a case where uptake of radiolabelled antibody was observed in a choroidal melanoma of the right eye and a benign choroidal naevus of the left.

  6. Biodistribution and planar gamma camera imaging of {sup 123}I- and {sup 131}I-labeled F(ab'){sub 2} and Fab fragments of monoclonal antibody 14C5 in nude mice bearing an A549 lung tumor

    Energy Technology Data Exchange (ETDEWEB)

    Burvenich, Ingrid J.G. [Laboratory of Radiopharmacy, University of Ghent, B-9000 Ghent (Belgium)]. E-mail:; Schoonooghe, Steve [Department of Biomedical Research, Flanders Institute of Biotechnology (VIB), University of Ghent, B-9000 Ghent (Belgium); Blanckaert, Peter [Laboratory of Radiopharmacy, University of Ghent, B-9000 Ghent (Belgium); Bacher, Klaus [Department of Medical Physics and Radiation Protection, Ghent University, B-9000 Ghent (Belgium); Vervoort, Liesbet [Laboratory of Radiopharmacy, University of Ghent, B-9000 Ghent (Belgium); Coene, Elisabeth [N. Goormaghtigh Institute of Pathology, Ghent University, B-9000 Ghent (Belgium); Mertens, Nico [Department of Biomedical Research, Flanders Institute of Biotechnology (VIB), University of Ghent, B-9000 Ghent (Belgium); Vos, Filip de [Laboratory of Radiopharmacy, University of Ghent, B-9000 Ghent (Belgium); Slegers, Guido [Laboratory of Radiopharmacy, University of Ghent, B-9000 Ghent (Belgium)


    Detection of antigen 14C5, involved in substrate adhesion and highly expressed on the membrane of many carcinomas, including lung cancer, provides important diagnostic information that can influence patient management. The aim of this study was to evaluate the biodistribution and planar gamma camera imaging characteristics of radioiodinated F(ab'){sub 2} and Fab fragments of monoclonal antibody (mAb) 14C5 in tumor-bearing mice. Methods: F(ab'){sub 2} and Fab 14C5 fragments were radioiodinated using the Iodo-Gen method. In vitro stability, binding specificity and affinity of {sup 125}I-labeled 14C5 fragments were studied in A549 lung carcinoma cells. Biodistribution, blood clearance and tumor-targeting characteristics of {sup 131}I-labeled 14C5 fragments and intact mAb 14C5 were studied in Swiss nu/nu mice bearing A549 lung carcinoma tumors. Planar gamma imaging illustrated the potential use of these {sup 123}I-labeled 14C5 fragments for radioimmunodetection (RID). Results: Saturation binding experiments showed highest affinity for {sup 125}I-labeled F(ab'){sub 2} fragments (K {sub d}=0.37{+-}0.10 nmol/L) and lowest affinity for {sup 125}I-labeled Fab fragments (K {sub d}=2.25{+-}0.44 nmol/L). Blood clearance studies showed that the alpha half-life (t1/2{alpha}) value for Fab, F(ab'){sub 2} and mAb 14C5 was 14.9, 21 and 118 min, respectively. The beta half-life t1/2{beta} value for Fab, F(ab'){sub 2} and mAb 14C5 was 439, 627 and 4067 min, respectively. {sup 131}I-Fab fragments showed highest tumor uptake 3 h after injection (2.4{+-}0.8 %ID/g), {sup 131}I-labeled F(ab'){sub 2} showed highest tumor uptake 6 h after injection (4.7{+-}0.7 %ID/g) and for {sup 131}I-labeled mAb highest tumor uptake was observed at 24 h (10.7{+-}2.3 %ID/g). In planar gamma imaging, both labeled fragments gave better tumor-to-background contrast than {sup 123}I-mAb 14C5. Conclusion: Fab and F(ab'){sub 2} fragments derived from intact mAb 14C5 have

  7. Magnetic Affinity Immunoassay Based Enzyme-Labeled Phage Displayed Antibody%基于酶标噬菌体抗体的磁分离免疫分析方法

    Institute of Scientific and Technical Information of China (English)

    穆晞惠; 童朝阳; 黄启斌; 刘冰; 刘志伟; 郝兰群; 张金平


    A new magnetic affinity immunoassay (MAIA) strategy based on enzyme-labeled phage displayed antibody was developed. The assay consisted of a sandwich format in which immobilized polyclonal antibody (pcAb) on magnetic microparticle was used for capture probe, and enzyme-labeled phage displayed antibody for specific detection probe to increase enzyme amount and enhance detection signal. By the proposed method,β-bungarotoxin (β-BGT) was successfully detected. A linear relationship between absorbance value and the concentration of β-BGT in the range of 0. 016-62. 5 μg / L was obtained. The linear regression equation was Y=0. 641X+1. 355 (R =0. 9925, n = 13, p<0. 0001) with a detection limit of 0. 016 μg / L. In comparison with the traditional ELISA, this method gave a 10-fold better sensitivity in β-BGT detection. This strategy also gave a 4-fold better sensitivity comparing with the MAIA based on enzyme labeled monoclonal antibody (mcAb). Due to low detection limit, acceptable reproducibility and high specificity, this method holds great promise in toxin trace detection.%以磁微粒偶联多抗为磁性捕获探针,酶标噬菌体抗体为特异信号检测探针,采用“磁性捕获探针-待测物-酶标噬菌体抗体探针”的检测模式,成功建立了一种基于酶标噬菌体抗体的磁分离免疫分析方法。本方法检测β-银环蛇毒素线性范围为0.016~62.5μg/ L,回归方程为 Y =0.641X+1.355( R =0.9925,n =13, p<0.0001),检出限为0.016μg/ L。本方法比传统 ELISA 法检测灵敏度提高了10倍,与采用酶标单抗复合物探针的双抗体夹心磁分离免疫分析法相比,检测灵敏度提高4倍。本方法灵敏度高,具有较好重现性与特异性,在毒素的痕量检测方面具有广阔的应用前景。

  8. 牛病毒性腹泻病毒荧光标记单克隆抗体的制备及鉴定%Preparation and identification of fluorescein-labelled monoclonal antibodies against bovine viral diarrhea virus

    Institute of Scientific and Technical Information of China (English)

    徐树兰; 李少英; 赵俊; 武华


    To obtain fluorescein-labelled monoclonal antibodies against different genotypes of bovine viral diarrhea virus(BVDV),specific monoclonal antibodies(McAbs) against BVDV type Ⅰ(BVDV-Ⅰ),type Ⅱ(BVDV-Ⅱ) or both were prepared.And in order to obtain large amount of the McAbs for fluorescein-labelling,BALB/c mice were intraperitoneally injected with hybridomas BV1,BV2,or BV12,respectively.Ascites for the three McAbs were purified with caprylic acid-ammonium method,and then identified by immunocompetence,protein concentration,and purity test.The purified McAbs were conjugated with fluorescein isothiocyanate(FITC) by stirring method.The F/P values of the three labeled McAbs were between 2.0 and 4.0,and their DFA titers to the respective BVDV strains were≥1∶200.Result of antigen cross reaction showed fine specificity of labeled-antibodies to their respective types of BVDV.In BVDV titration TCID50 test,fluorescent assay using the labeled McAbs can detect 1 TCID50 unit of BVDV virion.Preliminary diagnostic test to clinical samples showed that the fluorescence antibodies detected ≥95% of the positive samples,with a 100% concordance rate between the self-made and the imported fluorescence antibodies.Therefore,the three fluorescence antibodies established in the present study will be very useful for the identification of BVDV genotype,detection of NCP-BVDV and differential diagnosis of BVDV and CSFV,and worthy to be researched and developed in future.%为制备可以区分牛病毒性腹泻病毒(BVDV)基因型的荧光标记单克隆抗体,分别利用分泌抗牛病毒性腹泻Ⅰ型病毒(BVDV-Ⅰ)、牛病毒性腹泻Ⅱ型病毒(BVDV-Ⅱ)、牛病毒性腹泻病毒(BVDV-Ⅰ和BVDV-Ⅱ)单克隆抗体的杂交瘤细胞株BV1、BV2、BV12,腹腔接种BALB/c小鼠,大量制备单克隆抗体,用辛酸-硫酸铵法提纯,经活性、浓度、纯度检测合格后,利用搅拌法与异硫氰酸荧光素(FITC)偶联,制备成3

  9. Uptake of 111In-labeled fully human monoclonal antibody TSP-A18 reflects transferrin receptor expression in normal organs and tissues of mice. (United States)

    Sugyo, Aya; Tsuji, Atsushi B; Sudo, Hitomi; Nomura, Fumiko; Satoh, Hirokazu; Koizumi, Mitsuru; Kurosawa, Gene; Kurosawa, Yoshikazu; Saga, Tsuneo


    Transferrin receptor (TfR) is an attractive molecule for targeted therapy of cancer. Various TfR-targeted therapeutic agents such as anti-TfR antibodies conjugated with anticancer agents have been developed. An antibody that recognizes both human and murine TfR is needed to predict the toxicity of antibody-based agents before clinical trials, there is no such antibody to date. In this study, a new fully human monoclonal antibody TSP-A18 that recognizes both human and murine TfR was developed and the correlation analysis of the radiolabeled antibody uptake and TfR expression in two murine strains was conducted. TSP-A18 was selected using extracellular portions of human and murine TfR from a human antibody library. The cross-reactivity of TSP-A18 with human and murine cells was confirmed by flow cytometry. Cell binding and competitive inhibition assays with [111In]TSP-A18 showed that TSP-A18 bound highly to TfR-expressing MIAPaCa-2 cells with high affinity. Biodistribution studies of [111In]TSP-A18 and [67Ga]citrate (a transferrin-mediated imaging probe) were conducted in C57BL/6J and BALB/c-nu/nu mice. [111In]TSP-A18 was accumulated highly in the spleen and bone containing marrow component of both strains, whereas high [67Ga]citrate uptake was only observed in bone containing marrow component and not in the spleen. Western blotting indicated the spleen showed the strongest TfR expression compared with other organs in both strains. There was significant correlation between [111In]TSP-A18 uptake and TfR protein expression in both strains, whereas there was significant correlation of [67Ga]citrate uptake with TfR expression only in C57BL/6J. These findings suggest that the difference in TfR expression between murine strains should be carefully considered when testing for the toxicity of anti-TfR antibody in mice and the uptake of anti-TfR antibody could reflect tissue TfR expression more accurately compared with that of transferrin-mediated imaging probe such as [67Ga]citrate.

  10. Label-free Fab and Fc affinity/avidity profiling of the antibody complex half-life for polyclonal and monoclonal efficacy screening. (United States)

    Read, Thomas; Olkhov, Rouslan V; Williamson, E Diane; Shaw, Andrew M


    A unified approach to affinity screening for Fab and Fc interactions of an antibody for its antigen and FcγR receptor has been developed. An antigen array is used for the Fab affinity and cross-reactivity screening and protein A/G proxy is the FcγR receptor. The affinities are derived using a simple 1:1 binding model with a consistent error analysis. The association and dissociation kinetics are measured over optimised times for accurate determination. The Fab/Fc affinities are derived for ten antibodies: mAb-actin (mouse), pAb-BSA (sheep), pAb-collagen V (rabbit), pAb-CRP (goat), mAb-F1 (mouse), mAbs (mouse) 7.3, 12.3, 29.3, 36.3 and 46.3 raised against LcrV in Yersinia pestis. The rate of the dissociation of antigen-antibody complexes relates directly to their immunological function as does the Fc-FcγR complex and a new half-life plot has been defined with a Fab/Fc half-life range of 17-470 min. The upper half-life value points to surface avidity. Two antibodies that are protective as an immunotherapy define a Fab half-life >250 min and an Fc half-life >50 min as characteristics of ideal interactions which can form the basis of an antibody screen for immunotherapy.

  11. Three monoclonal antibodies with specific binding activity to the excretory system of Schistosoma mansoni: an immunoelectron microscopic study using the gold labeling technique. (United States)

    de Water, R; Fransen, J A; Schut, D W; Deelder, A M


    This study describes three monoclonal antibodies against the excretory system of Schistosoma mansoni. Immunofluorescence revealed antigens forming part of the excretory system of cercariae, adult worms, and miracidia, which were located on the luminal membranes of flame and first tubule cells by immunoelectron microscopy. These antigens are either structural components of the membranes or they derive from excretory fluid and are absorbed during transport and ultrafiltration. Binding specificity of the monoclonal antibodies was tested by immunoelectrophoresis and competitive immunofluorescence; one or two antigens were recognized by each. Reactivity of the antigens after treatment with 7.5% trichloroacetic acid or Rossman's fixative demonstrates at least partial polysaccharide content.

  12. Optimization of IGF-1R SPECT/CT Imaging Using In-111-Labeled F(ab ')(2) and Fab Fragments of Article the Monoclonal Antibody R1507

    NARCIS (Netherlands)

    Heskamp, S.; Laarhoven, H.W.M. van; Molkenboer-Kuenen, J.D.M.; Bouwman, W.H.; Graaf, W.T. van der; Oyen, W.J.G.; Boerman, O.C.


    The insulin-like growth factor 1 receptor (IGF-1R) is a potential new target for the treatment of breast cancer. Patients with breast cancer lesions that express IGF-1R may benefit from treatment with anti-IGF-IR antibodies. IGF-1R expression can be visualized using radiolabeled R1507, a monoclonal

  13. Substituent-specific antibody against glucuronoxylan reveals close association of glucuronic acid and acetyl substituents and distinct labeling patterns in tree species

    DEFF Research Database (Denmark)

    Koutaniemi, Sanna; Guillon, Fabienne; Tranquet, Olivier


    Immunolabeling can be used to locate plant cell wall carbohydrates or other components to specific cell types or to specific regions of the wall. Some antibodies against xylans exist; however, many partly react with the xylan backbone and thus provide limited information on the type of substituen...

  14. Optimization of radioimmunotherapy of renal cell carcinoma: labeling of monoclonal antibody cG250 with 131I, 90Y, 177Lu, or 186Re.

    NARCIS (Netherlands)

    Brouwers, A.H.; Eerd-Vismale, J.E.M. van; Frielink, C.; Oosterwijk, E.; Oyen, W.J.G.; Corstens, F.H.M.; Boerman, O.C.


    Radioimmunotherapy (RIT) can be performed with various radionuclides. We tested the stability, biodistribution, and therapeutic efficacy of various radioimmunoconjugates ((131)I, (88/90)Y, (177)Lu, and (186)Re) of chimeric antirenal cell cancer monoclonal antibody G250 (mAb cG250) in nude mice with


    NARCIS (Netherlands)



    Human IgM monoclonal antibody 16.88 recognised an intracellular antigen strongly expressed in colorectal cancer tissue in 51% of our patients. Tumour localisation was carried out with 185 MBq I-131-16.88 (8 mg) in 20 of these patients with advanced disease. In 16 patients (80%) immunoscintigraphy wa

  16. In vitro and in vivo evaluation of direct rhenium-188-labeled anti-CD52 monoclonal antibody alemtuzumab for radio immunotherapy of B-cell chronic lymphocytic leukemia

    NARCIS (Netherlands)

    De Decker, Mario; Bacher, Klaus; Thierens, Hubert; Slegers, Guido; Dierckx, Rudi A.; De Vos, Filip


    Alemtuzumab (Campath, Berlex) is a humanized IgG1 rat monoclonal antibody directed against the cell surface CD52 antigen, found on lymphocytes and monocytes. It is being developed for the treatment of chronic lymphocytic leukemia (CLL), autoinumme disease and for the prevention of transplant rejecti

  17. Supplemental treatment of rheumatoid arthritis with natural milk antibodies against enteromicrobes and their toxins: results of an open-labelled pilot study

    Directory of Open Access Journals (Sweden)

    Matsuno Takeo


    Full Text Available Abstract Background Environmental factors, particularly commensal bacteria in the gastrointestinal tract, may be involved in the pathogenesis of rheumatoid arthritis (RA. The aim of this study was to evaluate whether natural milk antibodies against a wide spectrum of pathogenic enteromicobes and their toxins modify the disease activity in RA. Methods Twenty patients with RA, whose disease activity was uncontrolled by authentic medications due to drug resistance, complications and/or risk factors were treated for 3 months with an oral administration of a whey protein concentrate (WPC containing high levels of natural milk antibodies. Eighteen background-matched RA patients, not supplemented with milk antibody adjunct, were used as controls. Results Statistically significant reduction of arthritis symptoms and improvement of intestinal disorders were observed only in the test group: effective in 8 (44%, possibly effective in 2 (12% and not effective in 8 (44% of 18 patients treated (2 patients withdrew based on an ad hoc "evaluation point", the sum of variables that are improved more than 20% among the 8 core variables used for the American College of Rheumatology (ACR response criteria. This disease modifying effect of the WPC disappeared upon cessation of treatment, but was reappeared upon reintroduction of it. Importantly, 7 of 8 non-responders carry DR15 haplotype (DRB1-1501 and 1502, whereas only 1 of 7 responders was DR15 positive (risk ratio: 6.1. Furthermore, the pre-clinical serum anti-LPS and anti-type II collagen antibody levels in the responders were higher or tended to be higher than those in the non-responders, suggesting that there are 2 sub-types of RA based on an interaction between gastrointestinal pathogens and MHC class II haplotypes. Conclusions The natural milk antibody preparation containing high levels antibodies against pathogenic enteromicrobes and their toxins seems to be effective in a certain RA subset, and deserves

  18. Site-directed immobilization of a genetically engineered anti-methotrexate antibody via an enzymatically introduced biotin label significantly increases the binding capacity of immunoaffinity columns. (United States)

    Davenport, Kaitlynn R; Smith, Christopher A; Hofstetter, Heike; Horn, James R; Hofstetter, Oliver


    In this study, the effect of random vs. site-directed immobilization techniques on the performance of antibody-based HPLC columns was investigated using a single-domain camelid antibody (VHH) directed against methotrexate (MTX) as a model system. First, the high flow-through support material POROS-OH was activated with disuccinimidyl carbonate (DSC), and the VHH was bound in a random manner via amines located on the protein's surface. The resulting column was characterized by Frontal Affinity Chromatography (FAC). Then, two site-directed techniques were explored to increase column efficiency by immobilizing the antibody via its C-terminus, i.e., away from the antigen-binding site. In one approach, a tetra-lysine tail was added, and the antibody was immobilized onto DSC-activated POROS. In the second site-directed approach, the VHH was modified with the AviTag peptide, and a biotin-residue was enzymatically incorporated at the C-terminus using the biotin ligase BirA. The biotinylated antibody was subsequently immobilized onto NeutrAvidin-derivatized POROS. A comparison of the FAC analyses, which for all three columns showed excellent linearity (R(2)>0.999), revealed that both site-directed approaches yield better results than the random immobilization; the by far highest efficiency, however, was determined for the immunoaffinity column based on AviTag-biotinylated antibody. As proof of concept, all three columns were evaluated for quantification of MTX dissolved in phosphate buffered saline (PBS). Validation using UV-detection showed excellent linearity in the range of 0.04-12μM (R(2)>0.993). The lower limit of detection (LOD) and lower limit of quantification (LLOQ) were found to be independent of the immobilization strategy and were 40nM and 132nM, respectively. The intra- and inter-day precision was below 11.6%, and accuracy was between 90.7% and 112%. To the best of our knowledge, this is the first report of the AviTag-system in chromatography, and the first

  19. Studies of monoclonal antibodies IOR-CEA-1 and IOR-EGF/R3 labelled with {sup 99m}Tc; Estudo de marcacao dos anticorpos monoclonais IOR-CEA-1 e IOR-EGF/R3 com {sup 99m}Tc

    Energy Technology Data Exchange (ETDEWEB)

    Dias, Carla Roberta de Barros Rodrigues


    Nuclear Medicine is a speciality that uses radioisotopes for the diagnosis or treatment of diseases and it is considered one of the best tools among the diagnostic modalities for detection of cancer. {sup 99m}Tc is one of the main isotopes for labelling antibodies and in Nuclear Medicine in general, due to its adequate physical properties, availability and low cost. Labelled monoclonal antibodies have shown promising results for diagnosis and therapy of cancer and their use has brought great experimental and clinical advances in the field of oncology. The main clinical applications of immunoscintigraphy with monoclonal antibodies are staging and evaluation of tumoral reappearance. The antibodies employed in this work were: OIR-CEA-1, a murine monoclonal antibody that acts directly against CEA expressed in several neoplasia in particular those from the gastrointestinal tract (colorectal cancer) and IOR-EGF/R3, a murine monoclonal antibody that binds to the external domain of EGF-R and it has been used in the diagnosis of tumors of epithelial origin. The objectives of this work were the development and optimization of the reduction and purification processes, the radiolabelling techniques and quality control procedures (radiochemical, immunoreactivity and cystein challenge) and imaging studies of monoclonal antibodies OIR-CEA-1 and IOR-EGF/R3, using the simple, fast and efficient method of direct labelling of the antibody with {sup 99m}Tc. The final results was the definition of the best conditions for the preparation of lyophilized reactive kits of OIR-CEA-1 and IOR- EGF/R3 for an efficient diagnostic application in Nuclear Medicine. The most adequate conditions for the labelling of the antibodies were: 1.0 mg Ab, 29 {mu}L MDP, 3.0 {mu}g Sn{sup 2+}, 1 mL of {sup 99m}Tc and 30 min. reaction time. With these conditions the labelling yield was always higher than 95% and the maximum activity of {sup 99m}Tc was about 2220 MBq (60 mCi). The evidences of the efficiency and

  20. Structural Characterization of Peptide Antibodies

    DEFF Research Database (Denmark)

    Chailyan, Anna; Marcatili, Paolo


    The role of proteins as very effective immunogens for the generation of antibodies is indisputable. Nevertheless, cases in which protein usage for antibody production is not feasible or convenient compelled the creation of a powerful alternative consisting of synthetic peptides. Synthetic peptides...... can be modified to obtain desired properties or conformation, tagged for purification, isotopically labeled for protein quantitation or conjugated to immunogens for antibody production. The antibodies that bind to these peptides represent an invaluable tool for biological research and discovery...

  1. Food Labels (United States)

    ... Loss Surgery? A Week of Healthy Breakfasts Shyness Food Labels KidsHealth > For Teens > Food Labels Print A ... have at least 95% organic ingredients. continue Making Food Labels Work for You The first step in ...

  2. sup 99m Tc-labeled monoclonal antibodies against granulocytes (BW 250/183) for the detection of appendicitis. sup 99m Tc-anti-Granulozyten-Antikoerper (BW 250/183) zum Nachweis der Appendizitis

    Energy Technology Data Exchange (ETDEWEB)

    Overbeck, B.; Briele, B.; Hotze, A.; Biersack, H.J. (Bonn Univ. (Germany). Klinik fuer Nuklearmedizin); Kania, U. (Bonn Univ. (Germany). Chirurgische Klinik); Vogel, J. (Bonn Univ. (Germany). Pathologisches Inst.); Lange, L.; Ott, G. (Evangelisches Krankenhaus, Bonn (Germany). Chirurgische Abt.)


    Scintigraphy with {sup 99m}Tc-labeled anti-granulocyte antibodies (AGAb) was performed in 50 patients with suspected appendicitis. Sequential and static imaging as well as SPECT of the pelvis and abdomen was performed 2 hp.i. In all patients the diagnosis was confirmed either histologically or by long-term follow-up. 13 patients had histologically proven acute appendicitis. In 11 patients the appendix scan had been positive and in 2 patients the scan had shown no significant tracer uptake in the right lower abdomen. The remaining 37 patients turned out not to have acute appendicitis. 29 out of these patients had negative and 3 had positive scan findings. In 5 patients the scan was equivocal. Out of these patients 2 had pathologic findings on the left side of the abdomen which turned out to be acute diverticulitis in one patient and acute peritonitis in the other. The remaining 3 patients with unclear scintigraphic findings had no acute appendicitis. Scintigraphy with AGAb is fast and easy to perform and thus superior to cell labeling methods for diagnosing acute appendicitis. Sensitivity for the diagnosis of acute appendicitis was 85% with a specifitiy of 91%. Chronic or scarred non-granulocytic appendicitis - in which there is often no definite indication for surgery - was negative in our study expect for two cases. (orig.).

  3. An Ultrasensitive Electrochemiluminescent Immunoassay for Aflatoxin M1 in Milk, Based on Extraction by Magnetic Graphene and Detection by Antibody-Labeled CdTe Quantumn Dots-Carbon Nanotubes Nanocomposite

    Directory of Open Access Journals (Sweden)

    Ning Gan


    Full Text Available An ultrasensitive electrochemiluminescent immunoassay (ECLIA for aflatoxins M1 (ATM1 in milk using magnetic Fe3O4-graphene oxides (Fe-GO as the absorbent and antibody-labeled cadmium telluride quantum dots (CdTe QDs as the signal tag is presented. Firstly, Fe3O4 nanoparticles were immobilized on GO to fabricate the magnetic nanocomposites, which were used as absorbent to ATM1. Secondly, aflatoxin M1 antibody (primary antibody, ATM1 Ab1, was attached to the surface of the CdTe QDs-carbon nanotubes nanocomposite to form the signal tag (ATM1 Ab1/CdTe-CNT. The above materials were characterized. The optimal experimental conditions were obtained. Thirdly, Fe-GO was employed for extraction of ATM1 in milk. Results indicated that it can adsorb ATM1 efficiently and selectively within a large extent of pH from 3.0 to 8.0. Adsorption processes reached 95% of the equilibrium within 10 min. Lastly, the ATM1 with a serial of concentrations absorbed on Fe-GO was conjugated with ATM1 Ab1/CdTe-CNT signal tag based on sandwich immunoassay. The immunocomplex can emit a strong ECL signal whose intensity depended linearly on the logarithm of ATM1 concentration from 1.0 to 1.0 × 105 pg/mL, with the detection limit (LOD of 0.3 pg/mL (S/N = 3. The method was more sensitive for ATM1 detection compared to the ELISA method. Finally, ten samples of milk were tested based on the immunoassay. The method is fast and requires very little sample preparation, which was suitable for high-throughput screening of mycotoxins in food.

  4. An Ultrasensitive Electrochemical Immunosensor for HIV p24 Based on Fe3O4@SiO2 Nanomagnetic Probes and Nanogold Colloid-Labeled Enzyme–Antibody Copolymer as Signal Tag

    Directory of Open Access Journals (Sweden)

    Tianhua Li


    Full Text Available An ultrasensitive portable electrochemical immunosensor for human immunodeficiency virus p24 (HIV p24 antigen detection has been developed, whereby the detection sensitivity was 1000 times higher than that of the ELISA method. Firstly, a novel HRP enzyme–antibody copolymer (EV-p24 Ab2 was synthesized through an EnVision regent (EV, a dextrin amine skeleton anchoring more than 100 molecules of HRP and 15 molecules of anti IgG, then incubated in the secondary antibody of p24. Secondly, the copolymer was immobilized on the gold nanocolloids (AuNPs to fabricate a novel signal tag (AuNPs/EV-p24 Ab2. Subsequently, a sandwich-type immunoreaction would take place between the capture probe (silicon dioxide-coated magnetic Fe3O4 nanoparticles (MNPs labeled with the primary p24 antibody (MNPs-p24 Ab1, p24 (different concentrations and the signal tag [AuNPs/EV-p24 Ab2] to form the immunocomplex. Finally, the immunocomplex was absorbed on the surface of screen printed carbon electrode (SPCE by a magnet and immersed in the o-hydroxyl phenol (HQ and H2O2. The large amounts of HRP on the signal tag can catalyze the oxidation of HQ by H2O2, which can induce an amplified reductive current. Moreover, the capture probe could improve the accumulation ability of p24 and facilitate its separation from the substrate through the magnet. Under optimal conditions, the proposed immunoassay exhibited good sensitivity to p24 within a certain concentration range from 0.001 to 10.00 ng/mL, with a detection limit of 0.5 pg/mL (S/N = 3. The proposed method can be used for real-time and early detection of HIV-infected people.

  5. [VGKC-complex antibodies]. (United States)

    Watanabe, Osamu


    Various antibodies are associated with voltage-gated potassium channels (VGKCs). Representative antibodies to VGKCs were first identified by radioimmunoassays using radioisotope-labeled alpha-dendrotoxin-VGKCs solubilized from rabbit brain. These antibodies were detected only in a proportion of patients with acquired neuromyotonia (Isaacs' syndrome). VGKC antibodies were also detected in patients with Morvan's syndrome and in those with a form of autoimmune limbic encephalitis. Recent studies indicated that the "VGKC" antibodies are mainly directed toward associated proteins (for example LGI-1 and CASPR-2) that complex with the VGKCs themselves. The "VGKC" antibodies are now commonly known as VGKC-complex antibodies. In general, LGI-1 antibodies are most commonly detected in patients with limbic encephalitis with syndrome of inappropriate secretion of antidiuretic hormone. CASPR-2 antibodies are present in the majority of patients with Morvan's syndrome. These patients develop combinations of CNS symptoms, autonomic dysfunction, and peripheral nerve hyperexcitability. Furthermore, VGKC-complex antibodies are tightly associated with chronic idiopathic pain. Hyperexcitability of nociceptive pathways has also been implicated. These antibodies may be detected in sera of some patients with neurodegenerative diseases (for example, amyotrophic lateral sclerosis and Creutzfeldt-Jakob disease).

  6. Preparation and HRP Labeling of Rabbit Polyclonal Antibodies against Seventeen Mammals%十七种哺乳动物二级抗体的制备和辣根过氧化物酶标记

    Institute of Scientific and Technical Information of China (English)

    林树柱; 刘先菊; 杨帆; 张连峰


    目的 制备兔抗17种哺乳动物的二级抗体,并进行辣根过氧化物酶标记,为哺乳血清学检测系统的建立提供工具.方法 利用饱和硫酸铵沉淀法粗纯抗体后,再利用protein A或protein G亲和层析的方法进一步纯化哺乳动物的IgG,免疫大耳白兔制备抗血清,利用免疫双扩散来检测抗血清的效价,并用Protein A亲和层析的方法来纯化兔抗哺乳动物的二级抗体,采用简易过碘酸钠法对纯化的兔抗哺乳动物的二级抗体进行辣根过氧化物酶标记,通过ELlSA方法测定标记抗体的效价,并利用Western blot方法考察标记抗体的特异性.结果 纯化了恒河猴、东北虎、布氏田鼠、黑线姬鼠、斑羚、原驼、果子狸、食蟹猴、梅花鹿、长爪沙鼠、马鹿、骆驼、大仓鼠、豚鹿、熊猴、大耳羊和雪貂等17种哺乳动物的血清IgG,分别免疫大耳白兔制备了这17种哺乳动物的兔抗血清,免疫双扩散法测定兔抗这17种哺乳动物的抗血清效价均达到1:32,并对纯化的兔抗恒河猴IgG、兔抗东北虎IgG、兔抗布氏田鼠IgG、兔抗黑线姬鼠IgG、兔抗斑羚IgG、兔抗原驼IgG、兔抗果子狸IgG、兔抗食蟹猴IgG、兔抗梅花鹿IgG、兔抗长爪沙鼠IgG、兔抗马鹿IgG、兔抗骆驼IgG、兔抗大仓鼠IgG、兔抗豚鹿IgG、兔抗熊猴IgG、兔抗大耳羊IgG和兔抗雪貂IgG等17种哺乳动物的二级抗体进行了辣根过氧化物酶标记,ELISA测定标记抗体的效价达到1:(2000~60000)左右,Western blots显示标记抗体具有很好的特异性.结论 成功制备了HRP标记的兔抗17种哺乳动物的二级抗体,为哺乳动物血清学检测体系的建立提供了工具.%Objective To prepare the rabbit polyclonal antibodies against seventeen mammals and to label the secondary antibodies with horseradish peroxidase for developing the serological test system of mammals. Methods IgGs of mammals were purified by precipitation with ammonium sulfate

  7. Preliminary radioimmunoimaging and biodistribution of 131iodine-labeled single-chain antibody fragment against progastrin-releasing peptide(31-98) in small cell lung cancer xenografts

    Institute of Scientific and Technical Information of China (English)

    Hong Zhihui; Shi Yizhen; Liu Zengli; Zhou Xiaolin; Yang Yi; Tang Jun


    Background Monoclonal antibodies (mAbs) such as DD3,raised against progastrin-releasing peptide(31-98) (ProGRP(31-98)) antigen,have been used to target small cell lung cancer (SCLC).However,as an intact mAb,DD3 is cleared slowly from the body,with an optimal radioimmunoimaging time of 72 hours.More recently,a singlechain antibody fragment has demonstrated reduced excretion time in blood and normal tissues and is increasingly used in diagnostic cancer research.Thereby,it potentially increases the radioimmunoimaging efficacy.However,there have been few studies with this antibody fragment.The aim of this study was to characterize the preliminary radioimmunoimaging and biodistribution of 1311I-anti-ProGRP(31-98)scFv in nude mice bearing SCLC xenografts.Methods Anti-ProGRP(31-98) scFv was used to detect ProGRP expression by flow cytometry analysis and immunohistochemistry.131I-anti-ProGRP(31-98) scFv was injected intravenously into healthy Kunming mice and the percentage injected dose per gram (%ID/g) in various organs was calculated.Similarly,the %ID/g and tumor/non-tumor ratio in xenograft-bearing mice was calculated.After injection of 131I-anti-ProGRP(31-98) scFv,treated mice were imaged at 1,24,and 30 hours.Then the tumor/base ratios were calculated.Results ProGRP was highly expressed in NCI-H446 cells and xenograft tissue.The metabolism of 131I-anti-ProGRP(31-98) scFv in healthy mice was consistent with a first-order and two-compartment model; T1/2α and T1/2β were 10.2 minutes and 5 hours 18 minutes,respectively.The %ID/g of 131I-anti-ProGRP(31-98) scFv in xenografts was much higher than in healthy tissues at 12 hours after injection,reaching a maximum of (5.38±0.92) %ID/g at 24 hours.Successful imaging of xenograft tissue was achieved as early as 1 hour post-injection and persisted until 30 hours,with 24 hours proving optimal.Conclusion 131I-anti-ProGRP(31-98)scFv shows highly selective tumor uptake with low accumulation in normal tissues and rapid

  8. Safety of Repeated Open-Label Treatment Courses of Intravenous Ofatumumab, a Human Anti-CD20 Monoclonal Antibody, in Rheumatoid Arthritis

    DEFF Research Database (Denmark)

    Quattrocchi, Emilia; Ostergaard, Mikkel; Taylor, Peter C.


    OBJECTIVES: To investigate the safety of ofatumumab retreatment in rheumatoid arthritis. METHODS: Patients with active rheumatoid arthritis participating in two phase III trials (OFA110635 and OFA110634) and a phase II extension trial (OFA111752) received individualised open-label ofatumumab...... retreatment (700 mg X 2 intravenous infusions two weeks apart) ≥24 weeks following the first course and ≥16 weeks following further courses. Retreatment required evidence of clinical response followed by disease relapse. These studies were prematurely terminated by the sponsor to refocus development...... was 463, 182 and 175 patient-years, respectively. Mean time between courses was 17-47 weeks. Ofatumumab induced a profound depletion of peripheral B-lymphocytes. Retreated patients derived benefit based on improvement in DAS28. Adverse events were reported for 93% (226/243), 91% (134/148) and 76% (70...

  9. Use of Radioactive Ion Beams for Biomedical Research 1. in vivo labelling of monoclonal antibodies with radio-lanthanides and $^{225}$Ac

    CERN Multimedia


    % IS330 \\\\ \\\\\\begin{enumerate} \\item The aim of this study was to contribute to developments of new radiopharmaceuticals for tumour diagnosis and therapy. CERN-ISOLDE is the leading facility in the world to provide radioactive ion beams with high selectivity, purity and intensity. Radioisotope production by spallation makes available a complete range of rare earth isotopes having as complete a diversity of types and energy of radiation, of half-life, and of ionic properties as one would wish. The availability of exotic nuclei, e.g. radionuclides of rare earth elements and $^{225}$Ac, opens new possibilities for the development of radiopharmaceuticals for diagnosis and therapy.\\\\ \\\\ \\item Two approaches were followed within the experimental program. The radioactive metal ions are bound either to bio-specific ligands (monoclonal antibodies or peptides) or to unspecific low molecular weight form. The aim of the experimental program is to evaluate relationships between physico-chemical parameters of the tracer m...

  10. Evaluation of a new DTPA-derivative chelator: comparative biodistribution and imaging studies of [sup 111]In-labeled B3 monoclonal antibody in athymic mice bearing human epidermoid carcinoma xenografts. [Diethylenetriaminpentaacetic acid

    Energy Technology Data Exchange (ETDEWEB)

    Camera, L.; Kinuya, S.; Garmestani, K.; Pai, L.H.; Brechbiel, M.W.; Gansow, O.A.; Paik, C.H.; Pastan, I.; Carrasquillo, J.A. (National Cancer Inst., Bethesda, MD (United States))


    Biodistribution and imaging characteristics of monoclonal antibody (MAb) B3 conjugated to either the 2-(p-isothiocvanatobenzyl)-cyclohexyl-DTPA (CHX-B) or 2-(p-isothiocyanatobenzyl)-6-methyl-DTPA (1B4M) and labeled with [sup 111]In, were evalulated in nude mice bearing A431 human epidermoid carcinoma xenografts. MAb B3, is a murine IgG1k reacting with a carbohydrate antigen abundantly expressed by most carcinomas. Both [sup 111]In-(CHX-B)-B3 and [sup 111]In-(1B4M)-B3 showed good tumor targeting with peak values observed at 72 h with 27.6 [+-] 7.6 and 25.4 [+-] 1.7% ID/g, respectively (P > 0.05). High tumor-to-organ ratios were also observed and, confirmed by the imaging results. In particular, tumor-to liver ratios increased from 5.0 [+-] 0.9 at 24 h to 9.2 [+-] 2.0 at 168 h for [sup 111]In-(CHX-B)-B3 and from 4.5 [+-] 0.6 to 8.9 [+-] 3.5 for [sup 111]In-(1B4M)-B3. This was mainly the result of low liver accumulation of both [sup 111]In-(CHX-B)-B3 and [sup 111]In-(1B4M)-B3, with only 2.48 [+-] 0.46 and 2.5 [+-] 0.9% ID/g at 168h, respectively (P > 0.05). Our findings indicate that either CHX-B or 1B4M can be successfully used for [sup 111]In-labeling of MAbs and that [sup 111]In-B3 may represent a promising radioimmunoimaging agent. (Author).

  11. Human pharmacokinetics, biodistribution and dosimetry of the kit of monoclonal antibody IOR EGF/R3 labelled with {sup 99m} Tc

    Energy Technology Data Exchange (ETDEWEB)

    Torres, L.A.; Ramos, M.; Perera, A.; Hernandez, A.; Iznaga, M.E. N. [Solano, Ivette Alvarez, Jose L. Rodriguez. Centro de InvestigacionesClinicas. 34 no.4501 e/45 y 47 Kohly, Playa, C. Habana (Cuba)


    The aim of this work was to assess the human pharmacokinetics, biodistribution and dosimetry of the {sup 99m} Tc-labeled MAb ior egf/r3. Five patients were included in the biodistribution and dosimetric studies and three in the pharmacokinetic analysis. Multiple blood and urine samples we recollected and sequential anterior and posterior whole-body scintigraphies u pto 24 hr post-injection were performed to all patients . The internal radiation dosimetry was estimated from gamma camera imaging data using the methods developed by the Medical Internal radiation dosimetry (MIRD)committee. Raw data were computed from operations between gamma graphic images and regions of interest (ROI) using the Bio-Dose software and time-activity curves were calculated in order to determine the residence times of the source organs. The Pharmacokinetics and Biodistribution results showed that this compound have a bio exponential plasmatic and blood clearance with a rapid biodistribution phase of 9.1 {+-} 8.4 min and 12.2{+-}4.4 min, respectively, and a slower elimination phase of 6.6 {+-} 1.6 hr and 10.8 {+-} 6.8 hr. respectively. The urinary and hepatobiliary excretion showed 4.7 {+-} 0.4 % and 9.9 {+-} 1.8 % of the total administered dose,eliminated by these ways. Liver was the target organ of this product and had an uptake peak at 1 hr post-injection (61.2%) and a great retention of the MAb(T 1/2 eff = 5.3 hr, T 1/2 Biol. = 45.0 hr). The dosimetric results showed that liver, gallbladder and spleen received the higher absorbed. The effective dose and the effective equivalent dose were 1,2E-01 mSv/MBq and 9,2E-02 mSv/MBq respectively. These results allow to see the i or egf/r3 kit in a safe and controlled way. (Author)

  12. Efficacy of Rituximab in Refractory Inflammatory Myopathies Associated with Anti- Synthetase Auto-Antibodies: An Open-Label, Phase II Trial.

    Directory of Open Access Journals (Sweden)

    Yves Allenbach

    Full Text Available Anti-synthetase syndrome (anti-SS is frequently associated with myositis and interstitial lung disease (ILD. We evaluated prospectively, in a multicenter, open-label, phase II study, the efficacy of rituximab on muscle and lung outcomes.Patients were enrolled if they were refractory to conventional treatments (prednisone and at least 2 immunosuppressants. They received 1 g of rituximab at D0, D15, and M6. The primary endpoint was muscular improvement based on manual muscular testing (MMT10, Kendall score in 10 muscles at M12. Secondary endpoints were normalization of creatine kinase (CK level, ILD improvement based on forced vital capacity and/or diffuse capacity for carbon monoxide, and number and/or doses of associated immunosuppressants.Twelve patients were enrolled, and 10 completed the study. Only 2 patients presented an improvement of at least 4 points on at least two muscle groups (primary end-point. Overall, seven patients had an increase of at least 4 points on MMT10. CK level decreased from 399 IU/L (range, 48-11,718 to 74.5 IU/L (range, 40-47,857. Corticosteroid doses decreased from 52.5 mg/d (range, 10-70 to 9 mg/d (range, 7-65 and six patients had a decrease in the burden of their associated immunosuppressants. At baseline, all 10 patients presented with ILD. At M12, improvement of ILD was observed in 5 out of the 10 patients, stabilization in 4, and worsening in 1.This pilot study of rituximab treatment in patients with refractory anti-SS provided data on evolution of muscular and pulmonary parameters. Rituximab should now be evaluated in a larger, controlled study for this homogenous group of NCT00774462.

  13. Vitamin D supplementation reduces thyroid peroxidase antibody levels in patients with autoimmune thyroid disease: An open-labeled randomized controlled trial

    Directory of Open Access Journals (Sweden)

    Sandeep Chaudhary


    Full Text Available Background and Aims: Although Vitamin D deficiency has been linked to autoimmune thyroid disorders (AITD, the impact of Vitamin D supplementation on thyroid autoimmunity is not known. This study aimed to evaluate the impact of Vitamin D supplementation on thyroid autoimmunity (thyroid peroxidase antibody [TPO-Ab] titers in patients with newly diagnosed AITD in a randomized controlled trial. Materials and Methods: One hundred two patients with newly diagnosed AITD (TPO-Ab > 34 kIU/L and/or sonographic evidence of thyroiditis patients were randomized into Group-1 (intervention group and Group-2 (control group. Group-1 received cholecalciferol 60,000 IU weekly and calcium 500 mg/day for 8 weeks; Group-2 received calcium 500 mg/day for 8 weeks. Responders were defined as ≥25% fall in TPO-Ab titers. Individuals with at least 3-month follow-up were analyzed. Trial is registered at (CTRI/2015/04/005713. Results: Data from 100 AITD patients (68 with thyroid stimulating hormone [TSH] ≤10 mIU/L, 32 with TSH > 10 mIU/L, 93% having Vitamin D insufficiency, were analyzed. TPO-Ab titers were highest among patients in the lowest 25-hydroxyvitamin D quartile (P = 0.084. At 3 months follow-up, there was significant fall in TPO-Ab in Group-1 (−46.73% as compared to Group-2 (−16.6% (P = 0.028. Sixty-eight percentage patients in Group-1 were responders compared to 44% in Group-2 (P = 0.015. Kaplan–Meier analysis revealed significantly higher response rate in Group-1 (P = 0.012. Significantly greater reduction in TPO-Ab titers was observed in AITD with TSH ≤ 10 mIU/L compared to TSH > 10 mIU/L. Cox regression revealed Group-1 followed by TPO-Ab and free tetraiodothyronine levels to be a good predictor of response to therapy (P = 0.042, 0.069, and 0.074, respectively. Conclusion: Vitamin D supplementation in AITD may have a beneficial effect on autoimmunity as evidence by significant reductions in TPO-Ab titers.

  14. Nutrition Labeling

    DEFF Research Database (Denmark)

    Grunert, Klaus G


    Nutrition labeling refers to the provision of information on a food product’s nutritional content on the package label. It can serve both public health and commercial purposes. From a public health perspective, the aim of nutrition labeling is to provide information that can enable consumers...... choices, but the nutritional content of food products may not always be clear to consumers, nutrition labeling can contribute to making the nutritional content more transparent, thus reducing the frequency of unhealthy choices. Nutrition labeling is sometimes also motivated by consumers’ right to know......, implying that the availability of information on the nutritional content on food products is of value in itself, no matter how this impacts consumer choices. Another argument for nutrition labeling is that making information about nutritional content transparent will lead to healthier products, partly...

  15. Antimitochondrial antibody (United States)

    ... page: // Antimitochondrial antibody To use the sharing features on this page, please enable JavaScript. Antimitochondrial antibodies (AMA) are substances ( antibodies ) that form against mitochondria. ...

  16. High-throughput epitope binning assays on label-free array-based biosensors can yield exquisite epitope discrimination that facilitates the selection of monoclonal antibodies with functional activity.

    Directory of Open Access Journals (Sweden)

    Yasmina Noubia Abdiche

    Full Text Available Here, we demonstrate how array-based label-free biosensors can be applied to the multiplexed interaction analysis of large panels of analyte/ligand pairs, such as the epitope binning of monoclonal antibodies (mAbs. In this application, the larger the number of mAbs that are analyzed for cross-blocking in a pairwise and combinatorial manner against their specific antigen, the higher the probability of discriminating their epitopes. Since cross-blocking of two mAbs is necessary but not sufficient for them to bind an identical epitope, high-resolution epitope binning analysis determined by high-throughput experiments can enable the identification of mAbs with similar but unique epitopes. We demonstrate that a mAb's epitope and functional activity are correlated, thereby strengthening the relevance of epitope binning data to the discovery of therapeutic mAbs. We evaluated two state-of-the-art label-free biosensors that enable the parallel analysis of 96 unique analyte/ligand interactions and nearly ten thousand total interactions per unattended run. The IBIS-MX96 is a microarray-based surface plasmon resonance imager (SPRi integrated with continuous flow microspotting technology whereas the Octet-HTX is equipped with disposable fiber optic sensors that use biolayer interferometry (BLI detection. We compared their throughput, versatility, ease of sample preparation, and sample consumption in the context of epitope binning assays. We conclude that the main advantages of the SPRi technology are its exceptionally low sample consumption, facile sample preparation, and unparalleled unattended throughput. In contrast, the BLI technology is highly flexible because it allows for the simultaneous interaction analysis of 96 independent analyte/ligand pairs, ad hoc sensor replacement and on-line reloading of an analyte- or ligand-array. Thus, the complementary use of these two platforms can expedite applications that are relevant to the discovery of therapeutic

  17. Immunohistology of carcinoembryonic antigen (CEA)-expressing tumors grafted in nude mice after radioimmunotherapy with 131I-labeled bivalent hapten and anti-CEA x antihapten bispecific antibody. (United States)

    Gautherot, E; Kraeber-Bodéré, F; Daniel, L; Fiche, M; Rouvier, E; Saï-Maurel, C; Thedrez, P; Chatal, J F; Barbet, J


    We have developed a pretargeting strategy, called the Affinity Enhancement System (AES), which uses bispecific antibodies (BsF(ab')2) to target radiolabeled bivalent haptens to tumor cells. We performed several radioimmunotherapy (RIT) experiments in nude mice grafted with LS174T colon carcinoma or TT medullary thyroid cancer. Mice were treated with 131I-labeled di-DTPA-indium-tyrosyl-lysine bivalent hapten (75-112 MBq) administered 15-48 h after anti-CEA x anti-DTPA-indium BsF(ab')2. Immunohistological studies were performed on tumors at their minimal relative volume (TT), on stabilized tumor nodules (LS174T), and on regrowing tumors (TT and LS174T). Untreated tumors were used as controls. On microscopic examination, regrowing tumors (2 months posttherapy) were similar to untreated tumors with cells showing their respective typical morphology (large cells with a high nucleocytoplasmic ratio for TT, small and very undifferentiated cells for LS174T). However, regrowing tumors showed larger necrotic areas and a higher mitotic index correlated with Ki-67 antigen staining. Immunostaining for CEA was as strong as for controls. By contrast, the immunohistology of TT tumors at their minimal relative volume (1 month posttherapy) or of LS174T residual nodules (8 months posttherapy) showed decreased mitotic indices correlated with poor Ki-67 antigen staining. Some clusters of LS174T presented with features of glandular lumen, which suggested a more differentiated and less aggressive status. In TT tumors, CEA expression remained unchanged (80-100% membrane and cytoplasmic staining), whereas only 70% of the LS174T tumors were stained, with 58% loss of the membrane expression. Repeated treatment early after the tumor has reached its minimal relative volume should thus be efficient and improve the overall efficacy of AES RIT.

  18. PF-03446962, a fully-human monoclonal antibody against transforming growth-factor β (TGFβ) receptor ALK1, in pre-treated patients with urothelial cancer: an open label, single-group, phase 2 trial. (United States)

    Necchi, A; Giannatempo, P; Mariani, L; Farè, E; Raggi, D; Pennati, M; Zaffaroni, N; Crippa, F; Marchianò, A; Nicolai, N; Maffezzini, M; Togliardi, E; Daidone, M G; Gianni, A M; Salvioni, R; De Braud, F


    Despite a compelling preclinical rationale for the use of anti-angiogenic drugs in urothelial cancer (UC), short-living responses have been observed in clinical trials. PF-03446962 is a novel monoclonal antibody against Activin Receptor-Like Kinase-1 (ALK1), a type I subclass of the TGFβ receptor, with dose-dependent anti-angiogenic activity. An open label, single-group, phase 2 trial of PF-03446962 was conducted in salvage setting. Patients failing at least one chemotherapy regimen were eligible. Design provided PF-03446962 10 mg/Kg intravenously fortnightly until disease progression (PD) or unacceptable toxicity. Two-month progression-free survival (PFS) was the primary endpoint. The trial was registered with, number NCT01620970. Fourteen patients were enrolled from October 2012 to July 2013. Median age was 64 years (interquartile range [IQR]: 58.2-69.5), 9 patients had a Bellmunt score of 1-2, median number of prior drugs was 3. One stable disease and 13 PD were recorded and the study met the futility stopping rule of interim analysis. Median PFS was 1.8 months (95 %CI, 1.4-2.0). After a median follow up of 7.4 months (IQR 4.5-10.9), 8 patients are alive. Median overall survival (OS) was 8 months (95 %CI, 2.9-not estimable). Most common toxicities were thrombocytopenia (G1-2 in 5 cases, persistent G3 in one, with 3 dose delays and 1 dose interruption), fatigue and abdominal pain (G1-2 in 4 cases each). Impairment of quality of life (ESAS score) was observed as well as an increase from baseline to +2 month median levels of vascular endothelial growth factor (VEGF) and interleukin-8. PF-03446962 had no activity as single drug in refractory UC and we do not recommend further investigation outside of the combination with agents targeting the VEGF receptor axis.

  19. 21 CFR 660.35 - Labeling. (United States)


    ... donor code in the product labeling each donor of peripheral blood used for detection or identification... IDENTIFICATION OF UNEXPECTED ANTIBODIES” or “NOT FOR USE IN DETECTION OR IDENTIFICATION OF UNEXPECTED ANTIBODIES... antibodies shall identify the number of donors contributing to the pool. Products designed exclusively...

  20. Radioimmunotherapy with Tenarad, a {sup 131}I-labelled antibody fragment targeting the extra-domain A1 of tenascin-C, in patients with refractory Hodgkin's lymphoma

    Energy Technology Data Exchange (ETDEWEB)

    Aloj, Luigi [Istituto Nazionale Tumori ' ' Fondazione G. Pascale' ' - IRCCS, Struttura Complessa di Medicina Nucleare, Napoli (Italy); D' Ambrosio, Laura; Aurilio, Michela; Morisco, Anna; Caraco' , Corradina; Di Gennaro, Francesca; Lastoria, Secondo [Istituto Nazionale Tumori ' ' Fondazione G. Pascale' ' - IRCCS, Struttura Complessa Medicina Nucleare, Napoli (Italy); Frigeri, Ferdinando; Capobianco, Gaetana; Pinto, Antonio [Istituto Nazionale Tumori ' ' Fondazione G. Pascale' ' - IRCCS, Struttura Complessa di Ematologia Oncologica, Napoli (Italy); Giovannoni, Leonardo; Menssen, Hans D. [Philogen, SpA, Siena (Italy); Neri, Dario [Institute of Pharmaceutical Sciences, ETH, Zurich (Switzerland)


    The extra-domain A1 of tenascin-C (TC-A1) is highly expressed in the extracellular matrix of tumours and on newly formed blood vessels and is thus a valuable target for radionuclide therapy. Tenarad is a fully human miniantibody or small immunoprotein (SIP, molecular weight 80 kDa) labelled with {sup 131}I that is derived from a TC-A1-binding antibody. Previous phase I/II studies with a similar compound ({sup 131}I-L19SIP) used for radioimmunotherapy (RIT) have shown preliminary efficacy in a variety of cancer types. In this ongoing phase I/II trial, Tenarad was administered to patients with recurrent Hodgkin's lymphoma (HL) refractory to conventional treatments. Eight patients (four men, four women; age range 19 - 41) were enrolled between April 2010 and March 2011. All patients had received a median of three previous lines of chemotherapy (range three to six) and seven had also undergone autologous stem cell transplantation (ASCT) or bone marrow transplantation. In addition, seven patients received external beam radiation. All patients had nodal disease, constitutional B symptoms and some showed extranodal disease in skeletal bone (four patients), lung (three), liver (two) and spleen (one). Baseline assessments included whole-body FDG PET with contrast-enhanced CT and diagnostic Tenarad planar and SPECT studies. Patients were considered eligible to receive a therapeutic dose of Tenarad (2.05 GBq/m{sup 2}) if tumour uptake was more than four times higher than that of muscle. All patients were eligible and received the therapeutic dose of Tenarad. Only one patient developed grade 4 thrombocytopenia and leucocytopenia, requiring hospitalization and therapeutic intervention. All other patients had haematological toxicity of grade 3 or lower, which resolved spontaneously. At the first response assessment (4 - 6 weeks after therapy), one patient showed a complete response, one showed a partial response (PR) and five had disease stabilization (SD). Five patients

  1. Photonic crystal fiber based antibody detection

    DEFF Research Database (Denmark)

    Duval, A; Lhoutellier, M; Jensen, J B


    An original approach for detecting labeled antibodies based on strong penetration photonic crystal fibers is introduced. The target antibody is immobilized inside the air-holes of a photonic crystal fiber and the detection is realized by the means of evanescent-wave fluorescence spectroscopy...

  2. Photonic crystal fiber based antibody detection


    Duval, A.; Lhoutellier, M; Jensen, J. B.; Hoiby, P E; Missier, V; Pedersen, L. H.; Hansen, Theis Peter; Bjarklev, Anders Overgaard; Bang, Ole


    An original approach for detecting labeled antibodies based on strong penetration photonic crystal fibers is introduced. The target antibody is immobilized inside the air-holes of a photonic crystal fiber and the detection is realized by the means of evanescent-wave fluorescence spectroscopy and the use of a transversal illumination setup.

  3. [Neuroimmunological diseases associated with VGKC complex antibodies]. (United States)

    Watanabe, Osamu


    Antibodies to voltage-gated potassium channels(VGKC) were first identified by radioimmunoassay of radioisotope labeled alpha-dendrotoxin-VGKCs solubilized from rabbit brain. These antibodies were found only in a proportion of patients with acquired neuromyotonia (Isaacs' syndrome). VGKC antibodies were also detected in Morvan's syndrome and in a form of autoimmune limbic encephalitis. Recent studies indicated that the "VGKC" antibodies are mainly directed toward associated proteins(for example LGI-1, Caspr-2) that complex with the VGKCs themselves. The "VGKC" antibodies are now usually known as VGKC-complex antibodies. In general, LGI-1 antibodies are most common in limbic encephalitis with SIADH. Caspr-2 antibodies are present in the majority of patients with Morvan's syndrome. These patients develop combinations of CNS symptoms, autonomic dysfunction, and peripheral nerve hyperexcitability.

  4. Fluorescent antibody labeling for experimental choroidal neovascularization in mice%荧光抗体标记法在小鼠脉络膜新生血管中的应用

    Institute of Scientific and Technical Information of China (English)

    顾丽萍; 陈莉; 陈辉; 庹旌生; 高小伟


    impairment in many retinal diseases.To create an ideal CNV animal model is very important for the experimental and clinical study of CNV.The assessment method of repeatable and reliable for CNV model is still seldom.Objective This experiment was to explore the label value of fluorescent antibody for visualizing and quantifying the morphologic changes associated with laser-induced CNV.Methods Laser-induced CNV models were created in 30 eyes of 15 male SPF C57BL/6J mice by Krypton red laser irradiating fundus 2 spots around the optical disc with the wavelength 647.1nm,power 260 mW,spot diameter 50μm and exposure time 0.05 seconds.The CNV was evaluated at 5 minutes,4,7,14 and 28 days after laser injury by using fundus photography and fundus fluorescein angiography (FFA),and the successful models were identified as the rupture of Bruch's membrane.The mice were then immediately sacrificed and the eyeballs were enucleated to prepare the choroidal flatmounts.The posterior eye cups were fluorescently labeled with markers of cell nuclei (DAPI,4',6'-diamino-2-phenylindole),endothelial cells (isolectin-B4),and filamentous actin (phalloidin).The CNV areas from specimens were measured by Image pro plus 6.0.Two eyes from one matched mouse without receiving photocoagulation were used as the controlls.This study followed the Standard of Association for Research in Vision and Ophthalmology.Results No any CNV was seen in photocoagulated eyes in 5 minutes and 4 days after laser irradiation.The first sign of CNV appeared at 7 days following photocoagulation.The incidence of fluorescein leakage was 76.47% (26/34),81.81% (18/22),50.00% (5/10) at 7,14 and 28 days,respectively.The fluomicroscope examination showed that in unphotocoagulated areas,retinal pigment epithelial (RPE) cells were visualized with a uniform hexagonal array.Immediately after laser exposure,a circular area devoid of fluorescent labeling was observed,indicating disruption of the choroid-Bruch membrane-RPE complex.On the fourth

  5. Antibody Based Surgical Imaging and Photodynamic Therapy for Cancer

    NARCIS (Netherlands)

    de Boer, Esther


    In 1944 Albert Coons was the first to show that a fluorescent molecule could be conjugated directly to an antibody made against a target site of interest. This binding does not affect antibody specificity so that labeled antibodies can be used to visualize the location and distribution of the target

  6. 中性红、吖啶橙及PE标记的LAMP-2抗体在B16F10细胞溶酶体检测中的应用与比较%Application and comparison of neutral red, acridine orange, or PE labeled LAMP-2 antibodies in the detection of lysosomes in B16F10 cells

    Institute of Scientific and Technical Information of China (English)

    翟晓峰; 施文; 李国兴; 孙永强; 赵文静; 钱红燕; 李静; 陈橼; 何向锋


    We aimed to investigate the application and value of neutral red, acridine orange, or PE labeled anti-LAMP-2 antibodies in the detection of lysosomes in B16F10 cells. Firstly, we labeled the lysosomes of B16F10 cells with neutral red, acridine orange, and PE labeled anti-LAMP-2 antibodies respectively and detect the labeled lysosomes by optical and fluorescent microscopy. The results showed that the distribution and numbers of lysosomes in B16F10 cells could be clearly observed in optical microscope through the staining of neutral red, and the cytoplasm and lysosome could be stained in green and red respectively by acridine orange, but the quenching of red fluorescence in lysosome was so fast that the detection window was too narrow. The location and numbers of lysosomes in B16F10 cells could be clearly revealed with red fluorescence after the application of PE-labeled anti-LAMP-2 antibody, and the relative location of lysosomes and nucleus could be presented directly along with DAPI staining. In conclusion, the neutral red, acridine orange and PE labeled LAMP-2 antibody staining have their own advantage and characteristics in the detection of lysosomes. The choice of the effective lysosomes detection method should be based on the aim of study.%目的 探讨中性红、吖啶橙及PE标记的LAMP-2抗体在小鼠黑色素瘤B16F10细胞溶酶体检测中的应用与价值.方法 分别用中性红、吖啶橙和PE标记的LAMP-2抗体标记B16F10细胞溶酶体,通过光学和荧光显微镜进行检测.结果 中性红染色法能够在光镜下清晰显示溶酶体在细胞中的分布与数量,并能够反应溶酶体的功能;吖啶橙能够同时将细胞质和溶酶体分别用绿色和红色荧光标示出来,但溶酶体的红色荧光淬灭很快,观测窗口较窄;PE标记的LAMP-2抗体能够将溶酶体在细胞内的位置和数量清晰以红色荧光呈现出来,配合DAPI染色,可以直观显示溶酶体同细胞核

  7. Introduction to Pesticide Labels (United States)

    Pesticide product labels provide critical information about how to safely and legally handle and use pesticide products. Unlike most other types of product labels, pesticide labels are legally enforceable. Learn about pesticide product labels.

  8. On Online Labeling with Polynomially Many Labels

    DEFF Research Database (Denmark)

    Babka, Martin; Bulánek, Jan; Cunat, Vladimír


    In the online labeling problem with parameters n and m we are presented with a sequence of nkeys from a totally ordered universe U and must assign each arriving key a label from the label set {1,2,…,m} so that the order of labels (strictly) respects the ordering on U. As new keys arrive it may be...

  9. Assay for the specificity of monoclonal antibodies in crossed immunoelectrophoresis

    DEFF Research Database (Denmark)

    Skjødt, K; Schou, C; Koch, C


    A method is described based on crossed immunoelectrophoresis of a complex antigen mixture in agarose gel followed by incubation of the gel with the monoclonal antibody. The bound monoclonal antibody is detected by the use of a secondary enzyme-labelled antibody. Using this technique we have been...... I molecules. In other experiments using the same technique we demonstrated the reaction of a monoclonal antibody specific for chicken Ig light chains. Udgivelsesdato: 1984-Aug-3...

  10. Thyroid Antibodies (United States)

    ... e.g., at regular intervals after thyroid cancer treatment) Thyroid stimulating hormone receptor antibody, Thyroid Stimulating Immunoglobulin TRAb, TSHR Ab, TSI Graves disease When a person has symptoms of hyperthyroidism If a pregnant woman has a known autoimmune ...


    NARCIS (Netherlands)



    Two different fluorescein isothiocyanate (FITC) conjugates were used to analyze the effect of labeling intensity on the flow cytometric appearance of marine dinoflagellates labeled with antibodies that specifically recognized the outer cell wall. Location of the labeling was revealed by epifluoresce

  12. The labelling of antibody anti-PBP2a with {sup 99m}Tc; Estudo de marcacao do anticorpo monoclonal anti-PBP2a com {sup 99m}Tc

    Energy Technology Data Exchange (ETDEWEB)

    Mororo, Janio da Silva


    Staphylococcus aureus is a major cause of life-threatening infections such as bacteraemia and endocarditis. Unfortunately, many strains of this bacterial species have become resistant to certain antibiotics, including methicillin and amoxicillin. These strains are known as methicillin-resistant S. aureus (MRSA). The penicillin binding protein 2a (PBP2a) is the enzyme responsible for conferring resistance p-lactams antibiotics for MRSA, being one promising molecule for therapy with mAb. However, besides the therapy, the methods of diagnosis are also inefficient because the diagnosis currently takes several days to produce a reliable result. Taking into account, the objective of this research was radiolabeling one anti-PBP2a mAb developed by Bio-Manguinhos/FioCruz-RJ, utilizing {sup 99m}Tc, for in situ diagnostic of the infectious caused by MRSA. First, anti-PBP2a mAb was reduced utilizing 2-mecaptoethanol (2-ME) for generate sulphydryl groups (-SH) and after to be labeled with {sup 99m}Tc. In this work, were utilized two techniques of direct method: Method 1, using tartrate and gentisic acid reagents, acting like transchelant and stabilizer agents, respectively; and Method 2, using one commercial kit of MDP. Besides the radiolabeling, the mAb reduced and mAb labeled with {sup 99m}Tc were submitted to immunoreactivity analysis, with SDS-PAGE non-reducing, Immunoblotting, ELISA and neutralization assay in vitro methods. The quantity produced of sulphydryl groups by mAb was satisfactory, approximately 5 per mAb, utilizing 6.500:1 of 2-ME:mAb molar ratio. The better labeling method was Method 2, with labeling yield of 73.5%, and showed a good stability after 2 hours (73.2%). The better formulation was: 0.5 mg of mAb anti- PBP2a, 10 {mu}U of MDP kit, after resuspended with 5 mL of saline, and 75.48 MBq (2.04 mCi) of {sup 99m}Tc, reacting by 15 minutes. The labeled mAb maintained the immunoreactivity, utilizing immunologic and in vitro experiments. (author)

  13. Production of Alexa Fluor 488-labeled reovirus and characterization of target cell binding, competence, and immunogenicity of labeled virions. (United States)

    Fecek, Ronald J; Busch, Ryan; Lin, Hong; Pal, Kasturi; Cunningham, Cynthia A; Cuff, Christopher F


    Respiratory enteric orphan virus (reovirus) has been used to study many aspects of the biology and genetics of viruses, viral infection, pathogenesis, and the immune response to virus infection. This report describes the functional activity of virus labeled with Alexa Fluor 488, a stable fluorescent dye. Matrix assisted laser desorption-time of flight analysis indicated that Alexa Fluor 488 labeled the outer capsid proteins of reovirus. Labeled virus bound to murine L929 fibroblasts as determined by flow cytometry and fluorescence microscopy, and the specificity of binding were demonstrated by competitive inhibition with non-labeled virus. Labeled reovirus induced apoptosis and cytopathic effect in infected L929 cells. Mice infected with labeled virus mounted robust serum antibody and CD8(+) T-cell responses, indicating that labeled virus retained immunogenicity in vivo. These results indicate that Alexa Fluor 488-labeled virus provides a powerful new tool to analyze reovirus infection in vitro and in vivo.

  14. Scintigraphy to antimyosin antibodies labelled with Indium 111 in the diagnosis of acute myocarditis: first results; La scintigraphie aux anticcorps antimyosine marques a l`Indium 111 dans le diagnostique des myocardites aigues: premiers resultats

    Energy Technology Data Exchange (ETDEWEB)

    Hossein-Foucher, Claude [Medecine Nucleaire CHRU Lille, 59037 Lille Cedex (France); Millaire, Alain; De Groote, Pascal [Cardiologie C CHRU Lille, 59037 Lille Cedex (France); Marchandise, Xavier [Medecine Nucleaire CHRU Lille, 59037 Lille Cedex (France)


    The utility of antimyosin antibody scintigraphy is rather well established in severe myocarditis in which the certainty of diagnosis is based on endo-myocardial biopsies. However, only a few studies concern the moderated forms of myocarditis in which the biopsy was generally not realised. In our study the myocarditis diagnosis is based on a bunch of clinical, electrocardiographic and echocardiographic arguments. Eighteen patients were explored (13 males, 5 females of 37 {+-} 17 years old). The scintigraphy was achieved 48 hours after injecting 2 mCi of Myoscint and compared with a thallium scintigraphy at rest. Zones of interest were established in order to calculate a heart/lung index. An ischemic cardiopathy was excluded either by coronary-graphy or thallium scintigraphy. In 11 patients diagnosis of myocarditis or myo-pericarditis was thought as certain when the the rate of CPK mb increased significantly. The other 7 patients presented either isolated pericarditis or another heart pathology. ROC curves were established in order to determine the best threshold to pose a myocarditis diagnosis. An index of 1.55 represents the best compromise between sensitivity (91%) and specificity (100%). When the scintigraphy was positive the positioning was more often localised (80% of cases) than smeared out. In conclusion, our results indicate that the antimyosin antibody heart scintigraphy is a precise and reliable investigation in myocarditis diagnosis

  15. Quantitative assessment of antibody internalization with novel monoclonal antibodies against Alexa fluorophores.

    Directory of Open Access Journals (Sweden)

    Sindy Liao-Chan

    Full Text Available Antibodies against cell surface antigens may be internalized through their specific interactions with these proteins and in some cases may induce or perturb antigen internalization. The anti-cancer efficacy of antibody-drug conjugates is thought to rely on their uptake by cancer cells expressing the surface antigen. Numerous techniques, including microscopy and flow cytometry, have been used to identify antibodies with desired cellular uptake rates. To enable quantitative measurements of internalization of labeled antibodies, an assay based on internalized and quenched fluorescence was developed. For this approach, we generated novel anti-Alexa Fluor monoclonal antibodies (mAbs that effectively and specifically quench cell surface-bound Alexa Fluor 488 or Alexa Fluor 594 fluorescence. Utilizing Alexa Fluor-labeled mAbs against the EphA2 receptor tyrosine kinase, we showed that the anti-Alexa Fluor reagents could be used to monitor internalization quantitatively over time. The anti-Alexa Fluor mAbs were also validated in a proof of concept dual-label internalization assay with simultaneous exposure of cells to two different mAbs. Importantly, the unique anti-Alexa Fluor mAbs described here may also enable other single- and dual-label experiments, including label detection and signal enhancement in macromolecules, trafficking of proteins and microorganisms, and cell migration and morphology.

  16. Validating Antibodies to the Cannabinoid CB2 Receptor: Antibody Sensitivity Is Not Evidence of Antibody Specificity. (United States)

    Marchalant, Yannick; Brownjohn, Philip W; Bonnet, Amandine; Kleffmann, Torsten; Ashton, John C


    Antibody-based methods for the detection and quantification of membrane integral proteins, in particular, the G protein-coupled receptors (GPCRs), have been plagued with issues of primary antibody specificity. In this report, we investigate one of the most commonly utilized commercial antibodies for the cannabinoid CB2 receptor, a GPCR, using immunoblotting in combination with mass spectrometry. In this way, we were able to develop powerful negative and novel positive controls. By doing this, we are able to demonstrate that it is possible for an antibody to be sensitive for a protein of interest-in this case CB2-but still cross-react with other proteins and therefore lack specificity. Specifically, we were able to use western blotting combined with mass spectrometry to unequivocally identify CB2 protein in over-expressing cell lines. This shows that a common practice of validating antibodies with positive controls only is insufficient to ensure antibody reliability. In addition, our work is the first to develop a label-free method of protein detection using mass spectrometry that, with further refinement, could provide unequivocal identification of CB2 receptor protein in native tissues.

  17. Evaluating the Efficacy of Three Different Fluorochrome-labeled CD4 Antibodies in Studying T Helper Cells by Flow Cytometry%三种荧光色素标记的CD4抗体在流式细胞术研究T辅助细胞中的有效性评价

    Institute of Scientific and Technical Information of China (English)

    郑翠玲; 韩晓红


    To determine the preferred and effective CD4 antibody used for T helper (Th) cell study, Th cells before and after stimulation were investigated by flow cytometry with three different fluorochrome-labeled CD4 antibodies. Before and after stimulation with PMA and Ion, the percentage of Th cells (CD3 + CD8-T cells) was determined by CD3-APC and CD8-PerCP, while the percentage of Th cells (CD4 + T cells) was detected by CD4PerCP, CD4-FITC and CD4-APC. Furthermore, CD4 + T cells sorted by immunomagnetic beads were used to confirm the efficacy of CD4-APC antibody in Th cell research. The results showed that there was no significant difference in the percentages of Th cells ( CD3 + CD8- T cells) before and after stimulation (P >0.05). The percentages of Th cells (CD4 + CD8-T cells) before and after stimulation detected by three fluorochrome-labeled CD4 antibodies were all declined. Among them, CD4-PerCP declined significantly (P < 0. 05 ), while CD4-FITC and CD4-APC declined slightly (P > 0. 05). The MFI of CD4-APC was the strongest among three fluorochrome-labeled CD4 antibodies and did not decrease significantly after stimulation (P >0.05), while the MFI of CD4-PerCP and CD4-FITC decreased sharply (P < 0.05 ). Cells sorted by immunomagnetic beads also confirmed that the stimulation had little effect on the expression of CD4-APC. It is thus showed that the preferred CD4-APC antibody was effective in Th cell research among the three fluorochrome-labeled CD4 antibodies.%探讨三种荧光色素标记的CD4抗体在流式细胞术检测培养刺激前后的T 辅助(Th)细胞中的有效性,从而确定Th细胞研究时优选且有效的CD4抗体.将佛波脂(PMA)和离子霉素(Ion)培养刺激前后的正常人外周血单个核细胞(PBMC)分别用CD3-APC和CD8-PerCP确定Th细胞(CD3+CD8- T细胞)百分率,用CD4-PerCP、CD4-FITC和CD4-APC确定Th细胞(CD4+ T细胞)百分率.并用免疫磁珠分选后的CD4+ T细胞验证CD4-APC抗体在Th细胞研究中的

  18. Quantitative localization microscopy: effects of photophysics and labeling stoichiometry.

    Directory of Open Access Journals (Sweden)

    Robert P J Nieuwenhuizen

    Full Text Available Quantification in localization microscopy with reversibly switchable fluorophores is severely hampered by the unknown number of switching cycles a fluorophore undergoes and the unknown stoichiometry of fluorophores on a marker such as an antibody. We overcome this problem by measuring the average number of localizations per fluorophore, or generally per fluorescently labeled site from the build-up of spatial image correlation during acquisition. To this end we employ a model for the interplay between the statistics of activation, bleaching, and labeling stoichiometry. We validated our method using single fluorophore labeled DNA oligomers and multiple-labeled neutravidin tetramers where we find a counting error of less than 17% without any calibration of transition rates. Furthermore, we demonstrated our quantification method on nanobody- and antibody-labeled biological specimens.

  19. Pretargeting CD45 enhances the selective delivery of radiation to hematolymphoid tissues in nonhuman primates

    Energy Technology Data Exchange (ETDEWEB)

    Green, Damian J.; Pagel, John M.; Nemecek, Eneida R.; Lin, Yukang; Kenoyer, Aimee L.; Pantelias, Anastasia; Hamlin, Donald K.; Wilbur, D. S.; Fisher, Darrell R.; Rajendran, Joseph G.; Gopal, Ajay K.; Park, Steven I.; Press, Oliver W.


    Pretargeted radioimmunotherapy (PRIT) is designed to enhance the directed delivery of radionuclides to malignant cells. Through a series of studies in nineteen nonhuman primates (M. fascicularis) the potential therapeutic advantage of anti-CD45 PRIT was evaluated. Anti-CD45 PRIT demonstrated a significant improvement in target-to-normal organ ratios of absorbed radiation when compared to directly radiolabeled bivalent antibody (conventional radioimmunotherapy [RIT]). Radio-DOTA-biotin administered 48 hours after anti-CD45 streptavidin fusion protein (FP) [BC8 (scFv)4SA] produced markedly lower concentrations of radiation in non-target tissues when compared to conventional RIT. PRIT generated superior target:normal organ ratios in the blood, lung and liver (10.3:1, 18.9:1 and 9.9:1 respectively) when compared to the conventional RIT controls (2.6:1, 6.4:1 and 2.9:1 respectively). The FP demonstrated superior retention in target tissues relative to comparable directly radiolabeled bivalent anti-CD45 RIT. The time-point of administration of the second step radiolabeled ligand (radio-DOTA-biotin) significantly impacted the biodistribution of radioactivity in target tissues. Rapid clearance of the FP from the circulation rendered unnecessary the addition of a synthetic clearing agent in this model. These results support proceeding to anti-CD45 PRIT clinical trials for patients with both leukemia and lymphoma.

  20. Antiparietal cell antibody test (United States)

    APCA; Anti-gastric parietal cell antibody; Atrophic gastritis - anti-gastric parietal cell antibody; Gastric ulcer - anti-gastric parietal cell antibody; Pernicious anemia - anti-gastric parietal cell antibody; ...

  1. Pre-clinical evaluation of a 213Bi-labeled 2556 antibody to HIV-1 gp41 glycoprotein in HIV-1 mouse models as a reagent for HIV eradication.

    Directory of Open Access Journals (Sweden)

    Ekaterina Dadachova

    Full Text Available BACKGROUND: Any strategy for curing HIV infection must include a method to eliminate viral-infected cells. Based on our earlier proof-of-principle results targeting HIV-1 infected cells with radiolabeled antibody (mAb to gp41 viral antigen, we embarked on identifying a suitable candidate mAb for preclinical development. METHODOLOGY/PRINCIPAL FINDINGS: Among the several human mAbs to gp41 tested, mAb 2556 was found to have high affinity, reactivity with multimeric forms of gp41 present on both the surface of virus particles and cells expressing HIV-1 Env, and recognition of a highly conserved epitope of gp41 shared by all HIV-1 subtypes. Also, mAb 2556 was the best in competition with HIV-1+ serum antibodies, which is an extremely important consideration for efficacy in the treatment of HIV patients. When radiolabeled with alpha-emitting radionuclide 213-Bismuth ((213Bi - (213Bi-2556 efficiently and specifically killed ACH-2 human lymphocytes chronically infected with HIV-1, and HIV-1 infected human peripheral blood mononuclear cells (hPBMCs. The number of binding sites for (213Bi-2556 on the surface of the infected cells was >10(6. The in vivo experiments were performed in two HIV-1 mouse models--splenic and intraperitoneal. In both models, the decrease in HIV-1 infected hPBMCs from the spleens and peritoneum, respectively, was dose-dependent with the most pronounced killing of hPBMCs observed in the 100 µCi (213Bi-2556 group (P = 0.01. Measurement of the blood platelet counts and gross pathology of the treated mice demonstrated the lack of toxicity for (213Bi-2556. CONCLUSIONS/SIGNIFICANCE: We describe the preclinical development of a novel radiolabeled mAb reagent that could potentially be part of an HIV eradication strategy that is ready for translation into the clinic as the next step in its development. As viral antigens are very different from "self" human antigens - this approach promises high selectivity, increased efficacy and low

  2. Preparation of radiopharmaceuticals labeled with metal radionuclides

    Energy Technology Data Exchange (ETDEWEB)

    Welch, M.J.


    We recently developed a useful zinc-62/copper-62 generator and are presently evaluating copper-62 radiopharmaceuticals for clinical studies. While developing these copper-62 radiopharmaceuticals, in collaboration with the University of Missouri Research Reactor, Columbia we have also explored copper-64 radiopharmaceuticals. The PET images we obtained with copper-64 tracers were of such high quality that we have developed and evaluated copper-64 labeled antibodies for PET imaging. The major research activities described herein include: the development and assessment of gallium-68 radiopharmaceuticals; the development and evaluation of a new zinc-62/copper-62 generator and the assessment of copper-62 radiopharmaceuticals; mechanistic studies on proteins labeled with metal radionuclides.

  3. A single-arm, open-label, phase 2 clinical trial evaluating disease response following treatment with BI-505, a human anti-intercellular adhesion molecule-1 monoclonal antibody, in patients with smoldering multiple myeloma (United States)

    Wichert, Stina; Juliusson, Gunnar; Johansson, Åsa; Sonesson, Elisabeth; Teige, Ingrid; Wickenberg, Anna Teige; Frendeus, Björn; Korsgren, Magnus; Hansson, Markus


    Background Smoldering multiple myeloma (SMM) is an indolent disease stage, considered to represent the transition phase from the premalignant MGUS (Monoclonal Gammopathy of Undetermined Significance) state towards symptomatic multiple myeloma (MM). Even though this diagnosis provides an opportunity for early intervention, few treatment studies have been done and the current standard of care is observation until progression. BI-505, a monoclonal antibody directed against intercellular adhesion molecule 1 (ICAM-1) with promising anti-myeloma activity in preclinical trials, is a possible treatment approach for this patient category with potential to eliminate tumor cells with minimal long-term side effects. BI-505 was well tolerated in an earlier phase 1 trial. Methods and findings In this phase 2 trial the effects of BI-505 in patients with SMM were studied. Four patients were enrolled and three of them completed the first cycle of treatment defined as 5 doses of BI-505, a total of 43 mg/kg BW, over a 7-week period. In the three evaluable patients, BI-505 showed a benign safety profile. None of the patients achieved a response as defined per protocol. EudraCT number: 2012-004884-29. Conclusions The study was conducted to assess the efficacy, safety and pharmacodynamics of BI-505 in patients with SMM. BI-505 showed no clinically relevant efficacy on disease activity in these patients with SMM, even if well tolerated. Trial registration Identifier: NCT01838369. PMID:28158311

  4. Antibody-based biological toxin detection

    Energy Technology Data Exchange (ETDEWEB)

    Menking, D.E.; Goode, M.T. [Army Edgewood Research, Development and Engineering Center, Aberdeen Proving Ground, MD (United States)


    Fiber optic evanescent fluorosensors are under investigation in our laboratory for the study of drug-receptor interactions for detection of threat agents and antibody-antigen interactions for detection of biological toxins. In a direct competition assay, antibodies against Cholera toxin, Staphylococcus Enterotoxin B or ricin were noncovalently immobilized on quartz fibers and probed with fluorescein isothiocyanate (FITC) - labeled toxins. In the indirect competition assay, Cholera toxin or Botulinum toxoid A was immobilized onto the fiber, followed by incubation in an antiserum or partially purified anti-toxin IgG. These were then probed with FITC-anti-IgG antibodies. Unlabeled toxins competed with labeled toxins or anti-toxin IgG in a dose dependent manner and the detection of the toxins was in the nanomolar range.

  5. Labeled Antimicrobial Peptides for Detection of Microorganisms (United States)


    Beer , C, Koka, R. and Wang, X., 2001: Solid-Phase Capture of Proteins, Spores, and Bacteria. Applied and Environmental Microbiology , 67: 1300-1307. Yu...detection but often lack stability and sensitivity. Current biosensor technologies using fluorescently labeled antibodies are generally capable of...biosensor. J. ofRapid Methods and Automation in Microbiology , 9: 241- 257. Demarco, D. R. and Lim, D. Y., 2002: Detection of Escherichia coli 0157:H7

  6. Semiotic labelled deductive systems

    Energy Technology Data Exchange (ETDEWEB)

    Nossum, R.T. [Imperial College of Science, Technology and Medicine, London (United Kingdom)


    We review the class of Semiotic Models put forward by Pospelov, as well as the Labelled Deductive Systems developed by Gabbay, and construct an embedding of Semiotic Models into Labelled Deductive Systems.

  7. Mental Labels and Tattoos (United States)

    Hyatt, I. Ralph


    Discusses the ease with which mental labels become imprinted in our system, six basic axioms for maintaining negative mental tattoos, and psychological processes for eliminating mental tattoos and labels. (RK)

  8. Pesticide Product Label System (United States)

    U.S. Environmental Protection Agency — The Pesticide Product Label System (PPLS) provides a collection of pesticide product labels (Adobe PDF format) that have been approved by EPA under Section 3 of the...

  9. Radiolabeled monoclonal antibodies for imaging and therapy: Potential, problems, and prospects: Scientific highlights

    Energy Technology Data Exchange (ETDEWEB)

    Srivastava, S.C.; Buraggi, G.L.


    This meeting focused on areas of research on radiolabeled monoclonal antibodies. Topics covered included the production, purification, and fragmentation of monoclonal antibodies and immunochemistry of hybridomas; the production and the chemistry of radionuclides; the radiohalogenation and radiometal labeling techniques; the in-vivo pharmacokinetics of radiolabeled antibodies; the considerations of immunoreactivity of radiolabeled preparations; the instrumentation and imaging techniques as applied to radioimmunodetection; the radiation dosimetry in diagnostic and therapeutic use of labeled antibodies; the radioimmunoscintigraphy and radioimmunotherapy studies; and perspectives and directions for future research. Tutorial as well as scientific lectures describing the latest research data on the above topics were presented. Three workshop panels were convened on ''Methods for Determining Immunoreactivity of Radiolabeled Monoclonal Antibodies - Problems and Pitfalls,'' Radiobiological and Dosimetric Considerations for Immunotherapy with Labeled Antibodies,'' and ''The Human Anti-Mouse Antibody Response in Patients.''

  10. Labeling Schemes with Queries



    We study the question of ``how robust are the known lower bounds of labeling schemes when one increases the number of consulted labels''. Let $f$ be a function on pairs of vertices. An $f$-labeling scheme for a family of graphs $\\cF$ labels the vertices of all graphs in $\\cF$ such that for every graph $G\\in\\cF$ and every two vertices $u,v\\in G$, the value $f(u,v)$ can be inferred by merely inspecting the labels of $u$ and $v$. This paper introduces a natural generalization: the notion of $f$-...

  11. 99mTc: Labeling Chemistry and Labeled Compounds (United States)

    Alberto, R.; Abram, U.

    This chapter reviews the radiopharmaceutical chemistry of technetium related to the synthesis of perfusion agents and to the labeling of receptor-binding biomolecules. To understand the limitations of technetium chemistry imposed by future application of the complexes in nuclear medicine, an introductory section analyzes the compulsory requirements to be considered when facing the incentive of introducing a novel radiopharmaceutical into the market. Requirements from chemistry, routine application, and market are discussed. In a subsequent section, commercially available 99mTc-based radiopharmaceuticals are treated. It covers the complexes in use for imaging the most important target organs such as heart, brain, or kidney. The commercially available radiopharmaceuticals fulfill the requirements outlined earlier and are discussed with this background. In a following section, the properties and perspectives of the different generations of radiopharmaceuticals are described in a general way, covering characteristics for perfusion agents and for receptor-specific molecules. Technetium chemistry for the synthesis of perfusion agents and the different labeling approaches for target-specific biomolecules are summarized. The review comprises a general introduction to the common approaches currently in use, employing the N x S4-x , [3+1] and 2-hydrazino-nicotinicacid (HYNIC) method as well as more recent strategies such as the carbonyl and the TcN approach. Direct labeling without the need of a bifunctional chelator is briefly reviewed as well. More particularly, recent developments in the labeling of concrete targeting molecules, the second generation of radiopharmaceuticals, is then discussed and prominent examples with antibodies/peptides, neuroreceptor targeting small molecules, myocardial imaging agents, vitamins, thymidine, and complexes relevant to multidrug resistance are given. In addition, a new approach toward peptide drug development is described. The section

  12. Succesful labelling schemes

    DEFF Research Database (Denmark)

    Juhl, Hans Jørn; Stacey, Julia


    It is usual practice to evaluate the success of a labelling scheme by looking at the awareness percentage, but in many cases this is not sufficient. The awareness percentage gives no indication of which of the consumer segments that are aware of and use labelling schemes and which do not. In the ......It is usual practice to evaluate the success of a labelling scheme by looking at the awareness percentage, but in many cases this is not sufficient. The awareness percentage gives no indication of which of the consumer segments that are aware of and use labelling schemes and which do not....... In the spring of 2001 MAPP carried out an extensive consumer study with special emphasis on the Nordic environmentally friendly label 'the swan'. The purpose was to find out how much consumers actually know and use various labelling schemes. 869 households were contacted and asked to fill in a questionnaire...... it into consideration when I go shopping. The respondent was asked to pick the most suitable answer, which described her use of each label. 29% - also called 'the labelling blind' - responded that they basically only knew the recycling label and the Government controlled organic label 'Ø-mærket'. Another segment of 6...

  13. [Antinuclear antibodies]. (United States)

    Cabiedes, Javier; Núñez-Álvarez, Carlos A


    Anti-nuclear antibodies (ANA) are immunoglobulin directed against autologous cell nuclear and cytoplasmic components. Besides the autoimmune ANA there are other ANA that can be detected in circulation, like natural and infectious ANA. Because of its high sensibility, detection of the ANA must be done by indirect immunofluorescence (IIF) as screening test and all of those positive samples are convenient to confirm its specificity by ELISA, western blot or other techniques. Positive ANA detected by IIF must be evaluated taking in to account the pattern and titer. The following recommended step is the specificity characterization (reactivity against extractable nuclear antigens [ENA], dsDNA, etc.) which is useful for the diagnosis and follow up of patients with autoimmune diseases, and by such reasoning, its detection must be performed in an orderly and reasonable way using guides or strategies focused to the good use and interpretation of the autoantibodies. The objective of this review is to present a compilation of the literature and our experience in the detection and study of the ANA.

  14. A Strategy for Screening Monoclonal Antibodies for Arabidopsis Flowers (United States)

    Shi, Qian; Zhou, Lian; Wang, Yingxiang; Ma, Hong


    The flower is one of the most complex structures of angiosperms and is essential for sexual reproduction. Current studies using molecular genetic tools have made great advances in understanding flower development. Due to the lack of available antibodies, studies investigating the localization of proteins required for flower development have been restricted to use commercial antibodies against known antigens such as GFP, YFP, and FLAG. Thus, knowledge about cellular structures in the floral organs is limited due to the scarcity of antibodies that can label cellular components. To generate monoclonal antibodies that can facilitate molecular studies of the flower, we constructed a library of monoclonal antibodies against antigenic proteins from Arabidopsis inflorescences and identified 61 monoclonal antibodies. Twenty-four of these monoclonal antibodies displayed a unique band in a western blot assay in at least one of the examined tissues. Distinct cellular distribution patterns of epitopes were detected by these 24 antibodies by immunofluorescence microscopy in a flower section. Subsequently, a combination of immunoprecipitation and mass spectrometry analysis identified potential targets for three of these antibodies. These results provide evidence for the generation of an antibody library using the total plant proteins as antigens. Using this method, the present study identified 61 monoclonal antibodies and 24 of them were efficiently detecting epitopes in both western blot experiments and immunofluorescence microscopy. These antibodies can be applied as informative cellular markers to study the biological mechanisms underlying floral development in plants. PMID:28293248

  15. Baculovirus display of functional antibody Fab fragments. (United States)

    Takada, Shinya; Ogawa, Takafumi; Matsui, Kazusa; Suzuki, Tasuku; Katsuda, Tomohisa; Yamaji, Hideki


    The generation of a recombinant baculovirus that displays antibody Fab fragments on the surface was investigated. A recombinant baculovirus was engineered so that the heavy chain (Hc; Fd fragment) of a mouse Fab fragment was expressed as a fusion to the N-terminus of baculovirus gp64, while the light chain of the Fab fragment was simultaneously expressed as a secretory protein. Following infection of Sf9 insect cells with the recombinant baculovirus, the culture supernatant was analyzed by enzyme-linked immunosorbent assay using antigen-coated microplates and either an anti-mouse IgG or an anti-gp64 antibody. A relatively strong signal was obtained in each case, showing antigen-binding activity in the culture supernatant. In western blot analysis of the culture supernatant using the anti-gp64 antibody, specific protein bands were detected at an electrophoretic mobility that coincided with the molecular weight of the Hc-gp64 fusion protein as well as that of gp64. Flow cytometry using a fluorescein isothiocyanate-conjugated antibody specific to mouse IgG successfully detected the Fab fragments on the surface of the Sf9 cells. These results suggest that immunologically functional antibody Fab fragments can be displayed on the surface of baculovirus particles, and that a fluorescence-activated cell sorter with a fluorescence-labeled antigen can isolate baculoviruses displaying specific Fab fragments. This successful baculovirus display of antibody Fab fragments may offer a novel approach for the efficient selection of specific antibodies.

  16. Selection of antibodies from synthetic antibody libraries. (United States)

    Harel Inbar, Noa; Benhar, Itai


    More than 2 dozen years had passed since the field of antibody engineering was established, with the first reports of bacterial [1-3] and mammalian cells [4] expression of recombinant antibody fragments, and in that time a lot of effort was dedicated to the development of efficient technological means, intended to assist in the creation of therapeutic monoclonal antibodies (mAbs). Research focus was given to two intertwined technological aspects: the selection platform and the recombinant antibody repertoires. In accordance with these areas of interest, it is the goal of this chapter to describe the various selection tools and antibody libraries existing, with emphasis on the later, and their applications. This chapter gives a far from exhaustive, subjective "historic account" of the field, describing the selection platforms, the different formats of antibody repertoires and the applications of both for selecting recombinant antibodies. Several excellent books provide detailed protocols for constructing antibody libraries and selecting antibodies from those libraries [5-13]. Such books may guide a newcomer to the field in the fine details of antibody engineering. We would like to offer advice to the novice: although seemingly simple, effective library construction and antibody isolation provide best benefits in the hands of professionals. It is an art as much as it is science.

  17. Study on biodistribution of 131 iodine labeled monoclonal antibody D-D3 against pro-gastrin-releasing peptide(31-98) in healthy Kunming mice%抗ProGRP(31-98)单克隆抗体D-D3的131I标记及其体内生物学分布研究

    Institute of Scientific and Technical Information of China (English)

    陈传新; 石怡珍; 杨仪; 唐军; 刘增礼; 徐巧玲


    Objective To study the 131I labeling methods, stability, and biological distribution pattern of D-D3 antibody against pro-gastrin-releasing peptide 31-98(ProGRP(31-98) ). Methods The radioiodination of D-D3 antibody was performed using the chloramine-T method. The radiochemical purity was determined through thin-layer chromotography. 131I-D-D3 was injected into the healthy Kunming mice via a tail vein, and the % ID/g for various organs was obtained, and then, the biodistribution and pharmacokinetics of 131 I-D-D3 antibody in healthy Kunming mice were studied. Results The 131 I-D-D3 labeling rate was (86.56 ± 3.8) %. The radiochemical purity of 131 I-D-D3 was (99.27 ± 0. 6)%. After 48 h incubating in 37 ℃ water bath, the radiochemical purity was (88.38 ± 0.4)%. While being mixed 24 h with healthy human serum, the radiochemical purity was still more than (64.43 ± 0.7)%. The metabolism of 131I-D-D3 in healthy Kunming mice was consistent with a two-compartment model with first-order absorption, T1/2α and T1/2β was 0.25,37.89 h, respectively. Conclusion The labeling efficiency and radiochemical purity of 131 I-D-D-D3 are high and stable. 131I-D-D3 is a promising radioimmunoimaging reagent for small cell lung cancer(SCLC).%目的 探讨抗胃泌素释放肽前体(ProGRP)(31-98)单克隆抗体D-D3的131I标记方法 及其在健康昆明小鼠体内的生物学分布规律与特点.方法 采用氯胺-T法用131I标记单克隆抗体D-D3,利用纸层析法测定其标记率、放化纯度和稳定性.取健康昆明小鼠50只,随机分为10组,每组5只,自昆明小鼠尾静脉注射131I-D-D3 1.48 kBq/100 μL(4 μCi/100 μL),各组小鼠分别于注射后5、15、30 min及1、2、4、8、12、24、48 h处死,取血液、心脏、肝脏、脾脏、肺脏、肾脏、胃、小肠、骨骼(右下肢)、肌肉(右下肢)和脑组织,称质量(g)后测放射性计数(cpm),计算各脏器组织的每克组织百分注射剂量率(%ID

  18. Production and Characterization of Monoclonal Antibodies of Shrimp White Spot Syndrome Virus Envelope Protein VP28

    Institute of Scientific and Technical Information of China (English)

    Wan-gang GU; Jun-fa YUAN; Ge-lin XU; Li-juan LI; Ni LIU; Cong ZHANG; Jian-hong ZHANG; Zheng-li SHI


    BALB/c mice were immunized with purified White spot syndrome virus (WSSV).Six monoclonal antibody cell lines were selected by ELISA with VP28 protein expressed in E.coll in vitro neutralization experiments showed that 4 of them could inhibit the virus infection in crayfish.Westernblot suggested that all these monoclonal antibodies were against the conformational structure of VP28.The monoclonal antibody 7B4 was labeled with colloidal gold particles and used to locate the VP28 on virus envelope by immunogold labeling.These monoclonal antibodies could be used to develop immunological diagnosis methods for WSSV infection.

  19. Study on 125I Labeled OxLDL Monoclonal Antibody

    Institute of Scientific and Technical Information of China (English)


    <正>Atherosclerosis is a common disease, which is a severe threat to human health and life. However, as it has been notoriously difficult to detected early and treat timely. The aim of this study focuses on the

  20. Antibody microarrays for native toxin detection. (United States)

    Rucker, Victor C; Havenstrite, Karen L; Herr, Amy E


    We have developed antibody-based microarray techniques for the multiplexed detection of cholera toxin beta-subunit, diphtheria toxin, anthrax lethal factor and protective antigen, Staphylococcus aureus enterotoxin B, and tetanus toxin C fragment in spiked samples. Two detection schemes were investigated: (i) a direct assay in which fluorescently labeled toxins were captured directly by the antibody array and (ii) a competition assay that employed unlabeled toxins as reporters for the quantification of native toxin in solution. In the direct assay, fluorescence measured at each array element is correlated with labeled toxin concentration to yield baseline binding information (Langmuir isotherms and affinity constants). Extending from the direct assay, the competition assay yields information on the presence, identity, and concentration of toxins. A significant advantage of the competition assay over reported profiling assays is the minimal sample preparation required prior to analysis because the competition assay obviates the need to fluorescently label native proteins in the sample of interest. Sigmoidal calibration curves and detection limits were established for both assay formats. Although the sensitivity of the direct assay is superior to that of the competition assay, detection limits for unmodified toxins in the competition assay are comparable to values reported previously for sandwich-format immunoassays of antibodies arrayed on planar substrates. As a demonstration of the potential of the competition assay for unlabeled toxin detection, we conclude with a straightforward multiplexed assay for the differentiation and identification of both native S. aureus enterotoxin B and tetanus toxin C fragment in spiked dilute serum samples.

  1. Performance limitations of label-free sensors in molecular diagnosis using complex samples (United States)

    Varma, Manoj


    Label-free biosensors promised a paradigm involving direct detection of biomarkers from complex samples such as serum without requiring multistep sample processing typical of labelled methods such as ELISA or immunofluorescence assays. Label-free sensors have witnessed decades of development with a veritable zoo of techniques available today exploiting a multitude of physical effects. It is appropriate now to critically assess whether label-free technologies have succeeded in delivering their promise with respect to diagnostic applications, particularly, ambitious goals such as early cancer detection using serum biomarkers, which require low limits of detection (LoD). Comparison of nearly 120 limits of detection (LoD) values reported by labelled and label-free sensing approaches over a wide range of detection techniques and target molecules in serum revealed that labeled techniques achieve 2-3 orders of magnitude better LoDs. Data from experiments where labelled and label-free assays were performed simultaneously using the same assay parameters also confirm that the LoD achieved by labelled techniques is 2 to 3 orders of magnitude better than that by label-free techniques. Furthermore, label-free techniques required significant signal amplification, for e.g. using nanoparticle conjugated secondary antibodies, to achieve LoDs comparable to labelled methods substantially deviating from the original "direct detection" paradigm. This finding has important implications on the practical limits of applying label-free detection methods for molecular diagnosis.

  2. From Label to Practice

    DEFF Research Database (Denmark)

    Byrkjeflot, Haldor; Strandgaard, Jesper; Svejenova, Silviya


    This article examines the process of creation of new Nordic cuisine (NNC) as a culinary innovation, focusing on the main stages, actors, and mechanisms that shaped the new label and its practices and facilitated its diffusion in the region and internationally. Fast-paced diffusion was possible...... because NNC was conceived as an identity movement, triggered by active involvement of entrepreneurial leaders from the culinary profession, high-profile political supporters, legitimating scientists, disseminating media, and interpreting audiences. It was facilitated by three mechanisms: First, the use...... actors and institutions to develop practices associated with the NNC label. Third, organized dissemination allowed the excitement and engagement with the new label to spread quickly....

  3. Visualizing dengue virus through Alexa Fluor labeling. (United States)

    Zhang, Summer; Tan, Hwee Cheng; Ooi, Eng Eong


    The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors that influence this interaction could lead to improved understanding of disease pathogenesis and thus influence vaccine or therapeutic design. Hence, the development of methods to probe this interaction would be useful. Recent advancements in fluorophores development and imaging technology can be exploited to improve our current knowledge on dengue pathogenesis and thus pave the way to reduce the millions of dengue infections occurring annually. The enveloped dengue virus has an external scaffold consisting of 90 envelope glycoprotein (E) dimers protecting the nucleocapsid shell, which contains a single positive strand RNA genome. The identical protein subunits on the virus surface can thus be labeled with an amine reactive dye and visualized through immunofluorescent microscopy. Here, we present a simple method of labeling of dengue virus with Alexa Fluor succinimidyl ester dye dissolved directly in a sodium bicarbonate buffer that yielded highly viable virus after labeling. There is no standardized procedure for the labeling of live virus and existing manufacturer's protocol for protein labeling usually requires the reconstitution of dye in dimethyl sulfoxide. The presence of dimethyl sulfoxide, even in minute quantities, can block productive infection of virus and also induce cell cytotoxicity. The exclusion of the use of dimethyl sulfoxide in this protocol thus reduced this possibility. Alexa Fluor dyes have superior photostability and are less pH-sensitive than the common dyes, such as fluorescein and rhodamine, making them ideal for studies on cellular uptake and endosomal transport of the virus. The conjugation of Alexa Fluor dye did not affect the recognition of labeled dengue virus by virus-specific antibody and its putative receptors in host cells. This method could have useful applications in virological studies.

  4. Biopatterning for label-free detection. (United States)

    Goddard, Julie M; Mandal, Sudeep; Nugen, Sam R; Baeumner, Antje J; Erickson, David


    We present a biopatterning technique suitable for applications which demand a high degree of surface cleanliness, such as immobilization of biological recognition elements onto label-free biosensors. In the case of label-free biosensing, the mechanism of signal transduction is based on surface bound matter, making them highly sensitive to surface contamination including residues left during the biopatterning process. In this communication we introduce a simple, rapid processing step that removes 98% of the residues that often remain after standard parylene lift-off patterning. Residue-free parylene biopatterning is combined with microfluidics to localize biomolecule immobilization onto the sensing region and to enable multiplexed biopatterning. We demonstrate the applicability of this method to multiplexed label-free detection platforms by patterning nucleic acid capture probes corresponding to the four different serotypes of Dengue virus onto parallel 1D photonic crystal resonator sensors. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) are used to quantify surface cleanliness and uniformity. In addition to label-free biosensors, this technique is well suited to other nanobiotechnology patterning applications which demand a pristine, residue-free surface, such as immobilization of enzymes, antibodies, growth factors, or cell cultures.

  5. Microfluidic Radiometal Labeling Systems for Biomolecules

    Energy Technology Data Exchange (ETDEWEB)

    Reichert, D E; Kenis, P J. A.


    In a typical labeling procedure with radiometals, such as Cu-64 and Ga-68; a very large (~ 100-fold) excess of the non-radioactive reactant (precursor) is used to promote rapid and efficient incorporation of the radioisotope into the PET imaging agent. In order to achieve high specific activities, careful control of reaction conditions and extensive chromatographic purifications are required in order to separate the labeled compounds from the cold precursors. Here we propose a microfluidic approach to overcome these problems, and achieve high specific activities in a more convenient, semi-automated fashion and faster time frame. Microfluidic reactors, consisting of a network of micron-sized channels (typical dimensions in the range 10 - 300¼m), filters, separation columns, electrodes and reaction loops/chambers etched onto a solid substrate, are now emerging as an extremely useful technology for the intensification and miniaturization of chemical processes. The ability to manipulate, process and analyze reagent concentrations and reaction interfaces in both space and time within the channel network of a microreactor provides the fine level of reaction control that is desirable in PET radiochemistry practice. These factors can bring radiometal labeling, specifically the preparation of radio-labeled biomolecules such as antibodies, much closer to their theoretical maximum specific activities.

  6. FDA Online Label Repository (United States)

    U.S. Department of Health & Human Services — The drug labels and other drug-specific information on this Web site represent the most recent drug listing information companies have submitted to the Food and Drug...

  7. Behind the Label "Alcoholic." (United States)

    Wright, Deborah M.


    Relates individual's personal story of her childhood influenced by her parent's alcoholism, her own alcoholism as a young adult, and her experiences with counseling. Asks others not to reject her because of the label "alcoholic." (ABL)

  8. Selective Serial Multi-Antibody Biosensing with TOPAS Microstructured Polymer Optical Fibers

    DEFF Research Database (Denmark)

    Emiliyanov, Grigoriy Andreev; Høiby, Poul E.; Pedersen, Lars H.


    We have developed a fluorescence-based fiber-optical biosensor, which can selectively detect different antibodies in serial at preselected positions inside a single piece of fiber. The fiber is a microstructured polymer optical fiber fabricated from TOPAS cyclic olefin copolymer, which allows...... for UV activation of localized sensor layers inside the holes of the fiber. Serial fluorescence-based selective sensing of Cy3-labelled α-streptavidin and Cy5-labelled α-CRP antibodies is demonstrated....

  9. Selective Serial Multi-Antibody Biosensing with TOPAS Microstructured Polymer Optical Fibers

    Directory of Open Access Journals (Sweden)

    Lars H. Pedersen


    Full Text Available We have developed a fluorescence-based fiber-optical biosensor, which can selectively detect different antibodies in serial at preselected positions inside a single piece of fiber. The fiber is a microstructured polymer optical fiber fabricated from TOPAS cyclic olefin copolymer, which allows for UV activation of localized sensor layers inside the holes of the fiber. Serial fluorescence-based selective sensing of Cy3-labelled α-streptavidin and Cy5-labelled α-CRP antibodies is demonstrated.

  10. Selective serial multi-antibody biosensing with TOPAS microstructured polymer optical fibers. (United States)

    Emiliyanov, Grigoriy; Høiby, Poul E; Pedersen, Lars H; Bang, Ole


    We have developed a fluorescence-based fiber-optical biosensor, which can selectively detect different antibodies in serial at preselected positions inside a single piece of fiber. The fiber is a microstructured polymer optical fiber fabricated from TOPAS cyclic olefin copolymer, which allows for UV activation of localized sensor layers inside the holes of the fiber. Serial fluorescence-based selective sensing of Cy3-labelled α-streptavidin and Cy5-labelled α-CRP antibodies is demonstrated.

  11. Considerations in producing preferentially reduced half-antibody fragments. (United States)

    Makaraviciute, Asta; Jackson, Carolyn D; Millner, Paul A; Ramanaviciene, Almira


    Half-antibody fragments are a promising reagent for biosensing, drug-delivery and labeling applications, since exposure of the free thiol group in the Fc hinge region allows oriented reaction. Despite the structural variations among the molecules of different IgG subclasses and those obtained from different hosts, only generalized preferential antibody reduction protocols are currently available. Preferential reduction of polyclonal sheep anti-digoxin, rabbit anti-Escherichia coli and anti-myoglobin class IgG antibodies to half-antibody fragments has been investigated. A mild reductant 2-mercaptoethylamine (2-MEA) and a slightly stronger reductant tris(2-carboxyethyl)phosphine (TCEP) were used and the fragments obtained were quantitatively determined by SDS-PAGE analysis. It has been shown that the yields of half-antibody fragments could be increased by lowering the pH of the reduction mixtures. However, antibody susceptibility to the reductants varied. At pH4.5 the highest yield of sheep anti-digoxin IgG half-antibody fragments was obtained with 1M 2-MEA. Conversely, rabbit IgG half-antibody fragments could only be obtained with the stronger reductant TCEP. Preferential reduction of rabbit anti-myoglobin IgG antibodies was optimized and the highest half-antibody yield was obtained with 35 mM TCEP. Finally, it has been demonstrated that produced anti-myoglobin half-IgG fragments retained their binding activity.

  12. Acetylcholine receptor antibody (United States)

    ... page: // Acetylcholine receptor antibody To use the sharing features on this page, please enable JavaScript. Acetylcholine receptor antibody is a protein found in the blood ...

  13. Antinuclear antibody panel (United States)

    ... page: // Antinuclear antibody panel To use the sharing features on this page, please enable JavaScript. The antinuclear antibody panel is a blood test that looks at ...

  14. Lyme disease antibody (United States)

    ... JavaScript. The Lyme disease blood test looks for antibodies in the blood to the bacteria that causes ... needed. A laboratory specialist looks for Lyme disease antibodies in the blood sample using the ELISA test . ...

  15. The antibody mining toolbox


    D'Angelo, Sara; Glanville, Jacob; Ferrara, Fortunato; Naranjo, Leslie; Gleasner, Cheryl D.; Shen, Xiaohong; Bradbury, Andrew RM; Kiss, Csaba


    In vitro selection has been an essential tool in the development of recombinant antibodies against various antigen targets. Deep sequencing has recently been gaining ground as an alternative and valuable method to analyze such antibody selections. The analysis provides a novel and extremely detailed view of selected antibody populations, and allows the identification of specific antibodies using only sequencing data, potentially eliminating the need for expensive and laborious low-throughput ...

  16. Selective detection of antibodies in microstructured polymer optical fibers

    DEFF Research Database (Denmark)

    Jensen, Jesper Bo Damm; Hoiby, P.E.; Emiliyanov, Grigoriy Andreev


    We demonstrate selective detection of fluorophore labeled antibodies from minute samples probed by a sensor layer of complementary biomolecules immobilized inside the air holes of microstructured Polymer Optical Fiber (mPOF). The fiber core is defined by a ring of 6 air holes and a simple procedure...... was applied to selectively capture either α-streptavidin or α-CRP antibodies inside these air holes. A sensitive and easy-to-use fluorescence method was used for the optical detection. Our results show that mPOF based biosensors can provide reliable and selective antibody detection in ultra small sample...

  17. Selective detection of antibodies in microstructured polymer optical fibers. (United States)

    Jensen, Jesper; Hoiby, Poul; Emiliyanov, Grigoriy; Bang, Ole; Pedersen, Lars; Bjarklev, Anders


    We demonstrate selective detection of fluorophore labeled antibodies from minute samples probed by a sensor layer of complementary biomolecules immobilized inside the air holes of microstructured Polymer Optical Fiber (mPOF). The fiber core is defined by a ring of 6 air holes and a simple procedure was applied to selectively capture either alpha-streptavidin or alpha-CRP antibodies inside these air holes. A sensitive and easy-to-use fluorescence method was used for the optical detection. Our results show that mPOF based biosensors can provide reliable and selective antibody detection in ultra small sample volumes.

  18. European consumers and nutrition labelling

    DEFF Research Database (Denmark)

    Wills, Josephine M.; Grunert, Klaus G.; Celemín, Laura Fernández


    Nutrition labelling of food in Europe is not compulsory, unless a nutrition or health claim is made for the product. The European Commission is proposing mandatory nutrition labelling, even front of pack labelling with nutrition information. Yet, how widespread is nutrition labelling in the EU...

  19. Distance labeling schemes for trees

    DEFF Research Database (Denmark)

    Alstrup, Stephen; Gørtz, Inge Li; Bistrup Halvorsen, Esben;


    We consider distance labeling schemes for trees: given a tree with n nodes, label the nodes with binary strings such that, given the labels of any two nodes, one can determine, by looking only at the labels, the distance in the tree between the two nodes. A lower bound by Gavoille et al. [Gavoill...

  20. A new method for the labelling of proteins with radioactive arsenic isotopes

    Energy Technology Data Exchange (ETDEWEB)

    Jennewein, M. [Institute of Nuclear Chemistry, Johannes Gutenberg University of Mainz, Fritz-Strassmann-Weg 2, 55128 Mainz (Germany); Hermanne, A. [VUB Cyclotron, University of Brussels, Laarbeeklaan 103, 1090 Brussels (Belgium); Mason, R.P. [Department of Radiology, Advanced Radiological Sciences, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas (United States); Thorpe, P.E. [Department of Pharmacology and Simmons and Hamon Cancer Centers, University of Texas Southwestern Medical Center at Dallas, Dallas, TX (United States); Roesch, F. [Institute of Nuclear Chemistry, Johannes Gutenberg University of Mainz, Fritz-Strassmann-Weg 2, 55128 Mainz (Germany)]. E-mail:


    Radioarsenic labelled radiopharmaceuticals could be a valuable asset to positron emission tomography. In particular, the long half-lives of {sup 72}As (T{sub 1/2}=26h) and {sup 74}As (T{sub 1/2}=17.8d) allow to investigate slow physiological or metabolical processes, like the enrichment and distribution of monoclonal antibodies (mab) in tumour tissue. In this work, a new method for the labelling of proteins with various radioactive arsenic isotopes was developed. For this purpose, two proteins, namely a chimeric IgG{sub 3} monoclonal antibody, ch3G4, directed against anionic phospholipids, and Rituxan (Rituximab), were labelled as a proof of principle with no-carrier-added radioarsenic isotopes ({sup 74}As and {sup 77}As). The developed labelling chemistry gives high yields (>99.9%), is reliable and could easily be transferred to automated labelling systems in a clinical environment. At least for the mab used in this work, this route of radioarsenic labelling does not affect the immunoreactivity of the product. The arsenic label stays stable for up to 72h at the molecular mass of the monoclonal antibody, which is in particular relevant to follow the pharmacology and pharmacokinetics of the labelled mab for several days.

  1. Semantic Role Labeling

    CERN Document Server

    Palmer, Martha; Xue, Nianwen


    This book is aimed at providing an overview of several aspects of semantic role labeling. Chapter 1 begins with linguistic background on the definition of semantic roles and the controversies surrounding them. Chapter 2 describes how the theories have led to structured lexicons such as FrameNet, VerbNet and the PropBank Frame Files that in turn provide the basis for large scale semantic annotation of corpora. This data has facilitated the development of automatic semantic role labeling systems based on supervised machine learning techniques. Chapter 3 presents the general principles of applyin

  2. Labeling of Patient Specimens (United States)


    noted during the event that the number of near miss incidmts reported monthly was low due to laboratory personnel performing rounds each...specimens never leaves label and if moved it is labeled), All orders in system and all near misses and errors reported to patient safety Purchase/Install...Meeting 14 Aug 09, 1400 in lab break room thru out Develop TICK sheet to track near misses .JDI Ms. Clark Clinics will provide toPS 1st working day of

  3. Real-time kinetic analysis applied to the production of bispecific monoclonal antibodies for radioimmunodetection of cancer. (United States)

    Horenstein, A L; Poiesi, C; DeMonte, L; Camagna, M; Mariani, M; Albertini, A; Malavasi, F


    An automated biosensor system designed for measuring molecular interactions in real-time and without labelling of the reactants has been used to evaluate the association/dissociation rate and affinity constants of bivalent monoclonal antibodies and a monovalent bispecific monoclonal antibody. Observed differences in affinity between parental and bispecific antibody produced were related to the association rate constants, since the dissociation rate constants were in the same range. Values were also closely related to radioimmunochemical data. These results indicate that the biosensor system, besides presenting several advantages for characterizing antigen-antibody interaction, is valuable for selecting monoclonal antibodies with properties which might be useful in the development of bispecific monoclonal antibodies.

  4. Figuring Out Food Labels (United States)

    ... should get 30%-40% of calories from fat. Saturated Fat and Trans Fat The amount of saturated fat appears beneath total fat. The FDA also requires ... to list trans fats separately on the label. Saturated fats and trans fats are often called "bad fats" ...

  5. Labelled Execution Systems (United States)


    This is most starkly evident in the classical example used to demonstrate that, in the case of infinitely branching systems, a transfinite number of... transfinite number of iterations to converge, what can be shown to fail by appropriately embedding the labelled transition systems of [46, prop. 10.5

  6. Multi-label

    Directory of Open Access Journals (Sweden)

    Neda Abdelhamid


    Full Text Available Generating multi-label rules in associative classification (AC from single label data sets is considered a challenging task making the number of existing algorithms for this task rare. Current AC algorithms produce only the largest frequency class connected with a rule in the training data set and discard all other classes even though these classes have data representation with the rule’s body. In this paper, we deal with the above problem by proposing an AC algorithm called Enhanced Multi-label Classifiers based Associative Classification (eMCAC. This algorithm discovers rules associated with a set of classes from single label data that other current AC algorithms are unable to induce. Furthermore, eMCAC minimises the number of extracted rules using a classifier building method. The proposed algorithm has been tested on a real world application data set related to website phishing and the results reveal that eMCAC’s accuracy is highly competitive if contrasted with other known AC and classic classification algorithms in data mining. Lastly, the experimental results show that our algorithm is able to derive new rules from the phishing data sets that end-users can exploit in decision making.

  7. Waisda?: video labeling game

    NARCIS (Netherlands)

    Hildebrand, M.; Brinkerink, M.; Gligorov, R.; Steenbergen, M. van; Huijkman, J.; Oomen, J.


    The Waisda? video labeling game is a crowsourcing tool to collect user-generated metadata for video clips. It follows the paradigm of games-with-a-purpose, where two or more users play against each other by entering tags that describe the content of the video. Players score points by entering the sa

  8. A microfluidics-based technique for automated and rapid labeling of cells for flow cytometry (United States)

    Patibandla, Phani K.; Estrada, Rosendo; Kannan, Manasaa; Sethu, Palaniappan


    Flow cytometry is a powerful technique capable of simultaneous multi-parametric analysis of heterogeneous cell populations for research and clinical applications. In recent years, the flow cytometer has been miniaturized and made portable for application in clinical- and resource-limited settings. The sample preparation procedure, i.e. labeling of cells with antibodies conjugated to fluorescent labels, is a time consuming (˜45 min) and labor-intensive procedure. Microfluidics provides enabling technologies to accomplish rapid and automated sample preparation. Using an integrated microfluidic device consisting of a labeling and washing module, we demonstrate a new protocol that can eliminate sample handling and accomplish sample and reagent metering, high-efficiency mixing, labeling and washing in rapid automated fashion. The labeling module consists of a long microfluidic channel with an integrated chaotic mixer. Samples and reagents are precisely metered into this device to accomplish rapid and high-efficiency mixing. The mixed sample and reagents are collected in a holding syringe and held for up to 8 min following which the mixture is introduced into an inertial washing module to obtain ‘analysis-ready’ samples. The washing module consists of a high aspect ratio channel capable of focusing cells to equilibrium positions close to the channel walls. By introducing the cells and labeling reagents in a narrow stream at the center of the channel flanked on both sides by a wash buffer, the elution of cells into the wash buffer away from the free unbound antibodies is accomplished. After initial calibration experiments to determine appropriate ‘holding time’ to allow antibody binding, both modules were used in conjunction to label MOLT-3 cells (T lymphoblast cell line) with three different antibodies simultaneously. Results confirm no significant difference in mean fluorescence intensity values for all three antibodies labels (p < 0.01) between the

  9. Tritium labeled amino acid conjugates of prostaglandins and thromboxanes as labeled ligands in prostanoid radioimmunoassay

    Energy Technology Data Exchange (ETDEWEB)

    Sautebin, L.; Kindahl, H.; Kumlin, M.; Granstroem, E.


    Conjugates of prostaglandins and thromboxanes with tritium labeled amino acids were prepared and employed as labeled ligands in prostaglandin and thromboxane radioimmunoassays. Assays for PGF2 alpha, 15-keto-13, 14-dihydro-PGF2 alpha, TXB2 and 15-keto-13,14-dihydro-TXB2 were evaluated in comparative studies using either these heterologous ligands or the corresponding homologous tritiated eicosanoid as tracers. Binding properties for the respective antibodies were found to be similar using either tracer. Three biological studies were also conducted, viz. study of the release of TXB2 during collagen induced platelet aggregation, of 15-keto-13,14-dihydro-TXB2 during guinea pig pulmonary anaphylaxis, and of PGF2 alpha during bovine luteolysis. The analyses gave comparable results using either the heterologous or the homologous assay. Thus, this type of labeled prostanoid conjugates may serve as a convenient alternative to homologous tracers in radioimmunoassay. Heterologous tracers may even in certain cases provide the only simple solution to the problem of preparing a labeled ligand of high specific activity.

  10. Simultaneous quantitation of diphtheria and tetanus antibodies by double antigen, time-resolved fluorescence immunoassay. (United States)

    Aggerbeck, H; Nørgaard-Pedersen, B; Heron, I


    A dual, double antigen, time-resolved fluorescence immunoassay (DELFIA) for the simultaneous detection and quantitation of diphtheria (D) and tetanus (T) antibodies in sera has been developed. In the double antigen format one arm of the antibody binds to antigen coated microtitre wells and the other arm binds to labelled antigen to provide a fluorescent signal. This assay was found to be functionally specific for IgG antibodies and showed a good correlation with established toxin neutralization assays. Furthermore, the double antigen set-up was species independent, permitting the direct use of existing international references of animal origin to measure protective antibody levels in humans in international units (IU/ml). The detection limit corresponded to 0.0003 IU/ml with Eu(3+)-labelled toxoids and to 0.0035 IU/ml using Sm(3+)-labelled toxoids. The assay was fast with a high capacity making it a suitable method for serological surveillance studies.

  11. Recombinant anti-human melanoma antibodies are versatile molecules. (United States)

    Neri, D; Natali, P G; Petrul, H; Soldani, P; Nicotra, M R; Vola, R; Rivella, A; Creighton, A M; Neri, P; Mariani, M


    The low cost, high versatility, and reliable production of bacterially produced recombinant antibody fragments speeds up the development of tumor-targeting agents. High-quality recombinant anti-melanoma antibodies are much sought after in the scientific community. We cloned the murine antibody 225.28S, currently used in radioimmunoimaging of human melanoma lesions, in single-chain Fv configuration (scFv) for soluble expression in bacteria. The recombinant antibody fragment conserved the binding specificity of the parental antibody. In order to arm the scFv(225.28S) with biologically useful effector functions, we developed vectors for soluble expression of scFv(225.28S) in bacteria that allow both covalent and noncovalent chemical antibody modification at positions that do not interfere with antigen binding. An expression vector was developed that appends a cysteine residue at the C-terminal extremity of the recombinant antibody, thus allowing reaction with thiol-specific reagents, including 99mTc labeling, at a position that does not interfere with antigen binding. The scFv(225.28S) was also successfully expressed with a casein kinase II substrate tag that enables efficient and stable 32P labeling. For noncovalent antibody modification, we developed an expression vector that appends the human calmodulin gene at the C-terminal extremity of scFv(225.28S). The calmodulin domain is poorly immunogenic and can be targeted with chemically modified high-affinity calmodulin ligands. The recombinant anti-human melanoma antibodies described in this article should prove useful "building blocks" for the development of anti-melanoma diagnostic and therapeutic strategies.

  12. Anti-fibrin antibody binding in valvular vegetations and kidney lesions during experimental endocarditis. (United States)

    Yokota, M; Basi, D L; Herzberg, M C; Meyer, M W


    In Streptococcus sanguinis (sanguis) induced experimental endocarditis, we sought evidence that the development of aortic valvular vegetation depends on the availability of fibrin. Endocarditis was induced in New Zealand white rabbits by catheter placement into the left ventricle and inoculation of the bacteria. Fibrin was localized in the developing vegetation with 99mTechnetium (Tc)-labeled anti-fibrin antibody one or three days later. When rabbit anti-fibrin antibody was given intravenously on day 1, the mass of aortic valvular vegetation was significantly reduced at day 3; infusion of non-specific rabbit IgG showed no effect. The 99mTc-labeled anti-fibrin antibody also labeled kidneys that showed macroscopic subcapsular hemorrhage. To learn if the deposition of fibrin in the kidneys was a consequence of endocarditis required a comparison of farm-bred and specific pathogen-free rabbits before and after the induction of endocarditis. Before induction, the kidneys of farm-bred rabbits were labeled, but specific pathogen-free rabbits were free of labeling and signs of macroscopic hemorrhage. After 3 days of endocarditis, kidneys of 10 of 14 specific pathogen-free rabbits labeled with 99mTc-labeled anti-fibrin antibody and showed hemorrhage. Kidney lesions were suggested to be a frequent sequellae of S. sanguinis infective endocarditis. For the first time, fibrin was shown to be required for the continued development of aortic valvular vegetations.

  13. Decode the Sodium Label Lingo (United States)

    ... For Preschooler For Gradeschooler For Teen Decode the Sodium Label Lingo Published January 24, 2013 Print Email Reading food labels can help you slash sodium. Here's how to decipher them. "Sodium free" or " ...

  14. Recent developments in monoclonal antibody radiolabeling techniques

    Energy Technology Data Exchange (ETDEWEB)

    Srivastava, S.C.; Mease, R.C.


    Monoclonal antibodies (MAbs) have shown the potential to serve as selective carriers of radionuclides to specific in vivo antigens. Accordingly, there has been an intense surge of research activity in an effort to develop and evaluate MAb-based radiopharmaceuticals for tumor imaging (radioimmunoscintigraphy) and therapy (radioimmunotherapy), as well as for diagnosing nonmalignant diseases. A number of problems have recently been identified, related to the MAbs themselves and to radiolabeling techniques, that comprise both the selectivity and the specificity of the in vivo distribution of radiolabeled MAbs. This paper will address some of these issues and primarily discuss recent developments in the techniques for radiolabeling monoclonal antibodies that may help resolve problems related to the poor in vivo stability of the radiolabel and may thus produce improved biodistribution. Even though many issues are identical with therapeutic radionuclides, the discussion will focus mainly on radioimmunoscintigraphic labels. 78 refs., 6 tabs.

  15. Chick embryo proliferation studies using EdU labeling. (United States)

    Warren, Michelle; Puskarczyk, Karolina; Chapman, Susan C


    Cell proliferation studies are an important experimental tool. The most commonly used thymidine analogues, tritiated thymidine and bromodeoxyuridine (BrdU) label cells during S-phase. Both methods have significant drawbacks: low sensitivity in the case of tritiated thymidine and a denaturation step during BrdU detection that destroys most cellular epitopes, requiring careful optimization. The antibody against BrdU is also large and tissue penetration can be difficult. EdU (5'-ethynyl-2'-deoxyuridine) is closely chemically related to BrdU, with detection achieved by a copper catalyzed reaction requiring a small fluorescently conjugated azide. Cell cultures, flow cytometry and high throughput studies using EdU-labeled cells is exceptionally fast and does not require denaturation or antibodies. We have developed a tissue-labeling technique in chick embryos using EdU. Following EdU chemistry to detect proliferating cells, the tissue can undergo immunolabeling. We demonstrate fluorescent EdU chemistry followed by Tuj1 antibody staining resulting in multiplex fluorescent tissues.

  16. Labelling GM-free Products

    DEFF Research Database (Denmark)

    Punt, Maarten; Venus, Thomas; Wesseler, Justus


    Food suppliers in the EU must comply with labelling regulations for genetically modified organisms (GMOs). However, excluded from mandatory labelling are food products derived from animals fed with GM feed (mainly GM soybean in the EU). Because of this labelling exemption, consumers are unable to...

  17. Scaffolding Visitors' Learning through Labels (United States)

    Wang, Joyce; Yoon, Susan


    In museum literature, labels have been found to increase visitor learning and contribute to greater cognitive gains. In this study, we seek to understand how various labels support the visitors' learning experience, and specifically in regards to conceptual and cognitive learning. We investigated the increasing use of three types of labels (visual…

  18. Food Labels Tell the Story! (United States)

    ... My World From the Label to the Table! Food Labels Tell the Story! What is in food? Food provides your body with all of the ... your food choices. Nutrition Facts—the Labels on Food Products Beginning in 1994, the US government began ...

  19. Limitations of Label-Free Sensors in Serum Based Molecular Diagnostics

    CERN Document Server

    Varma, Manoj M


    Immunoassay formats applicable for clinical or point-of-care diagnostics fall into two broad classes. One which uses labeled secondary antibodies for signal transduction and the other which does not require the use of any labels. Comparison of the limits of detection (LoD) reported by these two sensing approaches over a wide range of detection techniques and target molecules in serum revealed that labeled techniques achieve 2-3 orders of magnitude better LoDs. Further, a vast majority of commercial tests and recent examples of technology translations are based on labeled assay formats. In light of this data, it is argued that extension of traditional labeled approaches and enhancing their functionality may have better clinical impact than the development of newer label-free techniques.

  20. Interfacial polymerization for colorimetric labeling of protein expression in cells.

    Directory of Open Access Journals (Sweden)

    Jacob L Lilly

    Full Text Available Determining the location of rare proteins in cells typically requires the use of on-sample amplification. Antibody based recognition and enzymatic amplification is used to produce large amounts of visible label at the site of protein expression, but these techniques suffer from the presence of nonspecific reactivity in the biological sample and from poor spatial control over the label. Polymerization based amplification is a recently developed alternative means of creating an on-sample amplification for fluorescence applications, while not suffering from endogenous labels or loss of signal localization. This manuscript builds upon polymerization based amplification by developing a stable, archivable, and colorimetric mode of amplification termed Polymer Dye Labeling. The basic concept involves an interfacial polymer grown at the site of protein expression and subsequent staining of this polymer with an appropriate dye. The dyes Evans Blue and eosin were initially investigated for colorimetric response in a microarray setting, where both specifically stained polymer films on glass. The process was translated to the staining of protein expression in human dermal fibroblast cells, and Polymer Dye Labeling was specific to regions consistent with desired protein expression. The labeling is stable for over 200 days in ambient conditions and is also compatible with modern mounting medium.

  1. Interfacial polymerization for colorimetric labeling of protein expression in cells. (United States)

    Lilly, Jacob L; Sheldon, Phillip R; Hoversten, Liv J; Romero, Gabriela; Balasubramaniam, Vivek; Berron, Brad J


    Determining the location of rare proteins in cells typically requires the use of on-sample amplification. Antibody based recognition and enzymatic amplification is used to produce large amounts of visible label at the site of protein expression, but these techniques suffer from the presence of nonspecific reactivity in the biological sample and from poor spatial control over the label. Polymerization based amplification is a recently developed alternative means of creating an on-sample amplification for fluorescence applications, while not suffering from endogenous labels or loss of signal localization. This manuscript builds upon polymerization based amplification by developing a stable, archivable, and colorimetric mode of amplification termed Polymer Dye Labeling. The basic concept involves an interfacial polymer grown at the site of protein expression and subsequent staining of this polymer with an appropriate dye. The dyes Evans Blue and eosin were initially investigated for colorimetric response in a microarray setting, where both specifically stained polymer films on glass. The process was translated to the staining of protein expression in human dermal fibroblast cells, and Polymer Dye Labeling was specific to regions consistent with desired protein expression. The labeling is stable for over 200 days in ambient conditions and is also compatible with modern mounting medium.

  2. Expression of recombinant antibodies. (United States)

    Frenzel, André; Hust, Michael; Schirrmann, Thomas


    Recombinant antibodies are highly specific detection probes in research, diagnostics, and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transgenic plants and animals. Currently, almost all therapeutic antibodies are still produced in mammalian cell lines in order to reduce the risk of immunogenicity due to altered, non-human glycosylation patterns. However, recent developments of glycosylation-engineered yeast, insect cell lines, and transgenic plants are promising to obtain antibodies with "human-like" post-translational modifications. Furthermore, smaller antibody fragments including bispecific antibodies without any glycosylation are successfully produced in bacteria and have advanced to clinical testing. The first therapeutic antibody products from a non-mammalian source can be expected in coming next years. In this review, we focus on current antibody production systems including their usability for different applications.

  3. Therapeutic Recombinant Monoclonal Antibodies (United States)

    Bakhtiar, Ray


    During the last two decades, the rapid growth of biotechnology-derived techniques has led to a myriad of therapeutic recombinant monoclonal antibodies with significant clinical benefits. Recombinant monoclonal antibodies can be obtained from a number of natural sources such as animal cell cultures using recombinant DNA engineering. In contrast to…

  4. Recombinant renewable polyclonal antibodies. (United States)

    Ferrara, Fortunato; D'Angelo, Sara; Gaiotto, Tiziano; Naranjo, Leslie; Tian, Hongzhao; Gräslund, Susanne; Dobrovetsky, Elena; Hraber, Peter; Lund-Johansen, Fridtjof; Saragozza, Silvia; Sblattero, Daniele; Kiss, Csaba; Bradbury, Andrew R M


    Only a small fraction of the antibodies in a traditional polyclonal antibody mixture recognize the target of interest, frequently resulting in undesirable polyreactivity. Here, we show that high-quality recombinant polyclonals, in which hundreds of different antibodies are all directed toward a target of interest, can be easily generated in vitro by combining phage and yeast display. We show that, unlike traditional polyclonals, which are limited resources, recombinant polyclonal antibodies can be amplified over one hundred million-fold without losing representation or functionality. Our protocol was tested on 9 different targets to demonstrate how the strategy allows the selective amplification of antibodies directed toward desirable target specific epitopes, such as those found in one protein but not a closely related one, and the elimination of antibodies recognizing common epitopes, without significant loss of diversity. These recombinant renewable polyclonal antibodies are usable in different assays, and can be generated in high throughput. This approach could potentially be used to develop highly specific recombinant renewable antibodies against all human gene products.

  5. Use of commercially available rabbit monoclonal antibodies for immunofluorescence double staining

    DEFF Research Database (Denmark)

    Bzorek, M.; Stamp, I.M.; Frederiksen, L.


    Immunohistochemistry, that is, the use of polyclonal and monoclonal antibodies to detect cell and tissue antigens at a microscopical level is a powerful tool for both research and diagnostic purposes. Especially in the field of hematologic disease, there is often a need to detect several antigens...... synchronously, and we report here a fast and easy technique for demonstrating more than 1 antigen in 1 slide using immunofluorescence. We have used commercially available rabbit monoclonal antibodies (Cyclin D1, CD3, CD5, CD23, etc.) paired with mouse monoclonal antibodies (CD7, CD20, CD79a, Pax-5, etc.......) for double immunofluorescence labeling on paraffin-embedded tissue sections. Commercially available rabbit monoclonal antibodies in combination with mouse monoclonal antibodies proved useful in double immunofluorescence labeling on paraffin-embedded tissue, and all combinations used yielded excellent results...

  6. Anti-insulin antibody test (United States)

    Insulin antibodies - serum; Insulin Ab test; Insulin resistance - insulin antibodies; Diabetes - insulin antibodies ... You appear to have an allergic response to insulin Insulin no longer seems to control your diabetes

  7. Map labeling and its generalizations

    Energy Technology Data Exchange (ETDEWEB)

    Doddi, S. [New Mexico Univ., Albuquerque, NM (United States). Dept. of Computer Science]|[Los Alamos National Lab., NM (United States); Marathe, M.V. [Los Alamos National Lab., NM (United States); Mirzaian, A. [York Univ., Toronto, ON (Canada). Dept. of Computer Science; Moret, B.M.E. [New Mexico Univ., Albuquerque, NM (United States). Dept. of Computer Science; Zhu, B. [City Univ. of Hong Kong (Hong Kong). Dept. of Computer Science]|[Los Alamos National Lab., NM (United States)


    Map labeling is of fundamental importance in cartography and geographical information systems and is one of the areas targeted for research by the ACM Computational Geometry Impact Task Force. Previous work on map labeling has focused on the problem of placing maximal uniform, axis-aligned, disjoint rectangles on the plane so that each point feature to be labeled lies at the corner of one rectangle. Here, we consider a number of variants of the map labeling problem. We obtain three general types of results. First, we devise constant-factor polynomial-time-approximation algorithms for labeling point features by rectangular labels, where the feature may lie anywhere on the boundary of its label region and where labeling rectangles may be placed in any orientation. These results generalize to the case of elliptical labels. Secondly, we consider the problem of labeling a map consisting of disjoint rectilinear fine segments. We obtain constant-factor polynomial-time approximation algorithms for the general problem and an optimal algorithm for the special case where all segments are horizontal. Finally, we formulate a bicriteria version of the map-labeling problem and provide bicriteria polynomial- time approximation schemes for a number of such problems.

  8. Linerless label device and method

    KAUST Repository

    Binladen, Abdulkari


    This apparatus and method for applying a linerless label to an end user product includes a device with a printer for printing on a face surface of a linerless label, and a release coat applicator for applying a release coat to the face surface of the label; another device including an unwinder unit (103) to unwind a roll of printed linerless label; a belt (108); a glue applicator (102) for applying glue to the belt; a nip roller (106) for contacting and applying pressure to the face surface of the linerless label such that the glue on the belt transfers to the back surface of the linerless label; at least one slitting knife 105) positioned downstream the belt and a rewinder unit (104) positioned downstream the slitting knife; and a third device which die cuts and applies the linerless label to an end user object.

  9. Antibody response to the lipopolysaccharide and protein antigens of Salmonella typhi during typhoid infection. II. Measurement of intestinal antibodies by radioimmunoassay

    Energy Technology Data Exchange (ETDEWEB)

    Chau, P.Y.; Tsang, R.S.W.; Lam, S.K. (Hong Kong Univ.); La Brooy, J.T.; Rowley, D. (Adelaide Univ. (Australia))


    Antibodies to the lipopolysaccharide (LPS) and protein antigens of S. typhi in secretions of small intestine obtained from 12 typhoid patients, four typhoid carriers and 16 non-typhoid control subjects were measured by a solid-phase radioimmunoassay technique using /sup 125/I labelled anti-immunoglobulin antibody. Intestinal secretions obtained from typhoid patients as a group had significantly higher anti-LPS and anti-protein antibodies than those from the control group. These antibodies were both IgM and IgA classes. There was no correlation between the IgM or IgA antibody levels in serum and those in the intestinal secretions. In the intestinal secretions obtained from typhoid carriers, on the other hand, only IgA-class antibodies to the LPS and protein antigens of S. typhi were present at high levels.

  10. Monoclonal antibody "gold rush". (United States)

    Maggon, Krishan


    The market, sales and regulatory approval of new human medicines, during the past few years, indicates increasing number and share of new biologics and emergence of new multibillion dollar molecules. The global sale of monoclonal antibodies in 2006 were $20.6 billion. Remicade had annual sales gain of $1 billion during the past 3 years and five brands had similar increase in 2006. Rituxan with 2006 sales of $4.7 billion was the best selling monoclonal antibody and biological product and the 6th among the top selling medicinal brand. It may be the first biologic and monoclonal antibody to reach $10 billion annual sales in the near future. The strong demand from cancer and arthritis patients has surpassed almost all commercial market research reports and sales forecast. Seven monoclonal antibody brands in 2006 had sales exceeding $1 billion. Humanized or fully human monoclonal antibodies with low immunogenicity, enhanced antigen binding and reduced cellular toxicity provide better clinical efficacy. The higher technical and clinical success rate, overcoming of technical hurdles in large scale manufacturing, low cost of market entry and IND filing, use of fully human and humanized monoclonal antibodies has attracted funds and resources towards R&D. Review of industry research pipeline and sales data during the past 3 years indicate a real paradigm shift in industrial R&D from pharmaceutical to biologics and monoclonal antibodies. The antibody bandwagon has been joined by 200 companies with hundreds of new projects and targets and has attracted billions of dollars in R&D investment, acquisitions and licensing deals leading to the current Monoclonal Antibody Gold Rush.

  11. Antiphospholipid antibody syndrome. (United States)

    Kutteh, William H; Hinote, Candace D


    Antiphospholipid antibodies (aPLs) are acquired antibodies directed against negatively charged phospholipids. Obstetric antiphospholipid antibody syndrome (APS) is diagnosed in the presence of certain clinical features in conjunction with positive laboratory findings. Obstetric APS is one of the most commonly identified causes of recurrent pregnancy loss. Thus, obstetric APS is distinguished from APS in other organ systems where the most common manifestation is thrombosis. Several pathophysiologic mechanisms of action of aPLs have been described. This article discusses the diagnostic and obstetric challenges of obstetric APS, proposed pathophysiologic mechanisms of APS during pregnancy, and the management of women during and after pregnancy.

  12. Radio labeling with pre-assigned frequencies

    NARCIS (Netherlands)

    Bodlaender, H.L.; Broersma, H.J.; Fomin, F.V.; Pyatkin, A.V.; Woeginer, G.J.


    A radio labeling of a graph G is an assignment of pairwise distinct, positive integer labels to the vertices of G such that labels of adjacent vertices differ by at least 2. The radio labeling problem (RL) consists in determining a radio labeling that minimizes the maximum label that is used (the so

  13. Towards Multi Label Text Classification through Label Propagation

    Directory of Open Access Journals (Sweden)

    Shweta C. Dharmadhikari


    Full Text Available Classifying text data has been an active area of research for a long time. Text document is multifaceted object and often inherently ambiguous by nature. Multi-label learning deals with such ambiguous object. Classification of such ambiguous text objects often makes task of classifier difficult while assigning relevant classes to input document. Traditional single label and multi class text classification paradigms cannot efficiently classify such multifaceted text corpus. Through our paper we are proposing a novel label propagation approach based on semi supervised learning for Multi Label Text Classification. Our proposed approach models the relationship between class labels and also effectively represents input text documents. We are using semi supervised learning technique for effective utilization of labeled and unlabeled data for classification. Our proposed approach promises better classification accuracy and handling of complexity and elaborated on the basis of standard datasets such as Enron, Slashdot and Bibtex.

  14. Comparative study of the three different fluorophore antibody conjugation strategies. (United States)

    Shrestha, Dilip; Bagosi, Adrienn; Szöllősi, János; Jenei, Attila


    The progression in bioconjugational chemistry has significantly contributed to the evolution and success of protein biology. Mainly, antibody chemistry has been a subject of intensive study owing to the expansion of research areas warranted by using various derivatives of conjugated antibodies. Three reactive moieties (amine, sulfhydryl and carbohydrate) in the antibodies are chiefly favored for the conjugational purpose. This feature is known for decades, nevertheless, amine based conjugation is still the most preferred strategy despite the appreciation the other two methods receive in conserving the antigen binding affinity (ABA). No single report has been published, according to our knowledge, where these three conjugation strategies were applied to the same fluorophore antibody systems. In this study, we evaluated conjugation yield, time demand and cost efficiency of these conjugation procedures. Our results showed that amine based conjugations was by far the best technique due to its simplicity, rapidity, ease of operation, higher conjugate yield, cheaper cost and potential for larger fluorophore/protein labeling ratio without having much effect in ABA. Furthermore, sulfhydryl labeling clearly excelled in terms of reduced non-specific binding and mild effect in ABA but was usually complicated by an asymmetric antibody reduction due to mercaptoethylamine while carbohydrate oxidation based strategy performed the worst during our experiment.

  15. Labeling and preliminary in vivo assessment of niobium-labeled radioactive species: A proof-of-concept study. (United States)

    Radchenko, Valery; Bouziotis, Penelope; Tsotakos, Theodoros; Paravatou-Petsotas, Mari; la Fuente, Ana de; Loudos, George; Harris, Adrian L; Xanthopoulos, Stavros; Filosofov, Dmitry; Hauser, Harald; Eisenhut, Michael; Ponsard, Bernard; Roesch, Frank


    The application of radionuclide-labeled biomolecules such as monoclonal antibodies or antibody fragments for imaging purposes is called immunoscintigraphy. More specifically, when the nuclides used are positron emitters, such as zirconium-89, the technique is referred to as immuno-PET. Currently, there is an urgent need for radionuclides with a half-life which correlates well with the biological kinetics of the biomolecules under question and which can be attached to the proteins by robust labeling chemistry. (90)Nb is a promising candidate for in vivo immuno-PET, due its half-life of 14.6h and low β(+) energy of Emean=0.35MeV per decay. (95)Nb on the other hand, is a convenient alternative for longer-term ex vivo biodistribution studies, due to its longer half-life of (t½=35days) and its convenient, lower-cost production (reactor-based production). In this proof-of-principle work, the monoclonal antibody bevacizumab (Avastin(®)) was labeled with (95/90)Nb and in vitro and in vivo stability was evaluated in normal Swiss mice and in tumor-bearing SCID mice. Initial ex vivo experiments with (95)Nb-bevacizumab showed adequate tumor uptake, however at the same time high uptake in the liver, spleen and kidneys was observed. In order to investigate whether this behavior is due to instability of (⁎)Nb-bevacizumab or to the creation of other (⁎)Nb species in vivo, we performed biodistribution studies of (95)Nb-oxalate, (95)Nb-chloride and (95)Nb-Df. These potential metabolite species did not show any specific uptake, apart from bone accumulation for (95)Nb-oxalate and (95)Nb-chloride, which, interestingly, may serve as an "indicator" for the release of (90)Nb from labeled biomolecules. Concerning the initial uptake of (95)Nb-bevacizumab in non-tumor tissue, biodistribution of a higher specific activity radiolabeled antibody sample did show only negligible uptake in the liver, spleen, kidneys or bones. In-vivo imaging of a tumor-bearing SCID mouse after injection

  16. Tumor targeting of radiolabeled antibodies using HYNIC chelate

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Tae Sup; Chung, Wee Sup; Woo, Kwang Sun; Choi, Tae Hyun; Chung, Hye Kyung; Lee, Myung Jin; Kim, So Yeon; Jung, Jae Ho; Choi, Chang Woon; Lim, Sang Moo [KIRAMS, Seoul (Korea, Republic of); Darwati, Siti [National Nuclear Energy Agency, Tangerang (Indonesia)


    There is an increasing interest in the use of labeled antibodies for diagnosis of cancers as well as for therapy. Various radiolabeling methods have been used in order to obtain better tumor specific targeting for detection and therapy. It was generally used to tumor targeted immunotherapy and immunodetection that lym-1, mouse monoclonal antibody, was specific binding to surface antigen of Raji. The 3E8 antibody was produced from humanized anti-TAG-72 monoclonal antibody (AKA) by amino acid change in 95-99 residues of heavy chain complementary determinant regions (HCDRs) 3 using phage displayed library technology. In this study, we are investigating the usefulness of HYNIC chelate as a bifunctional chelating agent in radioimmunodetecton of tumor. Two types of antibodies, Lym-1 and 3E8, were used for the conjugation with HYNIC chelate. Lym-1 and 3E8 are specific antibodies to surface antigen of Non-Hogkin's lymphoma and TAG-72 antigen of colorectal carcinoma, respectively. We prepare HYNIC-antibody conjugates, determine radiolabeling yield with {sup 99m}Tc and evaluate tumor targeting in tumor bearing nude mice model.

  17. TU-F-12A-01: Quantitative Non-Linear Compartment Modeling of 89Zr- and 124I- Labeled J591 Monoclonal Antibody Kinetics Using Serial Non-Invasive Positron Emission Tomography Imaging in a Pre-Clinical Human Prostate Cancer Mouse Model

    Energy Technology Data Exchange (ETDEWEB)

    Fung, EK; Cheal, SM; Chalasani, S; Fareedy, SB; Punzalan, B; Humm, JL; Osborne, JR; Larson, SM; Zanzonico, PB [Memorial Sloan Kettering Cancer Center, New York, NY (United States); Otto, B; Bander, NH [Weill Cornell Medical College, New York, NY (United States)


    Purpose: To examine the binding kinetics of human IgG monoclonal antibody J591 which targets prostate-specific membrane antigen (PSMA) in a pre-clinical mouse cancer model using quantitative PET compartmental analysis of two radiolabeled variants. Methods: PSMA is expressed in normal human prostate, and becomes highly upregulated in prostate cancer, making it a promising therapeutic target. Two forms of J591, radiolabeled with either {sup 89}Zr or {sup 124}I, were prepared. {sup 89}Zr is a radiometal that becomes trapped in the cell upon internalization by the antigen-antibody complex, while radioiodine leaves the cell. Mice with prostate cancer xenografts underwent non-invasive serial imaging on a Focus 120 microPET up to 144 hours post-injection of J591. A non-linear compartmental model describing the binding and internalization of antibody in tumor xenograft was developed and applied to the PET-derived time-activity curves. The antibody-antigen association rate constant (ka), total amount of antigen per gram tumor (Ag-total), internalization rate of antibody-antigen complex, and efflux rate of radioisotope from tumor were fitted using the model. The surface-bound and the internalized activity were also estimated. Results: Values for ka, Ag-total, and internalization rate were found to be similar regardless of radiolabel payload used. The efflux rate, however, was ∼ 9-fold higher for {sup 124}I-J591 than for {sup 89}Zr-J591. Time-dependent surface-bound and internalized radiotracer activity were similar for both radiolabels at early times post-injection, but clearly differed beyond 24 hours. Conclusion: Binding and internalization of J591 to PSMA-expressing tumor xenografts were similar when radiolabeled with either {sup 89}Zr or {sup 124}I payload. The difference in efflux of radioactivity from tumor may be attributable to differential biological fate intracellularly of the radioisotopes. This has great significance for radioimmunotherapy and antibody

  18. Preparation and validation of radio iodinated recombinant human IL-10 for the measurement of natural human antibodies against IL-10

    DEFF Research Database (Denmark)

    de Lemos Rieper, Carina; Galle, Pia; Svenson, Morten;


    activity of 75 cpm/pg. Validation of the tracer confirmed preserved antibody epitopes and receptor binding ability. A robust Radio Immuno Assay (RIA) was developed and validated to detect natural human anti-IL-10 antibodies based on the formation of (125)I-labeled IL-10-IgG complexes in solution...

  19. Anti-cartilage antibody. (United States)

    Greenbury, C L; Skingle, J


    Antibody to cartilage has been demonstrated by indirect immunofluorescence on rat trachea in the serum of about 3% of 1126 patients with rheumatoid arthritis. Titres ranged from 1:20 to 1:640. The antibody was not found in 284 patients with primary or secondary osteoarthritis or in 1825 blood donors, nor, with the exception of two weak reactors, in 1314 paraplegic patients. In most cases the antibody appears to be specific for native type II collagen. Using this as an antigen in a haemagglutination test 94% of anti-cartilage sera were positive, whereas among 100 rheumatoid control sera there were only three weak positives. More than 80% of patients with antibody had some erosion of articular cartilage, but there was no correlation with age, sex, duration of disease, nor any recognisable clinical event or change.

  20. Antithyroid microsomal antibody (United States)

    ... to confirm the cause of thyroid problems, including Hashimoto thyroiditis . The test is also used to find ... positive test may be due to: Granulomatous thyroiditis Hashimoto thyroiditis High levels of these antibodies have also ...

  1. Serum herpes simplex antibodies (United States)

    ... 2. HSV-1 most often causes cold sores (oral herpes). HSV-2 causes genital herpes. How the Test ... whether a person has ever been infected with oral or genital herpes . It looks for antibodies to herpes simplex virus ...

  2. 78 FR 66826 - Prior Label Approval System: Generic Label Approval (United States)


    ... submitted for evaluation and approval before use under the final rule are: (1) Labels for chicken produced..., such as ``no antibiotics administered'' or ``vegetarian fed''; (4) instructional or...

  3. Preparation of Radiopharmaceuticals Labeled with Metal Radionuclides

    Energy Technology Data Exchange (ETDEWEB)

    Welch, M.J.


    The overall goal of this project was to develop methods for the production of metal-based radionuclides, to develop metal-based radiopharmaceuticals and in a limited number of cases, to translate these agents to the clinical situation. Initial work concentrated on the application of the radionuclides of Cu, Cu-60, Cu-61 and Cu-64, as well as application of Ga-68 radiopharmaceuticals. Initially Cu-64 was produced at the Missouri University Research Reactor and experiments carried out at Washington University. A limited number of studies were carried out utilizing Cu-62, a generator produced radionuclide produced by Mallinckrodt Inc. (now Covidien). In these studies, copper-62-labeled pyruvaldehyde Bis(N{sup 4}-methylthiosemicarbazonato)-copper(II) was studied as an agent for cerebral myocardial perfusion. A remote system for the production of this radiopharmaceutical was developed and a limited number of patient studies carried out with this agent. Various other copper radiopharmaceuticals were investigated, these included copper labeled blood imaging agents as well as Cu-64 labeled antibodies. Cu-64 labeled antibodies targeting colon cancer were translated to the human situation. Cu-64 was also used to label peptides (Cu-64 octriatide) and this is one of the first applications of a peptide radiolabeled with a positron emitting metal radionuclide. Investigations were then pursued on the preparation of the copper radionuclides on a small biomedical cyclotron. A system for the production of high specific activity Cu-64 was developed and initially the Cu-64 was utilized to study the hypoxic imaging agent Cu-64 ATSM. Utilizing the same target system, other positron emitting metal radionuclides were produced, these were Y-86 and Ga-66. Radiopharmaceuticals were labeled utilizing both of these radionuclides. Many studies were carried out in animal models on the uptake of Cu-ATSM in hypoxic tissue. The hypothesis is that Cu-ATSM retention in vivo is dependent upon the

  4. Antibody networks and imaging: elicitation of anti-fluorescein antibodies in response to the metatypic state of fluorescein-specific monoclonal antibodies. (United States)

    Cedergren, A M; Miklasz, S D; Voss, E W


    Studies are described regarding generation of anti-hapten antibodies starting with a monoclonal Ig immunogen in the ligand-induced conformation or metatypic state. Liganded monoclonal Ab1 antibodies represent the unique feature of the study since previous reports investigating internal imaging in the original Idiotype Network Hypothesis [Jerne, 1974 (Ann. Immun. 125C, 373-389)] were based on the non-liganded or idiotypic state [as reviewed in: Rodkey, 1980 (Microbiol. Rev. 44, 631-659); Kohler et al., 1979 (In: Methods in Enzymology: Antibodies, Antigens and Molecular Mimicry, pp. 3-35); Greenspan and Bona, 1993 (FASEB J. 7,437-444)]. Affinity-labeled liganded murine monoclonal anti-fluorescein antibodies served as immunogens administered both in the syngenic and xenogenic modes to determine if the metatypic state elicited anti-hapten antibodies through imaging-like mechanisms. Polyclonal and monoclonal anti-Ab1 reagents in various hosts were assayed for anti-fluorescein and/or anti-metatype specificity. Significant anti-fluorescein responses were measured indicating that the metatypic state directly or indirectly stimulates an anti-hapten antibody population.

  5. Characterization of Tumor-Avid Antibody Fragments Genetically Engineered for Mono-Specific Radionuclide Chelation

    Energy Technology Data Exchange (ETDEWEB)

    Quinn, T.P.


    The successful clinical application of targeted-radiopharmaceuticals depends on the development of molecules that optimize tumor specific radionuclide deposition and minimize non-specific organ irradiation. To this end, this proposal outlines a research effort to identify and evaluate novel antibodies and antibody fragments that bind breast tumors. The tumor-avid antibodies will be investigated for as imaging and therapeutic agents and to gain a better understanding of the pharmacokinetics and metabolism of radiolabeled tumor-avid antibody fragments through the use of site-specifically labeled molecules. Antibodies or antibody fragments, that bind breast carcinoma carbohydrate antigens, will be obtained from hybridoma or bacteriophage library screening. More specifically, antibody fragments that bind the carcinoma-associated Thomsen-Friedenreich (T) antigen will be radiolabeled with {sup 99m}Tc and {sup 188}Re at a natural amino acid chelation site and will be investigated in vivo for their abilities to target human breast tumors. In addition, site-specific radiolabeled antibody fragments will be biosynthesized using misacylated suppressor tRNAs. Homogeneously radiolabeled populations of antibody fragments will be used to investigate the effects of radionuclide location and chelation chemistries on their biodistribution and metabolism. It is hypothesized that site-specifically radiolabeled antibody fragments will possess enhanced tumor imaging and therapeutic properties due to optimal label location and conjugation chemistries. New insights into the factors that govern antibody metabolism in vivo are also expected from this work. Results from these studies should enhance our ability to design and synthesize radiolabeled antibody fragments that have improved pharmacokinetic properties. The studies in this proposal involve basic research into the development of antibody-based radiopharmaceuticals, with the ultimate goal of application in humans. This type of basic

  6. GEO label: The General Framework for Labeling and Certification (United States)

    Bye, B. L.; McCallum, I.; Maso, J.


    The Group on Earth Observations (GEO) is coordinating efforts to build a Global Earth Observation System of Systems, or GEOSS. As part of a strategy to increase the involvement of the science and technology community in GEOSS, both as users and developers of GEOSS itself, GEO decided to develop a GEO label concept related to the scientific relevance, quality, acceptance and societal needs for services and data sets of GEOSS. The development of a GEO label is included in the GEO work plan and several projects address the challenges of developing a GEO label concept. Within the different projects developing the GEO label, various perspectives and approaches are being applied. In order to arrive at a generally accepted GEO label concept, a common understanding and basic knowledge of labeling is necessary. Assessment of quality of internationally standardized Earth observation data products implies possible certification. A general understanding of the framework for international standards and certification will also contribute to a more coherent discussion and more efficient development of a GEO label. We will describe the general labeling and certification framework emphasizing the relation to the three elements of the GEO label: quality, user acceptance and relevance. Based on a survey of international labels done by the EGIDA project, we have analyzed the legal framework and organization of labels and certification. We will discuss the frameworks for certification, user ratings, registration and analysis of user requirements. Quality assessment is a particular focus of the analysis and is based on the work done by the GeoViQua project. A GEO label will function both as a data distribution strategy and as a general management system for data. Through a label users can compare different data sets and get access to more information about the relevant data, including quality. A label will provide traceability of data both in the interest of users as well as data

  7. Heparin-Induced Thrombocytopenia Antibody Test (United States)

    ... Global Sites Search Help? Heparin-induced Thrombocytopenia PF4 Antibody Share this page: Was this page helpful? Also known as: Heparin-PF4 Antibody; HIT Antibody; HIT PF4 Antibody; Heparin Induced Antibody; ...

  8. Sustainability labels on food products

    DEFF Research Database (Denmark)

    Grunert, Klaus G; Hieke, Sophie; Wills, Josephine


    This study investigates the relationship between consumer motivation, understanding and use of sustainability labels on food products (both environmental and ethical labels), which are increasingly appearing on food products. Data was collected by means of an online survey implemented in the UK......, human values as measured by the Schwartz value domains, and country differences. The results imply that sustainability labels currently do not play a major role in consumers’ food choices, and future use of these labels will depend on the extent to which consumers’ general concern about sustainability...

  9. [New antibodies in cancer treatment]. (United States)

    Pestalozzi, B C; Knuth, A


    Since the development of hybridoma technology in 1975 monoclonal antibodies with pre-defined specificity can be produced. Only twenty years later did it become possible to make therapeutic use of monoclonal antibodies in oncology. To this end it was necessary to attach the antigen-binding site of a mouse antibody onto the scaffold of a human antibody molecule. Such chimeric or "humanized" antibodies may be used in passive immunotherapy without eliciting an immune response. Rituximab and trastuzumab are such humanized antibodies. They are used today routinely in the treatment of malignant lymphoma and breast cancer, respectively. These antibodies are usually used in combination with conventional cytostatic anticancer drugs.

  10. Engineering antibodies for cancer therapy. (United States)

    Boder, Eric T; Jiang, Wei


    The advent of modern antibody engineering has led to numerous successes in the application of these proteins for cancer therapy in the 13 years since the first Food and Drug Administration approval, which has stimulated active interest in developing more and better drugs based on these molecules. A wide range of tools for discovering and engineering antibodies has been brought to bear on this challenge in the past two decades. Here, we summarize mechanisms of monoclonal antibody therapeutic activity, challenges to effective antibody-based treatment, existing technologies for antibody engineering, and current concepts for engineering new antibody formats and antibody alternatives as next generation biopharmaceuticals for cancer treatment.

  11. Synthesis and Preliminary Biological Evaluations of Fluorescent or 149Promethium Labeled Trastuzumab-Polyethylenimine

    Directory of Open Access Journals (Sweden)

    Jonathan Fitzsimmons


    Full Text Available Background: Radioimmunotherapy utilize a targeting antibody coupled to a therapeutic isotope to target and treat a tumor or disease. In this study we examine the synthesis and cell binding of a polymer scaffold containing a radiotherapeutic isotope and a targeting antibody. Methods: The multistep synthesis of a fluorescent or 149Promethium-labeled Trastuzumab-polyethyleneimine (PEI, Trastuzumab, or PEI is described. In vitro uptake, internalization and/or the binding affinity to the Her2/neu expressing human breast adenocarcinoma SKBr3 cells was investigated with the labeled compounds. Results: Fluorescent-labeled Trastuzumab-PEI was internalized more into cells at 2 and 18 h than fluorescent-labeled Trastuzumab or PEI. The fluorescent-labeled Trastuzumab was concentrated on the cell surface at 2 and 18 h and the labeled PEI had minimal uptake. DOTA-PEI was prepared and contained an average of 16 chelates per PEI; the compound was radio-labeled with 149Promethium and conjugated to Trastuzumab. The purified 149Pm-DOTA-PEI-Trastuzumab had a radiochemical purity of 96.7% and a specific activity of 0.118 TBq/g. The compound demonstrated a dissociation constant for the Her2/neu receptor of 20.30 ± 6.91 nM. Conclusion: The results indicate the DOTA-PEI-Trastuzumab compound has potential as a targeted therapeutic carrier, and future in vivo studies should be performed.

  12. 76 FR 75809 - Prior Label Approval System: Generic Label Approval (United States)


    ... Food Standards and Labeling Policy Book; does not bear any special claims, such as quality claims... addition, deletion, or amendment of recipe suggestions for the product; j. Any change in punctuation; k... Policy Book, and questions and answers on various topics, such as irradiation and the labeling...

  13. Quality Evaluation Model for Map Labeling

    Institute of Scientific and Technical Information of China (English)

    FAN Hong; ZHANG Zuxun; DU Daosheng


    This paper discusses and sums up the basic criterions of guaranteeing the labeling quality and abstracts the four basic factors including the conflict for a label with a label, overlay for label with the features, position's priority and the association for a label with its feature. By establishing the scoring system, a formalized four-factors quality evaluation model is constructed. Last, this paper introduces the experimental result of the quality evaluation model applied to the automatic map labeling system-MapLabel.

  14. C-kit-targeted imaging of gastrointestinal stromal tumor using radiolabeled anti-c-kit monoclonal antibody in a mouse tumor model

    Energy Technology Data Exchange (ETDEWEB)

    Sogawa, Chizuru [Diagnostic Imaging Group, Molecular Imaging Center, National Institute of Radiological Sciences, Chiba 263-8555 (Japan); Tsuji, Atsushi B. [Diagnostic Imaging Group, Molecular Imaging Center, National Institute of Radiological Sciences, Chiba 263-8555 (Japan)], E-mail:; Sudo, Hitomi [Diagnostic Imaging Group, Molecular Imaging Center, National Institute of Radiological Sciences, Chiba 263-8555 (Japan); Department of Pathology and Oncology, Juntendo University School of Medicine, Tokyo 113-8421 (Japan); Sugyo, Aya [Diagnostic Imaging Group, Molecular Imaging Center, National Institute of Radiological Sciences, Chiba 263-8555 (Japan); Yoshida, Chisato [Diagnostic Imaging Group, Molecular Imaging Center, National Institute of Radiological Sciences, Chiba 263-8555 (Japan); Department of Molecular Imaging and Radiotherapy, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 260-8675 (Japan); Odaka, Kenichi [Molecular Probe Group, Molecular Imaging Center, National Institute of Radiological Sciences, Chiba 263-8555 (Japan); Uehara, Tomoya; Arano, Yasushi [Department of Molecular Imaging and Radiotherapy, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 260-8675 (Japan); Koizumi, Mitsuru; Saga, Tsuneo [Diagnostic Imaging Group, Molecular Imaging Center, National Institute of Radiological Sciences, Chiba 263-8555 (Japan)


    Introduction: Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor arising from the gastrointestinal tract and highly expresses mutated c-kit. We aimed to develop a specific and sensitive method for detecting GISTs using radiolabeled anti-c-kit monoclonal antibody. Methods: A mutated c-kit-expressing cell clone was established by transfecting an expressing vector of mutated c-kit gene into HEK293 human embryonic kidney cells. The tumors were developed by inoculating c-kit-expressing cells into nude mice. {sup 125}I- and {sup 111}In-labeled anti-c-kit antibodies (12A8 and 41A11) were evaluated in vitro by cell binding, competitive inhibition and cellular internalization assays, and in vivo by biodistribution and imaging studies in tumor-bearing mice. Results: Both {sup 125}I- and {sup 111}In-labeled antibodies showed specific binding with c-kit-expressing cells with high affinity (dissociation constants = 2.2-7.1x10{sup 9} M{sup -1}). Internalization assay showed that {sup 125}I-labeled antibodies were rapidly internalized and dehalogenated, with the release of {sup 125}I from the cells, resulting in reduction of cell-associated radioactivity with time. In contrast, {sup 111}In-labeled antibody was internalized but did not result in the reduced radioactivity associated with tumor cells. Reflecting this phenomenon, the in vivo tumor uptake of {sup 125}I-labeled antibody was low on Day 1, further decreasing with time, while tumor uptake of {sup 111}In-labeled antibody was high on Day 1, further increasing with time. The xenografted tumor was clearly visualized by scintigraphy after injection of {sup 111}In-labeled antibody. Conclusion: The anti-c-kit monoclonal antibody labeled with a metal radionuclide would be promising for c-kit-targeted imaging of GISTs.

  15. Natural and Man-made Antibody Repertories for Antibody Discovery

    Directory of Open Access Journals (Sweden)

    Juan C eAlmagro


    Full Text Available Antibodies are the fastest-growing segment of the biologics market. The success of antibody-based drugs resides in their exquisite specificity, high potency, stability, solubility, safety and relatively inexpensive manufacturing process in comparison with other biologics. We outline here the structural studies and fundamental principles that define how antibodies interact with diverse targets. We also describe the antibody repertoires and affinity maturation mechanisms of human, mice and chickens, plus the use of novel single-domain antibodies in camelids and sharks. These species all utilize diverse evolutionary solutions to generate specific and high affinity antibodies and illustrate the plasticity of natural antibody repertoires. In addition, we discuss the multiple variations of man-made antibody repertoires designed and validated in the last two decades, which have served as tools to explore how the size, diversity and composition of a repertoire impact the antibody discovery process.

  16. Monoclonal antibody to native P39 protein from Borrelia burgdorferi.


    Sullivan, T J; Hechemy, K E; Harris, H L; Rudofsky, U H; Samsonoff, W A; Peterson, A J; Evans, B. D.; Balaban, S L


    We have produced, by using a sonicate of Borrelia burgdorferi, a monoclonal antibody (MAb), NYSP39H, that is specific for the P39 protein band. This MAb reacted with 13 isolates of B. burgdorferi but not with eight different spirochetes (four borrelias, two leptospiras, and two treponemas). Surface labeling of B. burgdorferi with biotin and subsequent treatment with Nonidet P-40 showed that P39 was not biotinylated but was extracted with Nonidet P-40, indicating that it is present within the ...

  17. Correlative microscopy of densely labeled projection neurons using neural tracers. (United States)

    Oberti, Daniele; Kirschmann, Moritz A; Hahnloser, Richard H R


    Three-dimensional morphological information about neural microcircuits is of high interest in neuroscience, but acquiring this information remains challenging. A promising new correlative technique for brain imaging is array tomography (Micheva and Smith, 2007), in which series of ultrathin brain sections are treated with fluorescent antibodies against neurotransmitters and synaptic proteins. Treated sections are repeatedly imaged in the fluorescence light microscope (FLM) and then in the electron microscope (EM). We explore a similar correlative imaging technique in which we differentially label distinct populations of projection neurons, the key routers of electrical signals in the brain. In songbirds, projection neurons can easily be labeled using neural tracers, because the vocal control areas are segregated into separate nuclei. We inject tracers into areas afferent and efferent to the main premotor area for vocal production, HVC, to retrogradely and anterogradely label different classes of projection neurons. We optimize tissue preparation protocols to achieve high fluorescence contrast in the FLM and good ultrastructure in the EM (using osmium tetroxide). Although tracer fluorescence is lost during EM preparation, we localize the tracer molecules after fixation and embedding by using fluorescent antibodies against them. We detect signals mainly in somata and dendrites, allowing us to classify synapses within a single ultrathin section as belonging to a particular type of projection neuron. The use of our method will be to provide statistical information about connectivity among different neuron classes, and to elucidate how signals in the brain are processed and routed among different areas.

  18. Correlative microscopy of densely labeled projection neurons using neural tracers

    Directory of Open Access Journals (Sweden)

    Daniele Oberti


    Full Text Available Three-dimensional morphological information about neural microcircuits is of high interest in neuroscience, but acquiring this information remains challenging. A promising new correlative technique for brain imaging is array tomography (Micheva and Smith, 2007, in which series of ultrathin brain sections are treated with fluorescent antibodies against neurotransmitters and synaptic proteins. Treated sections are repeatedly imaged in the fluorescence light microscope (FLM and then in the electron microscope (EM. We explore a similar correlative imaging technique in which we differentially label distinct populations of projection neurons, the key routers of electrical signals in the brain. In songbirds, projection neurons can easily be labeled using neural tracers, because the vocal control areas are segregated into separate nuclei. We inject tracers into areas afferent and efferent to the main premotor area for vocal production, HVC, to retrogradely and anterogradely label different classes of projection neurons. We optimize tissue preparation protocols to achieve high fluorescence contrast in the FLM and good ultrastructure in the EM (using osmium tetroxide. Although tracer fluorescence is lost during EM preparation, we localize the tracer molecules after fixation and embedding by using fluorescent antibodies against them. We detect signals mainly in somata and dendrites, allowing us to classify synapses within a single ultrathin section as belonging to a particular type of projection neuron. The use of our method will be to provide statistical information about connectivity among different neuron classes, and to elucidate how signals in the brain are processed and routed among different areas.

  19. Distance labeling schemes for trees

    DEFF Research Database (Denmark)

    Alstrup, Stephen; Gørtz, Inge Li; Bistrup Halvorsen, Esben;


    variants such as, for example, small distances in trees [Alstrup et al., SODA, 2003]. We improve the known upper and lower bounds of exact distance labeling by showing that 1/4 log2(n) bits are needed and that 1/2 log2(n) bits are sufficient. We also give (1 + ε)-stretch labeling schemes using Theta...

  20. Nutrition Marketing on Food Labels (United States)

    Colby, Sarah E.; Johnson, LuAnn; Scheett, Angela; Hoverson, Bonita


    Objective: This research sought to determine how often nutrition marketing is used on labels of foods that are high in saturated fat, sodium, and/or sugar. Design and Setting: All items packaged with food labels (N = 56,900) in all 6 grocery stores in Grand Forks, ND were surveyed. Main Outcome Measure(s): Marketing strategy, nutrient label…

  1. Targeted labeling of early-​stage tumor spheroid in chorioallantoic membrane model with upconversion nanoparticles

    NARCIS (Netherlands)

    K. Liu; J.A. Holz; Y. Ding; X. Liu; Y. Zhang; L. Tu; X. Kong; B. Priem; A. Nadort; S.A.G. Lambrechts; M.C.G. Aalders; W.J. Buma; Y. Liu; H. Zhang


    In vivo detection of cancer at early-​stage, i.e. smaller than 2 mm, is a challenge in biomedicine. In this work target labeling of early-​stage tumor spheroid (∼500 μm) is realized for the first time in chick embryo chorioallantoic membrane (CAM) model with monoclonal antibody functionalized upconv

  2. A new label-free strategy for a highly efficient chemiluminescence immunoassay. (United States)

    Yang, Zhanjun; Cao, Yue; Li, Juan; Wang, Juntao; Du, Dan; Hu, Xiaoya; Lin, Yuehe


    A new label-free chemiluminescence (CL) immunoassay method is proposed for the cheap, convenient and sensitive detection of proteins through the co-immobilization of a capture antibody and horseradish peroxidase on a sensing interface. The specific immunoreaction on the interface effectively inhibits the enzymatic CL reaction, thus causing a decrease in CL signals.

  3. Synthesis and properties of differently charged chemiluminescent acridinium ester labels. (United States)

    Natrajan, Anand; Sharpe, David


    Chemiluminescent acridinium dimethylphenyl esters containing N-sulfopropyl groups in the acridinium ring are highly sensitive, hydrophilic labels that are used in automated immunoassays for clinical diagnostics. Light emission from these labels is triggered with alkaline peroxide in the presence of a cationic surfactant. At physiological pH, N-sulfopropyl acridinium esters exist as water adducts that are commonly referred to as pseudobases. Pseudobase formation, which results from addition of water to the zwitterionic N-sulfopropyl acridinium ring, neutralizes the positive charge on the acridinium nitrogen and imparts a net negative charge to the label due to the sulfonate moiety. As a consequence, N-sulfopropyl acridinium ester conjugates of small molecule haptens as well as large molecules such as proteins gain negative charges at neutral pH. In the current study, we describe the synthesis and properties of two new hydrophilic acridinium dimethylphenyl ester labels where the net charge in the labels was altered. In one label, the structure of the hydrophilic N-alkyl group attached to the acridinium ring was changed so that the pseudobase of the label contains no net charge. In the second acridinium ester, two additional negative charges in the form of sulfopropyl groups were added to the acridinium ring to make this label's pseudobase strongly anionic. Chemiluminescence measurements of these labels, as well as their conjugates of an antibody with a neutral pI, indicate that acridinium ester charge while having a modest effect on emission kinetics has little influence on light output. However, our results demonstrate that acridinium ester charge can affect protein pI, apparent chemiluminescence stability and non-specific binding of protein conjugates to microparticles. These results emphasize the need for careful consideration of acridinium ester charge in order to optimize reagent stability and performance in immunoassays. In the current study, we observed that

  4. Ricin Detection Using Phage Displayed Single Domain Antibodies

    Directory of Open Access Journals (Sweden)

    Ellen R. Goldman


    Full Text Available Phage-displayed single domain antibodies (sdAb were compared to monomeric solubly expressed sdAb and llama polyclonal antibodies for the detection of ricin. SdAb are comprised of the variable domain derived from camelid heavy chain only antibodies (HcAb. Although HcAb lack variable light chains, they as well as their derivative sdAb are able to bind antigens with high affinity. The small size of sdAb (~16 kDa, while advantageous in many respects, limits the number of labels that can be incorporated. The ability to incorporate multiple labels is a beneficial attribute for reporter elements. Opportunely, sdAb are often selected using phage display methodology. Using sdAb displayed on bacteriophage M13 as the reporter element gives the potential for incorporating a very high number of labels. We have demonstrated the use of both sdAb and phage- displayed sdAb for the detection of ricin using both enzyme linked immunosorbent assays (ELISAs and Luminex fluid array assays. The phage-displayed sdAb led to five to ten fold better detection of ricin in both the ELISA and Luminex assays, resulting in limits of detection of 1 ng/mL and 64 pg/mL respectively. The phage-displayed sdAb were also dramatically more effective for the visualization of binding to target in nitrocellulose dot blot assays, a method frequently used for epitope mapping.

  5. A better carbon footprint label

    DEFF Research Database (Denmark)

    Thøgersen, John; Nielsen, Kristian S.


    Based on insights from behavioral economics, it is suggested to extend carbon footprint labeling with information about relative performance, using the well-known “traffic light” color scheme to communicate relative performance. To test this proposition, the impact of a carbon footprint label...... on Danish consumers’ choice of ground coffee was tested in a 3 price levels x 3 levels of carbon emission x 3 certifying organizations x 2 organic labeling conditions discrete choice experiment. Participants were randomly assigned to two slightly different variants of the experiment: In one condition......, participants saw the original Carbon Trust label and in the other condition they saw the same label, but with traffic light colors added to communicate the product’s relative performance in terms of carbon footprint. All included attributes were found to have a significant impact on consumer choices...

  6. Antibody affinity maturation

    DEFF Research Database (Denmark)

    Skjødt, Mette Louise

    surface expression of various antibody formats in the generated knockout strain. Functional scFv and scFab fragments were efficiently displayed on yeast whereas impaired chain assembly and heavy chain degradation was observed for display of full-length IgG molecules. To identify the optimal polypeptide...... linker for yeast surface display of scFv and scFab fragments, we compared a series of different Gly-Ser-based linkers in display and antigen binding proficiency. We show that these formats of the model antibody can accommodate linkers of different lengths and that introduction of alanine or glutamate...... fragments by in vivo homologous recombination large combinatorial antibody libraries can easily be generated. We have optimized ordered assembly of three CDR fragments into a gapped vector and observed increased transformation efficiency in a yeast strain carrying a deletion of the SGS1 helicase...

  7. Production of mouse anti-quail IgY and subsequent labeling with horseradish peroxidase using cyanuric chloride. (United States)

    Kassim, Neema; Mtenga, Adelard B; Shim, Won-Bo; Chung, Duck-Hwa


    Polyclonal antibodies labeled with a tracer have been commonly used as secondary antibodies in immunochemical assays to quantify the concentration of antibody-antigen complexes. The majority of these antibodies conjugated with a tracer are commercially available, with the exception of few untouched targets. This study focused on the production and application of mouse anti-quail IgY as an intermediate antibody to link between quail egg yolk IgY and goat anti-mouse IgG-HRP as primary and secondary antibodies, respectively. Subsequently, the produced mouse anti-quail IgY was labeled with horseradish peroxidase (HRP) and its efficiency on enzyme linked immunosorbent assay (ELISA) was compared with that of commercial rabbit anti-chicken IgY-HRP. As an intermediate antibody, mouse anti-quail IgY was successfully produced with good affinity and sensitivity (1:10,000) to the primary and secondary antibodies. Subsequently, mouse anti-quail IgY was effectively conjugated with HRP enzyme, resulting in a secondary antibody with good sensitivity (1:10,000) to quail anti-V. parahaemolyticus and V. vulnificus IgY. The detection limit was 10(5) CFU/ml for both V. parahaemolyticus and V. vulnificus. The efficiency of the produced conjugate to detect quail IgY on ELISA was comparable to that of the commercial rabbit anti-chicken IgY-HRP, and hence the produced and labeled mouse anti-quail IgY-HRP can be used as a secondary antibody to detect any antibody produced in quail.

  8. Antibody informatics for drug discovery

    DEFF Research Database (Denmark)

    Shirai, Hiroki; Prades, Catherine; Vita, Randi;


    to the antibody science in every project in antibody drug discovery. Recent experimental technologies allow for the rapid generation of large-scale data on antibody sequences, affinity, potency, structures, and biological functions; this should accelerate drug discovery research. Therefore, a robust bioinformatic...... infrastructure for these large data sets has become necessary. In this article, we first identify and discuss the typical obstacles faced during the antibody drug discovery process. We then summarize the current status of three sub-fields of antibody informatics as follows: (i) recent progress in technologies...... for antibody rational design using computational approaches to affinity and stability improvement, as well as ab-initio and homology-based antibody modeling; (ii) resources for antibody sequences, structures, and immune epitopes and open drug discovery resources for development of antibody drugs; and (iii...

  9. BRCAA1 antibody- and Her2 antibody-conjugated amphiphilic polymer engineered CdSe/ZnS quantum dots for targeted imaging of gastric cancer (United States)

    Li, Chao; Ji, Yang; Wang, Can; Liang, Shujing; Pan, Fei; Zhang, Chunlei; Chen, Feng; Fu, Hualin; Wang, Kan; Cui, Daxiang


    Successful development of safe and highly effective nanoprobes for targeted imaging of in vivo early gastric cancer is a great challenge. Herein, we choose the CdSe/ZnS (core-shell) quantum dots (QDs) as prototypical materials, synthesized one kind of a new amphiphilic polymer including dentate-like alkyl chains and multiple carboxyl groups, and then used the prepared amphiphilic polymer to modify QDs. The resultant amphiphilic polymer engineered QDs (PQDs) were conjugated with BRCAA1 and Her2 monoclonal antibody, and prepared BRCAA1 antibody- and Her2 antibody-conjugated QDs were used for in vitro MGC803 cell labeling and in vivo targeted imaging of gastric cancer cells. Results showed that the PQDs exhibited good water solubility, strong photoluminescence (PL) intensity, and good biocompatibility. BRCAA1 antibody- and Her2 antibody-conjugated QD nanoprobes successfully realized targeted imaging of in vivo gastric cancer MGC803 cells. In conclusion, BRCAA1 antibody- and Her2 antibody-conjugated PQDs have great potential in applications such as single cell labeling and in vivo tracking, and targeted imaging and therapeutic effects' evaluation of in vivo early gastric cancer cells in the near future.

  10. 21 CFR 201.71 - Magnesium labeling. (United States)


    ... 21 Food and Drugs 4 2010-04-01 2010-04-01 false Magnesium labeling. 201.71 Section 201.71 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.71 Magnesium labeling. (a) The labeling of over-the-counter (OTC) drug products intended for oral ingestion shall contain the magnesium...

  11. 16 CFR 460.12 - Labels. (United States)


    ... 16 Commercial Practices 1 2010-01-01 2010-01-01 false Labels. 460.12 Section 460.12 Commercial Practices FEDERAL TRADE COMMISSION TRADE REGULATION RULES LABELING AND ADVERTISING OF HOME INSULATION § 460.12 Labels. If you are a manufacturer, you must label all packages of your insulation. The labels...

  12. 21 CFR 610.60 - Container label. (United States)


    ... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Container label. 610.60 Section 610.60 Food and... GENERAL BIOLOGICAL PRODUCTS STANDARDS Labeling Standards § 610.60 Container label. (a) Full label. The following items shall appear on the label affixed to each container of a product capable of bearing a...

  13. Production of anti-idiotype antibodies for deoxynivalenol and their evaluation with three immunoassay platforms. (United States)

    Maragos, C M


    Immunoassays for deoxynivalenol (DON) that involve binding to DON-specific antibodies have been widely developed. In such assays, the responses of samples are generally compared with calibration curves generated by using DON in competition with labeled reagents such as enzymatic or fluorescent conjugates of the toxin. However, materials that mimic the toxin can also be used, provided that they compete effectively with the labeled reagents for the DON-specific antibodies. Examples include certain types of anti-idiotype antibodies, obtained by the immunization of animals with toxin-specific antibodies. In the present work, anti-idiotype antibodies were developed which mimicked DON in the ability to bind to a DON-specific monoclonal antibody (Mab). Fab fragments of the Mab (Ab1) were used to immunize rabbits. Sera were screened by competitive direct enzyme linked immunosorbent assay (CD-ELISA) for the presence of anti-idiotype antibodies (Ab2). In order to determine the most effective screening format and also the potential efficacy in various forms of biosensors, the sera were further evaluated in biolayer interferometry (BLI) and fluorescence polarization immunoassay (FPIA) formats. All three formats were used to demonstrate the presence of anti-idiotypes capable of binding to the paratope of the DON antibody (subtypes Ab2β or Ab2γ). Such materials have the potential to replace DON as calibrants in immunoassays for this toxin.

  14. Antithyroglobulin Antibodies and Antimicrosomal Antibodies in Various Thyroid Diseases

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Gwon Jun; Hong, Key Sak; Choi, Kang Won; Lee, Kyu; Koh, Chang Soon; Lee, Mun Ho; Park, Sung Hoe; Chi, Je Geun; Lee, Sang Kook [Seoul National University College of Medicine, Seoul (Korea, Republic of)


    The authors investigated the incidence of antithyroglobulin antibodies and antibodies and antimicrosomal antibodies measured by tanned red cell hemagglutination method in subjects suffering from various thyroid disorders. 1) In 15 normal patients, neither suffering from any thyroid diseases nor from any other autoimmune disorders, the antithyroglobulin antibodies were all negative, but the antimicrosomal antibody was positive only in one patient (6.7%). 2) The antithyroglobulin antibodies were positive in 31.5% (34 patients) of 108 patients with various thyroid diseases, and the antimicrosomal antibodies were positive in 37.0% (40 patients). 3) of the 25 patients with Graves' diseases, 7 patients (28.0%) showed positive for the antithyroglobulin antibodies, and 9 (36.0%) for the antimicrosomal antibodies. There was no definite differences in clinical and thyroid functions between the groups with positive and negative results. 4) Both antibodies were positive in 16 (88.9%) and 17 (94.4%) patients respectively among 18 patients with Hashimoto's thyroiditis, all of them were diagnosed histologically. 5) Three out of 33 patients with thyroid adenoma showed positive antibodies, and 3 of 16 patients with thyroid carcinoma revealed positive antibodies. 6) TRCH antibodies demonstrated negative results in 2 patients with subacute thyroiditis, but positive in one patient with idiopathic primary myxedema. 7) The number of patients with high titers(>l:802) was 16 for antithyroglobulin antibody, and 62.5% (10 patients) of which was Hashimoto's thyroiditis. Thirteen (65.0) of 20 patients with high titers (>l:802) for antimicrosomal antibody was Hashimoto's thyroiditis. TRCH test is a simple, sensitive method, and has high reliability and reproducibility. The incidences and titers of antithyroglobulin antibody and antimicrosomal antibody are especially high in Hashimoto's thyroiditis.

  15. Prediction of Antibody Epitopes

    DEFF Research Database (Denmark)

    Nielsen, Morten; Marcatili, Paolo


    Antibodies recognize their cognate antigens in a precise and effective way. In order to do so, they target regions of the antigenic molecules that have specific features such as large exposed areas, presence of charged or polar atoms, specific secondary structure elements, and lack of similarity...... to self-proteins. Given the sequence or the structure of a protein of interest, several methods exploit such features to predict the residues that are more likely to be recognized by an immunoglobulin.Here, we present two methods (BepiPred and DiscoTope) to predict linear and discontinuous antibody...

  16. Synthesis Of Labeled Metabolites (United States)

    Martinez, Rodolfo A.; Silks, III, Louis A.; Unkefer, Clifford J.; Atcher, Robert


    The present invention is directed to labeled compounds, for example, isotopically enriched mustard gas metabolites including: [1,1',2,2'-.sup.13 C.sub.4 ]ethane, 1,1'-sulfonylbis[2-(methylthio); [1,1',2,2'-.sup.13 C.sub.4 ]ethane, 1-[[2-(methylsulfinyl)ethyl]sulfonyl]-2-(methylthio); [1,1',2,2'-.sup.13 C.sub.4 ]ethane, 1,1'-sulfonylbis[2-(methylsulfinyl)]; and, 2,2'-sulfinylbis([1,2-.sup.13 C.sub.2 ]ethanol of the general formula ##STR1## where Q.sup.1 is selected from the group consisting of sulfide (--S--), sulfone (--S(O)--), sulfoxide (--S(O.sub.2)--) and oxide (--O--), at least one C* is .sup.13 C, X is selected from the group consisting of hydrogen and deuterium, and Z is selected from the group consisting of hydroxide (--OH), and --Q.sup.2 --R where Q.sup.2 is selected from the group consisting of sulfide (--S--), sulfone(--S(O)--), sulfoxide (--S(O.sub.2)--) and oxide (--O--), and R is selected from the group consisting of hydrogen, a C.sub.1 to C.sub.4 lower alkyl, and amino acid moieties, with the proviso that when Z is a hydroxide and Q.sup.1 is a sulfide, then at least one X is deuterium.

  17. Labeled Cocaine Analogs (United States)

    Goodman, Mark M.; Shi, Bing Zhi; Keil, Robert N.


    Novel compounds having the structure: ##STR1## where X in .beta. configuration is phenyl, naphthyl; 2,3 or 4-iodophenyl; 2,3 or 4-(trimethylsilyl)phenyl; 3,4,5 or 6-iodonaphthyl; 3,4,5 or 6-(trimethylsilyl)naphthyl; 2,3 or 4-(trialkylstannyl)phenyl; or 3,4,5 or 6-(trialkylstannyl)naphthyl Y in .beta. configuration is Y.sub.1 or Y.sub.2, where Y.sub.1 is 2-fluoroethoxy, 3-fluoropropoxy, 4-fluorobutoxy, 2-fluorocyclopropoxy, 2 or 3-fluorocyclobutoxy, R,S 1'-fluoroisopropoxy, R 1'-fluoroisopropoxy, S 1'-fluoroisopropoxy, 1',3'-difluoroisopropoxy, R,S 1'-fluoroisobutoxy, R 1'-fluoroisobutoxy, S 1'-fluoroisobutoxy, R,S 4'-fluoroisobutoxy, R 4'-fluoroisobutoxy, S 4'-fluoroisobutoxy, or 1',1'-di(fluoromethyl)isobutoxy, and Y.sub.2 is 2-methanesulfonyloxy ethoxy, 3-methanesulfonyloxy propoxy, 4-methanesulfonyloxy butoxy, 2-methanesulfonyloxy cyclopropoxy, 2 or 3-methanesulfonyloxy cyclobutoxy, 1'methanesulfonyloxy isopropoxy, 1'-fluoro, 3'-methanesulfonyloxy isopropoxy, 1'-methanesulfonyloxy, 3'-fluoro isopropoxy, 1'-methanesulfonyloxy isobutoxy, or 4'-methanesulfonyloxy isobutoxy bind dopamine transporter protein and can be labeled with .sup.18 F or .sup.123 I for imaging.

  18. Granulocyte antigen systems and antibodies and their clinical significance

    Energy Technology Data Exchange (ETDEWEB)

    McCullough, J.


    Granulocyte alloantibodies and autoantibodies have a key role in the pathophysiology of several clinical problems. These include febrile transfusion reactions, severe pulmonary reactions to transfusion, isoimmune neonatal neutropenia, failure of effective granulocyte transfusion, autoimmune neutropenia, drug-induced neutropenia, and neutropenias secondary to many other diseases. Although many techniques are available for detecting granulocyte antibodies, the optimal in-vitro tests for predicting the antibodies' clinical effects are not established. Use of indium-111-labeled granulocytes may provide valuable information regarding the in-vivo effects of different granulocyte antibodies. Granulocyte transfusions continue to be used for a limited number of severely infected neutropenic patients who do not respond to antibiotic therapy.

  19. 单克隆抗体与多克隆抗体配对ELISA方法比较%Study of the Optimum Pairing of Polyclonal Antibodies and Monoclonal Antibodies with Sandwich ELISA

    Institute of Scientific and Technical Information of China (English)

    张小兵; 邸禄芹; 吴萌; 闫静辉


    Using human chorionic gonadotrophin( HCG) as the antigen,polyclonal antibodies and monoclonal antibodies(McAbs) specific for HCG were generated, characterized and highly purified. Then these antibodies were labeled with horseradish peroxidase (HRP). By a double antibody sandwich enzyme-linked immunosorhent assay, several problems of the optimum pairing of polyclonal antibodies and monoclonal antibodies were discussed. The results showed that with monoclonal antibody as trapping antibody and polyclonal antibody labeling with enzyme as testing antibody which should be diluted with animal sera as diluent,the sandwich ELISA had a highly specificity and sensitivity to detect antigen.%以人绒毛膜促性腺激素(HCG)为抗原,制备出对HCG的多克隆抗体和特异性单克隆抗体,并进行抗体纯化和特性分析,利用辣根过氧化物酶(HRP)分别对其进行了标记.采用双抗夹心ELISA试验,探讨了多克隆抗体与单克隆抗体配对的若干事项.结果表明,利用单克隆抗体和酶标多克隆抗体配对,并用含动物血清的稀释液稀释酶标抗体,可实现对检测原的高特异性和高灵敏度检测.

  20. Compositions, antibodies, asthma diagnosis methods, and methods for preparing antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Jin, Hongjun; Zangar, Richard C.


    Methods for preparing an antibody are provided with the method including incorporating 3-bromo-4-hydroxy-benzoic acid into a protein to form an antigen, immunizing a mammalian host with the antigen, and recovering an antibody having an affinity for the antigen from the host. Antibodies having a binding affinity for a monohalotyrosine are provided as well as composition comprising an antibody bound with monohalotyrosine. Compositions comprising a protein having a 3-bromo-4-hydroxy-benzoic acid moiety are also provided. Methods for evaluating the severity of asthma are provide with the methods including analyzing sputum of a patient using an antibody having a binding affinity for monohalotyrosine, and measuring the amount of antibody bound to protein. Methods for determining eosinophil activity in bodily fluid are also provided with the methods including exposing bodily fluid to an antibody having a binding affinity for monohalotyrosine, and measuring the amount of bound antibody to determine the eosinophil activity.

  1. Rapid isolation of antibody from a synthetic human antibody library by repeated fluorescence-activated cell sorting (FACS.

    Directory of Open Access Journals (Sweden)

    Sung Sun Yim

    Full Text Available Antibodies and their derivatives are the most important agents in therapeutics and diagnostics. Even after the significant progress in the technology for antibody screening from huge libraries, it takes a long time to isolate an antibody, which prevents a prompt action against the spread of a disease. Here, we report a new strategy for isolating desired antibodies from a combinatorial library in one day by repeated fluorescence-activated cell sorting (FACS. First, we constructed a library of synthetic human antibody in which single-chain variable fragment (scFv was expressed in the periplasm of Escherichia coli. After labeling the cells with fluorescent antigen probes, the highly fluorescent cells were sorted by using a high-speed cell sorter, and these cells were reused without regeneration in the next round of sorting. After repeating this sorting, the positive clones were completely enriched in several hours. Thus, we screened the library against three viral antigens, including the H1N1 influenza virus, Hepatitis B virus, and Foot-and-mouth disease virus. Finally, the potential antibody candidates, which show K(D values between 10 and 100 nM against the target antigens, could be successfully isolated even though the library was relatively small (∼ 10(6. These results show that repeated FACS screening without regeneration of the sorted cells can be a powerful method when a rapid response to a spreading disease is required.

  2. Antibody response to the lipopolysaccharide and protein antigens of Salmonella typhi during typhoid infection. I. Measurement of serum antibodies by radioimmunoassay

    Energy Technology Data Exchange (ETDEWEB)

    Tsang, R.S.W.; Chau, P.Y.; Lam, S.K. (Hong Kong Univ.); La Brooy, J.T.; Rowley, D. (Adelaide Univ. (Australia))


    Serum antibody responses to the lipopolysaccharide and protein antigens of S. typhi in typhoid patients were studied using a solid-phase radioimmunoassay technique with /sup 125/I labelled anti-immunoglobulin antibody. Sera from 24 adult typhoid patients and 20 non-typhoid adult controls were compared. As a group, sera from typhoid patients showed increased IgA, IgG and IgM immunoglobulin levels and gave significantly higher anti-LPS and anti-protein antibody titres in all three major immunoglobulin classes than did non-typhoid controls. Levels of antibodies against LPS or protein in sera of typhoid patients were highly variable with a skew distribution. A good correlation was found between antibody titres to the LPS antigen and those to a protein antigen. No correlation, however, was found between the anti-LPS antibody titres measured by radioimmunoassay and the anti-O antibody titres measured by the Widal agglutination test. Titration of anti-LPS or anti-protein antibodies by radioimmunoassay was found to be more sensitive and specific than Widal test for the serological diagnosis of typhoid fever. The advantages of measuring antibody response by radioimmunoassay over conventional Widal test are discussed.

  3. Human germline antibody gene segments encode polyspecific antibodies. (United States)

    Willis, Jordan R; Briney, Bryan S; DeLuca, Samuel L; Crowe, James E; Meiler, Jens


    Structural flexibility in germline gene-encoded antibodies allows promiscuous binding to diverse antigens. The binding affinity and specificity for a particular epitope typically increase as antibody genes acquire somatic mutations in antigen-stimulated B cells. In this work, we investigated whether germline gene-encoded antibodies are optimal for polyspecificity by determining the basis for recognition of diverse antigens by antibodies encoded by three VH gene segments. Panels of somatically mutated antibodies encoded by a common VH gene, but each binding to a different antigen, were computationally redesigned to predict antibodies that could engage multiple antigens at once. The Rosetta multi-state design process predicted antibody sequences for the entire heavy chain variable region, including framework, CDR1, and CDR2 mutations. The predicted sequences matched the germline gene sequences to a remarkable degree, revealing by computational design the residues that are predicted to enable polyspecificity, i.e., binding of many unrelated antigens with a common sequence. The process thereby reverses antibody maturation in silico. In contrast, when designing antibodies to bind a single antigen, a sequence similar to that of the mature antibody sequence was returned, mimicking natural antibody maturation in silico. We demonstrated that the Rosetta computational design algorithm captures important aspects of antibody/antigen recognition. While the hypervariable region CDR3 often mediates much of the specificity of mature antibodies, we identified key positions in the VH gene encoding CDR1, CDR2, and the immunoglobulin framework that are critical contributors for polyspecificity in germline antibodies. Computational design of antibodies capable of binding multiple antigens may allow the rational design of antibodies that retain polyspecificity for diverse epitope binding.

  4. Prediction of antibody persistency from antibody titres to natalizumab

    DEFF Research Database (Denmark)

    Jensen, Poul Erik H; Koch-Henriksen, Nils; Sellebjerg, Finn Thorup;


    In a subgroup of patients with multiple sclerosis natalizumab therapy causes generation of anti-natalizumab antibodies that may be transient or persistent. It is recommended to discontinue natalizumab therapy in persistently antibody-positive patients.......In a subgroup of patients with multiple sclerosis natalizumab therapy causes generation of anti-natalizumab antibodies that may be transient or persistent. It is recommended to discontinue natalizumab therapy in persistently antibody-positive patients....

  5. An antibody toolkit for the study of membrane traffic in Drosophila melanogaster

    Directory of Open Access Journals (Sweden)

    Falko Riedel


    Full Text Available The use of Drosophila melanogaster as a model organism has been pivotal to understanding the developmental processes of metazoans. However, the use of flies for studying subcellular organization is hampered by a paucity of reliable reagents to label specific organelles. Here, we describe the generation of mouse monoclonal antibodies against a set of markers of the secretory and endocytic pathways, along with goat polyclonal antibodies against two Golgi proteins. We show that the monoclonal antibodies are highly specific and sufficiently sensitive to detect endogenous proteins in crude extracts by immunoblotting with little background staining. By immunofluorescence the major compartments of the membrane traffic system (including the endoplasmic reticulum, the Golgi, and early and late endosomes are labeled by at least one antibody. Moreover, the antibodies can be used to label organelles in fly tissues including salivary glands and wing imaginal discs. We anticipate that these antibodies will provide a useful tool kit to facilitate the investigation of how the endomembrane system functions and varies in the diverse tissue types of metazoans.

  6. Monoclonal antibodies in myeloma

    DEFF Research Database (Denmark)

    Sondergeld, P.; van de Donk, N. W. C. J.; Richardson, P. G.;


    The development of monoclonal antibodies (mAbs) for the treatment of disease goes back to the vision of Paul Ehrlich in the late 19th century; however, the first successful treatment with a mAb was not until 1982, in a lymphoma patient. In multiple myeloma, mAbs are a very recent and exciting add...

  7. Antibody Blood Tests (United States)

    ... What do I do if I have a negative blood test (or panel) but I’m still having symptoms? While it is rare, it is possible for patients to have a negative antibody test results and still have celiac disease. ...

  8. RBC Antibody Screen (United States)

    ... test also may be used to help diagnose autoimmune-related hemolytic anemia in conjunction with a DAT. This condition may be caused when a person produces antibodies against his or her own RBC antigens. This can happen with some autoimmune disorders , such as lupus , with diseases such as ...

  9. Algorithms for Labeling Focus Regions. (United States)

    Fink, M; Haunert, Jan-Henrik; Schulz, A; Spoerhase, J; Wolff, A


    In this paper, we investigate the problem of labeling point sites in focus regions of maps or diagrams. This problem occurs, for example, when the user of a mapping service wants to see the names of restaurants or other POIs in a crowded downtown area but keep the overview over a larger area. Our approach is to place the labels at the boundary of the focus region and connect each site with its label by a linear connection, which is called a leader. In this way, we move labels from the focus region to the less valuable context region surrounding it. In order to make the leader layout well readable, we present algorithms that rule out crossings between leaders and optimize other characteristics such as total leader length and distance between labels. This yields a new variant of the boundary labeling problem, which has been studied in the literature. Other than in traditional boundary labeling, where leaders are usually schematized polylines, we focus on leaders that are either straight-line segments or Bezier curves. Further, we present algorithms that, given the sites, find a position of the focus region that optimizes the above characteristics. We also consider a variant of the problem where we have more sites than space for labels. In this situation, we assume that the sites are prioritized by the user. Alternatively, we take a new facility-location perspective which yields a clustering of the sites. We label one representative of each cluster. If the user wishes, we apply our approach to the sites within a cluster, giving details on demand.

  10. How to Read a Nutrition Facts Label

    Medline Plus

    Full Text Available ... las etiquetas de datos nutricionales (video) Most packaged foods come with a Nutrition Facts label. These labels ... Watch this video for tips on figuring out food labels so you can make healthy choices. For ...

  11. Labelling of benzocaine with tritium

    Energy Technology Data Exchange (ETDEWEB)

    Malik, Sohail (Washington Univ., Seattle, WA (United States))


    A convenient method is described to label a local anesthetic, benzocaine, with tritium. The bromoester of para-aminobenzoic acid (PABA) was prepared from para-nitrotoluene and was reduced with tritium. The generation of isotopic hydrogen and labelling of benzocaine was achieved in one-step. A mixture of sodium borohydride (NaB[sup 3]H[sub 4]) with cobalt (II) chloride was used to generate tritium gas. 5% Pd/C was used as a catalyst. This constitutes the first report of tritium labelled benzocaine. (author).

  12. New labels for radiation therapy

    Energy Technology Data Exchange (ETDEWEB)

    Kubota, Susumu; Mukai, Minoru; Kato, Hirotoshi (National Inst. of Radiological Sciences, Chiba (Japan))


    In simulating radiotherapy, the bone and trachea identified by plain X-P and the other organs, such as the esophagus and bladder, outlined by contrast medium have so far been used as labels. However, irradiation with a high therapeutic ratio is required for an intracorporeal insertion of artificial labels that are identified by X-ray fluoroscopy. For this purpose, metal clips and seed dummies are available, although they cause artifacts in CT scans. Therefore, the authors are using an acupuncture needle and lipiodol for tracing as new artificial labels, since both are identified by X-ray fluoroscopy and CT scan and create few artifacts. (J.P.N.).

  13. Targeting Antibodies to Carbon Nanotube Field Effect Transistors by Pyrene Hydrazide Modification of Heavy Chain Carbohydrates

    Directory of Open Access Journals (Sweden)

    Steingrimur Stefansson


    Full Text Available Many carbon nanotube field-effect transistor (CNT-FET studies have used immobilized antibodies as the ligand binding moiety. However, antibodies are not optimal for CNT-FET detection due to their large size and charge. Their size can prevent ligands from reaching within the Debye length of the CNTs and a layer of charged antibodies on the circuits can drown out any ligand signal. In an attempt to minimize the antibody footprint on CNT-FETs, we examined whether pyrene hydrazide modification of antibody carbohydrates could reduce the concentration required to functionalize CNT circuits. The carbohydrates are almost exclusively on the antibody Fc region and this site-specific modification could mediate uniform antibody orientation on the CNTs. We compared the hydrazide modification of anti-E. coli O157:H7 polyclonal antibodies to pyrenebutanoic acid succinimidyl ester-coated CNTs and carbodiimide-mediated antibody CNT attachment. Our results show that the pyrene hydrazide modification was superior to those methods with respect to bacteria detection and less than 1 nM labeled antibody was required to functionalize the circuits.

  14. Ultrasensitive ELISA using enzyme-loaded nanospherical brushes as labels. (United States)

    Qu, Zhenyuan; Xu, Hong; Xu, Ping; Chen, Kaimin; Mu, Rong; Fu, Jianping; Gu, Hongchen


    Improving the detection sensitivity of enzyme-linked immunosorbent assay (ELISA) is of utmost importance for meeting the demand of early disease diagnosis. Herein we report an ultrasensitive ELISA system using horseradish peroxidase (HRP)-loaded nanospherical poly(acrylic acid) brushes (SPAABs) as labels. HRP was covalently immobilized in SPAABs with high capacity and activity via an efficient "chemical conjugation after electrostatic entrapment" (CCEE) process, thus endowing SPAABs with high amplification capability as labels. The periphery of SPAAB-HRP was further utilized to bind a layer of antibody with high density for efficient capture of analytes owing to the three-dimensional architecture of SPAABs. Using human chorionic gonadotrophin (hCG) as a model analyte, the SPAAB-amplified system drastically boosted the detection limit of ELISA to 0.012 mIU mL(-1), a 267-fold improvement as compared to conventional ELISA systems.

  15. General Compact Labeling Schemes for Dynamic Trees



    Let $F$ be a function on pairs of vertices. An {\\em $F$- labeling scheme} is composed of a {\\em marker} algorithm for labeling the vertices of a graph with short labels, coupled with a {\\em decoder} algorithm allowing one to compute $F(u,v)$ of any two vertices $u$ and $v$ directly from their labels. As applications for labeling schemes concern mainly large and dynamically changing networks, it is of interest to study {\\em distributed dynamic} labeling schemes. This paper investigates labelin...

  16. What Is Antiphospholipid Antibody Syndrome? (United States)

    ... page from the NHLBI on Twitter. What Is Antiphospholipid Antibody Syndrome? Antiphospholipid (AN-te-fos-fo-LIP-id) antibody ... weeks or months. This condition is called catastrophic antiphospholipid syndrome (CAPS). People who have APS also are at ...

  17. Red Blood Cell Antibody Identification (United States)

    ... ID, RBC; RBC Ab ID Formal name: Red Blood Cell Antibody Identification Related tests: Direct Antiglobulin Test ; RBC ... I should know? How is it used? Red blood cell (RBC) antibody identification is used as a follow- ...

  18. Lupus anticoagulants and antiphospholipid antibodies (United States)

    ... this page: // Lupus anticoagulants and antiphospholipid antibodies To use the sharing features on this page, please enable JavaScript. Lupus anticoagulants are antibodies against substances in the lining ...

  19. Anti-smooth muscle antibody (United States)

    ... gov/ency/article/003531.htm Anti-smooth muscle antibody To use the sharing features on this page, please enable JavaScript. Anti-smooth muscle antibody is a blood test that detects the presence ...

  20. Homogeneous plate based antibody internalization assay using pH sensor fluorescent dye. (United States)

    Nath, Nidhi; Godat, Becky; Zimprich, Chad; Dwight, Stephen J; Corona, Cesear; McDougall, Mark; Urh, Marjeta


    Receptor-mediated antibody internalization is a key mechanism underlying several anti-cancer antibody therapeutics. Delivering highly toxic drugs to cancer cells, as in the case of antibody drug conjugates (ADCs), efficient removal of surface receptors from cancer cells and changing the pharmacokinetics profile of the antibody drugs are some of key ways that internalization impacts the therapeutic efficacy of the antibodies. Over the years, several techniques have been used to study antibody internalization including radiolabels, fluorescent microscopy, flow cytometry and cellular toxicity assays. While these methods allow analysis of internalization, they have limitations including a multistep process and limited throughput and are generally endpoint assays. Here, we present a new homogeneous method that enables time and concentration dependent measurements of antibody internalization. The method uses a new hydrophilic and bright pH sensor dye (pHAb dye), which is not fluorescent at neutral pH but becomes highly fluorescent at acidic pH. For receptor mediated antibody internalization studies, antibodies against receptors are conjugated with the pHAb dye and incubated with the cells expressing the receptors. Upon binding to the receptor, the dyes conjugated to the antibody are not fluorescent because of the neutral pH of the media, but upon internalization and trafficking into endosomal and lysosomal vesicles the pH drops and dyes become fluorescent. The enabling attributes of the pHAb dyes are the hydrophilic nature to minimize antibody aggregation and bright fluorescence at acidic pH which allows development of simple plate based assays using a fluorescent reader. Using two different therapeutic antibodies--Trastuzumab (anti-HER2) and Cetuximab (anti-EGFR)--we show labeling with pHAb dye using amine and thiol chemistries and impact of chemistry and dye to antibody ration on internalization. We finally present two new approaches using the pHAb dye, which will be

  1. Characterization of Antibodies for Grain-Specific Gluten Detection. (United States)

    Sharma, Girdhari M; Rallabhandi, Prasad; Williams, Kristina M; Pahlavan, Autusa


    Gluten ingestion causes immunoglobulin E (IgE)-mediated allergy or celiac disease in sensitive individuals, and a strict gluten-free diet greatly limits food choices. Immunoassays such as enzyme-linked immunosorbent assay (ELISA) are used to quantify gluten to ensure labeling compliance of gluten-free foods. Anti-gluten antibodies may not exhibit equal affinity to gluten from wheat, rye, and barley. Moreover, because wheat gluten is commonly used as a calibrator in ELISA, accurate gluten quantitation from rye and barley contaminated foods may be compromised. Immunoassays utilizing grain-specific antibodies and calibrators may help improve gluten quantitation. In this study, polyclonal antibodies raised against gluten-containing grain-specific peptides were characterized for their immunoreactivity to gluten from different grain sources. Strong immunoreactivity to multiple gluten polypeptides from wheat, rye, and barley was observed in the range 34 to 43 kDa with anti-gliadin, 11 to 15 and 72 to 95 kDa with anti-secalin, and 30 to 43 kDa with anti-hordein peptide antibodies, respectively. Minimal or no cross-reactivity with gluten from other grains was observed among these antibodies. The anti-consensus peptide antibody raised against a repetitive amino acid sequence of proline and glutamine exhibited immunoreactivity to gluten from wheat, rye, barley, and oat. The antibodies exhibited similar immunoreactivity with most of the corresponding grain cultivars by ELISA. The high specificity and minimal cross-reactivity of grain-specific antibodies suggest their potential use in immunoassays for accurate gluten quantitation.

  2. Antibody Engineering and Therapeutics Conference


    Larrick, James W; Parren, Paul WHI; Huston, James S; Plückthun, Andreas; Bradbury, Andrew; Tomlinson, Ian M; Chester, Kerry A.; Burton, Dennis R.; Adams, Gregory P.; Weiner, Louis M.; Scott, Jamie K.; Alfenito, Mark R; Veldman, Trudi; Reichert, Janice M.


    The Antibody Engineering and Therapeutics conference, which serves as the annual meeting of The Antibody Society, will be held in Huntington Beach, CA from Sunday December 8 through Thursday December 12, 2013. The scientific program will cover the full spectrum of challenges in antibody research and development, and provide updates on recent progress in areas from basic science through approval of antibody therapeutics. Keynote presentations will be given by Leroy Hood (Institute of System Bi...

  3. Traceless affinity labeling of endogenous proteins for functional analysis in living cells. (United States)

    Hayashi, Takahiro; Hamachi, Itaru


    Protein labeling and imaging techniques have provided tremendous opportunities to study the structure, function, dynamics, and localization of individual proteins in the complex environment of living cells. Molecular biology-based approaches, such as GFP-fusion tags and monoclonal antibodies, have served as important tools for the visualization of individual proteins in cells. Although these techniques continue to be valuable for live cell imaging, they have a number of limitations that have only been addressed by recent progress in chemistry-based approaches. These chemical approaches benefit greatly from the smaller probe sizes that should result in fewer perturbations to proteins and to biological systems as a whole. Despite the research in this area, so far none of these labeling techniques permit labeling and imaging of selected endogenous proteins in living cells. Researchers have widely used affinity labeling, in which the protein of interest is labeled by a reactive group attached to a ligand, to identify and characterize proteins. Since the first report of affinity labeling in the early 1960s, efforts to fine-tune the chemical structures of both the reactive group and ligand have led to protein labeling with excellent target selectivity in the whole proteome of living cells. Although the chemical probes used for affinity labeling generally inactivate target proteins, this strategy holds promise as a valuable tool for the labeling and imaging of endogenous proteins in living cells and by extension in living animals. In this Account, we summarize traceless affinity labeling, a technique explored mainly in our laboratory. In our overview of the different labeling techniques, we emphasize the challenge of designing chemical probes that allow for dissociation of the affinity module (often a ligand) after the labeling reaction so that the labeled protein retains its native function. This feature distinguishes the traceless labeling approach from the traditional

  4. New Ideas on Labeling Schemes

    DEFF Research Database (Denmark)

    Rotbart, Noy Galil

    evaluation of fully dynamic labeling schemes. Due to a connection between adjacency labeling schemes and the graph theoretical study of induced universal graphs, we study these in depth and show novel results for bounded degree graphs and power-law graphs. We also survey and make progress on the related......With ever increasing size of graphs, many distributed graph systems emerged to store, preprocess and analyze them. While such systems ease up congestion on servers, they incur certain penalties compared to centralized data structure. First, the total storage required to store a graph...... in a distributed fashion increases. Second, attempting to answer queries on vertices of a graph stored in a distributed fashion can be significantly more complicated. In order to lay theoretical foundations to the first penalty mentioned a large body of work concentrated on labeling schemes. A labeling scheme...

  5. Off-Label Drug Use (United States)

    ... it’s not legal for drug companies to market (advertise or promote) their drugs for off-label uses. ... Statistics Center Volunteer Learning Center Follow Us Twitter Facebook Instagram Cancer Information, Answers, and Hope. Available Every ...

  6. Synthetic peptides for antibody production

    NARCIS (Netherlands)

    Zegers, N.D.


    Synthetic peptides are useful tools for the generation of antibodies. The use of antibodies as specific reagents in inununochemical assays is widely applied. In this chapter, the application of synthetic peptides for the generation of antibodies is described. The different steps that lead to the uni

  7. 131Ⅰ标记抗CD20单克隆抗体不同给药途径对荷瘤裸鼠的放射免疫治疗实验%Experimental Research on Radioimmunotherapy of 131Ⅰ-labeled Anti-CD20 Monoclonal Antibody to Nude Mice Xenografted Tumor

    Institute of Scientific and Technical Information of China (English)

    左强; 罗宇玲; 罗荣城


    Objective To investigate the therapeutic efficacy of radioimmunotherapy of iodine-131 labeled Rituximab using intratumor injection(IT)in nude mice with xenografted raji cells tumor. Methods Iodine-131 labeled Rituximab was carried out by IODO-GEN method. The nude mice bearing raji cells tumor were divided into six groups based on the injected marked-drugs. The size of the tumor was measured every 2~3 day and the inhibition rates of different groups were calculated. Results The tumor inhibition rates of 131 I-Rituximab IT group were higher than those of IP group, 131 I-IgG IT group and cell control group(P<0. 05). 131 I-Rituximab with intratumor injection in different dose showed that inhibition rate of low dose group was lower than that of high group, while there was no significant difference(P>0. 05). Conclusion lodine-131 labeled Rituximab with intratumor injection showed the highest radioimmunotherapy efficacy which offered the experimental evidence for clinical application in the futrue.%目的 探讨131Ⅰ-Rituximab经瘤内注射对荷人Burkitt's淋巴瘤细胞系Raji细胞移植瘤裸鼠放射免疫治疗疗效.方法 131Ⅰ标记物的标记采用IODO-GEN碘化标记;按预定治疗方案分别注入含有131Ⅰ标记物,开始治疗前及治疗后每天用游标卡尺测量肿瘤长、短径,计算肿瘤体积,依公式计算肿瘤生长抑制率.结果 131Ⅰ-Rituximab瘤内注射组肿瘤抑制率显著高于腹腔注射组、131Ⅰ-IgG瘤内注射组以及对照细胞组(P0.05).结论 131Ⅰ-Rituximab经瘤内途径给药可以获得更好的放射免疫治疗效果,为下一步临床应用奠定了基础.

  8. Recombinant anti-tenascin antibody constructs

    Energy Technology Data Exchange (ETDEWEB)



    The general objective of this research is to combine genetically derived molecular constructs reactive with tenascin, with appropriate radionuclides and labeling methods in order to generate more effective diagnostic and therapeutic reagents for oncologic nuclear medicine. Tenascin, a polymorphic extracellular matrix glycoprotein, is of interest because of its high expression on glioma, melanoma, as well as prostate and breast carcinoma. Recently, we have also documented high levels of tenascin in lymphomas, particularly those of higher grade, making the potential clinical impact of tenascin-specific radiodiagnostics and therapeutics even greater. An essential feature of our work plan is the ability to exploit our extensive clinical experience in order to design second-generation constructs with properties which could improve clinical efficacy. To date, we have treated over 150 brain tumor patients with 131I-labeled murine 81C6, an antibody which binds specifically to the alternatively spliced fibronectin type III repeats CD of the tenascin molecule. During the current grant period, we have made several observations which form the basis for our proposed specific aims. First, tissue distribution and catabolism experiments in animal models have demonstrated enhanced stability for a chimeric construct composed of murine variable regions and human IgG2 constant domains. Furthermore, pharmacokinetic studies in patients with 131I-labeled chimeric 81C6 have shown significantly longer retention in glioma tumor resection cavities compared with its murine parent. Second, we have initiated the first clinical trial of an endoradiotherapeutic labeled with the 7.2-hr -particle emitter 211At. Twelve glioma patients have received 211At-labeled chimeric 81C6 directly into their brain tumor resection cavity, and very encouraging results have been obtained. Now that the feasibility of human studies with 211At, has been demonstrated, the development and evaluation of anti

  9. New Insights into the Functional Behavior of Antibodies as Revealed by Binding Studies on an Anti-Uranium Monoclonal Antibody

    Energy Technology Data Exchange (ETDEWEB)

    Blake, Diane A.; Xia Li; Haini Yu; Blake, Robert C.


    As part of an ongoing effort to develop immunoassays for chelated uranium(VI) on a hand-held flow fluorimeter, an anti-uranium monoclonal antibody designated as 8A11 was fluorescently labeled using two different strategies. When 8A11 was coupled via reactive lysines to either ALEXATM 488 or Cy5TM, the resulting fluorescent antibody conjugate exhibited positive cooperativity in the presence of its antigen, U(VI) chelated with 2,9-dicarboxy-1,10-phenanthroline (U(VI)-DCP). That is, when one of the two binding sites on the covalently modified 8A11 was occupied with bound antigen, the affinity of the remaining site on the antibody for U(VI)-DCP appeared to increase. Unmodified 8A11 bound U(VI)-DCP with the expected hyperbolic dependence on the concentration of antigen, consistent with independent and equal binding of ligand at both sites. Proteolytic cleavage of the fluorescently conjugated 8A11 to produce the fluorescent monovalent Fab fragment yielded an active preparation that now bound U(VI)-DCP with no evidence of positive cooperativity. Although, in principle, any divalent antibody has the potential to exhibit positive cooperativity in its binding interactions with its antigen, very little literature precedent for this type of behavior exists. Native 8A11 was also noncovalently labeled with highly fluorescent ZENONTM reagents. These reagents are fluorescently-labeled Fab fragments of goat anti-mouse antibodies that bind to the Fc portion of 8A11. These high-affinity, monovalent fluorescent reagents permitted the intact 8A11 mouse antibody to be labeled in situ with no covalent modifications. Incubation of the 8A11 with ZENON 647 produced a fluorescent protein complex that showed an 8-fold higher affinity for U(VI)-DCP than did the free 8A11 alone. Again, very few literature precedents exist for this phenomenon, where agents that bind to the Fc portion of an intact antibody change the affinity of the antibody for the antigen at the structurally distant Fab portion

  10. Imaging studies of breast cancer xenografts. Monoclonal anti-mucin antibodies BM-7 and 12H12 labeled with {sup 99m}TC; Immunszintigraphie xenotransplantierter Mammakarzinome. {sup 99m}Tc-markierte monoklonale Anti-Muzinantikoerper BM-7 und 12H12

    Energy Technology Data Exchange (ETDEWEB)

    Bruemmendorf, T.H. [Onkologisches Labor, Universitaetsfrauenklinik, Heidelberg (Germany); Kaul, S. [Onkologisches Labor, Universitaetsfrauenklinik, Heidelberg (Germany); Schuhmacher, J. [Onkologisches Labor, Universitaetsfrauenklinik, Heidelberg (Germany); Baum, R.P. [Radiologische Diagnostik und Therapie, Deutsches Krebsforschungszentrum, Heidelberg (Germany); Kliveny, G. [Onkologisches Labor, Universitaetsfrauenklinik, Heidelberg (Germany); Matys, R. [Onkologisches Labor, Universitaetsfrauenklinik, Heidelberg (Germany); Eichler, A. [Onkologisches Labor, Universitaetsfrauenklinik, Heidelberg (Germany); Gueckel, B.; Beldermann, F.; Bastert, G.


    The present study was undertaken to evaluate the correlation of the favourable in vitro characteristics of the anit-mucin Mabs 12H12 and BM-7 with high tumor accumulation in vivo. They were labeled with {sup 99m}Tc; their biodistribution in nude mice bearing mammary tumor xenograft AR was examined and immunoscintigraphy was performed after 24 h. {sup 99m}Tc-labeling of the Mabs 12H12 and BM-7 led to tumor uptakes of 20.7% and 8.8% ID/g, respectively, after 48 h. Tumor-to-muscle ratios were 31 (12H12) and 18 (BM-7). Tumor xenografts were clearly visualized in immunoscintigrams. Combination of Mab 12H12 and {sup 99m}Tc provides high tumor-to-tissue ratios shortly after administration. (orig.) [Deutsch] Gegenstand dieser Arbeit waren tierexperimentelle Untersuchungen zur Biodistribution und Immunszintigraphie der Anti-Muzin-MoAbs 12H12 und BM-7 in thymusaplastischen Nacktmaeusen mit xenotransplantierten humanen Mammakarzinomen. Die MoAb waren mit {sup 99m}Tc markiert; ihre Biodistribution wurde im Tiermodell 24 und 48 h nach i. v. Injektion untersucht. Die {sup 99m}Tc-Markierungen der MoAbs 12H12 und BM-7 fuehrten zu einer Tumoranreicherung von 20,7% bzw. 8,8% ID/g und zu Tumor/Muskel-Quotienten von 31 bzw. 18 nach 48 h. Immunszintigraphisch liess sich mit beiden MoAbs nach 24 h das Mammakarzinom AR gut darstellen. Die beiden MoAbs erfahren im Tiermodell eine schnelle Tumoraufnahme und rasche Blut-Clearance. (orig.)

  11. Preclinical evaluation of (111)In-DTPA-INCA-X anti-Ku70/Ku80 monoclonal antibody in prostate cancer. (United States)

    Evans-Axelsson, Susan; Vilhelmsson Timmermand, Oskar; Welinder, Charlotte; Borrebaeck, Carl Ak; Strand, Sven-Erik; Tran, Thuy A; Jansson, Bo; Bjartell, Anders


    The aim of this investigation was to assess the Ku70/Ku80 complex as a potential target for antibody imaging of prostate cancer. We evaluated the in vivo and ex vivo tumor targeting and biodistribution of the (111)In-labeled human internalizing antibody, INCA-X ((111)In-DTPA-INCA-X antibody), in NMRI-nude mice bearing human PC-3, PC-3M-Lu2 or DU145 xenografts. DTPA-conjugated, non-labeled antibody was pre-administered at different time-points followed by a single intravenous injection of (111)In-DTPA-INCA-X. At 48, 72 and 96 h post-injection, tissues were harvested, and the antibody distribution was determined by measuring radioactivity. Preclinical SPECT/CT imaging of mice with and without the predose was performed at 48 hours post-injection of labeled DTPA-INCA-X. Biodistribution of the labeled antibody showed enriched activity in tumor, spleen and liver. Animals pre-administered with DTPA-INCA-X showed increased tumor uptake and blood content of (111)In-DTPA-INCA-X with reduced splenic and liver uptake. The in vitro and in vivo data presented show that the (111)In-labeled INCA-X antibody is internalized into prostate cancer cells and by pre-administering non-labeled DTPA-INCA-X, we were able to significantly reduce the off target binding and increase the (111)In-DTPA-INCA-X mAb uptake in PC-3, PC-3M-Lu2 and DU145 xenografts. The results are encouraging and identifying the Ku70/Ku80 antigen as a target is worth further investigation for functional imaging of prostate cancer.

  12. In-situ Detection of Squalane in Sedimentary Organic Matter Using Monoclonal Antibodies (United States)

    Bailey, J. V.; Corsetti, F. A.; Moldowan, J. M.; Fago, F.; Caron, D.


    Sedimentary geolipids can serve as powerful tools for reconstructing ancient ecosystems, but only if investigators can demonstrate that the hydrocarbons are indigenous to their host rocks. The association of molecules with primary sedimentary fabrics could indicate a syngenetic relationship. However, traditional biomarker analyses require extraction from large quantities of powdered rock, confounding detailed spatial correlations. Biological studies commonly use antibodies as extremely sensitive molecular probes. When coupled with fluorescent labels, antibodies allow for the visual localization of molecules. Here we show that monoclonal antibodies that bind specifically to geolipid compounds can be used for in situ detection and labeling of such compounds in mineral-bound organic macerals. Monoclonal antibodies to squalene, produced for human health studies, also react with the geolipid, squalane. We show that squalene antibodies do not react with other common sedimentary hydrocarbons. We also show that squalane antibodies bind specifically to isolated organic-rich lamina in Eocene-age, squalane-containing rocks. These results suggest that squalane is confined to discrete organo-sedimentary fabrics within those rocks, providing evidence for its syngeneity. The chemical similarity of squalane to other sedimentary hydrocarbons hints at the potential for developing monoclonal antibodies to a variety of biomarkers that could then be localized in rocks, sediments, and extant cells.

  13. Monoclonal antibodies directed against protoplasts of soybean cells : Generation of hybridomas and characterization of a monoclonal antibody reactive with the cell surface. (United States)

    Villanueva, M A; Metcalf, T N; Wang, J L


    Splenocytes, derived from mice that had been immunized with protoplasts prepared from suspension cultures of root cells of Glycine max (L.) Merr. (SB-1 cell line), were fused with a murine myeloma cell line. The resulting hybridoma cultures were screened for the production of antibodies directed against the soybean protoplasts and were then cloned. One monoclonal antibody, designated MVS-1, was found to bind to the outer surface of the plasma membrane on the basis of several criteria: (a) agglutination of the protoplasts; (b) binding of fluorescence-labeled immunoglobulin on protoplasts yielding a ring staining pattern with prominent intensity at the edges; and (c) saturable binding by protoplasts of (125)I-labeled Antibody MVS-1. The antigenic target of Antibody MVS-1, identified by immunoblotting techniques, contained a polypeptide of relative molecular mass (Mr) approx. 400000 under both reducing and non-reducing conditions. When the antigenic target of Antibody MVS-1 was chromatographed in potassium phosphate buffer, the position of elution corresponded to that of a high-molecular-weight species (Mr 400000). These results provide the protein characterization required for the analysis of the mobility of Antibody MVS-1 bound to the plasma membrane of SB-1 cells.

  14. Overcoming Instability of Antibody-Nanomaterial Conjugates: Next Generation Targeted Nanomedicines Using Bispecific Antibodies. (United States)

    Howard, Christopher B; Fletcher, Nicholas; Houston, Zachary H; Fuchs, Adrian V; Boase, Nathan R B; Simpson, Joshua D; Raftery, Lyndon J; Ruder, Tim; Jones, Martina L; de Bakker, Christopher J; Mahler, Stephen M; Thurecht, Kristofer J


    Targeted nanomaterials promise improved therapeutic efficacy, however their application in nanomedicine is limited due to complexities associated with protein conjugations to synthetic nanocarriers. A facile method to generate actively targeted nanomaterials is developed and exemplified using polyethylene glycol (PEG)-functional nanostructures coupled to a bispecific antibody (BsAb) with dual specificity for methoxy PEG (mPEG) epitopes and cancer targets such as epidermal growth factor receptor (EGFR). The EGFR-mPEG BsAb binds with high affinity to recombinant EGFR (KD : 1 × 10(-9) m) and hyperbranched polymer (HBP) consisting of mPEG (KD : 10 × 10(-9) m) and demonstrates higher avidity for HBP compared to linear mPEG. The binding of BsAb-HBP bioconjugate to EGFR on MDA-MB-468 cancer cells is investigated in vitro using a fluorescently labeled polymer, and in in vivo xenograft models by small animal optical imaging. The antibody-targeted nanostructures show improved accumulation in tumor cells compared to non-targeted nanomaterials. This demonstrates a facile approach for tuning targeting ligand density on nanomaterials, by modulating surface functionality. Antibody fragments are tethered to the nanomaterial through simple mixing prior to administration to animals, overcoming the extensive procedures encountered for developing targeted nanomedicines.

  15. Antiphospholipid Antibody and Antiphospholipid Syndrome

    Institute of Scientific and Technical Information of China (English)



    @@ Antiphospholipid antibodies (APA) APA is a big category for all kinds of negative charge phospholipid or lecithin - a protein complex autoantibodies or the same antibody, through its recognition of antigen (target protein) different, and phospholipids or lecithin - protein complex combination of various rely on the interference Phospholipid clotting and anti-coagulation factor, and promote endothelial cells, platelets, complement activation and play a role. APA including lupus anticoagulant(LA) and anticardiolipin antibody (ACA), In addition, there are anti-β2 glycoprotein-I (β2-GPI) antibody, anti-prothrombin (a- PT) antibody, anti-lysophosphatidic acid antibody and anti-phosphatidylserine antibody, and so on. APA as the main target of phospholipid-binding protein, including β2-GPI, prothrombin, annexin, protein C (PC) and protein S (PS), plasminogen, and so on.

  16. Engineering antibodies by yeast display. (United States)

    Boder, Eric T; Raeeszadeh-Sarmazdeh, Maryam; Price, J Vincent


    Since its first application to antibody engineering 15 years ago, yeast display technology has been developed into a highly potent tool for both affinity maturing lead molecules and isolating novel antibodies and antibody-like species. Robust approaches to the creation of diversity, construction of yeast libraries, and library screening or selection have been elaborated, improving the quality of engineered molecules and certainty of success in an antibody engineering campaign and positioning yeast display as one of the premier antibody engineering technologies currently in use. Here, we summarize the history of antibody engineering by yeast surface display, approaches used in its application, and a number of examples highlighting the utility of this method for antibody engineering.

  17. Isotopic labeling of mouse interferon by incorporation of radioactive amino acids during synthesis

    Energy Technology Data Exchange (ETDEWEB)

    DeMaeyer-Guignard, J.; Cachard, A.; DeMaeyer, E.


    Mouse interferon produced by C-243 cells induced with Newcastle disease virus was isotopically labeled by adding either (/sup 35/S)methionine or a /sup 14/C-labeled amino acid mixture to the culture medium. A method combining butyric acid and theophylline treatment and resulting in high interferon yields was used. Following purification by two-step affinity chromatography on poly(U) and antibody columns, the resulting material was analyzed on SDS-PAGE. The migration pattern of radioactivity and interferon coincided well and autoradiography revealed three major bands at migration distances corresponding, respectively, to 35, 28, and 22 K. Interferon represented 3.8% of all (/sup 35/S)methionine-labeled proteins and 2.6% of all /sup 14/C-amino acid-labeled proteins released into the medium.

  18. Library of monoclonal antibodies against brush border membrane epithelial antigens

    Energy Technology Data Exchange (ETDEWEB)

    Behar, M.; Katz, A.; Silverman, M.


    A purified fraction of proximal tubule brush border membranes (BBM) was prepared from dog kidney and used to immunize mice. The standard technique of hybridoma production was followed as described by Kohler and Milstein. Production of antibodies was detected by indirect immunofluorescence on dog kidney slices and by immunodot against the purified fraction on nitrocellulose. Five hybrids exhibited anti BBM activity. These were cloned twice and yielded stable cell lines producing IgG type monoclonal antibodies against BBM. They were designated A/sub 1/, C/sub 7/, D/sub 3/, D/sub 7/ and H/sub 4/. As a family these five monoclonals have broad tissue specificity, i.e. positive staining of the surface mucosa of intestinal kidney proximal tubules. D/sub 3/ exhibits even broader specificity for epithelium reacting with bile canaliculi and choroid plexus. The authors have verified that at least 4/5 antibodies are directed against BBM protein as revealed by immunoprecipitation of solubilized BBM and detected by Coomassie blue staining or autoradiography of lactoperoxidase labelled BBM. Most interestingly all antibodies bind to the surface of LL CPK/sub 1/ cells, a continuous pig kidney cell line of undefined origin but exhibiting many characteristics of proximal tubule cells. The library of monoclonal antibodies obtained provide important probes with which to study membrane biogenesis and polarization in epithelial cells.

  19. Indirect solid-phase immunosorbent assay for detection of arenavirus antigens and antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Ivanov, A.P.; Rezapkin, G.V.; Dzagurova, T.K.; Tkachenko, E.A. (Institute of Poliomyelitis anU Viral Encephalities of the U.S.S.R. Academy of Medical Sciences, Moscow)


    Indirect enzyme-linked immunosorbent assay (ELISA) and solid phase radioimmunoassay (SPRIA) using either enti-human or anti-mouse IgG labelled with horseradish peroxidase and /sup 125/I, respectively, were developed for the detection of Junin, Machupo, Tacaribe, Amapari, Tamiami, Lassa and LCM arenaviruses. Both methods allow high sensitivity detection of arenavirus antigens and antibodies.

  20. The antibody response in Lyme disease. (United States)

    Craft, J. E.; Grodzicki, R. L.; Shrestha, M.; Fischer, D. K.; García-Blanco, M.; Steere, A. C.


    We determined the antibody response against the Ixodes dammini spirochete in Lyme disease patients by indirect immunofluorescence and an enzyme-linked immunosorbent assay (ELISA). The specific IgM response became maximal three to six weeks after disease onset, and then declined, although titers sometimes remained elevated during later disease. Specific IgM levels correlated directly with total serum IgM. The specific IgG response, often delayed initially, was nearly always present during neuritis and arthritis, and frequently remained elevated after months of remission. Although results obtained by indirect immunofluorescence and the ELISA were similar, the ELISA was more sensitive and specific. Cross-reactive antibodies from patients with other spirochetal infections were blocked by absorption of sera with Borrelia hermsii, but titers of Lyme disease sera were also decreased. To further characterize the specificity of the humoral immune response against the I. dammini spirochete, 35S-methionine-labeled spirochetal antigens were identified by immunoprecipitation with sera from Lyme arthritis patients. These polypeptides had molecular weights of 62, 60, 47, 37, 22, 18, and 15 kDa, and were not recognized by control sera. We conclude that the ELISA, without absorption, is the best method to assay the humoral immune response in Lyme disease, and we have identified methionine-containing spirochetal polypeptides that may be important in Lyme arthritis. PMID:6393607

  1. How to Read a Nutrition Facts Label

    Medline Plus

    Full Text Available ... to 2-Year-Old How to Read a Nutrition Facts Label (Video) KidsHealth > For Parents > How to Read a Nutrition Facts Label (Video) Print A A A en ... nutricionales (video) Most packaged foods come with a Nutrition Facts label. These labels have a lot of ...

  2. 21 CFR 606.121 - Container label. (United States)


    ... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Container label. 606.121 Section 606.121 Food and... Container label. (a) The container label requirements are designed to facilitate the use of a uniform container label for blood and blood components (except Source Plasma) by all blood establishments. (b)...

  3. Enhancement of monoclonal antibody uptake in human colon tumor xenografts following irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Kalofonos, H.; Rowlinson, G.; Epenetos, A.A. (Royal Postgraduate Medical School, London (England))


    Indium-111-labeled AUA1 tumor-associated monoclonal antibody raised against an antigen of colon adenocarcinoma was used to evaluate the effect of ionizing radiation on antibody uptake by the LoVo adenocarcinoma cell line grown as a xenograft in nude mice. Tumors were exposed to single doses of external X-irradiation of between 400 and 1600 cGy followed, 24 h later, by administration of specific or nonspecific antibody. Animals were sacrificed 3 days after antibody administration. At doses higher than 400 cGy, tumor uptake with both specific and nonspecific antibody was significantly increased. No difference in changes in tumor volume was observed between the groups receiving irradiation and the controls. Specific antibody uptake by tumors was always significantly higher than nonspecific having an approximate 4-fold binding advantage. Vascular permeability and the vascular volume of irradiated and control tumors was measured 24 and 72 h after irradiation, using iodine-125-labeled nonspecific antibody and labelling of the red blood cells in vivo with 99mTcO4. At doses higher than 400 cGy, vascular permeability in the tumor 24 h after irradiation was significantly increased (P less than 0.05), while the vascular volume decreased (P less than 0.001) compared to control values. However at 72 h after irradiation there was no difference between treated and control groups. The results obtained in this study suggest a potential value of external irradiation to increase monoclonal antibody uptake by tumors governed mainly by the increased vascular permeability of the tumor vasculature soon after the irradiation exposure.

  4. Purification of mitochondrial proteins HSP60 and ATP synthase from ascidian eggs: implications for antibody specificity.

    Directory of Open Access Journals (Sweden)

    Janet Chenevert

    Full Text Available Use of antibodies is a cornerstone of biological studies and it is important to identify the recognized protein with certainty. Generally an antibody is considered specific if it labels a single band of the expected size in the tissue of interest, or has a strong affinity for the antigen produced in a heterologous system. The identity of the antibody target protein is rarely confirmed by purification and sequencing, however in many cases this may be necessary. In this study we sought to characterize the myoplasm, a mitochondria-rich domain present in eggs and segregated into tadpole muscle cells of ascidians (urochordates. The targeted proteins of two antibodies that label the myoplasm were purified using both classic immunoaffinity methods and a novel protein purification scheme based on sequential ion exchange chromatography followed by two-dimensional gel electrophoresis. Surprisingly, mass spectrometry sequencing revealed that in both cases the proteins recognized are unrelated to the original antigens. NN18, a monoclonal antibody which was raised against porcine spinal cord and recognizes the NF-M neurofilament subunit in vertebrates, in fact labels mitochondrial ATP synthase in the ascidian embryo. PMF-C13, an antibody we raised to and purified against PmMRF, which is the MyoD homolog of the ascidian Phallusia mammillata, in fact recognizes mitochondrial HSP60. High resolution immunolabeling on whole embryos and isolated cortices demonstrates localization to the inner mitochondrial membrane for both ATP synthase and HSP60. We discuss the general implications of our results for antibody specificity and the verification methods which can be used to determine unequivocally an antibody's target.

  5. An Optimal Labeling Scheme for Ancestry Queries



    An ancestry labeling scheme assigns labels (bit strings) to the nodes of rooted trees such that ancestry queries between any two nodes in a tree can be answered merely by looking at their corresponding labels. The quality of an ancestry labeling scheme is measured by its label size, that is the maximal number of bits in a label of a tree node. In addition to its theoretical appeal, the design of efficient ancestry labeling schemes is motivated by applications in web search engines. For this p...

  6. A brief history of cell labelling

    Energy Technology Data Exchange (ETDEWEB)

    Peters, A.M. [Royal Sussex Country Hospital, Brighton (United Kingdom)


    The term cell labelling is usually used in the context of labelled leukocytes for imaging inflammation and labelled platelets for imaging thrombosis. Erythrocyte labelling for in vitro measurements of red cell life span, in vivo measurements of splenic red cell pooling, radionuclide ventriculography and imaging sites of bleeding has developed rather separately and has a different history. Labelled platelets and leukocytes were originally developed for cell kinetic studies. Since the current-day applications of labelled platelets and leukocytes depend on a clear understanding of cell kinetics, these classical studies are important and relevant to the history of cell labelling.

  7. Photoaffinity Labeling of Plasma Proteins

    Directory of Open Access Journals (Sweden)

    Masaki Otagiri


    Full Text Available Photoaffinity labeling is a powerful technique for identifying a target protein. A high degree of labeling specificity can be achieved with this method in comparison to chemical labeling. Human serum albumin (HSA and α1-acid glycoprotein (AGP are two plasma proteins that bind a variety of endogenous and exogenous substances. The ligand binding mechanism of these two proteins is complex. Fatty acids, which are known to be transported in plasma by HSA, cause conformational changes and participate in allosteric ligand binding to HSA. HSA undergoes an N-B transition, a conformational change at alkaline pH, that has been reported to result in increased ligand binding. Attempts have been made to investigate the impact of fatty acids and the N-B transition on ligand binding in HSA using ketoprofen and flunitrazepam as photolabeling agents. Meanwhile, plasma AGP is a mixture of genetic variants of the protein. The photolabeling of AGP with flunitrazepam has been utilized to shed light on the topology of the protein ligand binding site. Furthermore, a review of photoaffinity labeling performed on other major plasma proteins will also be discussed. Using a photoreactive natural ligand as a photolabeling agent to identify target protein in the plasma would reduce non-specific labeling.

  8. An Electrochemiluminescence Immunosensor Based on Gold-Magnetic Nanoparticles and Phage Displayed Antibodies. (United States)

    Mu, Xihui; Tong, Zhaoyang; Huang, Qibin; Liu, Bing; Liu, Zhiwei; Hao, Lanqun; Dong, Hua; Zhang, Jinping; Gao, Chuan


    Using the multiple advantages of the ultra-highly sensitive electrochemiluminescence (ECL) technique, Staphylococcus protein A (SPA) functionalized gold-magnetic nanoparticles and phage displayed antibodies, and using gold-magnetic nanoparticles coated with SPA and coupled with a polyclonal antibody (pcAb) as magnetic capturing probes, and Ru(bpy)₃(2+)-labeled phage displayed antibody as a specific luminescence probe, this study reports a new way to detect ricin with a highly sensitive and specific ECL immunosensor and amplify specific detection signals. The linear detection range of the sensor was 0.0001~200 µg/L, and the limit of detection (LOD) was 0.0001 µg/L, which is 2500-fold lower than that of the conventional ELISA technique. The gold-magnetic nanoparticles, SPA and Ru(bpy)₃(2+)-labeled phage displayed antibody displayed different amplifying effects in the ECL immunosensor and can decrease LOD 3-fold, 3-fold and 20-fold, respectively, compared with the ECL immunosensors without one of the three effects. The integrated amplifying effect can decrease the LOD 180-fold. The immunosensor integrates the unique advantages of SPA-coated gold-magnetic nanoparticles that improve the activity of the functionalized capturing probe, and the amplifying effect of the Ru(bpy)₃(2+)-labeled phage displayed antibodies, so it increases specificity, interference-resistance and decreases LOD. It is proven to be well suited for the analysis of trace amounts of ricin in various environmental samples with high recovery ratios and reproducibility.

  9. An Electrochemiluminescence Immunosensor Based on Gold-Magnetic Nanoparticles and Phage Displayed Antibodies

    Directory of Open Access Journals (Sweden)

    Xihui Mu


    Full Text Available Using the multiple advantages of the ultra-highly sensitive electrochemiluminescence (ECL technique, Staphylococcus protein A (SPA functionalized gold-magnetic nanoparticles and phage displayed antibodies, and using gold-magnetic nanoparticles coated with SPA and coupled with a polyclonal antibody (pcAb as magnetic capturing probes, and Ru(bpy32+-labeled phage displayed antibody as a specific luminescence probe, this study reports a new way to detect ricin with a highly sensitive and specific ECL immunosensor and amplify specific detection signals. The linear detection range of the sensor was 0.0001~200 µg/L, and the limit of detection (LOD was 0.0001 µg/L, which is 2500-fold lower than that of the conventional ELISA technique. The gold-magnetic nanoparticles, SPA and Ru(bpy32+-labeled phage displayed antibody displayed different amplifying effects in the ECL immunosensor and can decrease LOD 3-fold, 3-fold and 20-fold, respectively, compared with the ECL immunosensors without one of the three effects. The integrated amplifying effect can decrease the LOD 180-fold. The immunosensor integrates the unique advantages of SPA-coated gold-magnetic nanoparticles that improve the activity of the functionalized capturing probe, and the amplifying effect of the Ru(bpy32+-labeled phage displayed antibodies, so it increases specificity, interference-resistance and decreases LOD. It is proven to be well suited for the analysis of trace amounts of ricin in various environmental samples with high recovery ratios and reproducibility.

  10. Label-free disposable immunosensor for detection of atrazine. (United States)

    Belkhamssa, Najet; Justino, Celine I L; Santos, Patrícia S M; Cardoso, Susana; Lopes, Isabel; Duarte, Armando C; Rocha-Santos, Teresa; Ksibi, Mohamed


    This work reports the construction of a fast, disposable, and label-free immunosensor for the determination of atrazine. The immunosensor is based on a field effect transistor (FET) where a network of single-walled carbon nanotubes (SWCNTs) acts as the conductor channel, constituting carbon nanotubes field effect transistors (CNTFETs). Anti-atrazine antibodies were adsorbed onto the SWCNTs and subsequently the SWCNTs were protected with Tween 20 to prevent the non-specific binding of bacteria or proteins. The principle of the immunoreaction consists in the direct adsorption of atrazine specific antibodies (anti-atrazine) to SWCNTs networks. After exposed to increasing concentrations of atrazine, the CNTFETs could be used as useful label-free platforms to detect atrazine. Under the optimal conditions, a limit of detection as low as 0.001 ng mL(-1) was obtained, which is lower than that of other methods for the atrazine detection, and in a working range between 0.001 and 10 ng mL(-1). The average recoveries obtained for real water samples spiked with atrazine varied from 87.3% to 108.0%. The results show that the constructed sensors display a high sensitivity and could be useful tools for detecting pesticides like atrazine at low concentrations. They could be also applied to the determination of atrazine in environmental aqueous samples, such as seawater and riverine water.

  11. 49 CFR 172.430 - POISON label. (United States)


    ... 49 Transportation 2 2010-10-01 2010-10-01 false POISON label. 172.430 Section 172.430... SECURITY PLANS Labeling § 172.430 POISON label. (a) Except for size and color, the POISON label must be as follows: EC02MR91.029 (b) In addition to complying with § 172.407, the background on the POISON label...

  12. Fibrinogen labelling with I-131

    Energy Technology Data Exchange (ETDEWEB)

    Seminario, C.; Capillo, T.; Montanez, J. (Instituto Peruano de Energia Nuclear, Lima)


    Of the different techniques of labelling liophylized human fibrinogen, the technique of mono-chloride with modified iodine was selected. The labelling of the molecule was performed in alkali media of buffalo glycine in which the solution of stable iodine will react as well as on a later stage will the radioactive isotope. The labelling processes which were undertaken with different activities had an efficiency of over 40%; when purification with resins amberlite was carried through, in none of the cases were the impurities over 5%. Daily controls till the seventh day showed that the average values of radiochemical purity decrease were lower than 1%. The specific activity as well as the concentration of I/sup 131/, the fibrinogen and other characteristics come up to the norms of the pharmacopoeia which are applied.

  13. Learning With Auxiliary Less-Noisy Labels. (United States)

    Duan, Yunyan; Wu, Ou


    Obtaining a sufficient number of accurate labels to form a training set for learning a classifier can be difficult due to the limited access to reliable label resources. Instead, in real-world applications, less-accurate labels, such as labels from nonexpert labelers, are often used. However, learning with less-accurate labels can lead to serious performance deterioration because of the high noise rate. Although several learning methods (e.g., noise-tolerant classifiers) have been advanced to increase classification performance in the presence of label noise, only a few of them take the noise rate into account and utilize both noisy but easily accessible labels and less-noisy labels, a small amount of which can be obtained with an acceptable added time cost and expense. In this brief, we propose a learning method, in which not only noisy labels but also auxiliary less-noisy labels, which are available in a small portion of the training data, are taken into account. Based on a flipping probability noise model and a logistic regression classifier, this method estimates the noise rate parameters, infers ground-truth labels, and learns the classifier simultaneously in a maximum likelihood manner. The proposed method yields three learning algorithms, which correspond to three prior knowledge states regarding the less-noisy labels. The experiments show that the proposed method is tolerant to label noise, and outperforms classifiers that do not explicitly consider the auxiliary less-noisy labels.

  14. Echinococcus granulosus: the potential use of specific radiolabelled antibodies in diagnosis by immunoscintigraphy

    Energy Technology Data Exchange (ETDEWEB)

    Rogan, M.T.; Morris, D.L.; Pritchard, D.I.; Perkins, A.C. (Nottingham Univ. (UK))


    Diagnosis of hydatid disease in man is frequently dependent on the imaging of cysts in situ by techniques such as ultrasonography and CAT scans. Such methods are useful but are not specific and can lead to errors in diagnosis. The present work reports preliminary experiments on the development of a specific imaging technique for hydatid cysts using radiolabelled antibodies. A purified preparation of antigen B of hydatid fluid was used to raise polyclonal antisera in rabbits and the resulting affinity-purified IgG labelled with {sup 131}I. Gerbils with an established Echinococcus granulosus infection were injected intraperitoneally with the labelled antibody and imaged 48 h later with a gamma camera. Hydatid cysts could be identified within the peritoneal cavity and post-mortem assessment of activity showed the cysts to contain approximately four times as much activity as the surrounding organs thereby indicating successful targeting of the antibody to the cysts. (author).

  15. How antibodies use complement to regulate antibody responses. (United States)

    Sörman, Anna; Zhang, Lu; Ding, Zhoujie; Heyman, Birgitta


    Antibodies, forming immune complexes with their specific antigen, can cause complete suppression or several 100-fold enhancement of the antibody response. Immune complexes containing IgG and IgM may activate complement and in such situations also complement components will be part of the immune complex. Here, we review experimental data on how antibodies via the complement system upregulate specific antibody responses. Current data suggest that murine IgG1, IgG2a, and IgG2b upregulate antibody responses primarily via Fc-receptors and not via complement. In contrast, IgM and IgG3 act via complement and require the presence of complement receptors 1 and 2 (CR1/2) expressed on both B cells and follicular dendritic cells. Complement plays a crucial role for antibody responses not only to antigen complexed to antibodies, but also to antigen administered alone. Lack of C1q, but not of Factor B or MBL, severely impairs antibody responses suggesting involvement of the classical pathway. In spite of this, normal antibody responses are found in mice lacking several activators of the classical pathway (complement activating natural IgM, serum amyloid P component (SAP), specific intracellular adhesion molecule-grabbing non-integrin R1 (SIGN-R1) or C-reactive protein. Possible explanations to these observations will be discussed.

  16. New Methods for Labeling RGD Peptides with Bromine-76

    Directory of Open Access Journals (Sweden)

    Lixin Lang, Weihua Li, Hong-Mei Jia, De-Cai Fang, Shushu Zhang, Xilin Sun, Lei Zhu, Ying Ma, Baozhong Shen, Dale O. Kiesewetter, Gang Niu, Xiaoyuan Chen


    Full Text Available Direct bromination of the tyrosine residues of peptides and antibodies with bromine-76, to create probes for PET imaging, has been reported. For peptides that do not contain tyrosine residues, however, a prosthetic group is required to achieve labeling via conjugation to other functional groups such as terminal α-amines or lysine ε-amines. The goal of this study was to develop new strategies for labeling small peptides with Br-76 using either a direct labeling method or a prosthetic group, depending on the available functional group on the peptides. A new labeling agent, N-succinimidyl-3-[76Br]bromo-2,6-dimethoxybenzoate ([76Br]SBDMB was prepared for cyclic RGD peptide labeling. N-succinimidyl-2, 6-dimethoxybenzoate was also used to pre-attach a 2, 6-dimethoxybenzoyl (DMB moiety to the peptide, which could then be labeled with Br-76. A competitive cell binding assay was performed to determine the binding affinity of the brominated peptides. PET imaging of U87MG human glioblastoma xenografted mice was performed using [76Br]-BrE[c(RGDyK]2 and [76Br]-BrDMB-E[c(RGDyK]2. An ex vivo biodistribution assay was performed to confirm PET quantification. The mechanisms of bromination reaction between DMB-c(RGDyK and the brominating agent CH3COOBr were investigated with the SCRF-B3LYP/6-31G* method with the Gaussian 09 program package. The yield for direct labeling of c(RGDyK and E[c(RGDyK]2 using chloramine-T and peracetic acid at ambient temperature was greater than 50%. The yield for [76Br]SBDMB was over 60% using peracetic acid. The conjugation yields for labeling c(RGDfK and c(RGDyK were over 70% using the prosthetic group at room temperature. Labeling yield for pre-conjugated peptides was over 60%. SDMB conjugation and bromination did not affect the binding affinity of the peptides with integrin receptors. Both [76Br]Br-E[c(RGDyK]2 and [76Br]BrDMB-E[c(RGDyK]2 showed high tumor uptake in U87MG tumor bearing mice. The specificity of the imaging tracers

  17. Dual-Quantum-Dots-Labeled Lateral Flow Strip Rapidly Quantifies Procalcitonin and C-reactive Protein (United States)

    Qi, XiaoPing; Huang, YunYe; Lin, ZhongShi; Xu, Liang; Yu, Hao


    In the article, a dual-quantum-dots-labeled (dual-QDs-labeled) lateral flow strip (LFS) method was developed for the simultaneous and rapid quantitative detection of procalcitonin (PCT) and C-reactive protein (CRP) in the blood. Two QD-antibody conjugates with different fluorescence emission spectra were produced and sprayed on the LFS to capture PCT and CRP in the blood. Furthermore, a double antibody sandwich method for PCT and, meanwhile, a competitive inhibition method for CRP were employed in the LFS. For PCT and CRP in serum assayed by the dual-QDs-labeled LFS, their detection sensitivities reached 0.1 and 1 ng/mL, respectively, and their linear quantitative detection ranges were from 0.3 to 200 ng/mL and from 50 to 250 μg/mL, respectively. There was little evidence that the PCT and CRP assays would be interfered with each other. The correlations for testing CRP and PCT in clinical samples were 99.75 and 97.02 %, respectively, between the dual-QDs-labeled LFS we developed and commercial methods. The rapid quantification of PCT and CRP on dual-QDs-labeled LFS is of great clinical value to distinguish inflammation, bacterial infection, or viral infection and to provide guidance for the use of antibiotics or other medicines.

  18. The antibody Hijikata Tatsumi

    Directory of Open Access Journals (Sweden)

    Éden Peretta


    Full Text Available Considered one of the most influential modern dance representatives in Japan, Tatsumi Hijikata’s work was a milestone in the Japanese post-war experimental artistic scene. Heretic son of his time, he staged a fertile mix of artistic and cultural influences, overlapping subversive elements of European arts and philosophy with radical references from pre-modern Japanese culture. In this way he built the foundations of its unstable antibody, its political-artistic project of dissolution of a organism, both physical and social.

  19. Denture labeling: A new approach

    Directory of Open Access Journals (Sweden)

    Pardeep K Bansal


    Full Text Available The need for denture labeling is important for forensic and social reasons in case patients need to be identified individually. The importance of denture marking has long been acknowledged by the dental profession. Over the years, various denture marking systems have been reported in the literature, but none till date fulfills all the prescribed ADA specifications. A simple, easy, inexpensive procedure for marking accurate identification marks on dentures with a lead foil is described here. The label caring the patient information is incorporated in the acrylic resin during the denture processing.

  20. Cancer imaging with radiolabeled antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Goldenberg, D.M. (Center for Molecular Medicine and Immunology, Newark, NJ (US))


    This book presents a perspective of the use of antibodies to target diagnostic isotopes to tumors. Antibodies with reasonable specificity can be developed against almost any substance. If selective targeting to cancer cells can be achieved, the prospects for a selective therapy are equally intriguing. But the development of cancer detection, or imaging, with radiolabeled antibodies has depended upon advances in a number of different areas, including cancer immunology and immunochemistry for identifying suitable antigen targets and antibodies to these targets, tumor biology for model systems, radiochemistry for he attachment of radionuclides to antibodies, molecular biology for reengineering the antibodies for safer and more effective use in humans, and nuclear medicine for providing the best imaging protocols and instrumentation to detect minute amounts of elevated radioactivity against a background of considerable noise. Accordingly, this book has been organized to address the advances that are being made in many of these areas.


    Directory of Open Access Journals (Sweden)

    S. Saidi


    Full Text Available One hundred sera from children 1 - 6 years of age, representative of a large serum collection, were tested for the prevalence of antibodies against different viruses. Hemagglutination-inhibition (HI antibodies were found in 68% for measles; 61 % for rubella; 75'% for influenza A2/Hong Kong/68, 16% for influenza B/Md./59, 0% for group A arboviruses, 10% for group B arboviruses, 3% for phlebotomus fever group and 4% for Congo-Crimean hemorrhagic fever (C-CHF group of arboviruses Poliomyelitis-neutralizing antibodies for type 1, 2 and 3 were 90%; 85% and 84%~ respectively. Antibody to EH virus was detected in 84% of the sera by immuno-fluorescence. None of the sera were positive for hepatitis-B antigen or antibody by immuno-precipitation test. The prevalence of some viral antibodies found in this survey are compared with results obtained from surveys in other parts of the country.

  2. Connected Component Labeling Using Components Neighbors-Scan Labeling Approach

    Directory of Open Access Journals (Sweden)

    Akmal Rakhmadi


    Full Text Available Problem statement: Many approaches have been proposed in previous such as the classic sequential connected components labeling algorithm which is relies on two subsequent raster-scans of a binary image. This method produced good performance in terms of accuracy, but because of the implementation of the image processing systems now requires faster process of the computer, the speed of this technique’s process has become an important issue. Approach: A computational approach, called components neighbors-scan labeling algorithm for connected component labeling was presented in this study. This algorithm required scanning through an image only once to label connected components. The algorithm started by scanning from the head of the component’s group, before tracing all the components neighbors by using the main component’s information. This algorithm had desirable characteristics, it is simple while promoted accuracy and low time consuming. By using a table of components, this approach also gave other advantages as the information for the next higher process. Results: The approach had been tested with a collection of binary images. In practically all cases, the technique had successfully given the desired result. Averagely, from the results the algorithm increased the speed around 67.4% from the two times scanning method. Conclusion: Conclusion from the comparison with the previous method, the approach of components neighbors-scan for connected component labeling promoted speed, accuracy and simplicity. The results showed that the approach has a good performance in terms of accuracy, the time consumed and the simplicity of the algorithm.

  3. Francisella tularensis detection using magnetic labels and a magnetic biosensor based on frequency mixing

    Energy Technology Data Exchange (ETDEWEB)

    Meyer, Martin H.F. [Institute for Pharmaceutical Chemistry, Philipps-Universitaet Marburg (Germany); Krause, Hans-Joachim [Institute of Bio-and Nanosystems (IBN-2), Research Center Juelich (Germany); Hartmann, Markus [Institute for Pharmaceutical Chemistry, Philipps-Universitaet Marburg (Germany); Miethe, Peter [SENOVA GmbH, Jena (Germany); Oster, Juergen [chemagen GmbH, Baesweiler (Germany); Keusgen, Michael [Institute for Pharmaceutical Chemistry, Philipps-Universitaet Marburg (Germany)]. E-mail:


    A biosensor that uses resonant coils with a special frequency-mixing technique and magnetic beads as detectable labels has been established for the detection of Francisella tularensis, the causative agent for tularemia. The detection principle is based on a sandwich immunoassay using an anti-Ft antibody for immunofiltration immobilized to ABICAP[reg] polyethylene filters, and biotinylated with streptavidin-coated magnetic beads as labels. The linear detection range of this biosensor was found to be 10{sup 4}-10{sup 6} cfu F. tularensis lipopolysaccharide (LPS) per ml. Tested sample matrices were physiological PBS buffer and rabbit serum.

  4. SLAP: Small Labeling Pair for Single-Molecule Super-Resolution Imaging. (United States)

    Wieneke, Ralph; Raulf, Anika; Kollmannsperger, Alina; Heilemann, Mike; Tampé, Robert


    Protein labeling with synthetic fluorescent probes is a key technology in chemical biology and biomedical research. A sensitive and efficient modular labeling approach (SLAP) was developed on the basis of a synthetic small-molecule recognition unit (Ni-trisNTA) and the genetically encoded minimal protein His6-10 -tag. High-density protein tracing by SLAP was demonstrated. This technique allows super-resolution fluorescence imaging and fulfills the necessary sampling criteria for single-molecule localization-based imaging techniques. It avoids masking by large probes, for example, antibodies, and supplies sensitive, precise, and robust size analysis of protein clusters (nanodomains).

  5. Preparation of radiopharmaceuticals labeled with metal radionuclides. Progress report, July 1, 1988--June 30, 1992

    Energy Technology Data Exchange (ETDEWEB)

    Welch, M.J.


    We recently developed a useful zinc-62/copper-62 generator and are presently evaluating copper-62 radiopharmaceuticals for clinical studies. While developing these copper-62 radiopharmaceuticals, in collaboration with the University of Missouri Research Reactor, Columbia we have also explored copper-64 radiopharmaceuticals. The PET images we obtained with copper-64 tracers were of such high quality that we have developed and evaluated copper-64 labeled antibodies for PET imaging. The major research activities described herein include: the development and assessment of gallium-68 radiopharmaceuticals; the development and evaluation of a new zinc-62/copper-62 generator and the assessment of copper-62 radiopharmaceuticals; mechanistic studies on proteins labeled with metal radionuclides.

  6. Microbial detection in microfluidic devices through dual staining of quantum dots-labeled immunoassay and RNA hybridization

    Energy Technology Data Exchange (ETDEWEB)

    Zhang Qing [Division of Environmental Science and Engineering, National University of Singapore, Blk E1A, 07-03, Engineering Drive 2, Singapore 117576 (Singapore); Zhu Liang [Division of Environmental Science and Engineering, National University of Singapore, Blk E1A, 07-03, Engineering Drive 2, Singapore 117576 (Singapore); Feng Hanhua [Institute of Microelectronics (Singapore); Ang, Simon [Department of Electrical Engineering, University of Arkansas, Fayetteville (United States); Chau, F.S. [Department of Mechanical Engineering, National University of Singapore (Singapore); Liu, W.-T. [Division of Environmental Science and Engineering, National University of Singapore, Blk E1A, 07-03, Engineering Drive 2, Singapore 117576 (Singapore)]. E-mail:


    This paper reported the development of a microfludic device for the rapid detection of viable and nonviable microbial cells through dual labeling by fluorescent in situ hybridization (FISH) and quantum dots (QDs)-labeled immunofluorescent assay (IFA). The coin sized device consists of a microchannel and filtering pillars (gap = 1-2 {mu}m) and was demonstrated to effectively trap and concentrate microbial cells (i.e. Giardia lamblia). After sample injection, FISH probe solution and QDs-labeled antibody solution were sequentially pumped into the device to accelerate the fluorescent labeling reactions at optimized flow rates (i.e. 1 and 20 {mu}L/min, respectively). After 2 min washing for each assay, the whole process could be finished within 30 min, with minimum consumption of labeling reagents and superior fluorescent signal intensity. The choice of QDs 525 for IFA resulted in bright and stable fluorescent signal, with minimum interference with the Cy3 signal from FISH detection.

  7. A comparative antibody analysis of Pannexin1 expression in four rat brain regions reveals varying subcellular localizations

    Directory of Open Access Journals (Sweden)

    Angela C Cone


    Full Text Available Pannexin1 (Panx1 channels release cytosolic ATP in response to signaling pathways. Panx1 is highly expressed in the central nervous system. We used four antibodies with different Panx1 anti-peptide epitopes to analyze four regions of rat brain. These antibodies labeled the same bands in Western blots and had highly similar patterns of immunofluorescence in tissue culture cells expressing Panx1, but Western blots of brain lysates from Panx1 knockout and control mice showed different banding patterns. Localizations of Panx1 in brain slices were generated using automated wide-field mosaic confocal microscopy for imaging large regions of interest while retaining maximum resolution for examining cell populations and compartments. We compared Panx1 expression over the cerebellum, hippocampus with adjacent cortex, thalamus and olfactory bulb. While Panx1 localizes to the same neuronal cell types, subcellular localizations differ. Two antibodies with epitopes against the intracellular loop and one against the carboxy terminus preferentially labeled cell bodies, while an antibody raised against an N-terminal peptide highlighted neuronal processes more than cell bodies. These labeling patterns may be a reflection of different cellular and subcellular localizations of full-length and/or modified Panx1 channels where each antibody is highlighting unique or differentially accessible Panx1 populations. However, we cannot rule out that one or more of these antibodies have specificity issues. All data associated with experiments from these four antibodies are presented in a manner that allows them to be compared and our claims thoroughly evaluated, rather than eliminating results that were questionable. Each antibody is given a unique identifier through the NIF Antibody Registry that can be used to track usage of individual antibodies across papers and all image and metadata are made available in the public repository, the Cell Centered Database, for on

  8. Food quality labels from the producers’ perspective

    Directory of Open Access Journals (Sweden)

    Šárka Velčovská


    Full Text Available The paper deals with analysing the food producer attitudes towards quality labels. The Klasa label, as the most known and the most frequently used food quality label in the Czech Republic, have become the subject of investigation. The aim of the research was to identify the benefits and problems arising from the certification process and the label use. Primary data were collected in online survey based on standardized questionnaire. In census, 86 respondents from the total 218 producers with the Klasa label in the Czech Republic completed the questionnaire. The most of producers (72% have a longer experience with the label, they are using the label for more than four years. The producers’ expectations from the label were fulfilled only partially. A poor state marketing support and missing marketing strategy were identified as general problems of the label. Specific perceived problems are formalities connected with the certification process and certification of poor-quality products. Correlation analysis, t-test and Pearson chi-square test were calculated to discover relations between variables. The results of the study can be beneficial to both, food producers as well as administrator of the label. Identified problems could help them to improve marketing strategy of the label in order to manage the label in effective way and use all benefits arising from the certification. Administrator of the label should make the certification process more effective and transparent, promotion should be focused on the explanation to consumers what the Klasa label guarantees.

  9. Novel method for the high-throughput production of phosphorylation site-specific monoclonal antibodies (United States)

    Kurosawa, Nobuyuki; Wakata, Yuka; Inobe, Tomonao; Kitamura, Haruki; Yoshioka, Megumi; Matsuzawa, Shun; Kishi, Yoshihiro; Isobe, Masaharu


    Threonine phosphorylation accounts for 10% of all phosphorylation sites compared with 0.05% for tyrosine and 90% for serine. Although monoclonal antibody generation for phospho-serine and -tyrosine proteins is progressing, there has been limited success regarding the production of monoclonal antibodies against phospho-threonine proteins. We developed a novel strategy for generating phosphorylation site-specific monoclonal antibodies by cloning immunoglobulin genes from single plasma cells that were fixed, intracellularly stained with fluorescently labeled peptides and sorted without causing RNA degradation. Our high-throughput fluorescence activated cell sorting-based strategy, which targets abundant intracellular immunoglobulin as a tag for fluorescently labeled antigens, greatly increases the sensitivity and specificity of antigen-specific plasma cell isolation, enabling the high-efficiency production of monoclonal antibodies with desired antigen specificity. This approach yielded yet-undescribed guinea pig monoclonal antibodies against threonine 18-phosphorylated p53 and threonine 68-phosphorylated CHK2 with high affinity and specificity. Our method has the potential to allow the generation of monoclonal antibodies against a variety of phosphorylated proteins. PMID:27125496

  10. C-C chemokine receptor-7 mediated endocytosis of antibody cargoes into intact cells

    Directory of Open Access Journals (Sweden)

    Xavier eCharest-Morin


    Full Text Available The C-C chemokine receptor-7 (CCR7 is a G protein coupled receptor that has a role in leukocyte homing, but that is also expressed in aggressive tumor cells. Preclinical research supports that CCR7 is a valid target in oncology. In view of the increasing availability of therapeutic monoclonal antibodies that carry cytotoxic cargoes, we studied the feasibility of forcing intact cells to internalize known monoclonal antibodies by exploiting the cycle of endocytosis and recycling triggered by the CCR7 agonist CCL19. Firstly, an anti-CCR7 antibody (CD197; clone 150503 labeled surface recombinant CCR7 expressed in intact HEK 293a cells and the fluorescent antibody was internalized following CCL19 treatment. Secondly, a recombinant myc-tagged CCL19 construction was exploited along the anti-myc monoclonal antibody 4A6. The myc-tagged ligand was produced as a conditioned medium of transfected HEK 293a cells that contained the equivalent of 430 ng/ml of immunoreactive CCL19 (average value, ELISA determination. CCL19-myc, but not authentic CCL19, carried the fluorophore-labeled antibody 4A6 into other recipient cells that expressed recombinant CCR7 (microscopy, cytofluorometry. The immune complexes were apparent in endosomal structures, colocalized well with the small GTPase Rab5 and progressed toward Rab7-positive endosomes. A dominant negative form of Rab5 (GDP-locked inhibited this endocytosis. Further, endosomes in CCL19-myc- or CCL19-stimulated cells were positive for β-arrestin2, but rarely for β-arrestin1. Following treatment with CCL19-myc and the 4A6 antibody, the melanoma cell line A375 that expresses endogenous CCR7 was specifically stained using a secondary peroxidase-conjugated antibody. Agonist-stimulated CCR7 can transport antibody-based cargoes, with possible therapeutic applications in oncology.

  11. The Labelling Approach to Deviance. (United States)

    Rains, Prudence M.; Kitsuse, John L.; Duster, Troy; Freidson, Eliot


    This reprint of one chapter from the 1975 text, "Issues in the Classification of Children" by Nicholas Hobbs and others, addresses the theoretical, methodological, and empirical issues involved in the "labeling" approach to the sociology of deviance. It examines the social process of classification, the use of classification in social agencies,…

  12. Psychological effectiveness of carbon labelling (United States)

    Beattie, Geoffrey


    Despite the decision by supermarket-giant Tesco to delay its plan to add carbon-footprint information onto all of its 70,000 products, carbon labelling, if carefully designed, could yet change consumer behaviour. However, it requires a new type of thinking about consumers and much additional work.

  13. On Labeled Traveling Salesman Problems

    DEFF Research Database (Denmark)

    Couetoux, Basile; Gourves, Laurent; Monnot, Jerome;


    We consider labeled Traveling Salesman Problems, defined upon a complete graph of n vertices with colored edges. The objective is to find a tour of maximum (or minimum) number of colors. We derive results regarding hardness of approximation, and analyze approximation algorithms for both versions...

  14. Improving the energy labelling scheme

    DEFF Research Database (Denmark)

    Gram-Hanssen, Kirsten; Christensen, Toke Haunstrup

    This report summarises the main results of an EU project on consumer response to energy labels in buildings. This report is mainly directed at Danish policy makers. The main focus is therefore on results that are relevant from a Danish point of view and on how they can be used to further strengthen...

  15. When Diagnostic Labels Mask Trauma (United States)

    Foltz, Robert; Dang, Sidney; Daniels, Brian; Doyle, Hillary; McFee, Scott; Quisenberry, Carolyn


    A growing body of research shows that many seriously troubled children and adolescents are reacting to adverse life experiences. Yet traditional diagnostic labels are based on checklists of surface symptoms. Distracted by disruptive behavior, the common response is to medicate, punish, or exclude rather than respond to needs of youth who have…

  16. Physical Characteristics of a Citrullinated Pro-Filaggrin Epitope Recognized by Anti-Citrullinated Protein Antibodies in Rheumatoid Arthritis Sera

    DEFF Research Database (Denmark)

    Trier, Nicole Hartwig; Holm, Bettina Eide; Slot, Ole;


    whether biotin labelling influence antibody recognition. The full-length cyclic pro-filaggrin peptide and a linear form with a N-terminal biotin, was recognized to the same level, whereas, a notable difference in ACPA reactivity to the linear peptides with a C-terminal biotin was found, probably due...... amino acid in position 4 C-terminal to citrulline. Collectively, peptide structure, length, the presence of charged amino acids and biotin labelling markedly influence antibody reactivity. In relation to the clinical diagnostics of ACPA, these findings may reflect the differences in diagnostic assays...

  17. Antibodies against antibodies: immunogenicity of adalimumab as a model

    NARCIS (Netherlands)

    van Schouwenburg, P.A.


    Upon repeated adalimumab exposure part of the patients start to produce ADA. The antibody response is polyclonal and consists mainly of antibodies of IgG1 and IgG4 isotype. In the majority of ADA positive patients ADA are already produced within the first 28 weeks of treatment and in part of the pat

  18. Near-infrared emitting fluorescent nanocrystals-labeled natural killer cells as a platform technology for the optical imaging of immunotherapeutic cells-based cancer therapy (United States)

    Taik Lim, Yong; Cho, Mi Young; Noh, Young-Woock; Chung, Jin Woong; Chung, Bong Hyun


    This study describes the development of near-infrared optical imaging technology for the monitoring of immunotherapeutic cell-based cancer therapy using natural killer (NK) cells labeled with fluorescent nanocrystals. Although NK cell-based immunotherapeutic strategies have drawn interest as potent preclinical or clinical methods of cancer therapy, there are few reports documenting the molecular imaging of NK cell-based cancer therapy, primarily due to the difficulty of labeling of NK cells with imaging probes. Human natural killer cells (NK92MI) were labeled with anti-human CD56 antibody-coated quantum dots (QD705) for fluorescence imaging. FACS analysis showed that the NK92MI cells labeled with anti-human CD56 antibody-coated QD705 have no effect on the cell viability. The effect of anti-human CD56 antibody-coated QD705 labeling on the NK92MI cell function was investigated by measuring interferon gamma (IFN- γ) production and cytolytic activity. Finally, the NK92MI cells labeled with anti-human CD56 antibody-coated QD705 showed a therapeutic effect similar to that of unlabeled NK92MI cells. Images of intratumorally injected NK92MI cells labeled with anti-human CD56 antibody-coated could be acquired using near-infrared optical imaging both in vivo and in vitro. This result demonstrates that the immunotherapeutic cells labeled with fluorescent nanocrystals can be a versatile platform for the effective tracking of injected therapeutic cells using optical imaging technology, which is very important in cell-based cancer therapies.

  19. Targeted labeling of an early-stage tumor spheroid in a chorioallantoic membrane model with upconversion nanoparticles

    NARCIS (Netherlands)

    K. Liu; J.A. Holz; Y. Ding; X. Liu; Y. Zhang; L. Tu; X. Kong; B. Priem; A. Nadort; S.A.G. Lambrechts; M.C.G. Aalders; W.J. Buma; Y. Liu; H. Zhang


    In vivo detection of cancer at an early-stage, i.e. smaller than 2 mm, is a challenge in biomedicine. In this work target labeling of an early-stage tumor spheroid (similar to 500 mu m) is realized for the first time in a chick embryo chorioallantoic membrane (CAM) model with monoclonal antibody fun

  20. Labelling schemes: From a consumer perspective

    DEFF Research Database (Denmark)

    Juhl, Hans Jørn; Stacey, Julia


    . A recent MAPP study has investigated the value consumers attach the Government-controlled labels 'Ø-mærket' and 'Den Blå Lup' and the private supermarket label 'Mesterhakket' when they purchase minced meat. The results reveal four consumer segments that use labelling schemes for food products very....... The remaining consumers, about 55%, trust the institutions guaranteeing the labels and they use the labels as a signal without actually knowing the content of each label. Segment composition will probably change depending on the food group studied. It is therefore recommended that the different consumer types...

  1. Initial study with Tc99m antigranulocyte antibody (MAK-47) in detection sources of infection

    Energy Technology Data Exchange (ETDEWEB)

    Cwikla, J.B.; Buscombe, J.R.; Hilson, A.J. [Dept. of Nuclear Medicine and Medical Physics, Royal Free Hospital and School of Medicine, London (United Kingdom); Janoki, G.A. [FJC National Research Inst. for Radiology and Radiohygene, Dept. of Applied Radioisotopes, Budapest (Hungary)


    Antigranulocytes antibodies (AGAB) are antibodies directed against glycoprotein on the surface of granulocytes and as such provides in vivo cell labelling. They are easily labelled with Tc99m and comes a one step labelling technique. 20 patients have been studied 1 h and 4-6 hours after administration of 200 MBq of Tc99m AGAB (MAK-47). Less then 0.5 mg of antibody was given to each patients. Sites of uptake and outside of the reticular-endothelial system were reported as showing positive accumulation. Clinical results were confirmed by microbiological, pathological examinations, clinical follow-up and autopsy. There were 8 patients who had sites of infection confirmed by additional examination. All patients were visualized by Tc99m AGAB (MAK-47). There were 4 cases of osteomyelitis and septic arthritis and 4 cases of focal intra-abdominal infection. Two patients had uptake in non-infected/inflammatory arthritis, both in the knee. The remaining patients had true negative studies. The diagnostic accuracy of this study was as follows: sensitivity 100%, specificity 83%, positive predictive value (PPV) was 80% and negative predictive value (NPV) was 100%. Antigranulocyte antibody (MAK-47) seems to by promising tool in detecting focal infection in bone and soft tissue, except physiological accumulation in some parts of the body. It should be considered that antibodies can have non-specific uptake in non-infected, inflammation sites. It is easy to use and no had allergic reaction and HAMA antibody (human antimouse antibody). (author) 18 refs, 4 figs, 2 tabs

  2. Antibody inhibition of polymorphonuclear phagocytosis. Dissociation of bacterial attachment and bacterial killing. (United States)

    Crowley, J P; Valeri, C R


    The inhibition of killing of Staphylococcus aureus 502A by PMNs treated with the IgG fraction of serum from a group of patients with demonstrable leukocyte antibodies was investigated. The uptake of opsonized thymidine-labeled S. aureus 502A by PMNs treated with allogeneic antibody was essentially unimpaired, despite significantly decreased killing. The findings were similar to bacteria opsonized by serum complement or bacteria opsonized with specific lapine antibody. An increased proportion of PMN-bound bacteria susceptible to lysis by lysostaphin indicated a reduced rate of translocation of bacteria from the surface of allogeneic antibody-treated PMNs. Antibody did not stimulate the basal oxidative metabolism, but the oxidative metabolism of antibody-treated PMNs during phagocytosis was increased. Although the precise mechanism of inhibition of PMN killing by antibody is uncertain, the data suggest that the impairment of bacterial killing by PMNs treated with allogeneic leukocyte antibody is associated with inefficient translocation of bacteria into phagolysosomes rather than by interference with the binding of bacteria to specific PMN opsonic receptors.

  3. A Model System for Concurrent Detection of Antigen and Antibody Based on Immunological Fluorescent Method

    Directory of Open Access Journals (Sweden)

    Yuan-Cheng Cao


    Full Text Available This paper describes a combined antigen/antibody immunoassay implemented in a 96-well plate using fluorescent spectroscopic method. First, goat anti-human IgG was used to capture human IgG (model antigen; goat anti-human IgG (Cy3 or FITC was used to detect the model antigen; a saturating level of model antigen was then added followed by unlabelled goat anti-human IgG (model antibody; finally, Cy3 labelled rabbit anti-goat IgG was used to detect the model antibody. Two approaches were applied to the concomitant assay to analyze the feasibility. The first approach applied FITC and Cy3 when both targets were present at the same time, resulting in 50 ng/mL of the antibody detection limit and 10 ng/mL of antigen detection limit in the quantitative measurements of target concentration, taking the consideration of FRET efficiency of 68% between donor and acceptor. The sequential approach tended to lower the signal/noise (S/N ratio and the detection of the model antigen (lower than 1 ng/mL had better sensitivity than the model antibody (lower than 50 ng/mL. This combined antigen/antibody method might be useful for combined detection of antigens and antibodies. It will be helpful to screen for both antigen and antibody particularly in the situations of the multiserotype and high-frequency mutant virus infections.

  4. Click Chemistry Immobilization of Antibodies on Polymer Coated Gold Nanoparticles. (United States)

    Finetti, Chiara; Sola, Laura; Pezzullo, Margherita; Prosperi, Davide; Colombo, Miriam; Riva, Benedetta; Avvakumova, Svetlana; Morasso, Carlo; Picciolini, Silvia; Chiari, Marcella


    The goal of this work is to develop an innovative approach for the coating of gold nanoparticles (AuNPs) with a synthetic functional copolymer. This stable coating with a thickness of few nanometers provides, at the same time, stabilization and functionalization of the particles. The polymeric coating consists of a backbone of polydimethylacrylamide (DMA) functionalized with an alkyne monomer that allows the binding of azido modified molecules by Cu(I)-catalyzed azide/alkyne 1,3-dipolar cycloaddition (CuAAC, click chemistry). The thin polymer layer on the surface stabilizes the colloidal suspension whereas the alkyne functions pending from the backbone are available for the reaction with azido-modified proteins. The reactivity of the coating is demonstrated by immobilizing an azido modified anti-mouse IgG antibody on the particle surface. This approach for the covalent binding of antibody to a gold-NPs is applied to the development of gold labels in biosensing techniques.

  5. Fluorescence polarization immunoassay for salinomycin based on monoclonal antibodies

    Institute of Scientific and Technical Information of China (English)


    A fluorescence polarization immunoassay(FPIA) for the determination of salinomycin(SAL) was developed by using anti-SAL monoclonal antibodies(mAb).Fluorescein labeled SAL(tracer) was synthesized by the N-hydroxysuccinimide active ester method and purified using thin layer chromatography(TLC).The developed FPIA for SAL had a dynamic range from 0.60 to 2193 ng/mL with an IC50 value of 33.2 ng/mL and a detection limit(LOD) of 0.08 ng/mL.No significant cross-reactivities were observed with other drugs but 67.6% with narasin.

  6. Bispecific Antibody Pretargeting for Improving Cancer Imaging and Therapy

    Energy Technology Data Exchange (ETDEWEB)

    Sharkey, Robert M.


    The main objective of this project was to evaluate pretargeting systems that use a bispecific antibody (bsMAb) to improve the detection and treatment of cancer. A bsMAb has specificity to a tumor antigen, which is used to bind the tumor, while the other specificity is to a peptide that can be radiolabeled. Pretargeting is the process by which the unlabeled bsMAb is given first, and after a sufficient time (1-2 days) is given for it to localize in the tumor and clear from the blood, a small molecular weight radiolabeled peptide is given. According to a dynamic imaging study using a 99mTc-labeled peptide, the radiolabeled peptide localizes in the tumor in less than 1 hour, with > 80% of it clearing from the blood and body within this same time. Tumor/nontumor targeting ratios that are nearly 50 times better than that with a directly radiolabeled Fab fragment have been observed (Sharkey et al., ''Signal amplification in molecular imaging by a multivalent bispecific nanobody'' submitted). The bsMAbs used in this project have been composed of 3 antibodies that will target antigens found in colorectal and pancreatic cancers (CEA, CSAp, and MUC1). For the ''peptide binding moiety'' of the bsMAb, we initially examined an antibody directed to DOTA, but subsequently focused on another antibody directed against a novel compound, HSG (histamine-succinyl-glycine).

  7. Production and purification of polyclonal antibody against melatonin hormone

    Directory of Open Access Journals (Sweden)

    Fooladsaz K


    Full Text Available Nowadays immunochemical techniques have played a very important and valuable role in quantitative and qualitative assays of liquid compounds of the body. Producing antibody against immunogenes is the first step to make immunochemical kits. In this study production and purification of polyclonal antibody against melatonin has been considered. This hormone which has several important functions in physiological conditions such as migraine, cirrhosis, mammary gland cancer and other diseases, is the most important pineal gland secretion. This gland is a circumventricular organ of brain and according to histological and anatomical studies, it is a high secretory organ, that secretes active biological substances like melatonin, oxytocin, serotonin and ect. In this study, melatonin has been considered as hapten and has become an immunogen by being linked to the bovine serum Albumin. Then, by the immunization of three white New Zeland rabbits that had the booster injections in regular intervals, the antibody titer was detected to be 1/2000, by using checkboard curves, and with the use of melatonin linked to penicillinase as a labeled antigen, the titer was detected 1/200. Finally an antibody with high purification rate has been obtained, which can be used in immunochemical assays like RIA, ELISA, and EIA.

  8. Pathogenic role of antiphospholipid antibodies

    NARCIS (Netherlands)

    Salmon, J. E.; de Groot, P. G.


    The antiphospholipid antibody syndrome (APS) is characterized by recurrent arterial and venous thrombosis and/or pregnancy in association with antiphospholipid (aPL) antibodies. The pathogenic mechanisms in APS that lead to in vivo injury are incompletely understood. Recent evidence suggests that AP

  9. Educational paper: Primary antibody deficiencies

    NARCIS (Netherlands)

    G.J.A. Driessen (Gertjan); M. van der Burg (Mirjam)


    textabstractPrimary antibody deficiencies (PADs) are the most common primary immunodeficiencies and are characterized by a defect in the production of normal amounts of antigen-specific antibodies. PADs represent a heterogeneous spectrum of conditions, ranging from often asymptomatic selective IgA a

  10. Targeting of Antibodies using Aptamers



    The chapter presents a methodology for the rapid selection of aptamers against antibody targets. It is a detailed account of the various methodological steps that describe the selection of aptamers, including PCR steps, buffers to be used, target immobilisation, partitioning and amplification of aptamers, clonning and sequencing, to results in high affinity and specificity ligands for the chosen target antibody.

  11. New engineered antibodies against prions (United States)

    Škrlj, Nives; Dolinar, Marko


    A number of recently developed and approved therapeutic agents based on highly specific and potent antibodies have shown the potential of antibody therapy. As the next step, antibody-based therapeutics will be bioengineered in a way that they not only bind pathogenic targets but also address other issues, including drug targeting and delivery. For antibodies that are expected to act within brain tissue, like those that are directed against the pathogenic prion protein isoform, one of the major obstacles is the blood-brain barrier which prevents efficient transfer of the antibody, even of the engineered single-chain variants. We recently demonstrated that a specific prion-specific antibody construct which was injected into the murine tail vein can be efficiently transported into brain tissue. The novelty of the work was in that the cell penetrating peptide was used as a linker connecting both specificity-determining domains of the antibody peptide, thus eliminating the need for the standard flexible linker, composed of an arrangement of three consecutive (Gly4Ser) repeats. This paves the road toward improved bioengineered antibody variants that target brain antigens. PMID:23941991

  12. Use of radiolabeled monoclonal antibodies for diagnostic imaging

    Energy Technology Data Exchange (ETDEWEB)

    Endo, Keigo (Kyoto Univ. (Japan). Faculty of Medicine)


    Monoclonal antibodies (MoAbs) are expected to carry radionuclides selectively to target tissues and to offer antigen-specific diagnosis. Indium (In)-111 has many favorable nuclear properties and is efficiently labeled with MoAbs using DAPA as a bifunctional chelating agent. In-111 labeled MoAbs are clinically employed for the diagnosis of malignant melanoma, colorectal cancer and acute myocardial infarction in Japan. Although non-specific deposit of In-111 was seen in liver and bone-marrow, scintigraphy using In-111 labeled MoAbs was encouraging, since it detected about 80% of tumors, tumors missed by conventional diagnostic methods such as CT, and tumors in patients with normal serum CEA values, and acute myocarditis as well as acute myocardial infarction was positive with In-111 labeled Fab fraction of anti-myosin Ab. Acute or subacute toxicity was not observed. Human anti-murine antibody (HAMA) was detected in 53 of 64 (82.8%) patients who were intravenously administered with 20 to 42 mg of anti-melanoma or anti-CEA MoAbs (whole IgG). In contrast, only 5 of 406 (1.2%) patients had detectable levels of HAMA in their serum after receiving 0.5 mg of Fab fraction of MoAb. Recently mouse-human chimeric Ab has been produced by recombinant DNA techniques, which localized well in xenografted tumors and seems to be promising for clinical use. Investigations are under way to increase the tumor to non-tumor ratio by modifying chelating agents for coupling MoAbs with radionuclides. (author).

  13. Ivabradine: A Review of Labeled and Off-Label Uses. (United States)

    Oliphant, Carrie S; Owens, Ryan E; Bolorunduro, Oluwaseyi B; Jha, Sunil K


    Ivabradine is a unique medication recently approved in the USA for the treatment of select heart failure patients. It was first approved for use in several countries around the world over a decade ago as an anti-anginal agent, with subsequent approval for use in heart failure patients. Since ivabradine has selective activity blocking the I f currents in the sinus node, it can reduce heart rate without appreciable effects on blood pressure. Given this heart-rate-specific effect, it has been investigated in many off-label indications as an alternative to traditional heart-rate-reducing medications such as beta blockers and calcium channel blockers. We conducted searches of PubMed and Google Scholar for ivabradine, heart failure, HFrEF, HFpEF, angina, coronary artery disease, inappropriate sinus tachycardia, postural orthostatic hypotension, coronary computed tomography angiography and atrial fibrillation. We reviewed and included studies, case reports, and case series published between 1980 and June 2016 if they provided information relevant to the practicing clinician. In many cases, larger clinical trials are needed to solidify the benefit of ivabradine, although studies indicate benefit in most therapeutic areas explored to date. The purpose of this paper is to review the current labeled and off-label uses of ivabradine, with a focus on clinical trial data.

  14. Metrics for antibody therapeutics development. (United States)

    Reichert, Janice M


    A wide variety of full-size monoclonal antibodies (mAbs) and therapeutics derived from alternative antibody formats can be produced through genetic and biological engineering techniques. These molecules are now filling the preclinical and clinical pipelines of every major pharmaceutical company and many biotechnology firms. Metrics for the development of antibody therapeutics, including averages for the number of candidates entering clinical study and development phase lengths for mAbs approved in the United States, were derived from analysis of a dataset of over 600 therapeutic mAbs that entered clinical study sponsored, at least in part, by commercial firms. The results presented provide an overview of the field and context for the evaluation of on-going and prospective mAb development programs. The expansion of therapeutic antibody use through supplemental marketing approvals and the increase in the study of therapeutics derived from alternative antibody formats are discussed.

  15. Determination of the labeling density of fluorophore-biomolecule conjugates with absorption spectroscopy. (United States)

    Grabolle, Markus; Brehm, Robert; Pauli, Jutta; Dees, Franziska M; Hilger, Ingrid; Resch-Genger, Ute


    Dye-biomolecule conjugation is frequently accompanied by considerable spectral changes of the dye's absorption spectrum that limit the use of the common photometrical method for the determination of labeling densities. Here, we describe an improvement of this method using the integral absorbance of the dye instead of its absorbance at the long wavelength maximum to determine the concentration of the biomolecule-coupled dye. This approach is illustrated for three different cyanine dyes conjugated to the antibody IgG.

  16. A Multi-Label Classification Approach Based on Correlations Among Labels

    Directory of Open Access Journals (Sweden)

    Raed Alazaidah


    Full Text Available Multi label classification is concerned with learning from a set of instances that are associated with a set of labels, that is, an instance could be associated with multiple labels at the same time. This task occurs frequently in application areas like text categorization, multimedia classification, bioinformatics, protein function classification and semantic scene classification. Current multi-label classification methods could be divided into two categories. The first is called problem transformation methods, which transform multi-label classification problem into single label classification problem, and then apply any single label classifier to solve the problem. The second category is called algorithm adaptation methods, which adapt an existing single label classification algorithm to handle multi-label data. In this paper, we propose a multi-label classification approach based on correlations among labels that use both problem transformation methods and algorithm adaptation methods. The approach begins with transforming multi-label dataset into a single label dataset using least frequent label criteria, and then applies the PART algorithm on the transformed dataset. The output of the approach is multi-labels rules. The approach also tries to get benefit from positive correlations among labels using predictive Apriori algorithm. The proposed approach has been evaluated using two multi-label datasets named (Emotions and Yeast and three evaluation measures (Accuracy, Hamming Loss, and Harmonic Mean. The experiments showed that the proposed approach has a fair accuracy in comparison to other related methods.


    Directory of Open Access Journals (Sweden)

    Oudone Phanalasy


    Full Text Available An antimagic labeling of a graph with q edges is a bijection from the set of edges to the set of positive integers {1,2,...,q} such that all vertex weights are pairwise distinct, where the vertex weight of a vertex is the sum of the labels of all the edges incident with that vertex. A graph is antimagic if it has an antimagic labeling. In this paper we construct antimagic labeling for the family of generalized sausage graphs.




    This article discusses economic issues related to public labeling. The main contributions in both the empirical and theoretical literatures are presented in order to motivate responses to the questions, when should a regulator promote public labeling, and what are the limits to and the possible market distortions from public labeling? Although the issues are complicated, there is already much economic guidance that can be given to inform the policy debate over food labeling.

  19. Automatic Labelling of Topics with Neural Embeddings


    Bhatia, Shraey; Lau, Jey Han; Baldwin, Timothy


    Topics generated by topic models are typically represented as list of terms. To reduce the cognitive overhead of interpreting these topics for end-users, we propose labelling a topic with a succinct phrase that summarises its theme or idea. Using Wikipedia document titles as label candidates, we compute neural embeddings for documents and words to select the most relevant labels for topics. Compared to a state-of-the-art topic labelling system, our methodology is simpler, more efficient, and ...

  20. Detection of functional groups and antibodies on microfabricated surfaces by confocal microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Nashat, A.H.; Ferrari, M. [Univ. of California, Berkeley, CA (United States); Moronne, M. [Lawrence Berkeley National Lab., CA (United States)


    Fluorescence confocal microscopy was used to characterize micron-sized microfabricated silicon particles and planar oxides surfaces after silanization and immobilization of IgG antibody. Surfaces treated with amino- and mercaptosilanes were tested by the presence of amine and sulfhydryl groups by labeling with specific fluorescein probes. In addition, human antibody (IgG) was immobilized to the thiol-coated microparticles using the heterobifunctional crosslinker succinimidyl 4-(N-maleimidolmthyl)-cyclohexane-1-carboxylate. Estimates of the surface density of IgG were consistent with 8.3% of a monolayer of covalently-bound antibody. Confocal images confirmed uniform layers of both silanes and antibodies on the microparticles. The sensitivity limit for the confocal measurements was determined to be as low as 1.5 x 10{sup {minus}5} fluors per nm{sup 2}.

  1. Labeling nuclear DNA using DAPI. (United States)

    Chazotte, Brad


    A number of fluorescent stains are available that label DNA and allow easy visualization of the nucleus in interphase cells and chromosomes in mitotic cells, including Hoechst, 4',6-diamidino-2-phenylindole (DAPI), ethidium bromide, propidium iodide, and acridine orange. Although not as bright as the vital Hoechst stains for DNA, DAPI has greater photostability. It is believed that DAPI associates with the minor groove of double-stranded DNA, with a preference for the adenine-thymine clusters. Cells must be permeabilized and/or fixed for DAPI to enter the cell and to bind DNA. Fluorescence increases approximately 20-fold when DAPI is bound to double-stranded DNA. This protocol describes the use of DAPI to label nuclear DNA of cells grown in culture.

  2. White Label Space GLXP Mission (United States)

    Barton, A.


    This poster presents a lunar surface mission concept and corresponding financing approach developed by the White Label Space team, an official competitor in the Google Lunar X PRIZE. The White Label Space team's origins were in the European Space Agency's ESTEC facility in the Netherlands. Accordingly the team's technical headquarters are located just outside ESTEC in the Space Business Park. The team has active partners in Europe, Japan and Australia. The team's goal is to provide a unique publicity opportunity for global brands to land on the moon and win the prestigious Google Lunar X PRIZE. The poster presents the main steps to achieve this goal, the cost estimates for the mission, describes the benefits to the potential sponsors and supporters, and details the progress achieved to date.

  3. Inhibition of Insulin Degradation by Hepatoma Cells after Microinjection of Monoclonal Antibodies to a Specific Cytosolic Protease (United States)

    Shii, Kozui; Roth, Richard A.


    Four monoclonal antibodies were identified by their ability to bind to 125I-labeled insulin covalently linked to a cytosolic insulin-degrading enzyme from human erythrocytes. All four antibodies were also found to remove more than 90% of the insulin-degrading activity from erythrocyte extracts. These antibodies were shown to be directed to different sites on the enzyme by mapping studies and by their various properties. Two antibodies recognized the insulin-degrading enzyme from rat liver; one inhibited the erythrocyte enzyme directly; and two recognized the enzyme after gel electrophoresis and transfer to nitrocellulose filters. By this latter procedure and immunoprecipitation from metabolically labeled cells, the enzyme from a variety of tissues was shown to be composed of a single polypeptide chain of apparent Mr 110,000. Finally, these monoclonal antibodies were microinjected into the cytoplasm of a human hepatoma cell line to assess the contribution of this enzyme to insulin degradation in the intact cell. In five separate experiments, preloading of cells with these monoclonal antibodies resulted in an inhibition of insulin degradation of 18-54% (average 39%) and increased the amount of 125I-labeled insulin associated with the cells. In contrast, microinjection of control antibody or an extraneous monoclonal antibody had no effect on insulin degradation or on the amount of insulin associated with the cells. Moreover, the monoclonal antibodies to the insulin-degrading enzyme caused no significant inhibition of degradation of another molecule, low density lipoprotein. Thus, these results support a role for this enzyme in insulin degradation in the intact cell.

  4. Kinetics of Label Retaining Cells in the Developing Rat Kidneys.

    Directory of Open Access Journals (Sweden)

    Jianwen Wang

    Full Text Available The kidney is a specialized low-regenerative organ with several different types of cellular lineages. The BrdU label-retaining cell (LRCs approach has been used as part of a strategy to identify tissue-specific stem cells in the kidney; however, because the complementary base pairing in double-stranded DNA blocks the access of the anti-BrdU antibody to BrdU subunits, the stem cell marker expression in BrdU-labeled cells are often difficult to detect. In this study, we introduced a new cell labeling and detection method in which BrdU was replaced with 5-ethynyl-2-deoxyuridine (EdU and examined the time-dependent dynamic changes of EdU-labeled cells and potential stem/progenitor markers in the development of kidney.Newborn rats were intraperitoneally injected with EdU, and their kidneys were harvested respectively at different time points at 1 day, 3 days, 1 week, 2 weeks, and 6 weeks post-injection. The kidney tissues were processed for EdU and cellular markers by immunofluorescence staining.At the early stage, LRCs labeled by EdU were 2176.0 ± 355.6 cells at day one in each renal tissue section, but dropped to 168 ± 48.4 cells by week 6. As time increased, the numbers of LRCs were differentially expressed in the renal cortex and papilla. At the postnatal day one, nearly twice as many cells in the cortex were EdU-labeled as compared to the papilla (28.6 ± 3.6% vs. 15.6 ± 3.4%, P<0.05, while there were more LRCs within the renal papilla since the postnatal week one, and at the postnatal week 6, one third as many cells in the cortex were EdU-labeled as compared to the papilla (2.5 ± 0.1% vs. 7.7 ± 2.7%, P<0.05. The long-term LRCs at 6-week time point were associated exclusively with the glomeruli in the cortex and the renal tubules in the papilla. At 6 weeks, the EdU-labeled LRCs combined with expression of CD34, RECA-1, Nestin, and Synaptopodin were discretely but widely distributed within the glomeruli; Stro-1 around the glomeruli; and

  5. Antibodies to Phospholipids and Liposomes: Binding of Antibodies to Cells (United States)


    LIPOSOMES: BINDING OF ANTIBODIES TO CELLS 12. PERSONAL AUTHOR(S) W.E. FOGLER , G. M. SWARTZ, AND C.R. ALVING 13a TYPE OF REPORT 13b. TIME COVERED 14. DATE...Elsevier BBA 73693 Antibodies to phospholipids and liposomes: binding of antibodies to cells William E. Fogler *, Glenn M. Swartz, Jr. and Carl R. Alving...Immunol. 21. Research Associateship from the U.S. National 12863-86812Hall. T. and Esser, K. (1984) 3. Immunol. 132. 2059-2063 Research Council. 13 Fogler

  6. Simultaneous expression of displayed and secreted antibodies for antibody screen.

    Directory of Open Access Journals (Sweden)

    Yuanping Zhou

    Full Text Available The display of full-length antibody on the cell surface was achieved by fusing a transmembrane domain of the platelet-derived growth factor receptor (PDGFR to the C-terminus of the heavy chain constant region. We also incorporated a furin cleavage site between the constant region and PDGFR transmembrane domain to obtain secreted antibodies. As a result, antibodies can be expressed simultaneously on the cell surface in a membrane-anchored version for screening and selecting through fluorescence-activated cell sorting (FACS analysis, as well as in conditioned medium in a secreted version for function analysis.

  7. Politique de label et commerce international



    This article investigates the effects of a public labelling system in a context of international trade. A simple framework allows to present some distortions linked to such a policy. A trade liberalisation situation may lead to a change in the label policy, where the foreign seller incurs the label cost. Classification JEL : F1, D8

  8. What determines consumer attention to nutrition labels?

    NARCIS (Netherlands)

    Bialkova, S.E.; Trijp, van J.C.M.


    To identify the key determinants of consumer attention to nutrition labels, visual search tasks (present – absent; one – two targets) were used as an effective experimental tool. The main manipulation concerned: set size (number of labels on front of pack); label characteristics (display size, posit


    Institute of Scientific and Technical Information of China (English)

    吕宗伟; 林争辉; 张镭


    DAG-MAP is an FPGA technology mapping algorithm for delay optimization and the labeling phase is the algorithm's kernel. This paper studied the labeling phase and presented an improved labeling method. It is shown through the experimental results on MCNC benchmarks that the improved method is more effective than the original method while the computation time is almost the same.

  10. Taste Bud Labeling in Whole Tongue Epithelial Sheet in Adult Mice. (United States)

    Venkatesan, Nandakumar; Boggs, Kristin; Liu, Hong-Xiang


    Molecular labeling in whole-mount tissues provides an efficient way to obtain general information about the formation, maintenance, degeneration, and regeneration of many organs and tissues. However, labeling of lingual taste buds in whole tongue tissues in adult mice has been problematic because of the strong permeability barrier of the tongue epithelium. In this study, we present a simple method for labeling taste buds in the intact tongue epithelial sheet of an adult mouse. Following intralingual protease injection and incubation, immediate fixation of the tongue on mandible in 4% paraformaldehyde enabled the in situ shape of the tongue epithelium to be well maintained after peeling. The peeled epithelium was accessible to taste bud labeling with a pan-taste cell marker, keratin 8, and a type II taste cell marker, α-gustducin, in all three types of taste papillae, that is, fungiform, foliate, and circumvallate. Overnight incubation of tongue epithelial sheets with primary and secondary antibodies was sufficient for intense labeling of taste buds with both fluorescent and DAB visualizations. Labeled individual taste buds were easy to identify and quantify. This protocol provides an efficient way for phenotypic analyses of taste buds, especially regarding distribution pattern and number.

  11. Fluorescent Labeling of Nanometer Hydroxyapatite

    Institute of Scientific and Technical Information of China (English)

    Yuan ZHANG; Yuan YUAN; Changsheng LIU


    A novel surface treatment method using 3-aminopropyltriethoxysilane (AMPTES), was developed to immobilize the fluorescein molecule on nano-HAP (nanometer hydroxyapatite) powders. By pretreating the nano-HAP powders surface with AMPTES, fluorescein, chosen on the basis of the chemical structure of the nano- HAP powders, could be bound to the nano-HAP powders surface. The chemical compositions of nano-HAP before and after being labeled were characterized by Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS). The morphology, phase composition, and the fluorescence characteristics of the nano-HAP powders with and without staining were also investigated. The FTIR and XPS results revealed that fiuorescein had been successfully immobilized on the surface of AMPTES-bound nano-HAP powders via the acylamide bond formation between the -COOH of fluorescein and the -NH2 of AMPTES. The labeled nano-HAP powders possessed strong fluorescent intensity with a little deviation from the maximum emission wavelength of fluorescein. But the morphology and phase composition had no obvious alteration. Under fluorescence microscopy, the labeled nano-HAP powders., even after 24 h cell incubation, exhibited strong fluorescence.

  12. The intensity of immunogold labeling of deplasticized acrylic sections compared to deplasticized epoxy sections-Theoretical deductions and experimental data. (United States)

    Brorson, Sverre-Henning; Reinholt, Finn P


    The purpose of this study was to compare the level of immunogold labeling of deplasticized acrylic sections and deplasticized epoxy sections. Pure protein gels of IgG, albumin and thyroglobulin were produced by glutaraldehyde fixation and embedded in non-crosslinked acrylic resin (Technovit 9100) and epoxy resin (Epon 812), respectively. Ultrathin sections of acrylic and epoxy resin were separately deplasticized in 2-methoxyethyl acetate (MEA) and sodium ethoxide. Quantitative immunogold labeling was performed with anti-IgG, anti-albumin and anti-thyroglobulin antibodies on sections of the corresponding protein gels. For all antibodies tested, the intensity of labeling for deplasticized acrylic sections was significantly higher (two to four times) than for the corresponding deplasticized epoxy sections. The results fit with a theoretically deduced relation: the quotient of the labeling of two deplasticized sections of different resins is equivalent to the square root of the quotient of the labeling of the similar sections not exposed to any kind of pre-treatment. The practical significance of the results is that immunolabeling of deplasticized non-crosslinked acrylic resin results in more intense immunogold labeling than deplasticized epoxy sections. Deplasticizing is most useful when the requirements for ultrastructural preservation according to conventional criteria are moderate. Our theoretically deduced results also indicate that deplasticized Technovit (or other non-crosslinked acrylic resins) sections will be significantly better suited for immunolabeling at the light microscopic level than deplasticized epoxy sections.

  13. Improved radioimmunoscintigraphy of human mammary carcinoma xenografts after injection of an anti-antibody

    Energy Technology Data Exchange (ETDEWEB)

    Senekowitsch, R.; Bode, W.; Glaessner, H.; Moellenstaedt, S.; Kriegel, H.; Reidel, G.; Pabst, H.W.


    The low tumor-to-background ratio obtained after administration of radiolabeled whole monoclonal antibodies (MAbs) is one of the major problems in immunoscintigraphy and -therapy. To reduce the blood pool label caused by the circulation of radiolabeled MAb we have investigated the advantage of injecting an anti-antibody after administration of tumor-specific MAb in nude mice bearing human mammary carcinoma xenografts. The MAb MA 10-11 of rat origin, used in these studies, had shown a high affinity to human mammary carcinoma tissue on frozen sections and low cross-reactivity with various normal human tissues. 24 h after injection of 1.5 MBq /sup 131/I-labeled MAb containing 10 IgG/sub 2a/ one group of mice received an additional injection of 100 anti-rat antibody. Blood taken 2 min after the second antibody injection showed nearly the whole activity bound to antibody aggregates, that cleared very rapidly from the circulation and accumulated in liver and spleen. The transitory high liver activity decreased within several hours because of rapid deiodination of the antibody-complex in this organ. The release of radioactivity from the spleen, however, was found to be much slower. The rapid excretion of the radioactivity from the blood pool combined with a nearly constant tumor activity allowed early tumor detection with tumor-to-blood ratios of 250:1 at 48 h after anti-antibody injection compared to 1.1:1 obtained for the control animals. In addition the results may explain the reported reduction of imaging quality and high uptake of radioactivity in the spleen of patients having repeated injections of mouse MAbs due to complex formation after development of human anti-mouse antibodies

  14. 78 FR 24211 - Draft Guidance for Industry on Safety Considerations for Container Labels and Carton Labeling... (United States)


    ... Container Labels and Carton Labeling Design To Minimize Medication Errors; Availability AGENCY: Food and... the availability of a draft guidance for industry entitled ``Safety Considerations for Container... aspects of the container label and carton labeling design for prescription drug and biological...

  15. Phthalocyanine dye as an extremely photostable and highly fluorescent near-infrared labeling reagent (United States)

    Peng, Xinzhan; Draney, Daniel R.; Volcheck, William M.; Bashford, Gregory R.; Lamb, Donald T.; Grone, Daniel L.; Zhang, Yonghong; Johnson, Craig M.


    Current organic fluorophores used as labeling reagents for biomolecule conjugation have significant limitations in photostability. This compromises their performance in applications that require a photostable fluorescent reporting group. For example, in molecular imaging and single molecule microscopy, photostable fluorescent labels are important for observing and tracking individual molecular events over extended period of time. We report in this paper an extremely photostable and highly fluorescent phthalocyanine dye, IRDye TM 700DX, as a near-infrared fluorescence labeling reagent to conjugate with biomolecules. This novel water-soluble silicon phthalocyanine dye has an isomericly pure chemical structure. The dye is about 45 to 128 times more photostable than current near-IR fluorophores, e.g. Alexa Fluor"R"680, Cy TM 5.5, Cy TM 7 and IRDye TM 800CW dyes; and about 27 times more photostable than tetramethylrhodamine (TMR), one of the most photostable organic dyes. This dye also meets all the other stringent requirements as an ideal fluorophore for biomolecules labeling such as excellent water solubility, no aggregation in high ionic strength buffer, large extinction coefficient and high fluorescent quantum yield. Antibodies conjugated with IRDye TM 700DX at high D/P ratio exist as monomeric species in high ionic buffer and have bright fluorescence. The IRDye TM 700DX conjugated antibodies generate sensitive, highly specific detection with very low background in Western blot and cytoblot assays.

  16. Stigma of a label: educational expectations for high school students labeled with learning disabilities. (United States)

    Shifrer, Dara


    Poorer outcomes for youth labeled with learning disabilities (LDs) are often attributed to the student's own deficiencies or cumulative disadvantage; but the more troubling possibility is that special education placement limits rather than expands these students' opportunities. Labeling theory partially attributes the poorer outcomes of labeled persons to stigma related to labels. This study uses data on approximately 11,740 adolescents and their schools from the Education Longitudinal Survey of 2002 to determine if stigma influences teachers' and parents' educational expectations for students labeled with LDs and labeled adolescents' expectations for themselves. Supporting the predictions of labeling theory, teachers and parents are more likely to perceive disabilities in, and hold lower educational expectations for labeled adolescents than for similarly achieving and behaving adolescents not labeled with disabilities. The negative effect of being labeled with LDs on adolescents' educational expectations is partially mechanized through parents' and particularly teachers' lower expectations.

  17. Antigen Binding and Site-Directed Labeling of Biosilica-Immobilized Fusion Proteins Expressed in Diatoms

    Energy Technology Data Exchange (ETDEWEB)

    Ford, Nicole R.; Hecht, Karen A.; Hu, Dehong; Orr, Galya; Xiong, Yijia; Squier, Thomas; Rorrer, Gregory L.; Roesijadi, Guritno


    The diatom Thalassiosira pseudonana was genetically modified to express biosilica-targeted fusion proteins incorporating a tetracysteine tag for site-directed labeling with biarsenical affinity probes and either EGFP or single chain antibody to test colocalization of probes with the EGFP-tagged recombinant protein or binding of biosilica-immobilized antibodies to large and small molecule antigens, respectively. Site-directed labeling with the biarsenical probes demonstrated colocalization with EGFP-encoded proteins in nascent and mature biosilica, supporting their use in studying biosilica maturation. Isolated biosilica transformed with a single chain antibody against either the Bacillus anthracis surface layer protein EA1 or small molecule explosive trinitrotoluene (TNT) effectively bound the respective antigens. A marked increase in fluorescence lifetime of the TNT surrogate Alexa Fluor 555-trinitrobenzene reflected the high binding specificity of the transformed isolated biosilica. These results demonstrated the potential use of biosilica-immobilized single chain antibodies as binders for large and small molecule antigens in sensing and therapeutics.

  18. Method and apparatus using selected superparamagnetic labels for rapid quantification of immunochromatographic tests. (United States)

    Laitinen, Mika Pa; Salmela, Jari; Gilbert, Leona; Kaivola, Risto; Tikkala, Topi; Oker-Blom, Christian; Pekola, Jukka; Vuento, Matti


    A rapid method and instrumentation for quantification of immunochromatographic tests (ICT) are described. The principle and performance of the method was demonstrated by measuring the levels of human chorionic gonadotropin (hCG) present in urine. The test format was a sandwich assay using two distinct monoclonal antibodies directed against hCG. The first anti-hCG antibody was labeled with superparamagnetic particles whereas the second was immobilized as a narrow detection zone on a porous membrane. The human urine sample was mixed with superparamagnetic particles coated with the first anti-hCG antibody, and the mixture was allowed to migrate past the detection zone containing the second anti-hCG antibody. Capillary forces facilitated migration of the immune complexes along the porous membrane. The amount of superparamagnetic particle-labelled monoclonal anti-hCG bound to the detection zone was directly proportional to the amount of hCG present in the sample as detected by measuring magnetization in the detector coil. The method had a practical detection limit of 20 U/l (54 nM) of hCG per 5 μl of human urine and a linear range of three decades from 20 U/l to 10 000 U/l. In addition, the analysis was completed within less than 10 minutes. Thus, the test format should be suitable for fast detection and monitoring of a large variety of clinically important parameters and analytes.

  19. Development of nano-particles labeled and enzyme free portable medical sensor

    Energy Technology Data Exchange (ETDEWEB)

    Uhm, Young Rang; Rhee, Chang Kyu; Lee, Min Ku; Kim, Jae Woo; Park, Jin Ju; Lee, Gung Ku; Lee, Gyoung Ja


    Development of the approach to the creation of new nonenzymatic biosensors based on immunocomplex 'antigen-antibody' using as signal generating compound Protein / Antibodies / Antigens, labeled with nanoparticles of metals, and using carbon containing nanomaterial as a transducer. Antigens of measles, forest-spring encephalitis or samonella serve as model systems. A technology for synthesis of magnetic Fe{sub 3}O{sub 4} nanoparticles with magnetite structure in 'nanoreactors' - inverse micelles - allowing the particle size regulation with in certain limit sand obtaining nanomaterials with reproducible properties was developed. A method for implementation of covered nanoparticles into microorganisms providing sorption of a reproducible number of nanoparticles on the cells was developed. A technology for production of conjugate of nanoparticles covered with polymeric layer with antibodies was developed.

  20. Tabhu: tools for antibody humanization

    DEFF Research Database (Denmark)

    Olimpieri, Pier Paolo; Marcatili, Paolo; Tramontano, Anna


    Antibodies are rapidly becoming essential tools in the clinical practice, given their ability to recognize their cognate antigens with high specificity and affinity, and a high yield at reasonable costs in model animals. Unfortunately, when administered to human patients, xenogeneic antibodies can...... elicit unwanted and dangerous immunogenic responses. Antibody humanization methods are designed to produce molecules with a better safety profile still maintaining their ability to bind the antigen. This can be accomplished by grafting the non-human regions determining the antigen specificity...

  1. Intracellular uptake and catabolism of anti-IgM antibodies and bi-specific antibody-targeted hapten by B-lymphoma cells. (United States)

    Manetti, C; Le Doussal, J M; Rouvier, E; Gruaz-Guyon, A; Barbet, J


    The efficiency of radioimmunotherapy with iodine-labelled antibodies is often limited by intracellular internalisation and catabolism after initial binding to the cellular targets. We have developed a technique called affinity enhancement system (AES) which uses bi-specific antibodies to target radiolabelled bivalent haptens to cells. This targeting method has been applied successfully to tumour imaging in colorectal cancer patients and is now considered for therapy. We have investigated the potential of this technique to target iodine radioisotopes by comparing it to targeting with covalently iodine-labelled antibodies in a rapidly internalising antigenic system, the surface IgM of a B-lymphoma cell line. A 5-fold increase in the intracellular retention time of activity as compared to 125I-labelled F(ab')2 or IgG was observed. The radiolabelled hapten did not undergo any catabolism after internalisation. Resistance to cellular proteases and failure of recognition of the hapten by amino acid transporter systems may be potential explanations for these observations. This should make non-covalent targeting, particularly the AES, a method of choice to target modulating antigens for the therapy of malignant hemopathies.

  2. An Optimal Labeling Scheme for Ancestry Queries

    CERN Document Server

    Fraigniaud, Pierre


    An ancestry labeling scheme assigns labels (bit strings) to the nodes of rooted trees such that ancestry queries between any two nodes in a tree can be answered merely by looking at their corresponding labels. The quality of an ancestry labeling scheme is measured by its label size, that is the maximal number of bits in a label of a tree node. In addition to its theoretical appeal, the design of efficient ancestry labeling schemes is motivated by applications in web search engines. For this purpose, even small improvements in the label size are important. In fact, the literature about this topic is interested in the exact label size rather than just its order of magnitude. As a result, following the proposal of a simple interval-based ancestry scheme with label size $2\\log_2 n$ bits (Kannan et al., STOC '88), a considerable amount of work was devoted to improve the bound on the size of a label. The current state of the art upper bound is $\\log_2 n + O(\\sqrt{\\log n})$ bits (Abiteboul et al., SODA '02) which is...

  3. Eye tracking and nutrition label use

    DEFF Research Database (Denmark)

    Graham, Dan J.; Orquin, Jacob Lund; Visschers, Vivianne H.M.


    Nutrition labels on food packages are among the most prominent and far-reaching policy measures related to diet and have the capacity to promote healthy eating. Unfortunately, certain nutrition label characteristics may impede consumer detection and comprehension of labels. Research using precise...... cameras monitoring consumer visual attention (i.e., eye tracking) has begun to identify ways in which label design could be modified to improve consumers’ ability to locate and effectively utilize nutrition information. The present paper reviews all published studies of nutrition label use that have...... utilized eye tracking methodology, identifies directions for further research in this growing field, and makes research-based recommendations for ways in which labels could be modified to improve consumers’ ability to use nutrition labels to select healthful foods....

  4. Nephritogenic lupus antibodies recognize glomerular basement membrane-associated chromatin fragments released from apoptotic intraglomerular cells. (United States)

    Kalaaji, Manar; Mortensen, Elin; Jørgensen, Leif; Olsen, Randi; Rekvig, Ole Petter


    Antibodies to dsDNA represent a classification criterion for systemic lupus erythematosus. Subpopulations of these antibodies are involved in lupus nephritis. No known marker separates nephritogenic from non-nephritogenic anti-dsDNA antibodies. It is not clear whether specificity for glomerular target antigens or intrinsic antibody-affinity for dsDNA or nucleosomes is a critical parameter. Furthermore, it is still controversial whether glomerular target antigen(s) is constituted by nucleosomes or by non-nucleosomal glomerular structures. Previously, we have demonstrated that antibodies eluted from murine nephritic kidneys recognize nucleosomes, but not other glomerular antigens. In this study, we determined the structures that bind nephritogenic autoantibodies in vivo by transmission electron microscopy, immune electron microscopy, and colocalization immune electron microscopy using experimental antibodies to dsDNA, to histones and transcription factors, or to laminin. The data obtained are consistent and point at glomerular basement membrane-associated nucleosomes as target structures for the nephritogenic autoantibodies. Terminal deoxynucleotidyl-transferase-mediated dUTP nick end-labeling or caspase-3 assays demonstrate that lupus nephritis is linked to intraglomerular cell apoptosis. The data suggest that nucleosomes are released by apoptosis and associate with glomerulus basement membranes, which may then be targeted by pathogenic anti-nucleosome antibodies. Thus, apoptotic nucleosomes may represent both inducer and target structures for nephritogenic autoantibodies in systemic lupus erythematosus.

  5. Development of Double Antibody Sandwich ELISA for Detection of Duck or Goose Flavivirus

    Institute of Scientific and Technical Information of China (English)

    NIU Hui-min; LI Xiang-rui; LI Yin; HUANG Xin-mei; HAN Kai-kai; LIU Yu-zhuo; ZHAO Dong-min; ZHANG Jing-feng; LIU Fei; LI Tong-tong; ZHOU Xiao-bo


    In order to establish double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) for detection of duck or goose flavivirus, polyclonal antibody against the flavivirus strain JS804 in geese and monoclonal antibody against the E protein of flavivirus strain JS804 in geese were used as the capture antibody and detection antibody, respectively. The optimal dilution of the capture antibody and detecting antibody capable of detecting the flavivirus strain JS804 in geese were 1:3 200 and 1:160 in the check-board titration, respectively. The reaction time of sample was 1 h, and the optimal working dilution of HRP-labeled goat-anti-mouse IgG was 1:10 000. The positive standard value was 0.247 (OD450 nm). The geese flavivirus could be detected at a minimal concentration of 1.875μg mL-1. The ELISA had no cross-reaction with Newcastle disease virus (NDV), Avian influenza virus (AIV), Infectious bronchitis virus (IBV), Infectious bursal disease virus (IBDV), Duck hepatitis virus (DHV), and Gosling plague virus (GPV). Twenty clinical samples were detected by the DAS-ELISA and RT-PCR respectively, with the agreement rate of 75%. The results revealed that the DAS-ELISA possessed favorable specificity and higher sensitivity, indicating a suitable method for rapid detection of the duck or goose flavivirus.

  6. DARPA Antibody Technology Program Standardized Test Bed for Antibody Characterization: Characterization of an MS2 ScFv Antibody (United States)



  7. Phase sensitive spectral domain interferometry for label free biomolecular interaction analysis and biosensing applications (United States)

    Chirvi, Sajal

    -channel label-free biosensing applications is introduced. Simultaneous interrogation of multiple biosensors is achievable with a single spectral domain phase sensitive interferometer by coding the individual sensograms in coherence-multiplexed channels. Experimental results demonstrating multiplexed quantitative biomolecular interaction analysis of antibodies binding to antigen coated functionalized biosensor chip surfaces on different platforms are presented.

  8. Whole-body effective half-lives for radiolabeled antibodies and related issues

    Energy Technology Data Exchange (ETDEWEB)

    Kaurin, D.G.L.; Carsten, A.L.; Baum, J.W.; Barber, D.E.


    Radiolabeled antibodies (RABs) are being developed and used in medical imaging and therapy in rapidly increasing numbers. Data on the whole body half effective half-lives were calculated from external dose rates obtained from attending physicians and radiation safety officers at participating institutions. Calculations were made using exponential regression analysis of data from patients receiving single and multiple administrations. Theses data were analyzed on the basis of age, sex, isotope label, radiation energy, antibody type, disease treated, administration method, and number of administrations.

  9. A radiomicroassay for cytotoxic antibody to human spermatozoa. Quantification by tritiated actinomycin d. (United States)

    Sung, J S; Shizuya, H; Black, D D; Mumford, D M


    A radiomicroassay for titration of spermocytotoxic antibody is described. The assay used [3H]AACTINOMYCIN D ([3H]Act D) to label damaged spermatozoa in a fashion analogous to penetration by vital dye. Optimal conditions for and some kinetics of the assay are presented. The assay is sensitive, reliable, simple to perform and uses only small amounts of serum and spermatozoa. Applied to sperm antibody positive human postvasectomy sera, the assay compared favourably in sensitivity eith vital dye microscopic observations and with parallel titration by the Isojima's immobilization tests.

  10. Evolution of antiphospholipid antibody syndrome. (United States)

    Baviskar, Rutuja R; Amonkar, Gayathri P; Chaudhary, Vinod A; Balasubramanian, Meenakshi; Mohite, Shailesh C; Puranik, Gururaj V


    Antiphospholipid antibody syndrome is a very important cause of cerebral infarction, myocardial infarction, and repeated pregnancy losses in women. We present an extremely rare case of a 44-year-old man with antiphospholipid syndrome who collapsed and died suddenly. At autopsy, he was found to have both cerebral and myocardial infarction. In all young patients with cerebral infarction, myocardial infarction, pulmonary embolism, recurrent miscarriages, and unexplained low platelet count, one must consider the strong possibility of antiphospholipid antibody syndrome.

  11. Antibodies to watch in 2016


    Reichert, Janice M


    The number of novel antibody therapeutics that received first marketing approvals in 2015 met expectations, with 6 (alirocumab (Praluent®), evolocumab (Repatha®), daratumumab (Darzalex®), dinutuximab (Unituxin®), idarucizumab (Praxbind®), mepolizumab (Nucala®)) granted first approvals as of mid-November*. Seven novel antibody therapeutics (begelomab, brodalumab, elotuzumab, ixekizumab, necitumumab, obiltoxaximab, reslizumab) are in regulatory review, and thus a similar number, if not more, ar...

  12. Hemoglobin Labeled by Radioactive Lysine (United States)

    Bale, W. F.; Yuile, C. L.; DeLaVergne, L.; Miller, L. L.; Whipple, G. H.


    This paper reports on the utilization of tagged epsilon carbon of DL-lysine by a dog both anemic and hypoproteinemic due to repeated bleeding plus a diet low in protein. The experiment extended over period of 234 days, a time sufficient to indicate an erythrocyte life span of at least 115 days based upon the rate of replacement of labeled red cell proteins. The proteins of broken down red cells seem not to be used with any great preference for the synthesis of new hemoglobin.

  13. Pretargeted PET Imaging Using a Site-Specifically Labeled Immunoconjugate. (United States)

    Cook, Brendon E; Adumeau, Pierre; Membreno, Rosemery; Carnazza, Kathryn E; Brand, Christian; Reiner, Thomas; Agnew, Brian J; Lewis, Jason S; Zeglis, Brian M


    In recent years, both site-specific bioconjugation techniques and bioorthogonal pretargeting strategies have emerged as exciting technologies with the potential to improve the safety and efficacy of antibody-based nuclear imaging. In the work at hand, we have combined these two approaches to create a pretargeted PET imaging strategy based on the rapid and bioorthogonal inverse electron demand Diels-Alder reaction between a (64)Cu-labeled tetrazine radioligand ((64)Cu-Tz-SarAr) and a site-specifically modified huA33-trans-cyclooctene immunoconjugate ((ss)huA33-PEG12-TCO). A bioconjugation strategy that harnesses enzymatic transformations and strain-promoted azide-alkyne click chemistry was used to site-specifically append PEGylated TCO moieties to the heavy chain glycans of the colorectal cancer-targeting huA33 antibody. Preclinical in vivo validation studies were performed in athymic nude mice bearing A33 antigen-expressing SW1222 human colorectal carcinoma xenografts. To this end, mice were administered (ss)huA33-PEG12-TCO via tail vein injection and-following accumulation intervals of 24 or 48 h-(64)Cu-Tz-SarAr. PET imaging and biodistribution studies reveal that this strategy clearly delineates tumor tissue as early as 1 h post-injection (6.7 ± 1.7%ID/g at 1 h p.i.), producing images with excellent contrast and high tumor-to-background activity concentration ratios (tumor:muscle = 21.5 ± 5.6 at 24 h p.i.). Furthermore, dosimetric calculations illustrate that this pretargeting approach produces only a fraction of the overall effective dose (0.0214 mSv/MBq; 0.079 rem/mCi) of directly labeled radioimmunoconjugates. Ultimately, this method effectively facilitates the high contrast pretargeted PET imaging of colorectal carcinoma using a site-specifically modified immunoconjugate.

  14. Tabhu: tools for antibody humanization.

    KAUST Repository

    Olimpieri, Pier Paolo


    SUMMARY: Antibodies are rapidly becoming essential tools in the clinical practice, given their ability to recognize their cognate antigens with high specificity and affinity, and a high yield at reasonable costs in model animals. Unfortunately, when administered to human patients, xenogeneic antibodies can elicit unwanted and dangerous immunogenic responses. Antibody humanization methods are designed to produce molecules with a better safety profile still maintaining their ability to bind the antigen. This can be accomplished by grafting the non-human regions determining the antigen specificity into a suitable human template. Unfortunately, this procedure may results in a partial or complete loss of affinity of the grafted molecule that can be restored by back-mutating some of the residues of human origin to the corresponding murine ones. This trial-and-error procedure is hard and involves expensive and time-consuming experiments. Here we present tools for antibody humanization (Tabhu) a web server for antibody humanization. Tabhu includes tools for human template selection, grafting, back-mutation evaluation, antibody modelling and structural analysis, helping the user in all the critical steps of the humanization experiment protocol. AVAILABILITY: CONTACT:, SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

  15. Avian Diagnostic and Therapeutic Antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Bradley, David Sherman [UND SMHS


    A number of infectious agents have the potential of causing significant clinical symptomology and even death, but dispite this, the number of incidence remain below the level that supports producing a vaccine. Therapeutic antibodies provide a viable treatment option for many of these diseases. We proposed that antibodies derived from West Nile Virus (WNV) immunized geese would be able to treat WNV infection in mammals and potential humans. We demonstrated that WNV specific goose antibodies are indeed successful in treating WNV infection both prophylactically and therapeutically in a golden hamster model. We demonstrated that the goose derived antibodies are non-reactogenic, i.e. do not cause an inflammatory response with multiple exposures in mammals. We also developed both a specific pathogen free facility to house the geese during the antibody production phase and a patent-pending purification process to purify the antibodies to greater than 99% purity. Therefore, the success of these study will allow a cost effective rapidly producible therapeutic toward clinical testing with the necessary infrastructure and processes developed and in place.

  16. Labeling and imaging cells in the zebrafish hindbrain. (United States)

    Jayachandran, Pradeepa; Hong, Elim; Brewster, Rachel


    Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish embryos, injection of plasmid DNA results in mosaic expression, allowing for the visualization of single cells or small clusters of cells (1) . We describe how injection of plasmid DNA encoding membrane-targeted Green Fluorescent Protein (mGFP) under the control of a ubiquitous promoter can be used for imaging cells undergoing neurulation. Central to this protocol is the methodology for imaging labeled cells at high resolution in sections and also in real time. This protocol entails the injection of mGFP DNA into young zebrafish embryos. Embryos are then processed for vibratome sectioning, antibody labeling and imaging with a confocal microscope. Alternatively, live embryos expressing mGFP can be imaged using time-lapse confocal microscopy. We have previously used this straightforward approach to analyze the cellular behaviors that drive neural tube formation in the hindbrain region of zebrafish embryos (2). The fixed preparations allowed for unprecedented visualization of cell shapes and organization in the neural tube while live imaging complemented this approach enabling a better understanding of the cellular dynamics that take place during neurulation.

  17. Link Label Prediction in Signed Citation Network

    KAUST Repository

    Akujuobi, Uchenna


    Link label prediction is the problem of predicting the missing labels or signs of all the unlabeled edges in a network. For signed networks, these labels can either be positive or negative. In recent years, different algorithms have been proposed such as using regression, trust propagation and matrix factorization. These approaches have tried to solve the problem of link label prediction by using ideas from social theories, where most of them predict a single missing label given that labels of other edges are known. However, in most real-world social graphs, the number of labeled edges is usually less than that of unlabeled edges. Therefore, predicting a single edge label at a time would require multiple runs and is more computationally demanding. In this thesis, we look at link label prediction problem on a signed citation network with missing edge labels. Our citation network consists of papers from three major machine learning and data mining conferences together with their references, and edges showing the relationship between them. An edge in our network is labeled either positive (dataset relevant) if the reference is based on the dataset used in the paper or negative otherwise. We present three approaches to predict the missing labels. The first approach converts the label prediction problem into a standard classification problem. We then, generate a set of features for each edge and then adopt Support Vector Machines in solving the classification problem. For the second approach, we formalize the graph such that the edges are represented as nodes with links showing similarities between them. We then adopt a label propagation method to propagate the labels on known nodes to those with unknown labels. In the third approach, we adopt a PageRank approach where we rank the nodes according to the number of incoming positive and negative edges, after which we set a threshold. Based on the ranks, we can infer an edge would be positive if it goes a node above the

  18. Label-free biosensor based on long period grating (United States)

    Baldini, Francesco; Chiavaioli, Francesco; Giannetti, Ambra; Brenci, Massimo; Trono, Cosimo


    Long period gratings have been recently proposed as label-free optical devices for biochemical sensing. A biochemical interaction along the grating region changes the biolayer refractive index and a change in the fiber transmission spectrum occurs. The fiber biofunctionalization was performed with a novel chemistry using Eudragit L100 copolymer as opposed to the commonly-used silanization procedure. An IgG/anti-IgG bioassay was carried out for studying the antigen/antibody interaction. The biosensor was fully characterized, monitoring the kinetics during the antibody immobilization and achieving the calibration curve of the assay. To compare the biosensor performance, two LPG-based biosensors with distinct grating periods were characterized following the same bioassay protocol. Experimental results demonstrated an enhancement of the biosensor performance when the fundamental core mode of a single-mode fiber couples with a higher order cladding mode. Considering an LPG manufactured on a bare optical fiber, in which the coupling occurs with the 7-th cladding mode, a dynamic signal range of 0.33 nm, a working range of 1.7 - 1450 mg L-1 and a LOD of 500 μg L-1 were achieved

  19. Label Space Reduction in MPLS Networks: How Much Can A Single Stacked Label Do?

    DEFF Research Database (Denmark)

    Solano, Fernando; Stidsen, Thomas K.; Fabregat, Ramon


    Most network operators have considered reducing LSR label spaces (number of labels used) as a way of simplifying management of underlaying virtual private networks (VPNs) and therefore reducing operational expenditure (OPEX). The IETF outlined the label merging feature in MPLS-allowing the config......Most network operators have considered reducing LSR label spaces (number of labels used) as a way of simplifying management of underlaying virtual private networks (VPNs) and therefore reducing operational expenditure (OPEX). The IETF outlined the label merging feature in MPLS...

  20. Biochemical subtypes of oligodendrocyte in the anterior medullary velum of the rat as revealed by the monoclonal antibody Rip. (United States)

    Butt, A M; Ibrahim, M; Ruge, F M; Berry, M


    Oligodendrocytes were studied in the anterior medullary velum (AMV) of the rat using the monoclonal antibody Rip, an oligodendrocyte marker of unknown function. Confocal microscopic imaging of double immunofluorescent labelling with antibodies to Rip and carbonic anhydrase II (CAII) revealed two biochemically and morphologically distinct populations of oligodendrocyte which were either Rip+CAII+ or Rip+CAII-. Double immunofluorescent labelling with Rip and myelin basic protein (MBP) or glial fibrillary acidic protein (GFAP) provided direct evidence that Rip-labelled cells were phenotypically oligodendrocytes and confirmed that Rip did not recognise astrocytes. Oligodendrocytes which were Rip+CAII+ supported numerous myelin sheaths for small diameter axons, whilst Rip+CAII- oligodendrocytes supported fewer myelin sheaths for large diameter axons. Morphologically, Rip+CAII+ oligodendrocytes corresponded to types I or II of classical nomenclature, whilst Rip+CAII- oligodendrocytes corresponded to types III and IV. The results demonstrated a biochemical difference between oligodendrocytes which myelinated small and large diameter fibres.

  1. Hybrid aptamer-antibody linked fluorescence resonance energy transfer based detection of trinitrotoluene. (United States)

    Sabherwal, Priyanka; Shorie, Munish; Pathania, Preeti; Chaudhary, Shilpa; Bhasin, K K; Bhalla, Vijayender; Suri, C Raman


    Combining synthetic macromolecules and biomolecular recognition units are promising in developing novel diagnostic and analysis techniques for detecting environmental and/or clinically important substances. Fluorescence resonance energy transfer (FRET) apta-immunosensor for explosive detection is reported using 2,4,6-trinitrotoluene (TNT) specific aptamer and antibodies tagged with respective FRET pair dyes in a sandwich immunoassay format. FITC-labeled aptamer was used as a binder molecule in the newly developed apta-immunoassay format where the recognition element was specific anti-TNT antibody labeled with rhodamine isothiocyanate. The newly developed sensing platform showed excellent sensitivity with a detection limit of the order of 0.4 nM presenting a promising candidate for routine screening of TNT in samples.

  2. Quantitative analysis of anti-CEA antibody accumulation in human colorectal carcinomas

    Energy Technology Data Exchange (ETDEWEB)

    Bares, R. (Technische Hochschule Aachen (Germany). Dept. of Nuclear Medicine); Fass, J. (Technische Hochschule Aachen (Germany). Dept. of Surgery); Hauptmann, S. (Technische Hochschule Aachen (Germany). Dept. of Pathology); Braun, J. (Technische Hochschule Aachen (Germany). Dept. of Surgery); Grehl, O. (Technische Hochschule Aachen (Germany). Dept. of Nuclear Medicine); Reinartz, R. (Technische Hochschule Aachen (Germany). Dept. of Surgery); Buell, U. (Technische Hochschule Aachen (Germany). Dept. of Nuclear Medicine); Schumpelick, V. (Technische Hochschule Aachen (Germany). Dept. of Surgery); Mittermayer, C. (Technische Hochschule Aachen (Germany). Dept. of Pathology)


    In order to obtain quantitative data on intratumoral anitbody accumulation we analyzed resected colorectal adenocarcinomas of 25 patients who had undergone immunoscintigraphy 4-14 days earlier. All had been injected with 0.5-1.0 mg intact anti-CEA antibody (BW 431/26) labelled with 70-80 MBq [sup 131]I. Correlation of tissue radioactivity concentrations with tumor characteristics revealed significantly higher values in necrotic compared to viable as well as in CEA-positive compared to CEA-negative viable tumor tissue indicating action of both specific and unspecific uptake mechanisms. In contrast, diagnostic results of immunoscintigraphy were influenced by tumor size only. 11 of 12 falsenegative findings were obtained in tumors <4 cm in diameter. Since ex-vivo scintigraphy of resected specimens correctly visualized all but one of these lesions, it is concluded that technical limitations of scintigraphy are the main cause of negative results of radioimmunoimaging with [sup 131]I-labelled antibodies. (orig.)

  3. Production and Purification of Polyclonal Antibodies. (United States)

    Nakazawa, Masami; Mukumoto, Mari; Miyatake, Kazutaka


    Polyclonal antibodies consist of a mixture of antibodies produced by multiple B-cell clones that have differentiated into antibody-producing plasma cells in response to an immunogen. Polyclonal antibodies raised against an antigen recognize multiple epitopes on a target molecule, which results in a signal amplification in indirect immunoassays including immune-electron microscopy. In this chapter, we present a basic procedure to generate polyclonal antibodies in rabbits.

  4. Simplified protocol for flow cytometry analysis of fluorescently labeled exosomes and microvesicles using dedicated flow cytometer

    Directory of Open Access Journals (Sweden)

    Vendula Pospichalova


    Full Text Available Flow cytometry is a powerful method, which is widely used for high-throughput quantitative and qualitative analysis of cells. However, its straightforward applicability for extracellular vesicles (EVs and mainly exosomes is hampered by several challenges, reflecting mostly the small size of these vesicles (exosomes: ~80–200 nm, microvesicles: ~200–1,000 nm, their polydispersity, and low refractive index. The current best and most widely used protocol for beads-free flow cytometry of exosomes uses ultracentrifugation (UC coupled with floatation in sucrose gradient for their isolation, labeling with lipophilic dye PKH67 and antibodies, and an optimized version of commercial high-end cytometer for analysis. However, this approach requires an experienced flow cytometer operator capable of manual hardware adjustments and calibration of the cytometer. Here, we provide a novel and fast approach for quantification and characterization of both exosomes and microvesicles isolated from cell culture media as well as from more complex human samples (ascites of ovarian cancer patients suitable for multiuser labs by using a flow cytometer especially designed for small particles, which can be used without adjustments prior to data acquisition. EVs can be fluorescently labeled with protein-(Carboxyfluoresceinsuccinimidyl ester, CFSE and/or lipid- (FM specific dyes, without the necessity of removing the unbound fluorescent dye by UC, which further facilitates and speeds up the characterization of microvesicles and exosomes using flow cytometry. In addition, double labeling with protein- and lipid-specific dyes enables separation of EVs from common contaminants of EV preparations, such as protein aggregates or micelles formed by unbound lipophilic styryl dyes, thus not leading to overestimation of EV numbers. Moreover, our protocol is compatible with antibody labeling using fluorescently conjugated primary antibodies. The presented methodology opens the

  5. Simplified protocol for flow cytometry analysis of fluorescently labeled exosomes and microvesicles using dedicated flow cytometer. (United States)

    Pospichalova, Vendula; Svoboda, Jan; Dave, Zankruti; Kotrbova, Anna; Kaiser, Karol; Klemova, Dobromila; Ilkovics, Ladislav; Hampl, Ales; Crha, Igor; Jandakova, Eva; Minar, Lubos; Weinberger, Vit; Bryja, Vitezslav


    Flow cytometry is a powerful method, which is widely used for high-throughput quantitative and qualitative analysis of cells. However, its straightforward applicability for extracellular vesicles (EVs) and mainly exosomes is hampered by several challenges, reflecting mostly the small size of these vesicles (exosomes: ~80-200 nm, microvesicles: ~200-1,000 nm), their polydispersity, and low refractive index. The current best and most widely used protocol for beads-free flow cytometry of exosomes uses ultracentrifugation (UC) coupled with floatation in sucrose gradient for their isolation, labeling with lipophilic dye PKH67 and antibodies, and an optimized version of commercial high-end cytometer for analysis. However, this approach requires an experienced flow cytometer operator capable of manual hardware adjustments and calibration of the cytometer. Here, we provide a novel and fast approach for quantification and characterization of both exosomes and microvesicles isolated from cell culture media as well as from more complex human samples (ascites of ovarian cancer patients) suitable for multiuser labs by using a flow cytometer especially designed for small particles, which can be used without adjustments prior to data acquisition. EVs can be fluorescently labeled with protein-(Carboxyfluoresceinsuccinimidyl ester, CFSE) and/or lipid- (FM) specific dyes, without the necessity of removing the unbound fluorescent dye by UC, which further facilitates and speeds up the characterization of microvesicles and exosomes using flow cytometry. In addition, double labeling with protein- and lipid-specific dyes enables separation of EVs from common contaminants of EV preparations, such as protein aggregates or micelles formed by unbound lipophilic styryl dyes, thus not leading to overestimation of EV numbers. Moreover, our protocol is compatible with antibody labeling using fluorescently conjugated primary antibodies. The presented methodology opens the possibility for routine

  6. Amphiregulin Antibody and Reduction of Axial Elongation in Experimental Myopia

    Directory of Open Access Journals (Sweden)

    Wen Jun Jiang


    Full Text Available To examine the mechanism of ocular axial elongation in myopia, guinea pigs (age: 2–3 weeks which either underwent unilateral or bilateral lens-induced myopization (group 1 or which were primarily myopic at baseline (group 2 received unilateral intraocular injections of amphiregulin antibody (doses: 5, 10, or 15 μg three times in intervals of 9 days. A third group of emmetropic guinea pigs got intraocular unilateral injections of amphiregulin (doses: 0.25, 0.50 or 1.00 ng, respectively. In each group, the contralateral eyes received intraocular injections of Ringer's solution. In intra-animal inter-eye comparison and intra-eye follow-up comparison in groups 1 and 2, the study eyes as compared to the contralateral eyes showed a dose-dependent reduction in axial elongation. In group 3, study eyes and control eyes did not differ significantly in axial elongation. Immunohistochemistry revealed amphiregulin labelling at the retinal pigment epithelium in eyes with lens-induced myopization and Ringer's solution injection, but not in eyes with amphiregulin antibody injection. Intraocular injections of amphiregulin-antibody led to a reduction of lens-induced axial myopic elongation and of the physiological eye enlargement in young guinea pigs. In contrast, intraocularly injected amphiregulin in a dose of ≤1 ng did not show a significant effect. Amphiregulin may be one of several essential molecular factors for axial elongation.

  7. Thrombus imaging in a primate model with antibodies specific for an external membrane protein of activated platelets

    Energy Technology Data Exchange (ETDEWEB)

    Palabrica, T.M.; Furie, B.C.; Konstam, M.A.; Aronovitz, M.J.; Connolly, R.; Brockway, B.A.; Ramberg, K.L.; Furie, B.


    The activated platelet is a potential target for the localization of thrombi in vivo since, after stimulation and secretion of granule contents, activated platelets are concentrated at sites of blood clot formation. In this study, we used antibodies specific for a membrane protein of activated platelets to detect experimental thrombi in an animal model. PADGEM (platelet activation-dependent granule-external membrane protein), a platelet alpha-granule membrane protein, is translocated to the plasma membrane during platelet activation and granule secretion. Since PADGEM is internal in unstimulated platelets, polyclonal anti-PADGEM and monoclonal KC4 antibodies do not bind to circulating resting platelets but do interact with activated platelets. Dacron graft material incubated with radiolabeled KC4 or anti-PADGEM antibodies in the presence of thrombin-activated platelet-rich plasma bound most of the antibody. Imaging experiments with 123I-labeled anti-PADGEM in baboons with an external arterial-venous Dacron shunt revealed rapid uptake in the thrombus induced by the Dacron graft; control experiments with 123I-labeled nonimmune IgG exhibited minimal uptake. Deep venous thrombi, formed by using percutaneous balloon catheters to stop blood flow in the femoral vein of baboons, were visualized with 123I-labeled anti-PADGEM. Thrombi were discernible against blood pool background activity without subtraction techniques within 1 hr. No target enhancement was seen with 123I-labeled nonimmune IgG. 123I-labeled anti-PADGEM cleared the blood pool with an initial half-disappearance time of 6 min and did not interfere with hemostasis. These results indicate that radioimmunoscintigraphy with anti-PADGEM antibodies can visualize thrombi in baboon models and is a promising technique for clinical thrombus detection in humans.

  8. Extending Modal Transition Systems with Structured Labels

    DEFF Research Database (Denmark)

    Bauer, Sebastian S.; Juhl, Line; Larsen, Kim Guldstrand


    We introduce a novel formalism of label-structured modal transition systems that combines the classical may/must modalities on transitions with structured labels that represent quantitative aspects of the model. On the one hand, the specification formalism is general enough to include models like...... study modal and thorough refinement, determinization, parallel composition, conjunction, quotient, and logical characterization of label-structured modal transition systems....

  9. Easy and Efficient 111Indium Labeling of Long-Term Stored DTPA Conjugated Protein

    DEFF Research Database (Denmark)

    Nalla, Amarnadh; Buch, Inge; Hesse, Birger


    targeting vehicles such as monoclonal antibodies and receptor directed peptides. The purpose of this study was to monitor the labelling efficiency of a DTPA-conjugate on long-term storage using 111In-chloride at two different temperatures and incubation times for the In-labelling. Method: Cyclic......-diethylene-triamine-pentaacetic acid (cDTAP) was conjugated to a polyclonal immunoglobulin-G (IgG) in borate buffer, pH 8.2 at +4?C for 4 hours. Then the DTPA-conjugate was dialyzed against 50 mmol/l sodium citrate buffer saline, pH 6.0 and stored at -80° C in aliquots of 1 mg/0.5 ml. The DTPA-conjugate was labeled with 111In...... months. Conclusion: Our study shows that 111In-labelling can easily be performed within 30 min at RT for thermo-stable proteins like polyclonal, DTPA-conjugated IgG stored long-term at -80°C with a high 111In-labelling efficiency....

  10. Easy and efficient (111)Indium labeling of long-term stored DTPA conjugated protein

    DEFF Research Database (Denmark)

    Nalla, Amarnadh; Buch, Inge; Hesse, Birger


    targeting vehicles such as monoclonal antibodies and receptor directed peptides. The purpose of this study was to monitor the labelling efficiency of a DTPA-conjugate on long-term storage using 111In-chloride at two different temperatures and incubation times for the In-labelling. Method: Cyclic......-diethylene-triamine-pentaacetic acid (cDTAP) was conjugated to a polyclonal immunoglobulin-G (IgG) in borate buffer, pH 8.2 at +4oC for 4 hours. Then the DTPA-conjugate was dialyzed against 50 mmol/l sodium citrate buffer saline, pH 6.0 and stored at -80o C in aliquots of 1 mg/0.5 ml. The DTPA-conjugate was labeled with 111In...... months. Conclusion: Our study shows that 111In-labelling can easily be performed within 30 min at RT for thermo-stable proteins like polyclonal, DTPA-conjugated IgG stored long-term at -80oC with a high 111In-labelling effi- ciency....

  11. Functional alterations of human platelets following indium-111 labelling using different incubation media and labelling agents

    Energy Technology Data Exchange (ETDEWEB)

    Isaka, Yoshinari; Imaizumi, Masatoshi (Osaka National Hospital (Japan). Dept. of Cardiovascular Medicine and Radiological Science); Kimura, Kazufumi (Osaka Univ. (Japan). Dept. of Nuclear Medicine); Matsumoto, Masayasu; Kamada, Takenobu (Osaka Univ. (Japan). 1. Dept. of Internal Medicine)


    Human platelets were labelled in the absence of presence of plasma using {sup 111}In-labelled oxine sulphate, tropolone or 2-mercaptopyridine-N-oxide (MPO). Under in vitro and in vivo conditions, platelet functions were evaluated by measuring their aggregability, survival, recovery and early distribution. High labelling efficiency was achieved in saline labelling, whereas with plasma labelling, it was necessary to concentrate the platelet-rich plasma to 4.8x10{sup 6} platelets/{mu}l. The aggregation of platelets labelled in plasma or saline was compared with that of controls; platelets labelled in saline showed lower aggregability in 2 {mu}M ADP but not in 5 {mu}M ADP nor with collagen. No significant differences in platelet survival and recovery were noted between platelets labelled in plasma and those labelled in saline. Our results indicate that partial loss of ADP aggregability in vitro does not influence the in vivo viability of platelets labelled in saline. Scintigraphic studies showed that platelets labelled in a saline medium were temporarily sequestrated in the liver but not in the spleen or heart. Thus, platelet labelling in saline does not affect platelet function adversely, but platelets labelled in plasma are more desirable for assessing the early distribution of platelets in the reticuloendothelial system. (orig.).

  12. ML-MG: Multi-label Learning with Missing Labels Using a Mixed Graph

    KAUST Repository

    Wu, Baoyuan


    This work focuses on the problem of multi-label learning with missing labels (MLML), which aims to label each test instance with multiple class labels given training instances that have an incomplete/partial set of these labels (i.e. some of their labels are missing). To handle missing labels, we propose a unified model of label dependencies by constructing a mixed graph, which jointly incorporates (i) instance-level similarity and class co-occurrence as undirected edges and (ii) semantic label hierarchy as directed edges. Unlike most MLML methods, We formulate this learning problem transductively as a convex quadratic matrix optimization problem that encourages training label consistency and encodes both types of label dependencies (i.e. undirected and directed edges) using quadratic terms and hard linear constraints. The alternating direction method of multipliers (ADMM) can be used to exactly and efficiently solve this problem. To evaluate our proposed method, we consider two popular applications (image and video annotation), where the label hierarchy can be derived from Wordnet. Experimental results show that our method achieves a significant improvement over state-of-the-art methods in performance and robustness to missing labels.

  13. Optimal design of isotope labeling experiments. (United States)

    Yang, Hong; Mandy, Dominic E; Libourel, Igor G L


    Stable isotope labeling experiments (ILE) constitute a powerful methodology for estimating metabolic fluxes. An optimal label design for such an experiment is necessary to maximize the precision with which fluxes can be determined. But often, precision gained in the determination of one flux comes at the expense of the precision of other fluxes, and an appropriate label design therefore foremost depends on the question the investigator wants to address. One could liken ILE to shadows that metabolism casts on products. Optimal label design is the placement of the lamp; creating clear shadows for some parts of metabolism and obscuring others.An optimal isotope label design is influenced by: (1) the network structure; (2) the true flux values; (3) the available label measurements; and, (4) commercially available substrates. The first two aspects are dictated by nature and constrain any optimal design. The second two aspects are suitable design parameters. To create an optimal label design, an explicit optimization criterion needs to be formulated. This usually is a property of the flux covariance matrix, which can be augmented by weighting label substrate cost. An optimal design is found by using such a criterion as an objective function for an optimizer. This chapter uses a simple elementary metabolite units (EMU) representation of the TCA cycle to illustrate the process of experimental design of isotope labeled substrates.

  14. Simultaneous Segmentation and Statistical Label Fusion. (United States)

    Asman, Andrew J; Landmana, Bennett A


    Labeling or segmentation of structures of interest in medical imaging plays an essential role in both clinical and scientific understanding. Two of the common techniques to obtain these labels are through either fully automated segmentation or through multi-atlas based segmentation and label fusion. Fully automated techniques often result in highly accurate segmentations but lack the robustness to be viable in many cases. On the other hand, label fusion techniques are often extremely robust, but lack the accuracy of automated algorithms for specific classes of problems. Herein, we propose to perform simultaneous automated segmentation and statistical label fusion through the reformulation of a generative model to include a linkage structure that explicitly estimates the complex global relationships between labels and intensities. These relationships are inferred from the atlas labels and intensities and applied to the target using a non-parametric approach. The novelty of this approach lies in the combination of previously exclusive techniques and attempts to combine the accuracy benefits of automated segmentation with the robustness of a multi-atlas based approach. The accuracy benefits of this simultaneous approach are assessed using a multi-label multi- atlas whole-brain segmentation experiment and the segmentation of the highly variable thyroid on computed tomography images. The results demonstrate that this technique has major benefits for certain types of problems and has the potential to provide a paradigm shift in which the lines between statistical label fusion and automated segmentation are dramatically blurred.


    DEFF Research Database (Denmark)


    ) transmitters. The payload and the label are encoded independently on optical carrier and subcarrier signals respectively, using electro-optical modulators. The invention applies single or double sideband carrier-suppressed modulation to generate subcarrier signals for encoding of the label. Thereby the payload......The present invention relates to optical labelling in WDM networks, in that it provides a method and a module to be used in subcarrier label generation and switching in network edge nodes and core switch nodes. The methods and modules are typically employed in Optical Subcarrier Multiplexing (OSCM...

  16. Organic labeling influences food valuation and choice. (United States)

    Linder, N S; Uhl, G; Fliessbach, K; Trautner, P; Elger, C E; Weber, B


    Everyday we choose between a variety of different food items trying to reach a decision that fits best our needs. These decisions are highly dependent on the context in which the alternatives are presented (e.g. labeling). We investigate the influence of cognition on food evaluation, using an fMRI experiment in which subjects saw and bid on different foods labeled with (or without) a widely known German emblem for organically produced food. Increased activity in the ventral striatum was found for foods labeled "organic" in comparison to conventionally labeled food. Between-subject differences in activity were related to actual everyday consumption behavior of organic food.

  17. Synthesis of tritium labelled 24-epibrassinolide

    Energy Technology Data Exchange (ETDEWEB)

    Kolbe, A.; Marquardt, V.; Adam, G. (Inst. of Plant Biochemistry Halle, Halle/Saale (Germany))


    Deuterium and tritium 5,7,7-tris-labelled 24-epibrassinolide were prepared by base catalyzed exchange reaction using 24-epicastasterone tetraacetate 1 or bis-isopropylidenedioxy-24-epicastasterone 8 and labelled water. Baeyer-Villiger oxidation of the obtained labelled 6-ketones 2 and 3 with CF[sub 3]CO[sub 3]H gave after alkaline deacetylation of the resulting 4 and 5 the desired tris-labelled 24-epibrassinolides 6 and 7, respectively, or starting from 9 under simultaneous oxidation and deprotection in one step the same final products. (author).

  18. Antibodies to watch in 2016. (United States)

    Reichert, Janice M


    The number of novel antibody therapeutics that received first marketing approvals in 2015 met expectations, with 6 (alirocumab (Praluent®), evolocumab (Repatha®), daratumumab (Darzalex®), dinutuximab (Unituxin®), idarucizumab (Praxbind®), mepolizumab (Nucala®)) granted first approvals as of mid-November*. Seven novel antibody therapeutics (begelomab, brodalumab, elotuzumab, ixekizumab, necitumumab, obiltoxaximab, reslizumab) are in regulatory review, and thus a similar number, if not more, are projected to gain first approvals in 2016. Commercial late-stage antibody therapeutics development exceeded expectations by increasing from 39 candidates in Phase 3 studies as of late 2014 to 53 as of late 2015. Of the 53 candidates, transitions to regulatory review by the end of 2016 are projected for 8 (atezolizumab, benralizumab, bimagrumab, durvalumab, inotuzumab ozogamicin, lebrikizumab, ocrelizumab, tremelimumab). Other "antibodies to watch" include 15 candidates (bavituximab, bococizumab, dupilumab, fasinumab, fulranumab, gevokizumab, guselkumab, ibalizumab, LY2951742, onartuzumab, REGN2222, roledumab, romosozumab, sirukumab, Xilonix) undergoing evaluation in Phase 3 studies that have estimated primary completion dates in 2016. As evidenced by the antibody therapeutics discussed in this perspective, the biopharmaceutical industry has a highly active late-stage clinical pipeline that may deliver numerous new products to the global market in the near future. *See Note added in proof for updates through December 31, 2015.

  19. Synthesis and labelling of epidepride

    Institute of Scientific and Technical Information of China (English)


    S-(-)-N-[(1-ethyl-2-pyrrolidinyl)methyl]-5-iodo-2,3-dimethoxybenzamide (proposed generic name, epidepride) is a very potent dopamine D2 antagonist. It was synthesized by five steps from 3-methoxysalicylic acid. [131I]epidepride was obtained in 97.3% radiochemical yields from the corresponding 5-(tributyltin) derivative using hydrogen peroxide as the oxidant. The aryltin precursor was prepared from non-labelled epidepride by palladium-catalyzed stannylation using bis(tri-n-butyltin) in triethylamine. [131I]epidepride was stable under 4℃, and partition coefficient was 72.3 at pH 7.40. The biodistribution study in rats exihibited high localization in the striatum of the brain with the striatum/cerebellum ratio reaching 237/1 at 320 min postinjection.All these results suggest that[131I]epidepride may be usedd widely as a useful dopamineD2 receptor imaging agent for SPECT.

  20. Principles of food product labelling

    Directory of Open Access Journals (Sweden)

    Krystyna Krysztofiak


    Full Text Available The purpose of the label of the food product is to provide information on ingredients and additionally on its origin, production method, storage conditions, date tagging, as well as to enable to identify the producer or distributor of this product. Legal regulations precisely give instructions on the range and the way of the presentation of these data, so they could be clear and understandable for the average consumer. Since 25th of November 2005, the information about allergens’ presence must be placed on the label, regardless of their content in the product (Directive 2003/89/WE... 2003 – Off. J. L 308: 15-18. The Regulation (WE No 1924/2006 about placing the nutritional information and medicinal claims concerning foods (Regulation (WE No 1924/2006... 2006 a is valid in all countries of European Union since 1st of July 2007 (Off. J. L 404: 9-25. It coordinates the legislative, executive and administrative regulations connected with this labelling. According to these regulations, “nutritional information” states, suggests or gives to understand that the food product has special properties concerning its ingredients. Those statements are of type: “the source of...”, “no... content”, “high content of...”, “low content of...”, “reduced content of...” with reference to calorie or selected ingredients’ content. “Medicinal claims” state, suggest or give to understand, that there is a connection between the food product or one of its ingredients and the health condition of the consumer. First type of these medicinal claims refers to the influence of the ingredient on the physiology. Such a statement is based on generally accepted scientific conclusions and could be properly understood by the average consumer, e.g. “calcium takes part in the process of building of strong bones”. “Statements about decreasing the risk of a disease” give information, that food product or one of its ingredients efficiently

  1. In vitro experimental {sup 211}At-anti-CD33 antibody therapy of leukaemia cells overcomes cellular resistance seen in vivo against gemtuzumab ozogamicin

    Energy Technology Data Exchange (ETDEWEB)

    Petrich, Thorsten; Korkmaz, Zekiye; Krull, Doris; Meyer, Geerd J.; Knapp, Wolfram H. [Hanover University School of Medicine, Department of Nuclear Medicine, Hanover (Germany); Froemke, Cornelia [Hanover University School of Medicine, Department of Biometry, Hanover (Germany)


    Monoclonal anti-CD33 antibodies conjugated with toxic calicheamicin derivative (gemtuzumab ozogamicin, GO) are a novel therapy option for acute myeloid leukaemia (AML). Key prognostic factors for patients with AML are high CD33 expression on the leukaemic cells and the ability to overcome mechanisms of resistance to cytotoxic chemotherapies, including drug efflux or other mechanisms decreasing apoptosis. Alpha particle-emitting radionuclides overwhelm such anti-apoptotic mechanisms by producing numerous DNA double-stranded breaks (DSBs) accompanied by decreased DNA repair. We labelled anti-CD33 antibodies with the alpha-emitter {sup 211}At and compared survival of leukaemic HL-60 and K-562 cells treated with the {sup 211}At-labelled antibodies, GO or unlabelled antibodies as controls. We also measured caspase-3/7 activity, DNA fragmentation and necrosis in HL-60 cells after treatment with the different antibodies or with free {sup 211}At. The mean labelling ratio of {sup 211}At-labelled antibodies was 1:1,090 {+-} 364 (range: 1:738-1:1,722) in comparison to 2-3:1 for GO. Tumour cell binding of {sup 211}At-anti-CD33 was high in the presence of abundant CD33 expression and could be specifically blocked by unlabelled anti-CD33. {sup 211}At-anti-CD33 decreased survival significantly more than did GO at comparable dilution (1:1,000). No significant differences in induction of apoptosis or necrosis or DNA DSB or in decreased survival were observed after {sup 211}At-anti-CD33 (1:1,090) versus GO (1:1) treatment. Our results suggest that {sup 211}At is a promising, highly cytotoxic radioimmunotherapy in CD33-positive leukaemia and kills tumour cells more efficiently than does calicheamicin-conjugated antibody. Labelling techniques leading to higher chemical yield and specific activities must be developed to increase {sup 211}At-anti-CD33 therapeutic effects. (orig.)

  2. F-18 labeled 3-fluorodiazepam

    Energy Technology Data Exchange (ETDEWEB)

    Luxen, A.; Barrio, J.R.; Bida, G.T.; Satyamurthy, N.; Phelps, M.E.


    3-Fluorodiazepam is a new and potent antianxiety agent with prolonged action. The authors found that molecular fluorine (0.5% in Ne) reacts cleanly with diazepam in freon or chloroform at room temperature to produce 3-fluorodiazepam in good yields. Successful syntheses have employed 2:1 to 5:1 molar ratios diazepam: fluorine to minimize the formation of byproducts. (/sup 18/F) 3-Fluorodiazepam, a potential candidate for PET studies, (specific activity 3-5 Ci/mmol) has been synthesized from /sup 18/F-F/sub 2/ using the same procedure, followed by column chromatographic purification (Silicagel, dichloromethane: ethyl acetate, 5:1) with a radiochemical yield of 12-20% (50% maximum) and a chemical and radiochemical purity >99% as judged by reversed-phase high pressure liquid chromatography analysis (Ultrasyl octyl column, 10 m, 4.6 x 250 mm i.d., 60% MeOH 40% water; flow rate, 1.0 ml/min; retention time for (/sup 18/F) fluorodiazepam, 11.4 min; for diazepam, 13.5 min; radioactivity and ultraviolet detectors). Lower radiochemical yields (5-7%), and significant formation of by-products were observed when (/sup 18/F)acetylhypofluorite, prepared in the gasphase, was used as the reagent. Readily accessible routes to /sup 18/F-labeled benzodiazepines of higher specific activity were also investigated. Approaches to the synthesis of high specific activity (>200 Ci/mmol) (/sup 18/F)3-fluorodiazepam involve nucleophilic displacement at carbon-3 (e.g. from 3-chlorodiazepam) with (/sup 18/F)fluoride ion. The results presented here demonstrate the synthetic accessibility of /sup 18/F-labeled benzodiazepines for application in neurotransmitter ligand studies with PET.

  3. Improved ELISA for determination of anti-diphtheria and/or anti-tetanus antitoxin antibodies in sera. (United States)

    Kristiansen, M; Aggerbeck, H; Heron, I


    Double-antigen ELISAs for detection and quantification of anti-tetanus or anti-diphtheria antibodies in serum have been developed. The assays showed good correlations with established toxin neutralizing assays and were functionally specific for IgG antibodies. The double-antigen set-up allows specific antibodies to bind to antigen-coated microtitre wells with one arm and the free arm to bind to biotin-labelled antigen. The amount of antibodies able to bind labelled antigen was assessed by adding enzyme-conjugated streptavidin and colour substrate followed by measurement of the colour using an ELISA reader. The double-antigen principle makes it possible to compare samples of different species on the same plate, permitting the direct use of existing international references of animal or human origin. The double-antigen ELISAs showed a detection limit of 0.00002 IU/ml for both antibodies and were suitable for quantifying antibodies in blood samples collected on filter paper as well as in serum. The assays required no special equipment compared to traditional ELISA.

  4. Antibodies to watch in 2013 (United States)

    Reichert, Janice M


    The transitions of antibody therapeutics to late-stage clinical development, regulatory review and the market are proceeding at a rapid pace in 2013. Since late 2012, two monoclonal antibody (mAb) therapeutics (itolizumab, trastuzumab emtansine) received their first approvals, first marketing applications for three mAbs (vedolizumab, ramucirumab, obinutuzumab) were submitted to regulatory agencies, and five mAbs (brodalumab, MABp1, moxetumomab pasudotox, tildrakizumab, rilotumumab) entered their first Phase 3 studies. The current total of commercially-sponsored antibody therapeutics undergoing evaluation in late-stage studies is 30. Recently announced study results for farletuzumab, naptumomab estafenatox, and tabalumab indicate that clinical endpoints were not met in some Phase 3 studies of these product candidates. PMID:23727858

  5. Epigenetics of the antibody response. (United States)

    Li, Guideng; Zan, Hong; Xu, Zhenming; Casali, Paolo


    Epigenetic marks, such as DNA methylation, histone post-translational modifications and miRNAs, are induced in B cells by the same stimuli that drive the antibody response. They play major roles in regulating somatic hypermutation (SHM), class switch DNA recombination (CSR), and differentiation to plasma cells or long-lived memory B cells. Histone modifications target the CSR and, possibly, SHM machinery to the immunoglobulin locus; they together with DNA methylation and miRNAs modulate the expression of critical elements of that machinery, such as activation-induced cytidine deaminase (AID), as well as factors central to plasma cell differentiation, such as B lymphocyte-induced maturation protein-1 (Blimp-1). These inducible B cell-intrinsic epigenetic marks instruct the maturation of antibody responses. Their dysregulation plays an important role in aberrant antibody responses to foreign antigens, such as those of microbial pathogens, and self-antigens, such as those targeted in autoimmunity, and B cell neoplasia.

  6. Autologous antibodies that bind neuroblastoma cells. (United States)

    Sun, Yujing; Sholler, Giselle S; Shukla, Girja S; Pero, Stephanie C; Carman, Chelsea L; Zhao, Ping; Krag, David N


    Antibody therapy of neuroblastoma is promising and our goal is to derive antibodies from patients with neuroblastoma for developing new therapeutic antibodies. The feasibility of using residual bone marrow obtained for clinical indications as a source of tumor cells and a source of antibodies was assessed. From marrow samples, neuroblastoma cells were recovered, grown in cell culture and also implanted into mice to create xenografts. Mononuclear cells from the marrow were used as a source to generate phage display antibody libraries and also hybridomas. Growth of neuroblastoma patient cells was possible both in vitro and as xenografts. Antibodies from the phage libraries and from the monoclonal hybridomas bound autologous neuroblastoma cells with some selectivity. It appears feasible to recover neuroblastoma cells from residual marrow specimens and to generate human antibodies that bind autologous neuroblastoma cells. Expansion of this approach is underway to collect more specimens, optimize methods to generate antibodies, and to evaluate the bioactivity of neuroblastoma-binding antibodies.

  7. Uses of monoclonal antibody 8H9 (United States)

    Cheung, Nai-Kong V.


    This invention provides a composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a suitable carrier. This invention provides a pharmaceutical composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a pharmaceutically acceptable carrier. This invention also provides an antibody other than the monoclonal antibody 8H9 comprising the complementary determining regions of monoclonal antibody 8H9 or a derivative thereof, capable of binding to the same antigen as the monoclonal antibody 8H9. This invention provides a substance capable of competitively inhibiting the binding of monoclonal antibody 8H9. This invention also provides an isolated scFv of monoclonal antibody 8H9 or a derivative thereof. This invention also provides the 8H9 antigen. This invention also provides different uses of the monoclonal antibody 8H9 or its derivative.

  8. A novel method for in Situ detection of hydrolyzable casein fragments in a cheese matrix by antibody phage display technique and CLSM

    DEFF Research Database (Denmark)

    Duan, Zhi; Brüggemann, Dagmar Adeline; Siegumfeldt, Henrik


    was successfully developed for the detection of scFvs binding to different alpha(s1)-casein fragments inside a cheese matrix by CLSM. To our knowledge, this is the first demonstrated immunofluorescent labeling method for in situ analysis of proteolysis phenomena in the cheese matrix. Additionally, this technique......A novel method to monitor in situ hydrolyzable casein fragments during cheese ripening by using immunofluorescent labeling and confocal laser scanning microscopy (CLSM) was developed. Monoclonal single chain variable fragments of antibody (scFvs) were generated by antibody phage display toward...

  9. Chain store management through private labels strategy

    Directory of Open Access Journals (Sweden)

    Martina Sopta


    Full Text Available The purpose of this paper is to examine the market shares of private labels in the European Union and on the global market, and to compare the results of the analysis with the level of presence of private labels on the Croatian market. Moreover, through the application of macro and microeconomic tools, the author tried to estimate the future trends of private labels in Croatia.For the purpose of the paper secondary and primary data was used in the research. Relevant scientific and professional literature of local and foreign authors was analyzed. In addition, a few recent research studies were analyzed and their results compared. Field research has been conducted by the survey method, with 225 respondents included in the intentional sample.The main hypothesis of the paper based on research is that, in total sales, private labels are gaining a growing share in all markets, regardless of the development level of those markets. Alongside the main hypothesis of the work, three supporting hypotheses were tested to see which private labels are a good alternative to other brands on the world market. Private labels are generally developed on generic products. The third supporting hypothesis starts from the assumption that the investments in the promotion of private labels are negligible, resulting in lower prices of thoseproducts. The results of research and analyses in the work indicate that the position of private labels will strengthen internationally, as part of the process of liberalization and globalization of trade flows. In the process of purchase of private labels the positioning of the point of sale and price have an increasing contribution. With the concentration of commerce in chain stores, the share of private labels grows, approaching a half of the total sales in some countries. Considering the Croatian market, according to the international product life cycle theory, the share of private labels in the total sales will grow in the future

  10. Use of AN Eosinophil Specific Monoclonal Antibody in Assessing Eosinophil Function. (United States)

    Minkoff, Marjorie Sue

    monoclonal antibody 2QD45 specifically immunoprecipitated the {rm M_ R} 67,000 ECF-binding protein from ^{125}{rm I}-labeled mouse, rat, and human eosinophils as assessed by SDS-PAGE and autoradiography. Two-dimensional gel electrophoresis showed that this ECF-binding protein has a lower PI point than either mouse or bovine albumin.

  11. Effects of dopamine concentration on energy transfer between dendrimer-QD and dye-labeled antibody

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Ki Rahm [Department of Chemical and Bio Engineering, Kyungwon University, Bokjung-dong, Sujung-gu, Sungnam-shi, Gyunggi-do 461-701 (Korea, Republic of); Kang, Ik-Joong, E-mail: [Department of Chemical and Bio Engineering, Kyungwon University, Bokjung-dong, Sujung-gu, Sungnam-shi, Gyunggi-do 461-701 (Korea, Republic of)


    The Unified Parkinson's Disease Rating Scale (UPDRS) is currently used to assess Parkinson's disease, and is a key method for determining the progression of disease based on the gross findings of patients. However, this method cannot quantify the extent of disease of patients, which means the administration of drugs cannot be determined on a real-time basis. Thalamotomy also causes discomfort and pain to the patients, and adversely affects treatment as it is performed following the onset of symptoms. Accordingly, the dopamine concentration, which is one of the key factors in determining this disease, needs to be detected quantitatively at ordinary times. Hence, the development of a bio-kit or a bio-sensor is essential for effectively prescribing the correct dopamine concentration in a customizable manner. In this study, the effect of dopamine level on this phenomenon was observed using the Forster resonance energy transfer (FRET) phenomenon generated between a donor and acceptor. By confirming the photoluminescence (PL) and lifetime data, it was demonstrated that the degree of energy transfer increased with increasing dopamine concentration. To apply this phenomenon to an optical sensor, a glass surface was modified with a quantum dot (QD)-encapsulated dendrimer, and analyzed using the contact angle and ATR-FTIR. The topology of surface was determined by an atomic force microscope (AFM).

  12. Comparative Analysis of Nanoparticle-Antibody Conjugations: Carbodiimide Versus Click Chemistry

    Directory of Open Access Journals (Sweden)

    Daniel L.J. Thorek


    Full Text Available The ability to modify the physical, chemical, and biologic properties of nanoparticles has led to their use as multifunctional platforms for drug delivery and diagnostic imaging applications. Typically, these applications involve functionalizing the nanoparticles with targeting agents. Antibodies remain an attractive choice as targeting agents because of their large epitope space and high affinity; however, implementation of antibody-nanoparticle conjugates is plagued by low coupling efficiencies and the high cost of reagents. Click chemistry may provide a solution to this problem, with reported coupling efficiencies nearing 100%. Although click chemistries have been used to functionalize nanoparticles with small molecules, they have not previously been used to functionalize nanoparticles with antibodies. Concerns associated with extending this procedure to antibodies are that reaction catalysts or the ligands required for cross-linking may result in loss of functionality. We evaluated the efficiency of conjugations between antibodies and superparamagnetic iron oxide nanoparticles using click chemistry as well as the functionality of the product. The results were compared with conjugates formed through carbodiimide cross-linking. The click reaction allowed for a higher extent and efficiency of labeling compared with carbodiimide, thus requiring less antibody. Further, conjugates prepared via the click reaction exhibited improved binding to target receptors.

  13. Antibody repertoire profiling using bacterial display identifies reactivity signatures of celiac disease. (United States)

    Spatola, Bradley N; Murray, Joseph A; Kagnoff, Martin; Kaukinen, Katri; Daugherty, Patrick S


    A general strategy to identify serum antibody specificities associated with a given disease state and peptide reagents for their detection was developed using bacterial display peptide libraries and multiparameter flow cytometry (MPFC). Using sera from patients with celiac disease (CD) (n = 45) or healthy subjects (n = 40), bacterial display libraries were screened for peptides that react specifically with antibodies from CD patients and not with those from healthy patients. The libraries were screened for peptides that simultaneously cross-react with CD patient antibodies present in two separate patient groups labeled with spectrally distinct fluorophores but do not react with unlabeled non-CD antibodies, thus affording a quantitative separation. A panel of six unique peptide sequences yielded 85% sensitivity and 91% specificity (AUC = 0.91) on a set of 60 samples not used for discovery, using leave-one-out cross-validation. Individual peptides were dissimilar with known CD-specific antigens tissue transglutaminase (tTG) and deamidated gliadin, and the classifier accuracy was independent of anti-tTG antibody titer. These results demonstrate that bacterial display/MPFC provides a highly effective tool for the unbiased discovery of disease-associated antibody specificities and peptide reagents for their detection that may have broad utility for diagnostic development.

  14. Recent Developments in Antibody-Based Assays for the Detection of Bacterial Toxins

    Directory of Open Access Journals (Sweden)

    Kui Zhu


    Full Text Available Considering the urgent demand for rapid and accurate determination of bacterial toxins and the recent promising developments in nanotechnology and microfluidics, this review summarizes new achievements of the past five years. Firstly, bacterial toxins will be categorized according to their antibody binding properties into low and high molecular weight compounds. Secondly, the types of antibodies and new techniques for producing antibodies are discussed, including poly- and mono-clonal antibodies, single-chain variable fragments (scFv, as well as heavy-chain and recombinant antibodies. Thirdly, the use of different nanomaterials, such as gold nanoparticles (AuNPs, magnetic nanoparticles (MNPs, quantum dots (QDs and carbon nanomaterials (graphene and carbon nanotube, for labeling antibodies and toxins or for readout techniques will be summarized. Fourthly, microscale analysis or minimized devices, for example microfluidics or lab-on-a-chip (LOC, which have attracted increasing attention in combination with immunoassays for the robust detection or point-of-care testing (POCT, will be reviewed. Finally, some new materials and analytical strategies, which might be promising for analyzing toxins in the near future, will be shortly introduced.

  15. Recent developments in antibody-based assays for the detection of bacterial toxins. (United States)

    Zhu, Kui; Dietrich, Richard; Didier, Andrea; Doyscher, Dominik; Märtlbauer, Erwin


    Considering the urgent demand for rapid and accurate determination of bacterial toxins and the recent promising developments in nanotechnology and microfluidics, this review summarizes new achievements of the past five years. Firstly, bacterial toxins will be categorized according to their antibody binding properties into low and high molecular weight compounds. Secondly, the types of antibodies and new techniques for producing antibodies are discussed, including poly- and mono-clonal antibodies, single-chain variable fragments (scFv), as well as heavy-chain and recombinant antibodies. Thirdly, the use of different nanomaterials, such as gold nanoparticles (AuNPs), magnetic nanoparticles (MNPs), quantum dots (QDs) and carbon nanomaterials (graphene and carbon nanotube), for labeling antibodies and toxins or for readout techniques will be summarized. Fourthly, microscale analysis or minimized devices, for example microfluidics or lab-on-a-chip (LOC), which have attracted increasing attention in combination with immunoassays for the robust detection or point-of-care testing (POCT), will be reviewed. Finally, some new materials and analytical strategies, which might be promising for analyzing toxins in the near future, will be shortly introduced.

  16. 76 FR 46671 - Food Labeling; Gluten-Free Labeling of Foods; Reopening of the Comment Period (United States)


    ... HUMAN SERVICES Food and Drug Administration 21 CFR Part 101 RIN 0910-ZA26 Food Labeling; Gluten-Free... the comment period for the proposed rule on the ``gluten-free'' labeling of foods, published in the... ``gluten- free,'' for voluntary use in the labeling of foods, to mean that the food does not contain...

  17. Portion Size Labeling and Intended Soft Drink Consumption: The Impact of Labeling Format and Size Portfolio (United States)

    Vermeer, Willemijn M.; Steenhuis, Ingrid H. M.; Leeuwis, Franca H.; Bos, Arjan E. R.; de Boer, Michiel; Seidell, Jacob C.


    Objective: To assess what portion size labeling "format" is most promising in helping consumers selecting appropriate soft drink sizes, and whether labeling impact depends on the size portfolio. Methods: An experimental study was conducted in fast-food restaurants in which 2 labeling formats (ie, reference portion size and small/medium/large…

  18. Antibody profiling sensitivity through increased reporter antibody layering

    Energy Technology Data Exchange (ETDEWEB)

    Apel, William A.; Thompson, Vicki S.


    A method for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an embodiment of the invention, the analyte is a drug, such as marijuana, Cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method comprises attaching antigens to a surface of a solid support in a preselected pattern to form an array wherein locations of the antigens are known; contacting the array with the biological sample such that a portion of antibodies in the sample reacts with and binds to the antigens in the array to form immune complexes; washing away antibodies that do form immune complexes; and detecting the immune complexes, to form an antibody profile. Forensic samples are identified by comparing a sample from an unknown source with a sample from a known source. Further, an assay, such as a test for illegal drug use, can be coupled to a test for identity such that the results of the assay can be positively correlated to the subject's identity.

  19. Antibody profiling sensitivity through increased reporter antibody layering

    Energy Technology Data Exchange (ETDEWEB)

    Apel, William A; Thompson, Vicki S


    A method for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an embodiment of the invention, the analyte is a drug, such as marijuana, Cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method comprises attaching antigens to a surface of a solid support in a preselected pattern to form an array wherein locations of the antigens are known; contacting the array with the biological sample such that a portion of antibodies in the sample reacts with and binds to the antigens in the array to form immune complexes; washing away antibodies that do form immune complexes; and detecting the immune complexes, to form an antibody profile. Forensic samples are identified by comparing a sample from an unknown source with a sample from a known source. Further, an assay, such as a test for illegal drug use, can be coupled to a test for identity such that the results of the assay can be positively correlated to the subject's identity.

  20. Antibody profiling sensitivity through increased reporter antibody layering

    Energy Technology Data Exchange (ETDEWEB)

    Apel, William A.; Thompson, Vicki S.


    A method for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an embodiment of the invention, the analyte is a drug, such as marijuana, Cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method comprises attaching antigens to a surface of a solid support in a preselected pattern to form an array wherein locations of the antigens are known; contacting the array with the biological sample such that a portion of antibodies in the sample reacts with and binds to the antigens in the array to form immune complexes; washing away antibodies that do form immune complexes; and detecting the immune complexes, to form an antibody profile. Forensic samples are identified by comparing a sample from an unknown source with a sample from a known source. Further, an assay, such as a test for illegal drug use, can be coupled to a test for identity such that the results of the assay can be positively correlated to the subject's identity.